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(\Eg LIBRARY Michigan State University This is to certify that the thesis entitled EVALUATION OF ALTERATIONS IN DNA METHYLATION ASSOCIATED WITH THE 2,3,7,8-TETRACHLORODlBENZO-p-DIOXIN (TCDD)- INDUCED INHIBITION OF DIFFERENTIATION IN LIPOPOLYSACCHARIDE (LPS)-STIMULATED MURINE SPLENOCYTES presented by Emily Ann McClure has been accepted towards fulfillment of the requirements for the Master of degree in Microbiology and Molecular Science Genetics MW Major Professor’ 5 Signature 577/” Date MSU is an Affirmative Action/Equal Opportunity Employer v n sic—z ‘ PLACE IN RETURN BOX to remove this checkout from your record. To AVOID FINES return on or before date due. MAY BE RECALLED with earlier due date if requested. DATE DUE DATE DUE DATE DUE 5/08 KrlProleocaPrelelRC/DateDue.indd EVALUATION OF ALTERATIONS IN DNA METHYLATION ASSOCIATED WITH THE 2,3,7,8-TETRACHLORODIBENZO—p-DIOXIN (TCDD)-INDUCED INHIBITION OF DIFFERENTIATION IN LIPOPOLYSACCHARIDE (LPS)—STIMULATED MURINE SPLENOCYTES By Emily Ann McClure A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree Of MASTER OF SCIENCE Microbiology and Molecular Genetics 2010 ABSTRACT EVALUATION OF ALTERATIONS IN DNA METHYLATION ASSOCIATED WITH THE 2,3,7,8-TETRACHLORODIBENZO-p-DIOXIN (TCDD)-INDUCED INHIBITION OF DIFFERENTIATION IN LIPOPOLYSACCHARIDE (LPS)—STIMULATED MURINE SPLENOCYTES By Emily Ann McClure Splenic B-cells isolated from mice injected with LPS, differentiate into antibody- producing plasma cells in vitro. Pretreatment with TCDD, a potent immunosuppressive agent, impairs B-cell differentiation. Altered DNA methylation, an epigenetic event, may play a key role in this impairment. DNA was isolated from splenocytes prepared 6 days post experiment initiation from 5-6wk old female C57BL/6 mice dosed with: TCDD, 3 or 30,ug/kg, on day 0; or LPS, Zing/mouse, on day 4; or sequentially with TCDD and then LPS on days 0 and 4, respectively. TO discern regions of altered DNA methylation (RAMs), DNA was restricted with HpaII (a methylation sensitive enzyme), followed by arbitrarily primed PCR and capillary electrophoresis. The mRNA expression of selected genes (annotated RAMs or genes closely affected by them) that might affect B-cell differentiation were analyzed using qRT-PCR. LPS, 3, or 30pg/kg TCDD alone resulted in 40, 43 and 42 RAMS, respectively, while LPS challenge subsequent to 3 or 30pg/kg TCDD resulted in 34 and 39 RAMS, respectively. Interestingly, the combined treatments lead to many RAMs Observed in single treatments but also many unique RAMs. Three patterns Of mRNA expression were observed: no change, similar changes in all groups, and different changes based upon treatment. Collectively, this research suggests a novel epigenetic mechanism potentially regulating gene expression stimulated by LPS and TCDD exposure and important in the differentiation of B—cells in the splenocyte population. I dedicate this work to all those who labor to the benefit Of others. There is no psychiatrist in the world like a puppy licking your face -Ben Williams iii ACKNOWLEDGMENTS Above all others, I wish to thank my ’family’, without whom my years at MSU would not be possible: My mother and father for supporting me through my entire education. My mother for being a sounding board when nothing seemed to go right. My siblings and extended family for pretending to be interested and continuing to ask about my research. The Waldos for feeding me, housing me, and adopting me as their own. The Lansing Paws family for listening to my rants and celebrating my successes. Thank you for helping me to raise Phoenix, Henna, Porter, and Scarlett. Carol for playdates, walks, chats, and the occassional beer. Susan Barman, for mentoring and support. I also thank my fellow microbiology graduate students and all those who ever taken time to study with me (especially members of BMB801 and 802). This work has been funded by National Institutes of Health superfund grant P42 E80491]. Finally, I thank Michigan State University for the Opportunity to study at this fine insti- tution. iv TABLE OF CONTENTS Page LIST OF TABLES ................................. vii LIST OF FIGURES ................................ viii LIST OF ABBREVIATIONS ........................... xvi 1 Introduction .................................. 1 1.1 B-cell Differentiation and Maturation .................. 3 1.1.1 Transcription Factors ....................... 6 1.1.2 Cytokines ............................ 9 1.1.3 Calcium ............................. 9 1.1.4 LPS Signaling .......................... 10 1.1.5 NFKB Signaling ......................... 15 1.1.6 Akt Signaling .......................... 15 1.1.7 BCR Signaling .......................... 17 1.2 TCDD .................................. 18 1.2.1 TCDD Signaling ......................... 18 1.3 Epigenetics ................................ 22 1.3.1 Small Non-coding RNA ..................... 23 1.3.2 Histone Code .......................... 24 1.3.3 Tissue-specific Transcription Factors ............... 24 1.3.4 DNA Methylation ........................ 25 1.4 Hypothesis ................................ 28 2 DNA methylation: a potential mechanism of crosstalk occurring in murine spleno- cytes exposed in vivo to lipopolysaccharide (LPS) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) .................. 30 2.1 Abstract ................................. 30 2.2 Introduction ............................... 31 2.3 Materials and Methods .......................... 35 2.3.1 Preparation of in vivo Splenocyte Samples ............ 35 2.3.2 Evaluation Of DNA Methylation Status by AP-PCR and CE . . . 36 2.3.3 Cloning and Annotation of RAMs ................ 38 2.4 Results .................................. 41 2.4.1 RAM Identification ....................... 41 2.4.2 RAM Annotation ........................ 45 2.4.3 DAVID and GO Analysis .................... 51 2.4.4 Annotated Gene Interaction Analysis .............. 51 2.5 Discussion ................................ 56 Page 3 Evaluation of alterations in gene expression in those genes exhibiting altered DNA methylation in murine splenocytes exposed in vivo to lipopolysaccharide (LPS) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) ............ 60 3.1 Abstract ................................. 60 3.2 Introduction ............................... 61 3.3 Materials and Methods .......................... 65 3.3.1 Preparation of in viva Splenocyte Samples ............ 65 3.3.2 qRT-PCR ............................. 66 3.4 Results and Discussion .......................... 70 3.4.1 Genes Involved in LPS Signaling ................ 71 3.4.2 Genes Involved in TCDD Signaling ............... 73 3.4.3 Genes Involved in LPS Signaling and Affected by TCDD exposure 76 3.4.4 Conclusion ............................ 81 4 Additional Results ............................... 83 5 Summary .................................... 85 Appendix A: Supplemental Tables ....................... 88 Appendix B: Supplemental Figures ...................... 113 LIST OF REFERENCES ............................. 116 vi LIST OF TABLES Table Page 2.1 Solo treatment annotated genes ....................... 47 2.2 Combined treatment annotated genes .................... 49 2.3 Functional groups represented by annotated genes ............. 52 3.1 Treatment-related changes in gene expression ................ 71 8.] Primer sequences .............................. 89 8.2 RAMs to which genes annotate ....................... 90 8.3 Signaling pathways and cellular processes represented by annotated genes . 103 8.4 Fold change in mRNA expression Of 14 selected genes as measured using qRT- PCR ..................................... 107 S5 Literature references for annotated genes .................. 109 vii LIST OF FIGURES Figure Page 1.1 Schematic representation of reciprocal repression of transcription factors regulating IgM expression in differentiating B-cells ................... 7 1.2 Signaling occurring in B-cells as a result of LPS binding to TLR4 ...... 12 1.3 Schematic representation of Akt signaling .................. 16 1.4 TCDD signaling through the AhR ...................... 19 2.1 AFCs as a function of treatment with LPS and TCDD ............ 32 2.2 RAMs in splenocytes ............................ 43 2.3 Common targets of annotated genes ..................... 54 2.4 Hypothesized regulatory interactions in splenocytes following LPS and 30pg/kg TCDD+LPS treatments ........................... 55 3.1 Treatment-related changes in Splenocyte gene expression .......... 72 3.2 Hypothesized significance of interactions occurring in differentiating spleno- cytes ..................................... 79 SJ Classification Of RAM annotation based upon genomic location, as determined by BLAT search ............................... 113 S2 Regulatory interactions of annotated genes in 25,ug LPS treatment ...... 114 83 Regulatory interactions of annotated genes in 30yg/kg TCDD treatment. . . 114 8.4 Regulatory interactions of annotated genes in 30pg/kg TCDD+LPS treatment. 115 viii LIST OF ABBREVIATIONS A AdcyS Adenylate cyclase type 5. AFCS Antigen Forming Colonies. Ag antigen. AhR aryl-hydrocarbon receptor. AIP aryl-hydrocarbon receptor interacting protein. Akt protein kinase B. A1dh3a1 aldehyde dehydrogenase 3 family, member A1. AP-l activator protein 1. AP-PCR Arbitrarily Primed PCR. ARE aryl-hydrocarbon response element. ARNT aryl-hydrocarbon nuclear translocator. ASC antibody-secreting cell. B BACH2 BTB and CNC homology 2. BAFF B-cell activating factor. BAFFR B-cell activating factor (BAFF) receptor. Bank] B-cell scaffold protein with ankyrin repeats. Bel-x1 B-cell lymphoma-extra large. Bc16 B-cell lymphoma 6 protein. Bcor Bcl6 co-repressor. BCR B-cell receptor. ix Bim Bc12-like protein 1 1. BLC B-lymphocyte chemoattractant. Blimp] B-lymphocyte—induced maturation protein 1. BLN K B-cell linker protein. Btk Bruton’s tyrosine kinase. C C32+ calcium. CadpsZ Calcium-dependent secretion activator 2. CaM calmodulin. CCLl chemokine (C-C motif) ligand 1. CD14 cluster of differentiation 14. CE Capillary Electrophoresis. CREB CAMP response element-binding protein. Cypl cytochrome P450, family 1. Cyplal cytochrome P450, family 1, subfamily A, polypeptide 1. D DAG diacylglycerol. DAVID Database for Annotation, Visualization and Integrated Discovery. DD death domain. Ddx54 Dead box 54. DNMT DNA methyltransferase. DRE dioxin response element. E EBF early B-cell factor. Ebfl transcription factor COEl. ER endoplasmic reticulum. EREG epiregulin. ERK extracellular signal-regulated kinase. EsR estrogen receptor. Etsl v-ets erythroblastosis virus E26 oncogene homolog 1. F Flil friend leukemia virus integration 1. FoxO forkhead box protein 0. G GO Gene Ontology. GPCR G-Protein Coupled Receptor. GSK-3B glycogen synthase kinase 30. Gstal glutathione S-transferase, alpha 1 (Ya). H HDAC histone deacetylase. Hie-1 hypermethylated in cancer- 1. Hrs hepatocyte growth factor-regulated tyrosine kinase substrate. Hsp90 heat shock protein 90kDa. IKB inhibitor of nuclear factor Of K light chain gene enhancer in B-cells. xi IgJ immunoglobulin J. IgM immunoglobulin M. [K inhibitor of nuclear factor of K light chain gene enhancer in B-cells (IKB) kinase. IL-l interleukin-1 . IL-8 interleukin-8. Ill7rd Interleukin-17 receptor D. ILL innate-like lymphocyte. 1P3 inositol triphosphate. IP3R inositol triphosphate receptor. IRAK interleukin-1 (IL-1) receptor associated kinase. IRF3 interferon regulatory factor 3. IRF4 interferon regulatory factor 4. JNK c-Jun N-terminal kinase. K Krrl small subunit processome component homolog. L LBP lipopolysaccharide (LPS) binding protein. LINE long interspersed nuclear element. LPS lipopolysaccharide. Lyn V-yes-l Yamaguchi sarcoma viral related oncogene homolog. M xii m¢ macrophages. MAPK mitogen-activated protein kinase. MDl lymphocyte antigen 86. MD2 lymphocyte antigen 96. Mdm2 murine double minute 2 oncogene. MHC major histocompatability complex. MITF microphthalmia-associated transcription factor. mTOR mammalian target of rapamycin. MyD88 Myeloid differentiation primary response gene 88. Ml marginal zone. N NFKB nuclear factor K-light—chain-enhancer of activated B-cells. NK natural killer cell. Nrf2 NF-E2 related factor 2. P p23 heat shock protein co-chaperone p23/prostaglandin E synthase 3. p27kip1 cyclin-dependent kinase inhibitor 18. p300 ElA binding protein p300. p38 p38 mitogen-activated protein kinase (MAPK). p53 tumor protein 53. PAMPS pathogen-associated molecular patterns. PaxS paired box protein 5. PBS phosphate buffered saline. xiii PDK pyruvate dehydrogenase kinase. PDKl 3-phosphoinositide dependent protein kinase- 1. Phlpp PH domain and leucine rich repeat protein phosphatase. PI3K phosphoinositide 3-kinase. PIP2 phosphatidylinositol (4,5)-bisphosphate. PIP3 phosphatidylinositol (3,4,5)-triphosphate. PKC protein kinase C. PLC phospholipase C. PLCY phospholipase C y. PRRs pattern recognition receptors. PTEN phosphatase and tensin homolog. Ptpn3 Tyrosine-protein phosphatase non-receptor type 3. PU.1 transcription factor PU. 1. Q qRT-PCR quantitative reverse transcription PCR. R RAG recombination activating gene. Ralgds Ral guanine nucleotide dissociation stimulator. RAM region of altered DNA methylation. RAMs regions of altered DNA methylation. Rara retinoic acid receptor 0t. RB retinoblastoma protein. REN renin. xiv RIP receptor-interacting serine-threonine kinase. RP105 lymphocyte antigen 64, CD180 molecule. S SAM S-adenosyl methionine. SpiB Spi-B transcription factor. SRBC sheep red blood cell. STATS signal transducer and activator of transcription 5. Syk spleen tyrosine kinase. T TAKl transforming growth factor B (TGFB) activated kinase 1. TBKl TANK-binding Kinase 1. TBP TATA-binding protein. TCDD 2,3,7,8-tetrachlorodibenzo-p-dioxin. ch T-cell factor/transcription factor. TGFI3 transforming growth factor [3. TI T-cell independent. TIR toll/interleukin-l receptor. TIRAP toll/interleukin-l receptor (TIR) domain containing adaptor protein. TLR4 toll-like receptor 4. TLRs toll-like receptors. TNF tumor necrosis factor. TRAF 6 tumor necrosis factor (TN F) receptor associated factor 6. XV TRAM adaptor molecule. TRIF TIR domain-containing adaptor-inducing interferon-B. TSC2 tuberous sclerosis complex 2. TSS transcriptional start site. U Ube216 Ubiquitin/ISGIS-conjugating enzyme E2 L6. Ube3A ubiquitin protein ligase E3A. W WT wild type. X Xbpl X-box binding protein 1. Z pr128 zinc finger protein 128. xvi TRIF-related 1. INTRODUCTION Foreign antigen protection in vertebrates is directed by a multicomponent immune system. Immune responses are divided into two main categories: innate and adaptive. These two immune responses work synergistically and in tandem to protect the animal from foreign pathogens and toxicants, e.g. bacteria, viral capsids, and venoms. Attenuation of immune response(s) results in increased incidence of illness and death in response to infection. Hypersensitivity results in varied responses ranging from inflammation and allergic reaction to (when allowed to continue out of control) death. The adaptive immune system is composed of T-cells and B-cells specialized to elimi- nate and neutralize antigens as well as to maintain memory of previous exposure. The response is highly specific, with each mature T- or B-cell recognizing only one antigen. Successful antigen recognition results in rapid activation and proliferation of stimulated lymphocytes. It is this response that is targeted in vaccinations and efficiently clears foreign antigens from the body due to the specificity of the response. However, an adaptive immune response is not fully triggered until 296hrs after initial infection. The one exception to this rule is allergic reaction, which is rapidly triggered by memory cells in response to antigen exposure after an initial sensitizing exposure. As the animals used in this study were kept in clean rooms and exposed to neither LPS nor 2,3,7,8-tetrachlorodibenzo—p-dioxin (TCDD) prior to experimental administration, it is unlikely that any of the reactions observed will be a result Of allergic reaction. Immune responses occurring before the adaptive response belong to the innate immune response. The innate immune response mounts the first response to foreign antigens. These first responses occur within 4hrs of exposure and are mediated by natural killer cells (NKS), dendritic cells, macrophagess (mOs), neutrophils, and innate-like lymphocytes (ILLS). LPS in the outer membrane of gram-negative bacteria, bacterial flagella, unmethylated CpG sequences in bacterial DNA, and viral double-stranded RNA contain repetitive structures known as pathogen—associated molecular patterns (PAMPS) which are recognized by cells of the innate immune system through pattern recognition receptors (PRRS). Many of the the cells involved in innate immunity mature and are initially activated within the spleen. The Spleen filters and recognizes foreign antigens from the blood while also serving as a repository for monocytes and a location for the maturation of B- and T- cells. In fact, the average spleen is composed of 45-50% B-cells (Crawford and Kaminski, unpublished data). Many of the pathways commonly hypothesized to occur in B-cells are not actually observed nor described in B-cell populations, although they are likely relevant as they have been Observed in multiple cell lineages (mOS, dendritic cells, and cultured cell lines) (Peng, 2005). For this reason, and the high concentration of B-cells regularly isolated from the spleen, this research will focus primarily on B-cells and the potential interactions occurring therein as a response to LPS challenge and TCDD exposure. Triggering of many signaling pathways is critical to mounting appropriate innate and adaptive immune responses. Inappropriate signaling through altered regulation of lineage, development, or differentiation-specific genes (especially transcription factors) leads to inappropriate immune responses varying from senescence to lymphoma. TCDD exposure prior to LPS stimulation causes animals and immune cell lines to fail in mounting appropriate response(s) (Agency for Toxic Substances and Disease Registry (ATSDR), 1998). TCDD’s efficient inhibition of innate (and adaptive) immunity can be used to assess mechanisms necessary for the differentiation of immunologic cells. Current knowledge indicates that TCDD-induced suppression of the primary humoral immune response occurs upstream of antibody (immunoglobulin M (IgM)) production and B-lymphocyte-induced maturation protein 1 (Blimpl) upregulation (North et al., 2009). 2 Where in the upstream signaling pathways TCDD inhibits signaling for future Blimpl expression and whether this inhibition is directed by genetic or epigenetic mechnanisms is not yet understood. Embryonic exposure to TCDD increases DNA methylation and decreases expression of imprinted H19 and Igf2 genes (Wu et al., 2004) while also inducing cytochrome P450, family 1, subfamily A, polypeptide l (Cyplal). These changes in DNA methylation and gene expression are accompanied by an increase in methyl transferase activity (Wu et al., 2004). This research will further these results by investigating changes in DNA methylation resulting from LPS and TCDD exposure and evaluating whether concomitant exposure alters the methylation patterns induced by either treatment in isolation. 1.1 B-cell Differentiation and Maturation B-cells are members Of the immune system whose different subsets perform various roles associated with innate immunity (within 4hrs of exposure), the early induced innate response (4-96hrs Of exposure), and the adaptive immune response (Z96hrs after exposure). B-cells begin development as hematopoeitic stem cells in the bone marrow and differen- tiate into multipotent progenitor cells. Multipotent progenitor cells differentiate into early lymphoid progenitor cells, which further differentiate into common lymphoid progenitors, the progenitor cell population from which B-cells, T-cells, and NKs derive. B-cells are further subdivided into two groups: Bl and 82 B-cells. Bl B-cells comprise ~5% of all B-cells in mice and appear first during fetal develop- ment. In adult mice, Bl B-cells are found in the spleen and intestine, as well as the peritoneal and pleural cavities (Kantor and Herzenberg, 1993). Because they represent such a small portion of B-cells and because they are mostly found in peritoneal and pleural cavities, the development of B1 B-cells is beyond the scope of this research. Non self-reactive, surface IgM-expressing B-cells (B2 B-cells) generated in the bone marrow migrate to the spleen and undergo further maturation into marginal zone (M2) and follicular B—cells (Pillai and Cariappa, 2009; Dorshkind and Montecino-Rodriguez, 2007). In secondary lymphoid tissues, differentiation is divided into 3 phases: pre-germinal center, germinal center, and post-germinal center stages. MZ B-cells do not recirculate like B1 B- cells, rather they react to pathogens trapped by m¢s in the spleen MZ and likely are uniquely adapted to provide the first response to pathogens reaching the bloodstream. These resting mature B-cells express low CD23, high MHCI, CD35, and CD21 and do not require T-cell help for activation. In rodents, IgM+CD27+ MZ B-cells are restricted to the spleen (Pillai and Cariappa, 2009). During development, immunoglobulin rearrangement begins in pro-B-cells in the bone marrow and regulates the formation Of plasma cells and memory cells. IL-7 signaling promotes E2A expression, which cooperates with transcription factor PU. 1 (PU. 1) to induce early B-cell factor (EBF) expression. E2A and EBF work together to drive expression Of pro-B proteins such as recombination activating gene (RAG)1, RAG2, and paired box protein 5 (Pax5) (Gupta et al., 2007). Pax5 induces B-cell linker protein (BLN K) expression (Gupta et al., 2007) (see section 1.1.5 for BLNK signaling). Transcription factors Pax5, B- cell lymphoma 6 protein (Bcl6), microphthalmia-associated transcription factor (MITF), v- ets erythroblastosis virus E26 oncogene homolog 1 (Etsl), and BTB and CNC homology 2 (BACH2) repress antibody-secreting cell (ASC) differentiation (Shapiro-Shelef and Calame, 2005). RAG genes induce V(D)J rearrangement in B- and T-cells and are expressed at two times during B-cell differentiation in order to rearrange the heavy and light chains Of the 4 B-cell receptor (BCR). VpreBl and AS gene protein products associate to form a surro- gate light chain which chaperones newly synthesized ,u chains to the cell surface in the form of the pre-BCR (Szutorisz et al., 2005). Signaling from this receptor then mediates Signaling leading to proliferation of pre-B-cells with productive heavy chain rearrange- ment. Immature B-cells expressing rearranged BCR are tested for self-reactivity in bone marrow before localization to the spleen to complete differentiation. Naive B-cells with appropriate in-frame immunoglobulin rearrangements travel to and mature in the spleen (Cariappa and Pillai, 2002). IgM+IgD— immature B-cells in bone marrow acquire the ability to emigrate to spleen red pulp and become IthiIgDIOCDzllOCDB— newly formed B-cells (Cariappa and Pillai, 2002). At high concentrations, T-cell independent (TI)—1 antigens (e.g. LPS) do not require T-cell help to induce polyclonal activation Of B-cells. TI-2 antigens (capsular polysaccharides) act by cross-linking the BCR, although excessive crosslinking causes B-cells to become anergic. Dendritic cells and macrophages provide co-stimulatory molecule BAFF which is recognized by the BAFF receptor (BAFFR) and plays an important role in directing follicular B—cell survival. Immature B-cells remain for ~l day in spleen red pulp, then colonize spleen lymphoid follicles where they express high levels of IgD and CD23 (Cariappa and Pillai, 2002). A proportion of follicular B—cells express intermediate levels Of CD21 and acquire the ability to recirculate, giving rise to IgDhiIgMIOCD23+CD21im mature recirculating naive follicular B-cells (Cariappa and Pillai, 2002). A subpopulation of IthiIgDhi folliclar B—cells express high levels of CD21 and CDld and represent extrafollicular IthiIgD_CD23_CD21hiCDldhi MZ B-cell precursors (Cariappa and Pillai, 2002). Due to selective stringency at many stages Of development, only an estimated ~10% of 15-20 million immature B-cells produced each day in the bone marrow emerge as mature cells in the periphery (Cariappa and Pillai, 2002). Terminally differentiated B-cells include plasmablasts, plasma cells, and memory cells and are found in lymphoid organ germinal centers or circulating. Plasmablasts express high levels of surface IgM, surface major histocompatability complex (MHC) II, and secrete high levels of IgM. Plasma cells express low levels of surface IgM, no surface MHC H, and secrete high levels of IgM. Development of plasma cells and plasmablasts is antigen (Ag) driven (Klein and Dalla-Favera, 2008). Especially important in regulating the production and appropriate folding of immunoglobulin proteins, endoplasmic reticulum (ER) resident folding factors (including X-box binding protein 1 (Xbpl )) and redox balance proteins are linearly upregulated until the end of plasma cell differentiation. IgM subunit expressions increase exponentially 2 days after initial Ag exposure, during the early induced innate immune response (van Anken et al., 2003). 1.1.1 'h'anscription Factors Waves of lineage-specific transcription factors regulate B-cell terminal differentiation. The main regulatory transcription factors controlling B-cell terminal differentiation (Pax5, Blimp] , and Bcl6) compose a reciprocally inhibiting transcriptional ‘Switch.’ Manipulation of mRNA levels or activation Status of any one of these three transcription factors results in significant changes in gene expression as well as cell fate. Initial entry of common lymphoid progenitors into the B-cell lineage depends on the appropriate expression of E2A, transcription factor COEl (Ebfl), and Pax5 (Nutt and Kee, 2007; Cobaleda et al., 2007). Friend leukemia virus integration 1 (Flil) positively regulates expression Of E2A proteins (Zhang et al., 2008). E2A proteins, in concert with ow \./ Figure 1.1.: Schematic Representation of Reciprocal Repression of Transcription Factors Regulating IgM Expression in Differentiating B-cells. Bc16 inhibits AP-l binding to the Blimpl promoter and so represses its transcription. Blimpl (a reciprocal repressor of Pax5 expression) is a transcription factor necessary for full induction of IgH and J chain and Xbpl mRNA in B—cells. Xbpl regulates the transcription of folding proteins as well as many B-cell receptor genes. . other transcription factors and signal transducer and activator of transcription 5 (STATS) promote Ebfl and c-Myc expression (Kee, 2009). Ebf 1, in turn, promotes Pax5 and E2A expression. Pax5 promotes Ebfl expression and represses Notchl transcription, preventing T-cell fate in pro-B-cells (active Notchl leads to E2A degradation and so inhibits B-cell transcription program maintenance) (Kee, 2009). Ebfl regulates Pax5 expression through chromatin remodeling of the entire Pax5 promoter region (Decker et al., 2009). The Pax5 promoter is also epigenetically regulated by DNA methylation in embryonic stem cells and mouse embryo fibroblasts. The Pax5 enhancer is demethylated upon the onset of hematopoeisis and organized into accessible chromatin at subsequent B-cell developmental stages (Decker etal., 2009). Blimpl (a reciprocal repressor of Pax5 expression) is a transcription factor necessary for full induction of IgH and J chain and Xbpl mRNA in B-cells (Savitslcy and Calame, 2006) (Figure 1.1). Upon activation, Blimpl represses genes required for cell cycle entry, DNA replication, and cell division (Savitsky and Calame, 2006). It is also a reciprocal repressor of Bcl6. Bcl6 inhibits AP-l binding to the Blimpl promoter and so represses its transcrip— tion (Vasanwala et al., 2002) (Figure 1.1). Mice deficient in Bcl6 lack germinal centers in secondary lymphoid organs, indicating a complete repression of B-cell differentiation (Dent et al., 1997). Bcl6 co-repressor (Bcor) functions in concert with Bc16 to repress transcription of many genes, potentially through epigenetic mechanisms such as histone methylation (Fan et al., 2009). These mechanisms direct gene silencing through chromatin modification by means Of histone ubiquitination and demethylation (Gearhart et al., 2006). When expressed, Pax5 works as a global regulatory element, controlling commitment tO B-cell development by repressing B-lineage inappropriate genes and activating B—lineage specific genes. It is expressed exclusively from the pro-B-cell to mature B-cell stages and subsequently repressed during terminal plasma cell differentiation (Fuxa and Busslinger, 2007). Reprogramming induced by Pax5 transcriptional activity facilitates pre-B-cell receptor signaling, promotes B-lymphocyte differentiation, and regulates B-cell adhesion and migration (Delogu et al., 2006; Schebesta et al., 2007). Much of this reprogramming occurs as a result of Pax5 inhibiting the expression of Blimpl (Mora—Lopez et al., 2007) (Shaffer et al., 2002) (Figure 1.1). Xbpl regulates the transcription of folding proteins as well as many B-cell receptor genes (Figure 1.1). Those B-cells lacking Xbpl fail to secrete IgM (a marker indicative of successful plasma cell differentiation) (Masciarelli et al., 2010). Expression levels of Xbpl reach high levels in primary Splenic B-cells and B-cell lymphoma lines after 3 days of activation with LPS (van Anken et al., 2003; Calfon et al., 2002). 1.1.2 Cytokines Most antigens are T—dependent, meaning that the B-cell requires T—cell help for maximal antibody production. Antigen cross linking to the BCR produces a signaling cascade modulated by spleen tyrosine kinase (Syk) and V—yes-l Yamaguchi sarcoma viral related oncogene homolog (Lyn) (see section 1.1.7). Processed proteins from T—dependent antigens are presented on B-cell MHC II for recognition by T H2 cells. The activated T-cell then secretes cytokines that activate the B-cell to trigger proliferation and differentiation into plasma cells. LPS activation induces T H1 cytokines such as TNF-0t, IFN-‘y and IL-12 (Mukherjee et al., 2009) and production Of interleukin-4 (IL-4) and interleukin-5 (IL-5) (Chiba et al., 2007). LPS-induced IL-4 production is TLR4 dependent, transcriptionally regulated, and requires de novo protein synthesis (Mukherjee et al., 2009). Splenocytes exposed to TCDD produce less IL-4 (responsible for stimulating activated B-cell and T-cell proliferation and upregulating MHC H production) and USS (responsible for stimulating B- cell growth and increasing immunoglobulin secretion) (Nohara et al., 2002). The inhibition of IL-4 and 5 production induced by TCDD exposure may, in part, be responsible for the suppression of humoral immune response to LPS. 1.1.3 Calcium A secondary signaling molecule with effects upon transcriptional elements, calcium is essential for effects elicited through BCR stimulation upon transcription factor expression including Pax5, Bcl6, MIT F, Etsl, Flil, interferon regulatory factor 4 (IRF4), Spi—B transcription factor (SpiB), and Blimpl (Hauser et al., 2009). Calcium-loaded calmod- ulin inhibits DNA-binding Of E2A, an inhibition that is essential for rapid down-regulation of immediate early genes after BCR activation (Saarikettu et al., 2004; Hauser et al., 2009). Active (calcium-loaded) calmodulin also reduces Pax5, Bcl6, Flil, and Etsl mRNA expression and increases Blimpl mRNA expression (Hauser et al., 2009). The Ca2+lcalmodulin-dependent protein kinase (CaMK) [a has been reported as a contributing factor to aryl-hydrocarbon receptor (AhR)-mediated genomic response (Monteiro et al., 2008b). However, more recent data indicates that the CaMKK inhibitor is an AhR ligand and that CaMK may not actually contribute to AhR signaling (Monteiro et al., 20083). However, further reports conflict as to whether increases in internal calcium levels are induced through release of internal calcium stores (Puga et al., 1997) or external calcium influx via T—type channels (Kim et al., 2009) and whether this increase occurs immediately or after a period of time. Studies of TCDD catalytic degradation in vitm show that degradation is increased in the presence of calcium, functioning as a catalyst (Mitoma et al., 2006). For this reason, it is still likely that cells increase internal calcium levels in response to TCDD exposure in order to increase degradation Of the toxicant, although whether the increase in cytosolic calcium is immediate or delayed may affect the efficiency of this process. 1.1.4 LPS Signaling Stimulatory The main receptor by which lipopolysaccharide (LPS) stimulates signaling in the cell is toll-like receptor 4 (TLR4). Soluble LPS binding protein (LBP) in the serum binds LPS and loads it onto cluster of differentiation 14 (CD14) in the plasma membrane. LPS- bound CD14 recruits the TLR4/lymphocyte antigen 96 (MD2) heterodimer and serves to initiate intracellular signaling through recruitment of cytoplasmic TIR domain containing adaptor proteins (TIRAPS) (Peng, 2005) (Figure 1.2). The TLR4/MD2 complex may 10 have additional discriminatory capability that confers specificity to the LPS recognition event, with B-cell lines and primary B-lymphocytes capable of differentiating between LPS chemotypes (Minguet et al., 2008). In fact, B-cell TLR4 may also recognize pathogenic antigens such as viral proteins or heat shock proteins (Peng, 2005). The role Of toll-like receptors (TLRS) in B-cell activation and antibody response appears to be dependent on antigen type and stimulation context, with different LPS chemotypes and stimulation events leading to unique signaling responses (Lanzavecchia and Sallusto, 2007). TLR4 stimulation leads to activation of two main cytoplasmic signaling cascades: TIRAP and TIR domain-containing adaptor-inducing interferon-[3 (TRIF)/TRIF-related adaptor molecule (TRAM). The activated cytoplasmic TIR domain of TLR4 recruits T IRAPs such as MyD88 and transduces signals through at least two signaling pathways. Through the DD, MyD88 recruits and activates IRAK which in turn recruits and activates TRAF6 in the plasma membrane. TRAF6 stimulates activation of TAKl, stimulating IKK (see section 1.1.5), JNK, and p38 activation (Figure 1.2). The second signaling pathway triggered by LPS-mediated TLR4 stimulation initiates with formation of a TRIF/T RAM heterodimer. The TRIF/TRAM complex regulates inter- feron regulatory factor 3 (IRF3) activation and subsequent induction of type I interferons and co-stimulatory molecules (Peng, 2005). IRF3 in turn stimulates IFN-B transcription. Alternative association of TRIF with TRAF6 stimulates TRAF6 signaling as described above (Figure 1.2). Unlike macrophages and dendritic cells, B-cells additionally utilize lymphocyte antigen 64, CD180 molecule (RP105)/lymphocyte antigen 86 (MDl) heterodimer (structurally related to TLR4/MD2) to recognize and respond to LPS (Pen g, 2005). RP105/MD1 plays a uniquely important role in B-cells, enhancing TLR4-dependent LPS response (Peng, 2005; Kawai and Akira, 2006). Subsequent to LPS stimulation and through an unknown mecha- ll Figure 1.2.: Signaling Occurring in B-cells as a Result of LPS binding to TLR4. Soluble LBP binds LPS and loads it onto CD14 in the plasma membrane. LPS-bound CD 14 recruits the TLR4/MD2 heterodimer and serves to initiate intracellular signaling through recruit- ment of cytoplasmic TIRAPs. The activated cytoplasmic TIR domain of TLR4 recruits TIRAPs such as MyD88 and transduces signals through at least two signaling pathways. Through the DD, MyD88 recruits and activates IRAK which in turn recruits and acivates TRAF6 in the plasma membrane. TRAF6 stimulates activation of TAKl , stimulating IKK (see section 1.1.5), JNK, and p38 activation. 12 nism, RP105 signaling activates phosphoinositide 3-kinase (PI3K). PI3K catalyzes the phosphorylation of phosphatidylinositol (4,5)-bisphosphate (PIPZ) to form phosphatidyl- inositol (3,4,5)-triphosphate (PIP3), which recruits protein kinase B (Akt) and pyruvate dehydrogenase kinase (PDK) to the plasma membrane. Once recruited to the plasma membrane, Akt is phosphorylated and activated by PDK. Phosphatase and tensin homolog (PTEN) catalyzes the opposite reaction in which PIP3 is dephosphorylated to form PIPZ. PIP2 is degraded by phospholipase C (PLC) to form inositol triphosphate (1P3) and diacyl- glycerol (DAG). 1P3 is recognized by the inositol triphosphate receptor (IP3R) to trigger release of internal calcium stores from the ER while DAG is capable of activating protein kinase C (PKC) to regulate Ras/Raf and MAPK signaling. PKC activation has also been shown to down-regulate DNA methylation activity in human lymphocytes, indicating a decrease in DNA methylation activity resulting from release of internal calcium stores (Bonilla-Henao et al., 2005) (Figure 1.3. Through TLR4 alone, there exist two distinct signaling pathways stimulated by LPS binding with a third signaling pathway in B-cells that is also stimulated by LPS recogni- tion. With so many pathways and interactions possible, it is small wonder that cells may have developed an increased ability to distinguish between LPS chemotypes and mount responses accordingly. The range of LPS chemotypes within a single preparation as well as the range Of possible cellular responses must all be taken into consideration when studying a heterogeneous population such as isolated splenocytes. Attenuation Crucial to any cellular signaling cascade is appropriate regulation and attenuation. The TLR4-ligand response is attenuated by increased tumor protein 53 (p53) levels (Liu et al., 13 2009a) and through a gradual decrease in plasma membrane TLR4 (Husebye et al., 2006). p53 is a transcription factor capable Of transactivating genes with functions including cell cycle arrest, apoptosis, and metabolic changes (Green and Kroemer, 2009). In rat liver, TCDD attenuates p53 phosphorylation (and so transcriptional activity) by increasing extra- cellular signal-regulated kinase (ERK)-mediated murine double minute 2 oncogene (Mdm2) phosphorylation (a post-translational negative regulator of p53) (Paajarvi et al., 2005; Womer and Schrenk, 1996; Green and Kroemer, 2009). Hepatocyte growth factor—regulated tyrosine kinase substrate (Hrs) directs the local- ization of LPS-bound TLR4 into endosomes-like structures after 1 hr of LPS stimulation (Husebye et al., 2006). This decrease in TLR4 at the plasma membrane is critical to regulate nuclear factor K-light—chain-enhancer of activated B-cells (NFKB) activation, with cellular Hrs knockdown eliciting a 70% increase in LPS-induced NFKB activation (Husebye et al., 2006). Without Hrs-mediated downregulation of TLR4 signaling, the cell may become hypersensitive to LPS-stimulus, producing large quantities of inflammation factors and inducing septic shock. Increased levels of receptor-interacting serine-threonine kinase (RIP) down-regulate PTEN through NFKB-independent pathways (Park et al., 2009) and so increases the activa- tion Of Akt while decreasing the production Of 1P3 and DAG from PIP3. RIP is essential for the TAKl-dependent TLR4 activation of PI3K (Vivarelli et al., 2004) and suppresses forkhead box protein 0 (FoxO) transcription factors, so downregulaing cyclin-dependent kinase inhibitor 1B (p27kip1) and favoring cell cycle progression (Park et al., 2009). However, the MyD88 mediated rapid NFKB activation and related inflammatory cytokine production is separate from the LPS-stimulated survival signals mediated by RIP in spleno- cytes (Vivarelli et al., 2004). 14 TLR4 gene expression is repressed by DNA methylation and histone deacetylation in the gene’s 5’ region (Takahashi et al., 2009), modifications that inhibit the recognition and binding of transcription factors through induction of a condensed chromatin conformation so repressing gene transcription. This may represent an epigenetic mechanism by which cells control sensitivity to an LPS stimulus. 1.1.5 NFKB Signaling One Of the Signaling cascades which is crucial to cell signaling and survival, the NFIcB pathway is stimulated through MAPK-mediated activation of IKK. Activated IKK induces IKB phosphorylation and degradation necessary for the release of NFKB. Functional IKK protein is crucial to B-cell survival, with conditional loss of IKK (on, B, or 7) resulting in decreased number Of B-cells with impaired survival (Claudio et al., 2006). IKBOL loss further results in an increased number of B-cells hyperresponsive to stimulation (Claudio et al., 2006). NFKB is a transcription factor, that when released from IKBOI accumulates in the nucleus where it is able to affect transcription of many genes (Heiss et al., 2001) with constitutive activation inducing cell proliferation and apoptotic resistance (Amit and Ben-Neriah, 2003). Activation of NFKB negatively regulates PTEN transcription and p53 transcriptional activity yet is insufficient to promote survival of LPS-stimulated B-cells (Vivarelli et al., 2004). 1.1.6 Akt Signaling Akt signaling is critical for survival and metabolic fitness (Patke et al., 2006) (Woodland et al., 2008). Appropriate Akt signaling prevents apoptosis through inhibition of Bim production, a process dependent upon mTOR (activated by Akt phosphorylation and inhibi- 15 M ®®®¢ 5:911? 0% wow? ® a @ Figure 1.3.: Schematic representation of Akt signaling. Appropriate Akt signaling prevents apoptosis through inhibition of Bim production, a process dependent upon mTOR (activated by Akt phosphorylation and inhibition of TSC2, a negative regulator of mTOR). Akt is also capable of inactivating FoxO transcription factors, so inhibiting p27kip1 transcription and regulating cell proliferation. Phlpp is a dominant phosphatase control— EsR ling cell cycle via specifically opposing Akt actions on p27klp1 hos ho lation state P P ry tion of TSC2, a negative regulator of mTOR) (Hahn—Windgassen et al., 2005) (Figure 1.3). Deregulation of Akt is highly associated with tumorigenesis in humans and mice (Blanco-Aparicio et al., 2010). Akt is also capable of inactivating FoxO transcrip- tion factors, so inhibiting p27kip1 transcription and regulating cell proliferation (Medema et al., 2000) (Tran et al., 2003). Specifically, FoxO3 promotes p53 cytoplasmic accumula- tion and so cell survival (Green and Kroemer, 2009). BCR crosslinking leads to Akt hyperphosphorylation and so activation (Ishiura et al., 2010). The BCR-induced activation of Akt requires PI3K and Syk (Li et al., 2002). IgM- bearing B-cells utilize B—cell scaffold protein with ankyrin repeats (Bankl) (a gene capable of limiting BCR—mediated Akt activation) to decrease signal strength and prevent a hyper- 16 IgM response (Aiba et al., 2006). Lyn is also a potent endogenous inhibitor of BCR- mediated activation of Akt (Li et al., 2002). 552 Upon activation, Akt can directly phosphorylate B-catenin at Ser in vitro and in vivo causing B-catenin to disassociate from cell-cell contacts and accumulate in the nucleus (Fang et al., 2007). In the nucleus, B-catenin complexes with T—cell factor/transcription factor (ch) to affect transcription of many genes. All three isoforms of Akt contain at least 6 putative ch/B-catenin binding sites (Dihlmann et al., 2005). Expression Of Akt is also regulated by AP-l and NFKB, as evidenced by the existence of putative binding elements within their promoters (Dihlmann et al., 2005). Akt signaling is specifically attenuated by Phlpp (a dominant phosphatase) isomers, with different Akt isomers individually targeted by Phlpp isomers. For example, Phlpp2 controls cell cycle via specifically opposing Akt3 actions on p27kip1 phosphorylation state (Brognard and Newton, 2008). 1.1.7 BCR Signaling The B-cell receptor (BCR) is composed of an immunoglobulin molecule in complex with, CD79a, and CD79b. Antigen binding to the immunoglobulin molecule induces signaling through the BCR to Syk and Lyn which activate Bruton’s tyrosine kinase (Btk). Syk has been shown to be important in B-cell emigration (Cariappa and Pillai, 2002). Btk activity is increased upon BCR crosslinking and its deficiency results in reduced B-cell proliferation in response to LPS-stimulation (Baba et al., 2001). In the absence of Btk, most mature follicular B-cells fail to survive (Cariappa and Pillai, 2002). Activation of Src-family kinases such as Btk requires CD45 (Cariappa and Pillai, 2002). B-cells from 17 mice deficient in Lyn, Btk, PI3K, BLNK, or phospholipase C y (PLCy) exhibit impaired proliferative responses to LPS-stimulation (Yang and Desiderio, 1997). Similar to variegated responses occurring through TLR4 signaling, BCR signaling alters the fate Of B-cell differentiation in response to different stimuli and environments. BCR inhibits some forms of B-cell differentiation, with increased BCR signaling decreasing differentiation of maturing B-cells into mid-zonal B-cells (Pillai and Cariappa, 2009) and persistant signaling abolishing LPS-induced plasma cell differentiation (Kurosaki, 1999). In other situations, strong BCR signaling favors follicular development (Pillai and Cariappa, 2009) and is required for maintenance of all peripheral B-cell populations (Cariappa and Pillai, 2002). 1.2 TCDD 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is a widespread environmental contam- inant with high lipophilicity, accumulating in the fat of carnivorous animals (including humans). TCDD is a polychlorinated dibenzodioxin and the most potent compound within its family. Mammalian effects of TCDD exposure include wasting, hepatotoxicity, cardio- toxicity, chloracne, death, and immunotoxicity. Specific immunologic aberrations Observed in mice exposed to TCDD include the inability to mount an innate immune response to LPS challenge. For this reason, it is a useful compound in the evaluation of events necessary to the differentiation of B-cells. 1.2.1 TCDD Signaling Cytoplasmic TCDD binds the AhR, which is commonly found in complex with AIP, p23, and 2 Hsp90 molecules. Binding of TCDD to the AhR induces release of all chaperone l8 C) Cl Cl Cl 0 Cl (3) TCDD -__--________/e _______________ Cytoplasm Transcription Factors (b) AhR Signaling Figure 1.4.: TCDD Signaling through the AhR. Cytoplasmic TCDD binds the AhR, which is commonly found in complex with AIP, p23, and 2 Hsp90 molecules. Binding of TCDD to the AhR induces release of all chaperone proteins and translocation of the TCDD-bound AhR to the nucleus where it binds ARNT. In the nucleus, the AhR/ARNT dimer binds to ARES in the promoter regions of genes and regulates their transcription. proteins and translocation of the TCDD-bound AhR to the nucleus where it binds ARNT and initiates transcription of several genes including Cyplal , NF-E2 related factor 2 (Nrf2), glutathione S-transferase, alpha 1 (Ya) (Gstal ), aldehyde dehydrogenase 3 family, member Al (Aldh3al), and epiregulin (EREG) (Nebert et al., 2000) (Figure 1.4). Phosphorylation of the charged linker region of Hsp90 modulates formation of a functional cytosolic AhR 19 complex (Puga et al., 2009). Although the chaperone protein complex maintains the AhR in a cytoplasmic state receptive to ligand binding, transcriptional functions of the AhR do not seem affected when the Hsp90 association with co-chaperone proteins is inhibited (Middendorp et al., 2003). In the nucleus, the AhR/ARNT dimer binds to dioxin response elements (DRES) in the promoter regions of genes and regulates their transcription (Figure 1.4). The prototypical gene used to assess AhR-regulated transcription is Cyplal. Within 15min of TCDD treat- ment, ElA binding protein p300 (p300) is recruited to DRES within the Cyplal promoter region (Sutter et al., 2009). p300 further serves to recruit other transcription factors to the gene and initiate transcription. AhR binding to DRES is reduced by DNA methylation within the gene enhancer region (Shen and Whitlock, 1989), so also reducing TCDD- induced gene transcription. Hypermethylation of the Cyplal regulatory region causes a decrease in TCDD-induced gene expression (Okino et al., 2006), suggesting the possibility of an epigenetic mechanism in regulating response to TCDD exposure. Global hyper- methylation upon embryonic exposure to TCDD is also accompanied by an increase in DNA methyl transferase activity (Wu and Sun, 2006), supplying additional evidence that TCDD may elicit epigenetic response within exposed cells. Other transcription factors are also recruited to DRES along with the AhR. Estrogen receptor (EsR)OL is recruited to AhR target genes in a TCDD—dependent manner and enhances target gene expression (MacPherson et al., 2009). Crosstalk occurring through the recruitment of EsR to DRES bound by activated AhR plays a role in tumor promotion (Matthews and Gstafsson, 2006). PKC inhibition blocks ligand-induced DNA-binding of AhR/ARNT heterodimer and leads to suppression of cytochrome P450, family 1 (Cypl) gene expression (Puga et al., 2009). 20 After activation, AhR is quickly exported to the cytosol where it is degraded by the 26s proteasome, preventing constitutive receptor activity (Puga et al., 2009). The AhR is not required to generate a normal immune response, yet is Obligatory in TCDD-induced immune response suppression (Vorderstrasse et al., 2001). Although immune response generation is normal in AhR—deficient animals, the doubling time of AhR-deficient cells is increased as compared to wild type (WT) cells (Ma et al., 2004). The functional AhR is not required for MAPK and PLC activation nor for JunB and c-Fos induction in response to TCDD (Puga et al., 2009) (Beebe et al., 1990), indicating that many important Signaling pathways affected by TCDD exposure are regulated in a non-classical (AhR-independent) manner. Some genes are expressed via a RelB/AhR transcriptional promoter recognition that does not require ARNT and involves signaling through the non-classical NFKB pathway (Matsumura, 2009). AhR activation also leads to time-dependent induction Of BAFF, B- lymphocyte chemoattractant (BLC), chemokine (C-C motif) ligand 1 (CCLl) and IRF3 in human m¢s, inductions that require RelB (Vogel and Matsumura, 2009) and likely occur through crosstalk between the non-classical NFKB and the AhR pathways. There is no evidence of direct DRE-mediated involvement in TCDD-induced alter- ations in Pax5 or Blimpl regulation (Schneider et al., 2009). Instead, activated AhR downregulates AP-l binding to the Blimpl promoter region (Schneider et al., 2009). This may be the mechanism by which TCDD affects JunB and c-Fos induction even in cells without a functional AhR. Blimpl binding to the Pax5 promoter is then suppressed by TCDD, resulting in decreased inhibition of Pax5 (Schneider et al., 2009). Activated AhR also interacts with the RB/E2F axis (Puga et al., 2009) in order to suppress E2F transcriptional activity. Hyperphosphorylated retinoblastoma protein (RB) protein cannot adequately repress E2F activity (Puga et al., 2009), so increasing transcrip- .21 tion of cell cycle regulatory and DNA replication genes necessary for B-cell differentiation or tumor generation. AhR activation further increases Nrf2 expression and subsequent binding to ARES (Yeager et al., 2009). In response to inflammation, a crosstalk may occur between Nrf2/ARE binding and NFKB signaling pathways (Prawan et al., 2008). Ablation of Nrf2 levels accelerates NFKB-mediated pro-inflammatory reactions (Li et al., 2008). The opposite is also true, in that Nrf2 activators attenuate LPS-induced NFKB activation (Li et al., 2008). Oxidants impede Nrf2 degradation, increasing its translocation to the nucleus, and likely reducing NFKB-mediated pro-inflammatory reactions through competition for binding to ARES (Li et al., 2008). LPS signaling through TLR4 exposure also induces oxidant stress through nitric-oxide generation, but the increase in oxidants due to LPS exposure induces septic shock and increases the incidence of septic shock (Zhang et al., 2000). Nrf2 signaling has thus been identified as a mechanism by which cells are protected from LPS-induced inflammatory response (Thimmulappa et al., 2006). However, when induced by TCDD exposure prior to LPS challenge the reduction in inflammatory response induced by Nrf2 Signaling may be exaggerated to such an extent that instead of protecting against LPS- induced septic shock, it actually inhibits response to the challenge and so limits appropriate B-cell differentiation. 1.3 Epigenetics Conrad Waddington originally defined epigenetics as the study of mechanisms by which genotypes give rise to phenotypes during development (Waddington, 1957). The definition was later modified by Arthur Riggs and colleagues to state “the study of mitotically and/or 22 meiotically heritable changes in gene function that cannot be explained by changes in DNA sequence” (Russo et al., 1996). Epigenetic patterns of regulation can be passed from parental to filial generations Of both cells and whole organisms. More transient occurrences are also identified in which various stages of cellular development and differentiation are regulated epigenetically, followed by complete erasure of the epi genetic mark(s) (Bird, 2007). Four specific forms Of epigenetic regulation are small non-coding RNA, histone code, tissue-specific transcription factors (Liu et al., 2009b), and DNA methylation. Although these mechanisms are labeled and defined separately, no epigenetic mechanism functions in total isolation and the study of any in isolation will not fully evaluate the interactions occurring within the cell. 1.3.1 Small Non-coding RNA Non-coding RNA’s are functional RNA molecules that are not translated into protein. These RNA genes code for functionally important RNAS such as transfer RNA, ribosomal RNA, micro RNA (miRNA), and silencing RNA. Transfer and ribosomal RNA’s are critical for transcription and translation processes in the cell. Through partial complement to messenger RNAS, miRNA downregulates the concentration of mature mRNA in higher order eukaryotic cells. Through an evolutionarily-conserved antiviral mechanism, double stranded RNA is recognized by the Dicer protein which digests the double stranded RNA into ~20 nucleotide segments, thus inhibiting translation into protein (Csorba et al., 2009). siRNA’S have recently been described as having longer lasting epigenetic effects than simple destruction of mRNA. These long lasting effects are mediated by DNA methylation of the targeted gene’s promoter region resulting in gene Silencing before mRNA transcrip- tion (Hawkins et al., 2009). 23 1.3.2 Histone Code Histones associate with DNA and form nucleosomes composed of two copies each of core histones H2A, H2B, H3 and H4 and wrapped by l46bp of DNA with flexible N- terminus histone tail. Often, the tail is modified in the form of methylation, acetylation, ubiquitination, and/or phosphorylation in order to affect chromatin structure. Acetylated histones tend to preferentially associate with transcriptionally active chromatin (Hebbes et al., 1988). Several transcriptional activators such as Gcn5, p300/CBP, PCAF, TAF250, and the p160 family of nuclear receptor coactivators contain intrinsic histone acetyl- transferase activity which is required for their transcriptional regulatory activity (Roth et al., 2001; Wang et al., 1998). Histone methylation, unlike acetylation, remains relatively static and the precise regulatory role of histone methylation has not yet been elucidated (Zhang and Reinberg, 2001). Histone ubiquitination plays a critical role in regulating processes such as transcription, silencing, and DNA repair (Weake and Workman, 2008). Histone phosphorylation results in disparate regulation, with H2A phosphorylation resulting in mitotic chromosome condensation and H3 phosphorylation occurring after DNA-damaging events or correlating with gene activation (Grant, 2001). Formation of a condensed chromatin state causes genes to become inaccessible to transcription factors and DNA replication proteins. 1.3.3 Tissue-specific Transcription Factors Often transcription factors are regulated in a developmental or tissue-specific manner. Recent evidence from the Tjian laboratory suggests that global changes in cell—type specific transcription may be facilitated by significant changes in general transcription factors such as TFIID, BAF, and Mediator (D’Alessio et al., 2009). The core transcription machinery, 24 that previously was assumed to operate in all cells, has been shown to be dramatically downregulated in some cell types while distinct transcription activating factors are thought to regulate distinct gene expression profiles (D’Alessio et al., 2009). Evidence suggests that tRNA and rRNA transcription, previously hypothesized to utilize TATA-binding protein (TBP) in the promoter recognition complex, may actually use related factors specific to the tissue in which the genes are transcribed. Disregulation of the appropriate expression of these cell-type Specific transcription factors may result in inappropriate gene regulation and so differentiation or development. 1.3.4 DNA Methylation DNA methylation, in the form of 5’-methylcytosine, normally occurs at ~70%-80% Of CpG dinucleotides (Naveh-Many and Cedar, 1981a; Craig and Bickmore, 1994), a sequence particularly abundant in gene promoter regions (Kristensen and Hansen, 2009). CpG dinucleotide methylation protects healthy cells from inappropriate transcription of repetitive elements such as long interspersed nuclear elements (LINES) and Alu repeats (Walsh et al., 1998) and may also help maintain chromosomal stability (Eden et al., 2003). Altered DNA-methylation, an epigenetic mechanism that plays a regulatory role in gene expression, has been proven to play multiple roles in carcinogenesis, development, and differentiation (Kurkjian et al., 2008) with unique methylomes observed in diverse cell types as well as gender-Specific imprinting. A decrease in DNA-methylation results in reduced global histone deacetylase (HDAC) recruitment, decreased chromatin condensa- tion, and increased gene expression (Martinowich et al., 2003). In addition to regulating the expression of genes, DNA methylation also reduces the mobility of transposons (Kato et al., 2003). 25 Four models of gene regulation through DNA methylation have been proposed: 1) Direct DNA-methylation: methylation in the promoter region of a gene inhibits binding of transcription factors while methylation in the intron(s) of a gene inhibits proper Splicing (Mares et al., 2001; Decker et al., 2009) 2) DNA-methylation of an enhancer’s promoter: methylation resulting in the increased binding of the enhancer to the genes promoter region and so increased transcription (Mares et al., 2001; Decker et al., 2009) 3) DNA-methylation of an insulator region: methylation upstream of an enhancer element results in neutralization of the insulator region and increased gene transcription (Hark et al., 2000) 4) Increase in DNA-methylation resulting in global recruitment of HDACs: induced condensed chromatin conformation reduces transcription factor access, so reducing gene expression (Baylin, 2005) Methyl groups are transferred to and from DNA via DNA methyltransferase (DNMT)s and putative demethylases. All DNMTS use S-adenosyl methionine (SAM) as a methyl group donor in an S N2 inversion reaction (Ulrey et al., 2005). DNMT] is the must abundant mammalian DNA methyltransferase and is key to DNA methylation mainte- nance through copying methylation patterns on newly-duplicated hemi-methylated DNA. DNMT3 DNA methyltransferases methylate hemimethylated and unmethylated CpG regions. Specifically, DNMT3a and DNMT3b mediate de novo DNA methylation while DNMT3L is required for establishing maternal genomic imprints (Fatemi et al., 2002). In MZ and follicular B-cells, DNMT3a expression is differentially regulated. DNA methyltransferases form a covalent bond between a cysteine residue in the enzyme’s active site and a cytosine’s C6 in DNA. Proper bond formation is followed by attack upon the methyl group Of SAM. Proton abstraction from the DNA cytosine’s C5 26 followed by B—elimination allows reformation of the C5-6 double bond and release Of the enzyme and methylated DNA. While the methyl group is transferred to the DNA, the target cysteine is actually flipped out Of line Of the other nucleotides. When 5’-azacytidine is targeted by DNA methyltransferases, the saturated C5—6 bond mimics the transition state 3 Of cytidine formed prior to methyl transfer and the sp character of C6 abrogates nucle- ophilic attack (Christrnan, 2002). Treatment with 5’-azacytidine (an inhibitor of methyltransferase activity when incorporated into DNA or RNA during replication, causing global demethylation) results in almost complete abolishment of follicular B-cells while leaving MZ B-cells numbers relatively intact, indicating the importance of differential DNA methylation mechanisms and states in regulating the localization and development of B-cells within the spleen (Wang et al., 2006). Studies of the AS-VpreBl locus methylation status have discovered an increase in methylation status during the pre-B-cell stage, followed by an almost complete attenuation of all DNA methylation in mature B—cells (Szutorisz et al., 2005). LPS challenge induces aberrant hypermethylation Of hypermethylated in cancer-1 (Hic- l) exon la in mouse embryonic fibroblasts lacking p53 and (unchallenged) human follicular lymphomas (Tatemichi et al., 2008; Guo et al., 2005). This would indicate that hyper- methylation of Hie-1 is important during rapid proliferation of cells. However, no further research suggesting altered DNA methylation in response to LPS challenge has been published. Because a clear genetic mechanism for crosstalk between LPS-regulated B cell differentiation and TCDD signaling has not yet been discerned, it is possible that this crosstalk may occur through epigenetic events. Hypermethylation of the PTEN promoter in cerebral cavernous malformations has been linked to a significant downregulation in protein expression (Zhu et al., 2009). Increased 27 methylation of PTEN has also been associated with increased alterations in the P13 K/Akt pathway and degree Of tumor aggression in thyroid tumors (Hou et al., 2008). 1.4 Hypothesis This work was designed to address two related but distinct hypotheses: 1) epigenetic events might, in part, underly B-cell differentiation to plasma cells 2) epigenetic events might, in part, underly TCDD-induced inhibition of LPS-induced B-cell differentiation To test these two hypotheses, a number of investigations were undertaken to investigate the following sub-hypotheses: 1) DNA is differentially methylated in treated populations 2) DNA is differentially methylated within 10kb of genes 3) DNA methylation alters expression of selected genes 4) Genes exhibiting altered expression are important to B-cell differentiation To assess the occurrence of potential epigenetic events, murine splenocytic DNA was probed via a non-biased AP-PCR, Capillary Electrophoresis (CE) method for RAMS where the DNA was hyper-, hypo-, or newly-methylated in comparison to DNA from control treated animals. This method is not limited by the regions already chosen on a PCR array and allows evaluation of total DNA for RAMS. PCR amplified DNA of the same length (in bp) as identified RAMS was sequenced and annotated to genes and CpG islands. Although this method precisely identifies potential RAMS, it is greatly limited by the ability of PCR products to be ligated into the plasmid and then amplified through clonal expansion of the host bacteria. Many promoters and enhancers with cis-acting regulatory abilities are located within 10kb of the transcriptional start site of genes, leading to the hypothesis that should 28 a RAM occur within this 10kb it may affect the regulatory capabilities Of the enhancer and promoter regions. A distance of 10kb up- and downstream of the sequenced PCR products was examined for gene coding sequences, miRNA coding sequences, and repeat elements. In this way, alterations in DNA methylation may have effects on gene expression were directly observed. Alterations in the pattern and sites of DNA methylation were identified in all treatment groups. Of particular interest were those RAMS identified within 10kb of (a) transcription factor-, (b) LINE repeat elements and (c) histone modifier-encoding sequences. When coding sequences are differentially methylated, it is reasonable to hypothesize that the expression patterns may also be altered. Therefore, through analysis of alterations in DNA methylation, this work describes new evidence indicating that many different epigenetic regulatory mechanisms may affect the differentiation of B-cells and the toxicity of TCDD. RAMS were identified as a result of LPS challenge that were different (in size, location, and methylation state) from those identified as a result Of TCDD exposure. Interestingly, over the course of preliminary evaluation, it became evident that the RAMS identified in response to LPS-mediated activation or TCDD exposure were not identical to those identified in response to LPS+TCDD treatment. This evidence suggests mechanisms regulating maintenance of the DNA-methylome is altered in LPS-induced signaling, TCDD exposure, and through a novel crosstalk mechanism occurring in concomitant treatment. 29 2. DNA METHYLATION: A POTENTIAL MECHANISM OF CROSSTALK OCCURRIN G IN MURINE SPLENOCYTES EXPOSED IN V1 V0 TO LIPOPOLYSACCHARIDE (LPS) AND 2,3,7 ,8-TETRACHLORODIBEN ZO-p-DIOXIN (TCDD) McClure, E.A., North, C.M., Kaminski, N., and Goodman, 1.1. a manuscript in preparation for submission to Toxicological Sciences, 2010 2.1 Abstract B cells within the Splenocyte population, isolated from mice treated with lipopoly- saccharide (LPS), differentiate into antibody-producing plasma cells in vivo. Pretreatment with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) inhibits differentiation. We hypothesize that altered DNA methylation, an epigenetic event, plays a key role in this inhibition. DNA was isolated from splenocytes prepared 6 days post experiment initiation from 5-6wk old female C57BL/6 mice dosed with: TCDD, 3 or 30pg/kg, on day 0; or LPS, 25yg/mouse, on day 4; or sequentially with TCDD and then LPS on days 0 and 4, respectively. To discern regions of altered DNA methylation (RAMs), DNA was restricted with HpaII (a methyl- ation sensitive enzyme), followed by arbitrarily primed PCR and capillary electrophoresis. LPS, 3, or 30pg/kg TCDD alone resulted in 40, 43 and 42 RAMS, respectively, while LPS challenge subsequent to 3 or 30pg/kg TCDD resulted in 34 and 39 RAMS, respectively. Interestingly, the combined treatment lead to 4 RAMS seen with LPS alone, 4 RAMs seen with TCDD alone, and 19 RAMS observed only in the co-treatment group. These data indicate treatment with LPS or TCDD can alter DNA methylation in Splenocytes. The combined TCDD+LPS leads to RAMS that are unique: we do not simply observe the sum of the RAMS that form from the individual treatments. PCR products representing a number 30 of the RAMs Observed were cloned, sequenced and annotated. Those unique to the LPS- TCDD combination include: Bankl (involved in B cell receptor-induced Ca2+ mobiliza- tion); Adcy5 (membrane-bound calcium-inhibitable adenylyl cyclase); Arvcf (involved in protein-protein interactions); CtBP2 (corepressor targeting diverse transcription regulators), Ling02 (leucine-rich repeat and immunoglobulin-like domain-containing nogO receptor- interacting protein 2; Pctk3 (may play a role in signal transduction cascades); Krrl (involved in nucleolar processing of pre-18S ribosomal RNA and ribosomal assembly). Unique RAMS in the TCDD+LPS group indicate crosstalk between the actions of LPS to induce _ B-cell differentiation and aryl-hydrocarbon receptor (AhR) signaling which, presumably, mediates the inhibitory effect of TCDD. TO our knowledge, this is the first largescale evalu- ation of changes in DNA methylation due to LPS exposure and the first identification of a DNA methylation-based crosstalk occurring in splenocytes exposed to TCDD prior to LPS-challenge. 2.2 Introduction TCDD is a widespread environmental contaminant with high lipophilicity and severe immunosuppressive effects. Specific immunologic aberrations observed in mice exposed to TCDD include the inability to mount an innate immune response to LPS challenge (Kerkvliet, 2002). LPS is a bacterial endotoxin and potent B-cell mitogen capable Of stimulating the innate humoral immune response in animals. The innate immune response occurs within 4 hrs of initial infection, with induced innate response occurring 4-96 hrs post initial infection. Upon stimulation with an antigen, B cells in peripheral lymphoid organs activate and begin rapidly proliferating, thereby increasing expression of surface and secreted immuno- 31 A :2 140 ., DayO Day4 ( Day6) Day7 '3 120 ' I I I g I l l I I- O 9 9° 0° ‘5’ 9° ‘é 100 a «(9 \9 <¢\Q& V Qo“ i a (5} Q3? 3 so i- 3 l u E 60 g l. 3 4o 3 3 75 c < Sesame Oil Sesame Oil Bug/kg long/kg 30ug/kg + PBS + LPS TCDD + LPS TCDD + LPS TCDD + LPS Figure 2.1.: AFCS as a Function of 'ITeatment with LPS and TCDD. All samples are from the same animals as used in (North et al., 2009). 6 female C57BL/6 mice per treatment group were treated on day 0 via oral gavage with O, 3, 10, or 30pg/kg TCDD; mice were further treated on day 4 via intraperitoneal injection with 0 or 25pg LPS in PBS; combined treatment included mice treated on days 0 and 4 as described above. AFCS was measured on day 7. Data are depicted :1: standard error. a. significantly different from control. b. significantly different from LPS treatment. Insert: Timeline showing treatment adminis- tration, Splenic tissue collection, and AFCS response measurement. Day 6 is highlighted as the day on which this study’s samples were obtained. Figure modified from data reported in (North et al., 2009) globulin M (IgM) (Dooley and Holsapple, 1988). A lack of increased IgM expression and secretion is indicative of B-cell activation and suppression of proliferation in response to LPS exposure. Murine TCDD-induced immunological suppression is observed both in vivo and in vitro (North et al., 2009) and is easily measured as a suppression of IgM secretion. Maximum in vitro suppression of primary sheep erythrocyte IgM response observed at 30pg/kg TCDD in C57BL/6 mice (Figure 2.1) (Vecchi et al., 1980). Immune suppression in splenocytes 32 treated in vivo with T CDD+LPS is also accompanied by decreased B-lymphocyte-induced maturation protein 1 (Blimpl) expression, decreased CD19+ cells, decreased expression of immunoglobulin J (IgJ), K, and p chains, and decreased major histocompatability complex (MHC) class H expression (North et al., 2009). Genomic effects of T CDD in animals are mediated through binding to the AhR. Upon TCDD binding, AhR releases all chaperone proteins [aryl-hydrocarbon receptor interacting protein (AIP), heat shock protein co-chaperone p23/prostaglandin E synthase 3 (p23), and 2 heat Shock protein 90kDa (Hsp90)] and translocates from the cytOplasm to the nucleus. Here it binds aryl-hydrocarbon nuclear translocator (ARNT) and functions as a transcrip- tional regulator binding aryl-hydrocarbon response element (ARE). The ARNT/AhR complex upregulates transcription of NF-E2 related factor 2 (Nrf2) (Yeager et al., 2009), which inhibits nuclear factor K—light-chain-enhancer of activated B-cells (NFKB) activation. Upon systemic challenge, LPS binding to toll-like receptor 4 (TLR4) stimulates an intracellular Signaling cascade terminating in the phosphorylation of interferon regulatory factor 4 (M4) (Takeda and Akira, 2004). IRF4 indirectly increases Blimpl expression by inhibiting B-cell lymphoma 6 protein (Bcl6) and paired box protein 5 (Pax5) expression. Together Bcl6, Blimpl, and Pax5 create a reciprocally repressing transcription factor ‘Switch’ which terminates in Pax5’s inhibiting IgM production. The ‘switch hypothesis’ anticipates an increase in IRF4 activity (as expected post TLR4 stimulation) to decrease Bcl6 and Pax5 transcription, thus upregulating Blimpl transcription and IgM expression. TCDD-induced inhibition of the primary humoral immune response likely occurs upstream Of antibody (IgM) production (North et al., 2009). Specifically, recently published evidence indicates negative regulation of IgM production resulting from failure of TCDD- exposed splenocytes to upregulate Blimpl expression (North et al., 2009). In which stage of upstream signaling pathways TCDD inhibits Blimpl expression is not well understood. 33 In 1989, Shen et al. published data suggesting DNA-methylation within the cytochrome P450, family 1, subfamily A, polypeptide 1 (Cyplal) promoter reduces the response to TCDD exposure (Shen and Whitlock, 1989). Epigenetic mechanisms of regulation, such as DNA methylation, occur through the addition Of mitotically and/or meiotically heritable changes to gene function that do not entail changes in DNA sequence (Wu and Sun, 2006). Epigenetic regulation can be transient, as identified in cellular development and differ- entiation. DNA methylation, in the form of 5’-methylcytosine, normally occurs at 40- 80% of CpG dinucleotides, with non-CpG methylation occurring as up to 55% of total cell methylation (Naveh-Many and Cedar, 1981a; Dupont et al., 2009; Ramsahoye et al., 2000). An increase in DNA-methylation results in the recruitment of histone deacetylase (HDAC) and induction of condensed chromatin conformation that decreases transcription factor binding, thus reducing gene expression. A decrease in DNA-methylation results in reduced HDAC recruitment, decreased chromatin condensation, and increased gene expression (MacDonald and Roskams, 2009). LPS challenge has been proven to induce aberrant hypermethylation of hypermethyl- ated in cancer—l (Hie-l) exon la in mouse embryonic fibroblasts lacking tumor protein 53 (p53) (Tatemichi et al., 2008). However, to the best of our knowledge, no further research suggesting altered DNA methylation in response to LPS challenge has been published. We hypothesize that signaling due to LPS, TCDD, or combined treatments is regulated, in part, by altered DNA methylation. TO test this hypothesis, we have assessed the methyl- ation status of splenocyte DNA treated in vivo with LPS, TCDD and LPS+TCDD. The genomically unbiased method chosen includes arbitrarily primed PCR followed by capillary electrophoresis, sequencing of PCR products, annotation to genes, and pathway analysis. This method has allowed the identification of many region Of altered DNA methylation (RAM)s resulting from LPS, TCDD, and TCDD+LPS treatments. Specifically, we have 34 demonstrated the occurrence of DNA methylation crosstalk, wherein the methylation status of combined treatments is different from that in either treatment alone, when mice are concomitantly treated with TCDD and LPS. Annotated genes include many important for cell survival, apoptosis, and B-cell signaling. Cumulatively, these Observations suggest a significant role of DNA methylation in regulating LPS and TCDD effects in vivo and suggest a role for DNA methylation in the TCDD-induced inhibition of B—cell stimulation by LPS. 2.3 Materials and Methods 2.3.1 Preparation of in viva Splenocyte Samples Chemicals TCDD was purchased from Accustandard (New Haven, CT) and prepared in sesame Oil (Sigma-Aldrich, St. Loius, MO). Salmonella typhasa LPS (Sigma-Aldrich, St. Louis, MO) was prepared in PBS immediately prior to administration. Animals Mice, treatments, and splenocyte collection were described previously (North et al., 2009). The same splenocyte samples from those animals sacrificed upon day 6 (post LPS exposure) in the previous study (North et al., 2009) were used for this study. Female 6-8 week old C57BL/6 mice were purchased from the National Cancer Institute and housed in accordance with Michigan State University Institutional Animal Care & Use Committee policy. On day 0, TCDD (0, 3, 10, or 30pg/kg in sesame Oil) was administered by single oral gavage. On Day 4, to initiate primary humoral immune response, LPS (0 or 25pg 35 in PBS) was administered by intraperitoneal injection. Spleen samples were collected on days 4-7 from all treatment groups (6 animals per group). Splenocytes were mechanically disrupted to form single-cell suspension and stored at —8OOC. Mice from day 4 that received only TCDD or vehicle treatment, were used to establish baseline TCDD effects (measured by AFCS response from samples collected on day 7) (Figure 2.1). 2.3.2 Evaluation of DNA Methylation Status by AP-PCR and CE Changes in DNA methylation status were evaluated using an Arbitrarily Primed PCR (AP-PCR) and Capillary Electrophoresis (CE) procedure (Bachman et al., 2006). This technique permits evaluation of genomic RAMS including hypomethylations (less methyl- ation than that observed in control), hypermethylations (more methylation than that Observed in control), and new methylations (methylation not Observed in control) simul- taneously. Most importantly, the procedure is unbiased in that it does not involve an evalu- ation of preselected genes but rather all genomic regions targeted by the restriction enzymes and arbitrary primer. DNA Isolation Single cell splenocyte suspensions were removed from -800C and mixed with lmL, 40C TRIzol® Reagent (Sigma-Aldrich, St. Louis, MO) before homogenizing completely using a Dounce homogenizer. DNA was isolated according to the manufacturers (Sigma- Aldrich, St. Louis, MO) protocol and precipitated with ethanol before dissolution in NaOH/HEPES buffer (pH=8.4) and storage at -200C. 36 Restriction Digest Each isolated DNA sample was subjected to double restriction digestion performed in duplicate as previously described (Bachman et al., 2006). Preliminary digestion with a methylation-insensitive enzyme, RsaI, ensures complete digestion by the methylation- sensitive enzyme, HpaII. RsaI recognizes 5’-GTAC-3’ sites and cuts between the guanine and adenine, but will not restrict DNA if the external cytosines (5’ and 3’) are methylated. HpaH recognizes 5’-CCGG—3’ sites and cuts between the internal cytosine and guanine when unmethylated. AP-PCR and CE Analysis of DNA Products AP-PCR and CE were performed as described previously (Phillips and Goodman, 2009). Data Analysis PCR products were evaluated with regard to size (in base pairs) and corresponding peak areas as measured by CE. An average peak area was calculated for each PCR product in treatment groups and compared to that of the control group. Regions of altered methyl- ation (RAMS) were identified as DNA regions in treatment groups with PCR products significantly (as determined by Students two-tailed t-test, p30.05) different in area than that Of the control group. RAMS include: a) complete hypomethylations (i.e. 100% decrease from methylation status Observed in control) and partial hypomethylations (Significant decrease in methylation when compared to control); b) hypermethylations (significant increase in methylation when compared to control); and c) new methylations (PCR product 37 formed in treatment that did not form in control). A detailed description of the data analysis procedure was provided previously (Bachman et al., 2006). Carry Forward and Unique RAMS One-way analysis of variance (ANOVA) was performed to compare RAMS (occur- ring at the same PCR product size in 22 treatment groups). Common RAMs with the same change in methylation status (one-way ANOVA, p30.05) in treatment groups were identified as Carry Forward RAMS. Unique RAMS include: 1) RAMS exhibiting different extents of methylation change in the same direction (one-way ANOVA, p30.05); 2) RAMS in common, which exhibited opposite directional changes; and 3) RAMs observed in only 1 treatment group. 2.3.3 Cloning and Annotation of RAMs Cloning and Sequencing of AP-PCR Products AP-PCR products were electrophoresed through a 3% High Resoluion agarose gel (Sigma-Aldrich, St. Loius, MO). PCR products were excised and DNA was isolated using Ultrafree-DA Columns (Millipore, Billerica, MA), and used for cloning reactions prepared with the pGEM-T Easy Vector System (Promega, Madison, WI) and Escherichia coli JM109 competent cells (Promega, Madison, WI). Clones that contained PCR product inserts were purified and sequenced using T7 sequencing primers as outlined in pGEM- T Easy Vector Technical Manual (Promega, Madison, WI) at the Research Technology and Support Facility (Michigan State University, East Lansing, MI) using an ABI 3730xl Genetic Analyzer. 38 Comparison of the sizes of cloned and sequenced AP-PCR products to the sizes of RAMS For sequenced inserts, the sizes of cloned products were compared with the sizes Of identified RAMS as described previously (Phillips and Goodman, 2009). Six animals per experimental group were used and restriction digestions performed in duplicate, followed by AP-PCR, for a total of 12 reactions. RAM Annotation to Genes BLAST like alignment tool (BLAT) database searches (UCSC Genome Browser, July 2007 mouse assembly, http://genome.ucsc.edu/cgi-bin/thlat?command=start &Org=mouse) determined in which regions of the genome sequenced RAMS occurred. RAMs were classified according to a scheme (Figure S. 1) that indicates location in relation to a gene (e.g., within an intron, overlapping an exon, overlapping the transcriptional start site, or within 10kb of a gene). Genes identified as being within 10kb of a RAM are referred to as annotated genes. RAMS were also categorized by chromosomal location and gene function (Table 82). PCR Products Annotating to 22 RAMs Those PCR products Of size (as measured post-sequencing) within 2bp of both a carry forward and unique RAM could not be definitively labeled and are designated uncertain (CF/U). For example: the 237bp PCR product sequenced from 30pg/kg TCDD+LPS treat- ment could annotate to a 239bp carry forward new-methylation or a 238bp unique new- methylation and is designated as uncertain. 39 DAVID and GO Analysis of Annotated Genes The Database for Annotation, Visualization and Integrated Discovery (DAVID) 2008 (Huang et al., 2008) was used to investigate the functions of annotated genes. With this program, Gene Ontology (GO) Ashbumer et al. (2000) information is efficiently examined for all genes annotating to particular cell processes. Major processes examined were apoptosis, calcium ion storage, cell cycle, differentiation, proliferation, chromatin modification, innate immunity, ion homeostasis and transport, kinase activity, protein trans- port, oxidoreductase activity, transcription, ubiquitin cycle, and vesicle-mediated transport (Tables 2.3 and 8.3). Pathway Analysis of Annotated Genes Pathway Studio 6.0® (Ariadne Genomics, Rockville, MD) was used to investigate the functions of annotated genes. With this program, common targets (genes, functional classes, and cellular processes affected by 22 genes annotated to RAMs) are identified. Treatments with 30pg/kg TCDD were used because this dose exhibited significant inhibi- tion of the primary IgM antibody response (Figure 2.1). Pathway Studio 6.0® was also utilized to uncover documented links between RAMS and B-cell or epigenetic processes including: chromatin remodeling, gene silencing, receptor internalization, mitogenesis, mRNA splicing and stabilization, NO biosynthesis, WNT signaling, differentiation, programmed cell death, protein degradation and folding, receptor mediated endocytosis, transcription initiation, xenobiotic clearance, cell development and fate, cell migration, DNA damage recognition, calcium ion homeostasis, DNA recombination, oxidative stress, translation, DNA replication, apoptosis, and survival (Figures 2.3 and 2.4). 40 2.4 Results 2.4.1 RAM Identification Hypo-, hyper-, and newly methylated RAMS were observed in splenocytes from all female C57BL/6 mice treated with (0, 3, or 30pg/kg) TCDD and (0 or 25pg) LPS when compared to those mice treated with only vehicle. Many individual RAMS occurred in more than one treatment group as carry forward RAMS, i.e., observed at both doses of LPS or TCDD, or observed following treatment with LPS or TCDD and also seen in an LPS+TCDD treatment (Figure 2.2). Treatment with LPS resulted in 32 hypo-, 3 hyper-, and 5 new-methylations. Treatment with 3pg/kg TCDD resulted in 12 hypo-, 13 hyper-, and 18 new- methylations while treat- ment with 30pg/kg TCDD resulted in 37 hypo-, l hyper-, and 4 new-methylations. RAMS that carried forward from 3 to 30pg/kg TCDD were limited to 6 hypomethylations. The l hyper- and all 4 newly methylated RAMS observed in 30pg/kg TCDD were unique to that treatment (Figure 2.2). Treatment with 3pg/kg TCDD+LPS resulted in 29 hypo-, 1 hyper-, and 4 new- methylations. RAMS that carried forward from a Single treatment group to 3yg/kg TCDD+LPS include 3 hypo- (LPS), 3 hypo- (3pg/kg TCDD), and l new-methylation (Bug/kg TCDD+LPS). RAMS that carried forward from two treatment groups to 3pg/kg TCDD+LPS were 2 hypomethylations (LPS and 3,ug/kg TCDD). A number of RAMS unique to 3pg/kg TCDD+LPS were identified: 1 hyper-, 21 hypo-, and 3 new-methylations. Of these unique RAMS, many were identified as having a different methylation status in LPS or 3pg/kg TCDD treatments: 3 unique hypomethylations in 3,ug/kg TCDD+LPS were new-methylations in 3pg/kg TCDD, 6 unique hypo- and 1 unique new-methylation in Bug/kg TCDD+LPS were hypermethylations in 3,ug/kg TCDD, 1 unique new-methylation 41 in 3pg/kg TCDD+LPS was a hypomethylation in both LPS and 3yg/kg TCDD, 1 unique new methylation in 3pg/kg TCDD+LPS was a hypomethylation in LPS, and the 1 unique hypermethylation in 3pg/kg TCDD+LPS was a hypomethylation in both LPS and 3pg/kg TCDD (Table 8.2 and data not Shown). Of the 12 remaining unique hypomethylated RAMS observed in 3pg/kg TCDD+LPS, none were the same size as RAMS observed in LPS or 3pg/kg TCDD (Figure 2.2). 42 38838 nouoaou ohm macaw—£86-30: 98 £093 roan wEEQmQEE m—Zé .mmxTn—QUH wfiwloM can .mmxTQQUH 933 do“; 3%? 9 3V have wfiém as .2; WEEQE mism .AOV m5 .fiv a8... 3%... use @320... 3:8 m2; .mEoEHmob mn3+QQUE wfiwloom can .mn—A+QDUH wfiwlm .QDUH $313 5 32390 803 mzé Esocm E2585 2.0 Eco E war—.588 25:5 98 995% E258: EOE do o5 E @5583 wagon £0 68365 mm. GOP—4%: no Awe—Elem S 0 DOOR. Aomsoctwlmmv ma: ”5; n83: 8:: 80¢ cod—20mm 838:2“? h.o <75. 5 3:586 803 m2¢¢<0 _H_ con—22.3.2 252 a Stefan—23%: a conm_>fim20n>: 2.7.32 9:28 53233.2 262 conmifimécmg: . _ _ _ n _ . _ _ _ 1 NDQZ: a coup 9.3% main—no... 9.3..» 01.5510“. bum-<0 2‘ 83235.2 >32 I 53558.2 262 ® 352235.59»: 203235.235 HN Ifl® fl -----JI------b conm_>£m$_._oa>z GD mac—z: D¢<>>¢Ou >¢¢<0 5:258:09»: ® ma.— Treatment with 30,11g/kg TCDD+LPS treatment resulted in 20 hypo-, 5 hyper-, and 14 new-methylations. RAMS that carried forward from a Single treatment group to 30pg/kg TCDD+LPS include 3 new- (LPS), 1 hyper- (3pg/kg TCDD), 3 hypo-(30pg/kg TCDD), and 3 hypomethylations (3pg/kg TCDD+LPS). RAMS that carried forward from two treat- ment groups to 30pg/kg TCDD+LPS include 2 hypo- (LPS and 3,ug/kg TCDD+LPS), 4 hypo- (LPS and 30pg/kg TCDD), 1 hypo- (30pg/kg TCDD and 3 pg/kg TCDD+LPS), and l new-methylation (3yg/kg TCDD and 30pg/kg TCDD). RAMS that carried forward from three treatment groups to 30pg/kg TCDD+LPS include 1 hypo- (LPS, 3pg/kg TCDD, and 30pg/kg TCDD), l hypo- (LPS, 30pg/kg TCDD, and 3pg/kg TCDD+LPS), and 1 hypomethylation (3pg/kg TCDD, 30pg/kg TCDD, and Brig/kg TCDD+LPS). A number of RAMS unique to 30pg/kg TCDD+LPS were identified including 4 hypo-, 4 hyper-, and 10 new-methylations. Of these unique RAMS, many hypermethylations were identified as having a methylation status different from that observed in LPS, 3pg/kg TCDD, 30,ug/kg TCDD, or 3pg/kg TCDD+LPS treatments: 2 were hypomethylations in both LPS and 30pg/kg TCDD, and l was a hypomethylation in LPS (Table S2 and data not Shown). Of the remaining unique RAMS observed in 30pg/kg TCDD+LPS, 4/4 hypo-, 1/4 hyper-, and 10/10 new-methylations were not the same size as RAMS observed in LPS, 3pg/kg TCDD, 30pg/kg TCDD, or mtg/kg TCDD+LPS (Figure 2.2). 2.4.2 RAM Annotation RAMS were annotated to sequenced PCR products within 2bp of RAM size. Treat- ment with LPS resulted in 40 RAMS, of which 63% were annotated to genes: 100% (3/3) of hyper-, 59% (19/32) of hypo-, and 80% (4/5) of new-methylations. Treatment with 3pg/kg TCDD resulted in 43 RAMS, of which 56% were annotated to genes: 54% (7/13) 45 of hyper-, 58% (7/ 12) of hypo-, and 56% (10/18) of new-methylations. Treatment with 30yg/kg TCDD resulted in 42 RAMS, of which 64% were annotated: 100% (III) of hyper- , 59% (22/37) of hypo-, and 100% (4/4) of new—methylations. Treatment with 3pglkg TCDD+LPS resulted in 34 RAMS, of which 62% were annotated to genes: 69% (20/29) of hypo— and 25% (1/4) of new-methylations. Treatment with 30yg/kg TCDD+LPS resulted in 39 RAMS, of which 78% were annotated: 100% (5/5) of hyper-, 73% (17/23) of hypo—, and 79% (1 1/l4) of new-methylations. Although a large percentage of RAMS were annotated, the chosen procedure limited complete annotation due to incomplete transfection. The probability also exists that those sequences found in one treatment group but not another may actually derive from both while not successfully transforming in both. However, to limit the number of sequences examined, we have chosen to limit annotation only to those sequences of the same size sequenced in the same treatment group(s) in which the RAM was originally observed (Figure 2.2 and Tables 2.1-2.2, 8.2). 46 6:258: E 3:0 888 8:88 mun: 88356830: 3 S .2888 8 889:8 8:3 88:38:. E 088.: Ewocmcwfiv coca—238883: 3 .2888 2 889:8 8:3 coca—>52: E 8888 EmoEcwmmv coca—388835 Am “8 8286.: mm was; coca—>52: Swain macaw—€88-38 do 3%; 82 Ba :25 .6 €88 08m v.53; co :5 82 ”858% as» S8 .888 88 $32 5 8582 225 S 2208 225235-38 .6 a :o a son as :an .6 85 98% :83 do 55 SE ”Basses 203 SE 8.58: 88 maxim 5 8582 £8 .2. 2: .5Q 8083:8830: ho A53 exsow 98 $093 mo AmQa—V 88% .3593 Go AQC 888— 68388 083 88$ 48:58: ma: 5 85:82 mg ow 2: “Cu 88w 83353 2.258.: Eom H _.~ 2an 47 LPSa 3ug/lrg rem)" , 30ug/kg Term" _Gene NCBI RefSeq Gene NCBI RefSeq Gene NCBI RefSelq Acyp2 NM_029344 Ap2al NM_001077264 Atg7 NM_028835 Cldn18 NM_019815 Clic6 NM_172469 Clstn2 NM_022319 Col4a5 NM_007736 Ddx54 NM_028041 ApZal NM_001077264 E2F8 NM_001013368 Cldn18 NM_019815 Fbxl7 NM_176959 Clic6 NM_172469 FOlr4 NM_176807 Clstn2 NM_022319 Illrapll NM_001160403 “a Dde4 NM_028041 Il3ra NM_008369 c ezrs NM001013368 Krrl NM_178610 g Krrl NM_178610 33:61 N§4MOO117021762964 Large NM_010687 g Limal NM_001113545 E2F8 NMO_01013368 Limal NM_001113545 *5 Myof NM_001099634 Pgm2 N 028132 Ling02 NM_175516 g Ntrk2 NM_001025074 Th NM_OO9377 Luzp2 NM_1‘78705 g; Pgm2 NM_028132 — Myof NM_001099634 1:: Prickle2 NM_001081146 Nostrin NM_181547 Six3 NM_011381 Nptl NM_009198 ch4 NM_OO9333 Npt4 NM_134069 Th NM_009377 Pgm2 NM_028132 Ubac2 NM_026861 Prickle2 NM_001081146 Six3 NM_011381 Spbc25 NM_025565 ch4 NM_OO9333 Th NM_OO9377 Ubac2 NM_026861 Unc5c NM_009472 Uspl3 NM_001013024 Wbpl NM_016757 “a Krrl NM_178610 . 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Putative positive (-—>), negative (-i), and binding (- - -)interactions of all annotated genes (white) with common elucidated genes (grey) and functional classes (black) are diagrammed. A. Interactions only occur- ring following 30pg/kg TCDD+LPS treatment. Annotated genes are identified as Unique or Carry Forward from LPS (El), TCDD (O), or both LPS and TCDD (it). B. Interac- tions only occurring following 25pg LPS treatment. References for interactions supplied in Figures 8.2-8.4 and Table 8.5. 55 forward annotated gene and downregulated by a 30,ug/kg TCDD+LPS unique annotated gene; Ras is upregulated by a LPS carry forward annotated gene and downregulated by a 30pg/kg TCDD+LPS unique annotated gene; Akt is upregulated by LPS and 30pg/kg TCDD carry forward annotated genes and downregulated by a 30,ug/kg TCDD+LPS unique annotated gene. Other carry forward annotated genes affect signaling genes and pathways in the same manner in which they are affected by 30pg/kg TCDD+LPS unique annotated genes. 2.5 Discussion Methylation—sensitive restriction digestion followed by arbitrarily primed PCR and capillary electrophoresis allows for the simultaneous and unbiased identification of treatment-related hypo-, hyper-, and new Methylations. We identified 40 RAMs resulting from LPS treatment, 43 RAMs resulting from 3pg/kg TCDD treatment, and 42 RAMs resulting from 30pg/kg TCDD treatment. We further identified 34 RAMs resulting from 3pg/kg TCDD+LPS treatment and 39 RAMs resulting from 30pg/kg TCDD+LPS. Many of the RAMs identified in TCDD+LPS treatments were carried forward from treatment with LPS and TCDD alone. However, almost half of the RAMs identified in TCDD+LPS treatments were unique (not carried forward from any other treatment). Because so many RAMs were unique to the TCDD+LPS treatments, it is unlikely that the phenomenon is random. We therefore propose that the methylation patterns observed in mice treated with LPS are a means by which the splenocytes regulate gene expression. We further propose that TCDD exposure may alter the normal splenocyte methylation pattern and interferes with processes by which normal LPS-induced methylation patterns arise. The interference of TCDD upon processes directing LPS-induced methylation patterns 56 results in a unique methylation pattern observed subsequent to either treatment alone. These results indicate the possibility of crosstalk occurring between TCDD and LPS treat- ments to uniquely affect methylation status of various DNA sequences. Some RAMs were also observed as having a different methylation status between treatment groups, indicating interference and differing influences upon methylation processes between treated groups (Figure 2.2). AP-PCR products, identified as RAMs, were subsequently sequenced to identify genes to which these RAMs annotated. Annotated genes include many involved in B cell signaling, differentiation, and proliferation. DAVID and GO analysis identified annotated genes involved in proliferation were affected by treatment with LPS but not by treatment with TCDD+LPS or TCDD. DAVID and GO analysis of annotated genes also identified genes involved in apoptosis and cytoskeletal rearrangement that are affected by treatment with TCDD+LPS but not by treatment with LPS. These results are consistent with that observed in vivo by North et a]. where LPS-injection increased the total number of B-cells producing IgM in sheep red blood cell (SRBC) assay, a result that is decreased by concomitant exposure to TCDD. Results from this study are consistent with those reported by North et a1 and indicate that changes in methylation may regulate some of the proliferative changes observed in mice treated in vivo with LPS and TCDD. Further analysis of annotated genes for potential interactions via Pathway Studio 6.0® elucidated common targets between treatment groups. LPS annotated genes included two genes (ch4 and Ddx54) that were not annotated from 30pg/kg TCDD+LPS treatment. ch4 positively affects Vegfa, inhibits AR, and inhibits Mmp9. These are interactions that occur only in response to LPS treatment and not to any treatment containing 30pg/kg TCDD. Ddx54 negatively affects ESRl which is an interaction that occurs in LPS and 30pg/kg TCDD treatments but not in 30,ug/kg TCDD+LPS treatment. Annotated genes 57 from LPS treatment inhibit Mmp9 and positively affect Vegfa. Regulation of these genes is observed only in LPS treatment and not in any other examined treatment. Jun and She] are positively affected by treatment with 30pg/kg TCDD+LPS but are not affected in any other examined treatment. Treatment with 30pg/kg TCDD+LPS resulted in annotated genes that potentially regulate common targets MAPK, SMAD, JNK, GAP, Ras, caspase, PI3K, and PLC signaling pathways. These pathways are critical in intracellular balance between cellular differentiation/proliferation and apoptosis, a balance regulated by LPS stimula- tion and deregulated upon concomitant exposure to TCDD. Those annotated genes and signaling pathways that Pathway Studio 6.0® has identified are prime candidates for cross- talk between LPS and TCDD signaling. Due to the large number of potential locations of interaction, the signaling occurring in LPS, TCDD, and TCDD+LPS treatments is compli- cated and likely is affected by many more extra/intracellular factors (Figures 2.3, 2.4, and 3.2). Protein kinase C (PKC) activation has previously been shown to down-regulate DNA methylation activity in human lymphocytes, indicating a decrease in DNA methylation activity resulting from release of internal calcium stores (Bonilla-Henao et al., 2005). Decreased methylation was observed in all three treatment groups, potentially indicating that PKC activity is increased in these splenocytes whether through calcium release (as occurs in LPS signaling) or other mechanisms. Results of this study provide compelling evidence for a DNA methylation based cross- talk between LPS and TCDD signaling in splenocytes. Because many annotated genes are closely involved in B-cell differentiation, we propose that alterations in DNA methylation in splenocytes also occur in B-cells and induce the genes expression changes implicated in controlling plasma cell differentiation and previously described by North et a1. Future studies are planned to assess the validity of these results in primary B—cells treated in vitro 58 with TCDD and LPS, as well as to confirm the correlation between changes in methylation status with alterations in mRNA expression of specific genes. 59 3. EVALUATION OF ALTERATIONS IN GENE EXPRESSION IN THOSE GENES EXHIBITIN G ALTERED DNA METHYLATION IN MURINE SPLENOCYTES EXPOSED IN VI V0 TO LIPOPOLYSACCHARIDE (LPS) AND 2,3,7,8-TETRACHLORODIBENZO-p-DIOXIN (TCDD) McClure, E.A., North, C.M., Kaminski, N., and Goodman, J .I. a manuscript in preparation for submission to Toxicology Letters, 2010 3.1 Abstract 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) inhibits lipopolysaccharide (LPS)- stimulated differentiation of B-cells within the splenocyte population into antibody- producing plasma cells. We hypothesize that altered DNA methylation, an epigenetic event, plays a key role in this inhibition. DNA was isolated from splenocytes prepared 6 days post experiment initiation from 5-6wk old female CS7BL/6 mice dosed with: TCDD, 30pg/kg, on day 0; or LPS, 25pg/mouse, on day 4; or with TCDD and then LPS on days 0 and 4, respectively. Regions of altered DNA methylation (RAMs) were discerned previ- ously by an arbitrarily primed PCR/capillary electrophoresis procedure (Bachman et al., 2006; McClure etal., 2010). The mRNA expression of selected genes (annotated RAMs or genes closely affected by them) that might affect B-cell differentiation were analyzed using qRT-PCR. Three patterns were observed: no change, similar changes in all groups, and different changes based upon treatment. AdcyS (calcium-dependent adenylyl cyclase) and Bankl (B-cell protein involved in calcium mobilization) increased mRNA expression in the LPS group and decreased mRNA expression in the TCDD and TCDD+LPS groups. Bcor (transcriptional regulator, can direct epi genetic modifications) decreased mRNA expression in the LPS group, did not change mRNA expression in the T CDD or TCDD+LPS groups. 60 Ddx54 (repressor of nuclear receptor transcriptional activity), Ralgds (affects ras signaling), Ube216 (ubiquitinates proteins) and Phlpp (inhibits Akt phosphorylation) decreased mRNA expression in the TCDD and TCDD+LPS groups, did not change mRNA expression in the LPS group. This study suggests that TCDD blocks LPS-induced B-cell differentiation by affecting both methylation status and gene expression. Further, genes likely important to LPS signaling and TCDD response as well as many LPS signaling genes that are affected by exposure to TCDD are identified. 3.2 Introduction TCDD is a widespread environmental contaminant with high lipophilicity, accumu- lating in the fat of carnivorous animals (including humans). Specific immunologic aberra- tions observed in mice exposed to TCDD include the inability to mount an innate immune response to LPS challenge. LPS is a bacterial endotoxin found in the outer membrane of gram—negative bacteria and is a potent B-cell mitogen capable of stimulating the humoral immune response. Upon stimulation, B-cells become activated and begin rapidly proliferating concomitant with increased expression of surface and secreted immunoglobulin M (IgM). Murine TCDD-induced immunological suppression is easily measured as a suppres- sion of IgM production with maximum in vitro suppression of primary sheep erythrocyte IgM response observed at Bong/kg TCDD in splenocytes isolated from C57BL/6 mice (Figure 2.1) (Vecchi et al., 1980). The immune suppression in splenocytes treated in vivo with TCDD+LPS is also accompanied with decreased B-lymphocyte-induced maturation protein 1 (Blimpl) expression, decreased CD 1 9+ cells, decreased expression of immuno- 61 globulin J (IgJ), K, and [1 chains, and decreased major histocompatability complex (MHC) class H expression (North et al., 2009). Genomic effects of TCDD are mediated almost entirely through binding to the ligand dependent transcription factor aryl-hydrocarbon receptor (AhR). The AhR is normally found in the cytoplasm of cells in complex with two heat shock protein 90kDa (Hsp90), aryl-hydrocarbon receptor interacting protein (AIP), and a heat shock protein co-chaperone p23/prostaglandin E synthase 3 (p23). Upon TCDD binding, AhR releases all chaperone proteins and translocates to the nucleus where it is bound by aryl-hydrocarbon nuclear translocator (ARNT) and functions as a transcriptional regulator which binds dioxin response element (DRE)s (5‘—TNGCGTG-3). The ARNT/AhR complex upregulates transcription of NF-E2 related factor 2 (Nrf2), which inhibits nuclear factor K—light-chain-enhancer of activated B-cells (NFKB) activation. Hsp90 is also a chaperone for inhibitor of nuclear factor of K light chain gene enhancer in B-cells (IKB) kinase (IKK), which inhibits IKK from inhibiting NFKB (so activating NFKB). NFKB is composed of heterodimers of subunits p50, RelA, p52, c-Rel, and RelB. Vogel and Matsumura (2009) have identified a RelB/AhR response element (RelBAhRE) which binds a RelB/AhR dimer complex and induces transcription in a TCDD-mediated manner. Binding of this dimer to RelBAhRE’s induces transcription of immunologically important genes, including interleukin-8 (IL-8) (Vogel and Matsumura, 2009). The RelB/AhR dimer is also capable of binding DRES, and NFKB binding sites. In the absence of exogenous ligands (TCDD), RelB/p52 binding sites are targeted by RelB/AhR complexes and induce normal trasncription of genes. Further endogenous activation of AhR is observed as a response to CAMP production. Upon systemic challenge, LPS is bound by soluble acute phase protein LPS binding protein (LBP). The LPS-LBP complex is then recognized and bound by the transmem- 62 brane CD14 receptor to interact with the lymphocyte antigen 96 (MD2)/toll-like receptor 4 (TLR4) complex. Binding of TLR4 results in its cytoplasmic toll/interleukin-l receptor (TIR) domain activation. TLR4’s TIR domain activates two separate pathways: 1) The Myeloid differentiation primary response gene 88 (MyD88) recruits interleukin-1 (IL-1) receptor associated kinase (IRAK) for phosphorylation and subsequent association with tumor necrosis factor (TNF) receptor associated factor 6 (TRAF6), leading to activation of two signaling pathways. These signaling pathways result in the activation of transcription factors NFKB and activator protein 1 (AP-l). 2) TIR domain-containing adaptor-inducing interferon-B (T RIF) and TRIF—related adaptor molecule (TRAM) activate TRAF6 which then activates TAN K-binding Kinase 1 (TBKl) to phosphorylate transcription factor interferon regulatory factor 4 (IRF4) (Takeda and Akira, 2004). Hsp90 is also (in high concentration) reported to induce T LR4 activation and positively contribute to antigen processing and presentation on MHC class II molecules (Tobian et al., 2004). TLR4 and B-cell receptor (BCR) signaling also induce Bruton’s tyrosine kinase (Btk) to bind phosphatidylinositol (3,4,5)-triphosphate (PIP3) and phosphorylate phospholipase C (PLC). Activated PLC hydrolyzes phosphatidylinositol (4,5)—bisphosphate (PIP2) to inositol triphosphate (1P3) and diacylglycerol (DAG) which regulate further signaling cascades in the cell (ie. calcium release). PIP3 is generated by phosphoinositide 3-kinase (PI3K) (regulated by Lyn of BCR signaling) and recruits Akt to the membrane for activation, with PIP3 binding to Akt identified as the rate-limiting step in Akt activa- tion. Akt signaling is important in regulating survival, cell cycle, metabolism, and apoptosis through regulation of Ras/Raf, mitogen-activated protein kinase (MAPK), and NFKB signaling cascades. 63 IRF4 is positively regulated by NFKB and indirectly increases Blimpl expression by inhibiting B-cell lymphoma 6 protein (Bcl6) and paired box protein 5 (Pax5) expression. Together, Bcl6, Blimpl, and Pax5 create a reciprocally repressing transcription factor switch, with Pax5 inhibiting IgM production (Figure 1.1). Epigenetic mechanisms of regulation occur through the addition of heritable, imperma— nent marks upon genetic material. Epigenetic regulation can be passed from parental to filial generations of both cells and whole organisms. More transient occurrences are also identified in which various stages of cellular development and differentiation are regulated epigenetically. DNA methylation, in the form of S-methylcytosine, normally occurs at 70% of CpG dinucleotides (Naveh-Many and Cedar, 1981b). Altered DNA-methylation, an epigenetic mechanism that plays a regulatory role in gene expression, has been proven to play multiple roles in carcinogenesis, development, differentiation and even TCDD response (Kurkjian et al., 2008; Okino et al., 2006; Wu et al., 2004). A change in DNA methylation due to LPS-challenge in vivo has previously been described as have alterations in DNA methylation due to TCDD exposure alone and prior to LPS-challenge (McClure et al., 2010). In this letter, evidence is presented that suggests some genes associated with regions of altered DNA methylation (RAMs) also exhibit changes in mRNA expression due to LPS and TCDD exposure. 64 3.3 Materials and Methods 3.3.1 Preparation of in viva Splenocyte Samples Chemicals TCDD was purchased from Accustandard (New Haven, CT) and prepared in sesame oil (Sigma-Aldrich, St. Loius, MO). Salmonella typhosa LPS (Sigma-Aldrich, St. Louis, MO) was prepared in phosphate buffered. saline (PBS) immediately prior to administration. Animals Mice, treatments, and splenocyte collection were described previously (North et al., 2009). The same splenocyte samples from those animals sacrificed upon day 6 (post LPS exposure) in the previous studies (North et al., 2009; McClure et al., 2010) were used for this study. Female 6-8 week old C57BL/6 mice were purchased from the National Cancer Institute and housed in accordance with Michigan State University Institutional Animal Care & Use Committee policy. On day 0, TCDD (0, 3, 10, or 30pg/kg in sesame oil) was administered by single oral gavage. On Day 4, to initiate primary humoral immune response, LPS (0 or 25pg in PBS) was administered by intraperitoneal injection. Spleen samples were collected on days 4-7 from all treatment groups (6 animals per group) and stored at -800C. Mice from day 4 that received only TCDD or vehicle treatment, were used to establish baseline TCDD effects (measured by Antigen Forming Colonies (AFCS) response from samples collected on day 7) (Figure 2.1). 65 3.3.2 qRT-PCR RNA Isolation RNA from splenocytes was isolated using TRIzol® Reagent (Sigma-Aldrich, St. Louis, MO) according to manufacturer’s protocol. Following isopropanol precipitation and ethanol wash, RNA pellets were resuspended in Promega SV RNA Lysis Solution and further purified according to the manufacturer’s protocol (Promega, Madison, WI). cDNA Generation cDNA was generated using Applied Biosystems High Capacity Archive kit according to manufacturer’s instructions (Applied Biosystems, Foster City, CA). Gene Selection Criteria Gene selection criteria is based upon data published in McClure et al. (2010). Each gene examined via quantitative reverse transcription PCR (qRT-PCR) was chosen for unique reasons as listed below: 0 Adenylate cyclase type 5 (Adcy5) is activated by calcium signaling. 0 In the previous study, protein kinase B (Akt) was uniquely identified as a common target of annotated genes occurring in groups treated with 25pg LPS, 30,ug/kg TCDD, or 30pg/kg TCDD+LPS. a Few studies have reported a change in Akt expression as a regulatory mechanism in cell development, while many report changes in the phosphorylation state (and so activity) of this kinase. For this reason, the expression of PH domain and leucine 66 rich repeat protein phosphatase (Phlpp), a dephosphorylase uniquely identified as specifically targeting Akt, was assessed. a B-cell scaflold protein with ankyrin repeats (Bank!) was selected due to its ability to regulate calcium signaling specifically in activated B-cells. o Bcl6 co—repressor (Bcor) functions in concert with Bcl6 in ‘switch’ regulatory sequence and was cloned from treatments although never annotated (unpublished data). 0 Calcium-dependent secretion activator 2 (Cadps2) participates in the priming step of dense-core vesicle exocytosis, an important function in various calcium-secreting cells (Sadakata et al., 2007). Study of mouse tissue homogenates indicates that CadpsZ is most highly expressed in the brain, pituitary, and lungs but only minimally expressed in the spleen and thymus (Sadakata et al., 2007). The pleckstrin homology domain of CadpsZ likely allows interaction with PIPz-rich microdomains in the plasma membrane in a calcium-dependent manner. Although it has not yet been studied nor proven, it is possible that CadpsZ helps in vesicle-mediated immuno- globulin secretion in differentiated B-cells. 0 Dead box 54 (Ddx54) was annotated in all treatments, but identified as having targets common with other annotated genes only in LPS and 30pg/kg TCDD and so was assessed as an anomalous representative of the annotated gene populations. 0 Interleukin-I 7 receptor D (Ill 7rd) affects Ras, MAPK, and Akt. 0 Small subunit processome component homolog (Krrl) is a ribosomal protein down- regulated in metastatic histiocytoma (Adrien et al., 2010). 67 o Tyrosine-protein phosphatase non-receptor type 3 (Ptpn3) is affected by ubiquitin protein ligase E3A (Ube3A) and inhibiting MAPK. o Ral guanine nucleotide dissociation stimulator (Ralgds) is important in G-Protein Coupled Receptor (GPCR) signaling. 0 Retinoic acid receptor 0t (RarOt) is a nuclear receptor which increases T—lymphocyte number, NO synthase activation, and LBP expression (Seguin-Devaux et al., 2002, 2005). o Ubiquitin/ISGI5-conjugating enzyme E2 L6 ( Ube216) functions in concert with Ube3A to affect Akt activity. 0 Zinc finger protein 128 (pr128) is a poorly studied zinc-finger protein with likely transcription factor activity. These genes have all been identified as potentially important in B-cell development, differ- entiation, and/or signaling. Primer Preparation Primers were designed using the web-based NCBI/Primer-BLAST (http://www.ncbi.nlm.nih.gov/tools/primer—blasU). Primers were synthesized by the Macro- molecular Structure Facility at Michigan State University. The UCSC In-Silico PCR web- based tool (July 2007 build, http://genome.ucsc.edu/cgi-bin/thcr?db=mm9) confirmed designed primers to preclude the possibility that expression data be attributed to genomic DNA contamination. Through designing primers to span an exon-exon junction and so that products span at least one intron we have increased stringency of primer selection to ensure 68 evaluation of changes in functional mRNA expression. Gene names, symbols, accession numbers, primer sequences, and amplicon size are listed in Table 8.]. mRNA Quantification According to the manufacturer’s protocol, each reaction contained lpL of cDNA from the aforementioned reverse transcription reaction (with the exception of 183 reactions, which contained lpL of 1:1000 cDNA in DEPC-treated water), lX Power SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA), 0.3pM of forward and reverse primers, and DEPC-treated water (Ambion, Austin, TX) to 50pL. QRT-PCR amplification of duplicate reactions was conducted as previously described (Phillips et al., 2009). Using the absolute quantitation method of determining mRNA expression levels, mRNA copy number of genes was standardized to that of the 188 rRNA gene copy number to control for differences in RNA quantity, quality, and reverse transcription efficiency between samples. Data Analysis Fold changes in the treatment groups (vs. control) were calculated by comparing: (1) 30pg/kg TCDD+LPS versus Control, (2) 30pg/kg TCDD versus Control, (3) LPS versus Control, (4) 3pg/kg TCDD+LPS versus Control, and (5) Bug/kg TCDD versus Control. Statistical outliers, identified by the Grubbs’s test (p30.05, http://www.graphpad.com/quickcalcs/Grubbsl.cfm) were excluded from the final fold change calculations. Expression was considered differentially regulated if it was statisti- cally different from the control group as determined by Students two-tailed t-test, p30.05. Cases in which there was no statistically significant change, but gene expression in _>_3 samples was outside of the 95% confidence interval (CI) of the control group, and all in the 69 same direction (either up or down), were considered to show an “indication of change” in expression. Results are presented in Tables 3.1 and 3.4. 3.4 Results and Discussion qRT-PCR analysis assessed mRNA expression of 14 genes (see section 3.3.2 for a description of selection criteria) which can affect crucial pathways involved in the differ- entiation and proliferation of B lymphocytes (Figures 3.1 and 3.2): 7 annotated genes plus 6 genes which interact with Akt-PI3K regulation of B-cell maturation plus Akt. Eleven of these genes exhibit altered expression in at least one treatment group, while the other 3 show an indication of altered expression (Figure 3.1, Tables 3.1 and 8.4). The only gene to show no change in expression due to treatment, Ill7rd mRNA expression is decreased in splenocytes of mice treated with 30pg/kg TCDD, 25pg LPS, or 130ng TCDD+LPS (Tables 3.1 and 8.4). Decreased Ill7rd mRNA expression may decrease its inhibition of Akt, increasing CAMP response element-binding protein (CREB), estrogen receptor (EsR), mammalian target of rapamycin (mTOR), IKK, and glycogen synthase kinase 3B (GSK-3B) activities. In concert, these changes increase Myc and Blimpl transcription, decreasing Pax5 activity and increasing IgM expression (Figure 3.2). Ill7rd is newly methylated in an intron in splenocytes of animals treated with 30pg/kg TCDD+LPS (McClure et al., 2010). The methylation status of this gene does not correlate with the decreased expression in all treatment groups. Because the decrease in Ill7rd mRNA expression is observed as a result of all three treatments, likely this downregulation is not the main regulatory mechanism by which TCDD inhibits the differentiation of plasma cells in response to LPS stimulation. 70 Table 3.1: Treatment-related changes in gene expression. G 3 Expression Changeb ene Gene Description/Function NCBI RefSeq SYIIIDOI LPS 3Ollg/kg 30Hg/kg TCDD TCDD+LPS Serine/Threonine Kinase Akt + — - Capable of regulating proliferation NM-OO9652 AdCYS ‘F W W Ca2+ dependent Adenylcyclase NM_001012765 _ B-Cell adaptor protein capable of inhibiting Bankl W W Akt activation NM_001033350l l 5‘ RNA Helicase able to repress nuclear D W W receptor transcriptional activity NM-028041 Phlpp - W W Inhibitor of Akt phosphorylation and activity NM_133 821 Stimulates dissociation of GDP from Ras- gds - W W related GTPases NM-OO9058 Ube216 - W W Ubiquitin-Conjugating Enzyme NM_019949 _ _ Bcl6 Co-Repressor, transcriptional regulator Bcor W May Affect Epigenetic Modifications NM-l75045 pr128 W _ _ Represses BBQ/£18112?) signaling through NM_l 53 802 Inhibits FGF signaling (and so proliferation) Ill7rd W it it through the FGFR NM_134437 Ptp n3 W _ W Protein Tyrosrne Phglsgehgtase, Non-Receptor NM_011207 aAnalyzed genes include 5 annotated genes (bold) and 6 other genes (selection criteria: see section 3.3.2), detailed data presented in Table 8.4 b All expression changes are statistically significant (as measured by Student’s, two-tailed, t-test, p_<_0.05) and indicated as: upregulation (T), downregulation (l), or no change (—) 3.4.1 Genes Involved in LPS Signaling Ptpn3 mRNA expression is not altered in splenocytes of mice treated with 30pg/kg TCDD, but decreased in those treated with 30pg/kg TCDD+LPS or 25pg LPS (Tables 3.] and 8.4). CadpsZ mRNA exhibits indication of a similar pattern of expression (Tables 8.4). Decreased Ptpn3 mRNA may increase MAPK signaling and so AP-l activity. Increased AP-l activity increases Blimpl transcription, so decreasing Pax5 activity and increasing IgM expression (Figure 3.2). Decreased CadpsZ mRNA may be also be an important component of LPS signaling that results in IgM secretion, that is not affected by TCDD exposure. 71 TCDD+LPS 4h 't [is .——— 1". 24 \s Figure 3.1.: 'h‘eatment-related changes in splenocyte gene expression. Expression of 5 annotated (bold) and 6 additional genes (selection criteria presented in section 3.3.2) was evaluated (upregulated: white, —>; downregulated: black, -1; or unchanged: grey) following treatment with 25pg LPS, 30pg/kg TCDD, or 30pg/kg TCDD+LPS. Genes exhibiting expression changes in the same direction (either up or down) are connected with a solid line, those exhibiting expression changes in opposite directions are connected with a dashed line. CadpsZ annotates to a new methylation, §2kb downstream of the gene, unique to 30pg/kg TCDD+LPS treatment and occurs within 2kb of the last exon/intron of the gene (McClure et al., 2010). Because the region of altered DNA methylation (RAM) annotated to Cadps2 is unique, it was not also observed in groups treated with LPS indicating that this change in DNA methylation is not specific to LPS signaling (like the gene expression data) and so cannot be correlated with gene expression. 72 Because Ptpn3 and Cadps2 expression are decreased only in the splenocytes of those animals treated with LPS, it is likely that these proteins help regulate IgM expression in response to LPS challenge. However, IgM expression, but neither Ptpn3 nor CadpsZ expression, is greatly diminished in those splenocytes of mice exposed to TCDD prior to LPS challenge (North et al., 2009), likely indicating that the downstream effects of Ptpn3 expression are also altered by TCDD exposure. 3.4.2 Genes Involved in TCDD Signaling Ddx54, Ralgds, Bankl, Phlpp, and Ube216 mRNA expression are not altered in spleno- cytes of mice treated with 25,11g LPS but decreased in those treated with 30pg/kg TCDD or 30pg/kg TCDD+LPS (Tables 3.1 and 8.4). Decreased Ddx54 mRNA may decrease its inhibition of EsR and so increase the transcription of genes regulated by EsR, including those with enhanced expression when AhR and EsR concomitantly bind the promoter region. Ddx54 also has helicase activity and so may regulate other genes epigenetically, an action that may be inhibited by Ddx54 downregulation (Figure 3.2). Decreased Ralgds mRNA may decrease 3-phosphoinositide dependent protein kinase-l (PDKl) and AP-l activity. In concert, these changes decrease Blimpl transcription, increasing Pax5 activity and decreasing IgM expression. Decreased Bankl mRNA may decrease release of calcium from internal stores, reducing calmodulin (CaM) inhibition of E2A (critical to promoting the expression of pro—B-cell proteins such as recombination activating gene (RAG)), increasing v-ets erythroblastosis virus E26 oncogene homolog l (Etsl) and EsR suppres- sion, reducing NFKB and IRF4 inhibition, and eventually activating Blimpl which results in inhibited Pax5 and increased IgM expression. Decreased Phlpp mRNA may decrease its inhibition of Akt, increasing CREB, EsR, mTOR, IKK, and GSK-3B activity. Decreased 73 UbeZl6 mRNA may increase Ptpn3s inhibition of MAPK and decrease Ube3As activation of Akt, decreasing AP-l activity while increasing CREB, EsR, mTOR, IKK, and GSK-3B activity (Figure 3.2). Raroc mRNA expression is not altered in splenocytes of mice treated with 25pg LPS but shows indication of increased expression in those treated with 30pg/kg TCDD or 30pg/kg TCDD+LPS (Tables 8.4). An increase in Raror mRNA may increase LBP expression (and so LPS binding and recognition by lymphocytes) as well as NO synthase activity (a process also induced by LPS signaling through TLR4). Oxidants impede Nrf2 degra- dation, increasing its translocation to the nucleus, and likely reducing NFKB-mediated pro-inflammatory reactions through competition for binding to aryl-hydrocarbon response elements (ARES) (Li et al., 2008). TCDD exposure induces Nrf2 transcription, thereby causing reduced cellular LPS-induced inflammatory response, a mechanism likely devel- oped to inhibit hypersensitive response to pathogens (Thimmulappa et al., 2006) (Figure 3.2). However, when induced by TCDD exposure prior to LPS challenge the reduction in inflammatory response induced by Nrf2 signaling may be exaggerated to such an extent that instead of protecting against LPS-induced septic shock, it actually inhibits response to the challenge and so limits appropriate B-cell differentiation. The upregulation of Rara in 30pg/kg T CDD and 30pg/kg TCDD+LPS increases both the recognition of LPS and the translocation of Nrf2 to the nucleus to inhibit inflammatory response. These actions work in opposition, however, because that reducing inflammatory response acts in cis to affect the originating cell while that increasing LBP expression acts in trans to affect other cells as well as the originating, it is likely that the former mechanism gains precedence and serves as a means by which T CDD exposure inhibits the differentiation of B-cells in response to LPS challenge. 74 . . . in I. .u. . : .- .... n , ; €:..::...;t.: 5.1,??? vii . _, .. Ralgds, Bankl, and Phlpp did not annotate to RAMs in any treatments. Ddx54 is hypomethylated at a location _>_2kb upstream of the transcriptional start site (TSS) in 25pg LPS, 30yg/kg TCDD, and 30pg/kg TCDD+LPS treatment groups. Ube216 is hypermethyl- ated at a location 22kb upstream of the T83 in 30pg/kg TCDD+LPS (McClure et al., 2010). This data suggests that the alterations in gene regulation observed as a result of TCDD treatment were not dependent upon methylation status in the examined regions. In concert, decreases in Ddx54, Ralgds, and Ube216 mRNA and increases in Rara expression result in increased EsR activity, increased Nrf2 activity, decreased Blimpl activity, and decreased IgM expression: a pattern observed in splenocytes of mice treated with 30pg/kg TCDD or 30,ug/kg TCDD+LPS. Decreases in Bankl and Phlpp mRNA expression are hypothesized to decrease EsR activity, decrease release of calcium from internal stores, increase Blimpl activity, and increase IgM expression. However, decreased Blimpl may also function by decreasing inositol triphosphate receptor (IP3R) mediated Ca2+ release from the endoplasmic reticulum, resulting in decreased protein kinase C (PKC) activity, leading to decreased ras/raf signaling, decreased MAPK signaling, decreased AP—l activity, which can downregulate Blimpl, activating Pax5 and decreasing levels of IgM mRNA. The described pattern (as well as that hypothesized to occur as a result of decreases in Ddx54, Ralgds, and Ube216 mRNA expression) of gene down- regulation in 30,ug/kg TCDD and 30pg/kg TCDD+LPS treatments is consistent with the observation that IgM expression is suppressed as a result of TCDD exposure. This result indicates that Raroc, Ddx54, Ralgds, Bankl, and Ube216 mRNA downregulation may be important signaling events occurring in T CDD-exposed splenocytes. 75 3.4.3 Genes Involved in LPS Signaling and Affected by TCDD exposure Akt mRNA expression is not altered in splenocytes of mice treated with 30pg/kg TCDD nor 30pg/kg TCDD+LPS but is increased in those treated with 25pg LPS. Bcor andepl28 mRNA is not altered in splenocytes of mice treated with 30pg/kg TCDD nor 30,ug/kg TCDD+LPS but decreased in those treated with 25pg LPS (Tables 3.1 and 8.4). Increased Akt mRNA may increase the concentration of active Akt and so CREB, EsR, mTOR, IKK, and GSK-3B activity. Decreased Bcor mRNA may decrease the activity of Bcl6, thereby increasing Blimpl and decreasing the activity of Pax5 and B-cell lymphoma-extra large (Bcl-xl), so increasing IgM expression and apoptosis (Figure 3.2). Krrl mRNA expression is not altered in splenocytes of mice treated with 30pg/kg TCDD nor 30pg/kg TCDD+LPS but shows indication of decreased expression in those treated with 25pg LPS (Table 8.4). This decrease in expression is correlated with hyper- methylation of the gene’s TSS (McClure et al., 2010) and suggests that LPS signaling induces methylation of Krrl’s TSS in response to LPS-challenge in order to decrease mRNA expression. Because Krrl is a subunit of a processome important in ribosomal biogenesis, it is likely that downregulation of this gene’s expression may have global impact in inhibiting cellular transcription and translation. In concert, these changes increase Myc and Blimpl transcription, decreasing Pax5 activity and increasing IgM expression (Figure 3.2). Because the hypothesized effects of the observed increase in Akt and Bcor expression are in accordance with those reported by North et al. (2009), it is reasonable to hypothesize that these gene expression alter- ations may be responsible for the increased IgM expression induced by LPS-challenge. The 76 lack of gene expression in those splenocytes of animals treated with 30pg/kg TCDD+LPS indicates that TCDD exposure inhibits appropriate gene regulation for LPS-signaling. AdcyS mRNA expression is increased in splenocytes of mice treated with 25pg LPS but decreased in those treated with 30pg/kg TCDD, or 30pg/kg TCDD+LPS (Tables 3.1 and 8.4). Decreased Adcy5 mRNA may decrease renin (REN) activity, acting upon down- stream elements to decrease Blimpl transcription, increasing Pax5 activity and decreasing IgM expression. Altered Bankl mRNA may affect calcium (Ca2+) release from internal stores, acting upon downstream elements to affect Blimpl transcription, affecting Pax5 activity and IgM expression (Figure 3.2). Because AdcyS expression is decreased only in the splenocytes of those animals treated with TCDD and is increased in those treated with only LPS, it is likely that this protein, as well as Akt, regulates pathways important in LPS response and disrupted in response to TCDD exposure. The similar pattern in mRNA expression of numerous genes, with similar regulation in TCDD and TCDD+LPS treatments but dissimilar regulation in LPS treatment, indicates that TCDD has a dominant influence upon the mRNA expression of these genes. Whether this influence is through many different affects of TCDD or simple alterations in the expression of specific transcription factors is yet to be determined, although the de- regulation of Blimpl expression is a likely candidate as it has been shown to be a master- regulator in the development of B cells and is a transcription factor capable of regulating many signaling pathways within the cell. Bankl, Ddx54, Phlpp, Ralgds, and Ube216 are all downregulated in 30,ug/kg TCDD and 30pg/kg TCDD+LPS treatments, but not in LPS treatment. Ptpn3 is downregulated in LPS and 30pg/kg TCDD+LPS treatments but not in 30,ug/kg TCDD treatment. This evidence would indicate that the regulation of mRNA expression of these genes is only by TCDD or LPS treatment, whether alone or in combination. Unfortunately, due to limitations in 77 the sequencing protocol, direct comparison of changes in methylation status to mRNA expression cannot be made. 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I Hvowmo 22 5332 52%: 32am... 8 win 88%: <21 I 6 co 0 9 I 6 mH o 9 I 6 on o emxuo NowmmHIzz 83% £9.95 9:35: 96555 mamoamm M H mad I M H 33 I m H Hod 9 «NHafi I 2038535. 23593 tutu >22 H 9 . l N 9 . | m 9 . mvcmmH 22 .3239: _mconflbmcmb w .ommmbawméu gum H 6 mo H N 6 om o I 6 ow o 9 Sum I 5%;an 9523mm: Ho 2338 H 9 . I H 9 . I I 9 . $88 :2 32; ascoefiwsam m e mm H N a. HH m m e mm H 6 §< $288922 8296353 “528% 18 m H med 9 m 6? mmd 9 w H m?» 6 £62 3m wmcm m. m wocm m. m wmcm u o u no u _ o u cu U _ a u so _onE>m 3&3. .32 coaucsicoratumwo @6365 28 .8229: Eon. _msgzus 28 3 9:59: «<28 88 9:28 w: 28 108 Table 85: Literature references for annotated genes. The numbered references correspond to the numbers above lines indicating interactions in figures 82-4. 1. McEntaffer, R. L., Natochin, M. and Artemyev, N. O. (1999). Modulation of Transducin thase Activity by Chimeric Rgsl6 and Rgs9 Regulators of G Protein Signaling and the Effector Molecule. Biochemistry 38, 4931-4937 2. Ippolito, D. L., Temkin, P. A., Rogalski, S. L. and Chavkin, C. (2002). N- Terminal Tyrosine Residues within the Potassium Channel Kir3 Modulate thase Activity of Gai. Journal of Biological Chemistry 277, 32692-32696 3. Xiong, S., Zhao, Q., Rong, Z., Huang, G., Huang, Y., Chen, P., Zhang, S., Liu, L. and Chang, Z. (2003). Hsef Inhibits Pc-12 Cell Differentiation by Interfering with Ras-Mitogen—Activated Protein Kinase Mapk Signaling. Journal of Biological Chemistry 278, 50273-50282 4. Gorski, J. A., Zeiler, S. R., Tamowski, S. and Jones, K. R. (2003). Brain- Derived Neurotrophic Factor Is Required for the Maintenance of Cortical Dendrites. J. Neurosci. 23, 6856-6865 S. MacFarlane, M., Kohlhaas, S. L., Sutcliffe, M. J., Dyer, M. J. S. and Cohen, G. M. (2005). Trail Receptor-Selective Mutants Signal to Apoptosis Via Trail-R1 in Primary Lymphoid Malignancies. Cancer Res 65, 11265-11270 6. Mota, M., Reeder, M., Chernoff, J. and Bazenet, C. E. (2001). Evidence for a Role of Mixed Lineage Kinases in Neuronal Apoptosis. J. Neurosci. 21, 4949-4957 7. Cowan, C. M., Thai, J ., Krajewski, 8., Reed, J. C., Nicholson, D. W., Kaufmann, S. H. and Roskams, A. J. (2001). Caspases 3 and 9 Send a Pro-Apoptotic Signal from Synapse to Cell Body in Olfactory Receptor Neurons. J. Neurosci. 21, 7099-7109 8. Gonzalez-Martinez, D., Kim, S.-H., Hu, Y., Guimond, S., Schofield, J ., Winyard, P., Vannelli, G. B., Turnbull, J. and Bouloux, P.-M. (2004). Anosmin-l Modulates Fibroblast Growth Factor Receptor 1 Signaling in Human Gonadotropin- Releasing Hormone Olfactory Neuroblasts through a Heparan Sulfate-Dependent Mechanism. J. Neurosci. 24, 10384-10392 9. Easton, J. B., Moody, N. M., Zhu, X. and Middlemas, D. S. (1999). Brain- Derived Neurotrophic Factor Induces Phosphorylation of Fibroblast Growth Factor Receptor Substrate 2. Journal of Biological Chemistry 274, 1 1321-11327 10. Preger, E., Ziv, I., Shabtay, A., Sher, I., Tsang, M., Dawid, I. B., Altuvia, Y. and Ron, D. (2004). Alternative Splicing Generates an Isoforrn of the Human Sef Gene with Altered Subcellular Localization and Specificity. Proceedings of the National Academy of Sciences of the United States of America 101, 1229-1234 109 11. Kanda, N., Koike, S. and Watanabe, S. (2005). Prostaglandin E2 Enhances Neurotrophin-4 Production Via Ep3 Receptor in Human Keratinocytes. Journal of Pharmacology and Experimental Therapeutics 315, 796-804 12. Le, S., Connors, T. J. and Maroney, A. C. (2001). C-Jun N-Terrninal Kinase Specifically Phosphorylates P66shca at Serine 36 in Response to Ultraviolet Irradiation. Journal of Biological Chemistry 276, 48332-48336 13. Atwal, J. K., Massie, B., Miller, F. D. and Kaplan, D. R. (2000). The Trkb-Shc Site Signals Neuronal Survival and Local Axon Growth Via Mek and Pi3-Kinase. Neuron 27, 265-277 14. Breit, A., Gagnidze, K., Devi, L. A., Lagace’, M. and Bouvier, M. (2006). Simultaneous Activation of the Delta Opioid Receptor (Deltaor)/ Sensory Neuron- Specific Receptor-4 (Snsr-4) Hetero-Oligomer by the Mixed Bivalent Agonist Bovine Adrenal Medulla Peptide 22 Activates Snsr-4 but Inhibits Deltaor Signaling. Molecular Pharmacology 70, 686-696 15. Zhang, L., Hu, Y., Sun, C., Huang, J. and Chu, Z. (2008). [Brain-Derived Neurotrophic Factor Promotes the Secretion of Mmp-9 in Human Myeloma Cell through Modulation of Nucleus Factor-Kappab]. Zhonghua X ue Ye Xue Za Zhi 29, 243- 6 16. Zi, X., Guo, Y., Simoneau, A. R., Hope, C., Xie, J ., Holcombe, R. F. and Hoang, B. H. (2005). Expression of Frzb/Secreted Frizzled-Related Protein 3, a Secreted Wnt Antagonist, in Human Androgen-Independent Prostate Cancer Pc-3 Cells Suppresses Tumor Growth and Cellular Invasiveness. Cancer Res 65, 9762-9770 17. Rajendran, R. R., Nye, A. C., F rasor, J ., Balsara, R. D., Martini, P. G. V. and Katzenellenbogen, B. S. (2003). Regulation of Nuclear Receptor Transcriptional Activity by a Novel Dead Box Rna Helicase (Dp97). Journal of Biological Chemistry 278, 4628-4638 18. Amir, A. L., Barua, M., McKnight, N. C., Cheng, S., Yuan, X. and Balk, S. P. (2003). A Direct S-Catenin-Independent Interaction between Androgen Receptor and T Cell Factor 4. Journal of Biological Chemistry 278, 30828-30834 19. Chen, X., Agate, R. J ., Itoh, Y. and Arnold, A. P. (2005). Sexually Dimorphic Expression of Trkb, a Z-Linked Gene, in Early Posthatch Zebra Finch Brain. Proceedings of the National Academy of Sciences of the United States of America 102, 7730-7735 20. Wilson, S. W. and Houart, C. (2004). Early Steps in the Development of the Forebrain. Developmental Cell 6, 167-181 21. Rena, V., Angeletti, S., Panzetta-Dutari, G. and Genti-Raimondi, S. (2009). Activation of S-Catenin Signalling Increases Stard7 Gene Expression in Jeg-3 Cells. Placenta 30, 876-883 110 22. Nakamura, K., Martin, K. C., Jackson, J. K., Beppu, K., Woo, C.-W. and Thiele, C. J. (2006). Brain-Derived Neurotrophic Factor Activation of Trkb Induces Vascular Endothelial Growth Factor Expression Via Hypoxia-Inducible Factor-1a in Neuroblastoma Cells. Cancer Res 66, 4249-4255 23. Holnthoner, W., Pillinger, M., Groger, M., Wolff, K., Ashton, A. W., Albanese, C., Neumeister, P., Pestell, R. G. and Petzelbauer, P. (2002). Fibroblast Growth Factor- 2 Induces Lef/ch-Dependent Transcription in Human Endothelial Cells. Journal of Biological Chemistry 277, 45847-45853 24. Tanaka, A., Itoh, F., Itoh, S. and Kato, M. (2009). Tall/Sc] Relieves the E2-2- Mediated Repression of Vegfr2 Promoter Activity. J Biochem 145, 129-135 25. Dentelli, P., Rosso, A., Garbarino, G., Calvi, C., Lombard, E., Di Stefano, P., Defilippi, P., Pegoraro, L. and Brizzi, M. F. (2005). The Interaction between Kdr and Interleukin-3 Receptor (ll-3r) Beta Common Modulates Tumor Neovascularization. Oncogene 24, 6394-6405 26. Panopoulou, E., Murphy, C., Rasmussen, H., Bagli, E., Rofstad, E. K. and Fotsis, T. (2005). Activin a Suppresses Neuroblastoma Xenograft Tumor Growth Via Antimitotic and Antiangiogenic Mechanisms. Cancer Res 65, 1877-1886 27. Dihlmann, S., Kloor, M., F allsehr, C. and von Knebel Doeberitz, M. (2005). Regulation of Aktl Expression by Beta-Catenin/ch/Lef Signaling in Colorectal Cancer Cells. Carcinogenesis 26, 1503-1512 28. Hasseine, L. K., Murdaca, J ., Suavet, F., Longnus, S., Giorgetti-Peraldi, S. and Van Obberghen, E. (2007). Hrs Is a Positive Regulator of Vegf and Insulin Signaling. Experimental Cell Research 313, 1927-1942 29. Zheng, F ., Soellner, D., Nunez, J. and Wang, H. (2008). The Basal Level of Intracellular Calcium Gates the Activation of Phosphoinositide 3-Kinase-Akt Signaling by Brain-Derived Neurotrophic Factor in Cortical Neurons. Journal of Neurochemistry 106, 1259-1274 30. Xie, Z.-H., Ambudkar, I. and Siraganian, R. P. (2002). The Adapter Molecule Gab2 Regulates F c{Epsilon}Ri-Mediated Signal Transduction in Mast Cells. J Immunol 168, 4682-4691 31. Cheng, L., Sapieha, P., Kittlerova, P., Hauswirth, W. W. and Di Polo, A. (2002). Trkb Gene Transfer Protects Retinal Ganglion Cells from Axotomy-Induced Death in Vivo. J. Neurosci. 22, 3977-3986 32. Zhang, H., Wu, W., Du, Y., Santos, S. J., Conrad, S. E., Watson, J. T., Grammatikakis, N. and Gallo, K. A. (2004). Hsp90/P500dc37 Is Required for Mixed- Lineage Kinase (Mlk) 3 Signaling. Journal of Biological Chemistry 279, 19457-19463 111 33. Yin, G., Zheng, Q., Yan, C. and Berk, B. C. (2005). Gitl Is a Scaffold for Erk1/2 Activation in Focal Adhesions. Journal of Biological Chemistry 280, 27705- 27712 34. Rayala, S. K., Hollander, P. d., Balasenthil, S., Molli, P. R., Bean, A. J., Vadlamudi, R. K., Wang, R.-A. and Kumar, R. (2006). Hepatocyte Growth F actor- Regulated Tyrosine Kinase Substrate (Hrs) Interacts with Pelpl and Activates Mapk. Journal of Biological Chemistry 281, 4395-4403 35. Thomas, M. K., Tsang, S. W., Yeung, M.-L., Leung, P. S. i. n. g. and Yao, K.- M. (2009). The Roles of the sz-Containing Proteins Bridge-1 and szd2 in the Regulation of Insulin Production and Pancreatic Beta-Cell Mass. Current Protein and Peptide Science 10, 30-36 36. Jung, H. S., Chung, K. W., Won Kim, J., Kim, J ., Komatsu, M., Tanaka, K., Nguyen, Y. H., Kang, T. M., Yoon, K.-H., Kim, J.-W., Jeong, Y. T., Han, M. S., Lee, M.-K., Kim, K.-W., Shin, J. and Lee, M.-S. (2008). Loss of Autophagy Diminishes Pancreatic S Cell Mass and Function with Resultant Hyperglycemia. Cell Metabolism 8, 318-324 37. Harrison, C. A., Gray, P. C., Koerber, S. C., Fischer, W. and Vale, W. (2003). Identification of a Functional Binding Site for Activin on the Type 1 Receptor Alk4. Journal of Biological Chemistry 278, 21 129-21 135 38. Wildey, G. M., Patil, S. and Howe, P. H. (2003). Smad3 Potentiates Transforming Growth Factor Beta (Tng)-Induced Apoptosis and Expression of the Bh3-Only Protein Bim in Wehi 231 B Lymphocytes. Journal of Biological Chemistry 278, 18069-18077 39. Oren, A., Herschkovitz, A., Ben-Dror, I., Holdengreber, V., Ben-Shaul, Y., Seger, R. and Vardimon, L. (1999). The Cytoskeletal Network Controls C-Jun Expression and Glucocorticoid Receptor Transcriptional Activity in an Antagonistic and Cell-Type-Specific Manner. Mol. Cell. Biol. 19, 1742-1750 40. Levenson, A. S., Svoboda, K. M., Pease, K. M., Kaiser, S. A., Chen, B., Simons, L. A., Jovanovic, B. D., Dyck, P. A. and Jordan, V. C. (2002). Gene Expression Profiles with Activation of the Estrogen Receptor A-Selective Estrogen Receptor Modulator Complex in Breast Cancer Cells Expressing Wild-Type Estrogen Receptor. Cancer Res 62, 4419-4426 41. Yang, X., Kovalenko, D., Nadeau, R. J ., Harkins, L. K., Mitchell, J ., Zubanova, 0., Chen, P.-Y. and Friesel, R. (2004). Sef Interacts with Takl and Mediates Jnk Activation and Apoptosis. Journal of Biological Chemistry 279, 38099-38102 42. Savinainen, A., Garcia, E. P., Dorow, D., Marshall, J. and Liu, Y. F. (2001). Kainate Receptor Activation Induces Mixed Lineage Kinase-Mediated Cellular Signaling Cascades Via Post-Synaptic Density Protein 95. Journal of Biological Chemistry 276, 11382-11386 112 Appendix B: Supplemental Figures 5' Transcriptional 3. St- Site 3 10 kb 2 kb A 2 kb 10 1 ' “-4—”. ' i” T I l T 1) Associated with a gene A. Within a gene i. Spans transcriptional start site and/or 5' untranslated region ii. Exon iii. intron B. Upstream from transcriptional start site i. sZkb ii. S10kb and >2kb C. Downstream from last exon/intron i. sZkb ii. $10kb and >2kb 2) >10kb from either transcriptional start site or last exon/intron 3) Repeat element: multiple ”top” hit scores and one/several are associated with gene(s) A. One site and one gene 8. Multiple sites in one gene C. Two or more genes 4) Repeat element: multiple ”top” hit scores and none associated with gene(s) Figure S. 1 .: Classification of RAM annotation based upon genomic location, as deter- mined by BLAT search. Our scheme used to classify annotated Regions of Altered Methylation (RAMs) according to information gleaned from BLAST-like Sequence Align- ment Tool (BLAT; http://genome.ucsc.edu/cgi-bin/thlat) analysis based on where, in relation to a gene, the PCR product aligned (i.e., the RAM was identified). For example, PCR products designated as lBii are located between 2 and 10kb upstream from an annotated gene. 113 Figure 82.: Regulatory interactions of annotated genes in 25pg LPS treatment. Replicate of Figure 2.3A with references designated as numbers above lines indicating regulation. Specific references corresponding to these numbers are listed in Table S5. WNT Figure S.3.: Regulatory interactions of annotated genes in 30pg/kg TCDD treatment. Replicate of Figure 2.3B with references designated as numbers above lines indicating regulation. Specific references corresponding to these numbers are listed in Table S5. 114 Ras SMADC 38 39 A? 9:5?99 B MAPK¢YV§ @Jik ®;.\ Figure S4: Regulatory interactions of annotated genes in 30pg/kg TCDD+LPS treat- ment. Replicate of Figure 2.3C with references designated as numbers above lines indicating regulation. Specific references corresponding to these numbers are listed in Table S5. ’caspase WAPlBll ’A 13 115 LIST OF REFERENCES Adrien, D., Ludger, K. H., Ingo, S., Oliver, M., Cornelius, K., Michael, C. A., Lars, 8., Ole, G., Hans-Ulrich, S. and Marcus, L. (2010). Malignant fibrous histiocytoma- pleomorphic sarcoma, NOS gene expression, histology, and clinical course. A pilot study. Langenbecks Arch Surg 395, 261—75. Agency for Toxic Substances and Disease Registry (ATSDR) (I998). Toxicological Profile for Chlorinated Dibenzo-p-Dioxins. Aiba, Y., Yamazaki, T., Okada, T., Gotoh, K., Sanjo, H., Ogata, M. and Kurosaki, T. (2006). BANK Negatively Regulates Akt Activation and Subsequent B Cell Responses. Immunity 24, 259—268. Amit, S. and Ben-Neriah, Y. (2003). NF-KB activation in cancer: a challenge for ubiquitination- and proteasome-based therapeutic approach. Seminars in Cancer Biology 13, 15—28. Ashbumer, M., Ball, C. A., Blake, J. A., Botstein, D., Butler, H., Cherry, J. M., Davis, A. P., Dolinski, K., Dwight, S. S., Eppig, J. T., Ham's, M. A., Hill, D. P., Issel-Tarver, L., Kasarskis, A., Lewis, S., Matese, J. C., Richardson, J. E., Ringwald, M., Rubin, G. M. and Sherlock, G. (2000). Gene ontology: tool for the unification of biology. The Gene Ontology Consortium. Nat Genet. 25, 25—9. Baba, Y., Hashimoto, S., Matsushita, M., Watanabe, D., Kishimoto, T ., Kurosaki, T. and Tsukada, S. (2001). BLNK mediates Syk-dependent Btk activation. Proceedings of the National Academy of Sciences of the United States of America 98, 2582—2586. Bachman, A. N., Phillips, J. M. and Goodman, J. I. (2006). Phenobarbital Induces Progressive Patterns of GC-Rich and Gene-Specific Altered DNA Methylation in the Liver of Tumor-Prone B6C3Fl Mice. Toxicol. Sci. 91, 393—405. Baylin, S. B. (2005). DNA methylation and gene silencing in cancer. Nat Clin Pract Oncol. 2, 84—1 1. Beebe, L., Park, S. S. and Anderson, L. M. (1990). Differential enzyme induction of mouse liver and lung following a single low or high dose of 2,3,7,8-tetrachlorodibenzo—p- dioxin (TCDD). J Biochem Toxicol. 5, 211-9. Bird, A. (2007). Perceptions of epigenetics. Nature 447, 396—398. Blanco-Aparicio, C., Caamero, M., Cecilia, Y., Pequeo, B., Renner, O., Ferrer, I. and Carnero, A. (2010). Exploring the Gain of Function Contribution of AKT to Mammary Tumorigenesis in Mouse Models. PLoS ONE 5. Bonilla-Henao, V., Martinez, R., Sobrino, F. and Pintado, E. (2005). Different signaling pathways inhibit DNA methylation activity and up-regulate IFN-gamma in human lymphocytes. J Leukoc Biol 78, 1339—1346. Brognard, J. and Newton, A. C. (2008). PHLiPPing the switch on Akt and protein kinase C signaling. Trends in Endocrinology & Metabolism 19, 223—230. Calfon, M., Zeng, H., Urano, F., Till, J. H., Hubbard, S. R., Harding, H. P., Clark, S. G. and Ron, D. (2002). IRE] couples endoplasmic reticulum load to secretory capacity by processing the XBP-l mRNA. Nature 415, 92—96. 116 Cariappa, A. and Pillai, S. (2002). Antigen—dependent B-cell development. Current Opinion in Immunology 14, 241—249. Chiba, N., Masuda, A., Yoshikai, Y. and Matsuguchi, T. (2007). Ceramide inhibits LPS- induced production of IL-5, IL-10, and H.- l 3 from mast cells. J Cell Physiol. 213, 126—36. Christman, J. K. (2002). S-Azacytidine and 5-aza-2’-deoxycytidine as inhibitors of DNA methylation: mechanistic studies and their implications for cancer therapy. Oncogene. 21 , 5483—95. Claudio, E., Brown, K. and Siebenlist, U. (2006). NF-kappaB guides the survival and differentiation of developing lymphocytes. Cell Death Differ. 13, 697—701. Cobaleda, C., Schebesta, A., Delogu, A. and Busslinger, M. (2007). Pax5: the guardian of B cell identity and function. Nat Immunol 8, 463—470. Craig, J. M. and Bickmore, W. A. (1994). The distribution of CpG islands in mammalian chromosomes. Nat Genet. 7, 376—82. Csorba, T., Pantaleo, V. and Burgya’n, J. (2009). RNA Silencing: An Antiviral Mecha- nism. In Natural and Engineered Resistance to Plant Viruses, (Loebenstein, G. and Carr, J. P., eds), vol. 75, of Advances in Virus Research pp. 35 — 71, 230. Academic Press. D’Alessio, J. A., Wright, K. J. and Tjian, R. (2009). Shifting Players and Paradigms in Cell-Specific Transcription. Molecular Cell 36, 924 — 931. Decker, T., Pasca di Magliano, M., McManus, S., Sun, Q., Bonifer, C., Tagoh, H. and Busslinger, M. (2009). Stepwise Activation of Enhancer and Promoter Regions of the B Cell Commitment Gene Pax5 in Early Lymphopoiesis. Immunity 30, 508—520. Delogu, A., Schebesta, A., Sun, Q., Aschenbrenner, K., Perlot, T. and Busslinger, M. (2006). Gene Repression by Pax5 in B Cells Is Essential for Blood Cell Homeostasis and Is Reversed in Plasma Cells. Immunity 24, 269—281. Dent, A. L., Shaffer, A. L., Yu, X., Allman, D. and Staudt, L. M. (1997). Control of Inflammation, Cytokine Expression, and Germinal Center Formation by BCL-6. Science 276, 589—592. Dihlmann, S., Kloor, M., Fallsehr, C. and von Knebel Doeberitz, M. (2005). Regulation of AKT] expression by beta-catenin/ch/Lef signaling in colorectal cancer cells. Carcino- genesis 26, 1503—1512. Dooley, R. K. and Holsapple, M. P. (1988). Elucidation of cellular targets responsible for tetrachlorodibenzo—p-dioxin (TCDD)-induced suppression of antibody responses: I. The role of the B lymphocyte. Immunophannacology. 16, 167—80. Dorshkind, K. and Montecino-Rodriguez, E. (2007). Fetal B-cell lymphopoiesis and the emergence of B-l-cell potential. Nat Rev Immunol 7, 213—219. Dupont, C., Armant, D. R. and Brenner, C. A. (2009). Epigenetics: definition, mecha- nisms and clinical perspective. Semin Reprod Med. 2 7, 351—7. Eden, A., Gaudet, F., Waghmare, A. and Jaenisch, R. (2003). Chromosomal Instability and Tumors Promoted by DNA Hypomethylation. Science 300, 455—. Fan, 2., Yamaza, T., Lee, J. S., Yu, J., Wang, S., Fan, G., Shi, S. and Wang, C.-Y. (2009). BCOR regulates mesenchymal stem cell function by epigenetic mechanisms. Nat Cell Biol 11, 1002-1009. 117 Fang, D., Hawke, D., Zheng, Y., Xia, Y., Meisenhelder, J., Nika, H., Mills, G. B., Kobayashi, R., Hunter, T. and Lu, Z. (2007). Phosphorylation of -Catenin by AKT Promotes -Catenin Transcriptional Activity. Journal of Biological Chemistry 282, 11221— 11229. Fasanaro, P., D’Alessandra, Y., Di Stefano, V., Melchionna, R., Romani, S., Pompilio, G., Capogrossi, M. C. and Martelli, F. (2008). MicroRNA-210 Modulates Endothelial Cell Response to Hypoxia and Inhibits the Receptor Tyrosine Kinase Ligand Ephrin-A3. Journal of Biological Chemistry 283, 15878—15883. Fatemi, M., Hermann, A., Gowher, H. and Jeltsch, A. (2002). Dnmt3a and Dnmtl functionally cooperate during de novo methylation of DNA. European Journal of Biochemistry 269, 4981—4984. Fuxa, M. and Busslinger, M. (2007). Reporter Gene Insertions Reveal a Strictly B Lymphoid-Specific Expression Pattern of Pax5 in Support of Its B Cell Identity Function. J Immunol 178, 3031—3037. Gearhart, M. D., Corcoran, C. M., Wamstad, J. A. and Bardwell, V. J. (2006). Polycomb group and SCF ubiquitin ligases are found in a novel BCOR complex that is recruited to BCL6 targets. Mol Cell Biol. 26, 6880—9. Grant, P. (2001). A tale of histone modifications. Genome Biology 2, reviews0003.l— reviews0003.6. Green, D. R. and Kroemer, G. (2009). Cytoplasmic functions of the tumour suppressor p53. Nature 458, 1127—1130. Guo, J., Burger, M., Nimmrich, I., Maier, S., Becker, E., Genc, B., Duff, D., Rahmat- panah, F., Chitma—Matsiga, R., Shi, H., Berlin, K., Huang, T. H. and Caldwell, C. W. (2005). Differential DNA methylation of gene promoters in small B-cell lymphomas. Am J Clin Pathol. 124, 430—9. Gupta, S., Alt, E, Cooper, M., Melchers, F. and Rajewsky, K. (2007). Gene Regulatory Networks that Orchestrate the Development of B Lymphocyte Precursors vol. 596, of Advances in Experimental Medicine and Biology pp. 57—62. : Springer US. Hahn-Windgassen, A., Nogueira, V., Chen, C.-C., Skeen, J. E., Sonenberg, N. and Hay, N. (2005). Akt Activates the Mammalian Target of Rapamycin by Regulating Cellular ATP Level and AMPK Activity. Journal of Biological Chemistry , 280, 32081—32089. Hark, A. T ., Schoenherr, C. J., Katz, D. J., Ingram, R. S., Levorse, J. M. and Tilghman, S. M. (2000). CT CF mediates methylation-sensitive enhancer-blocking activity at the H19/Igf2 locus. Nature , 405, 486—489. Hauser, J ., Verrna-Gaur, J ., Wallenius, A. and Grundstrom, T. (2009). Initiation of Antigen Receptor-Dependent Differentiation into Plasma Cells by Calmodulin Inhibition of E2A. J Immunol , 183, 1179—1187. Hawkins, P. G., Santoso, S., Adams, C., Anest, V. and Morris, K. V. (2009). Promoter targeted small RNAS induce long-term transcriptional gene silencing in human cells. Nucl. Acids Res., 37, 2984—2995. He, L., He, X., Lim, L. P., de Stanchina, E., Xuan, Z., Liang, Y., Xue, W., Zender, L., Magnus, J., Ridzon, D., Jackson, A. L., Linsley, P. S., Chen, C., Lowe, S. W., Cleary, M. A. and Hannon, G. J. (2007). A microRNA component of the p53 tumour suppressor network. Nature , 447, 1130—1134. 118 Hebbes, T. R., Thorne, A. W. and Crane-Robinson, C. (1988). A direct link between core histone acetylation and transcriptionally active chromatin. Embo J., 7, 1395—402. Heiss, E., Herhaus, C., Klimo, K., Bartsch, H. and Gerhuser, C. (2001). Nuclear Factor KB Is a Molecular Target for Sulforaphane-mediated Anti-inflammatory Mechanisms. Journal of Biological Chemistry , 276, 32008—32015. Hou, B, Ji, M. and Xing, M. (2008). Association of PT EN gene methylation with genetic alterations in the phosphatidylinositol 3-kinase/AKT signaling pathway in thyroid tumors. Cancer , 113, 2440—2447. Huang, D. W., Sherman, B. T. and Lempicki, R. A. (2008). Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat. Protocols , 4, 44—57. Husebye, H., Halaas, O., Stenmark, H., ’I‘unheim, G., Sandanger, O., Bogen, B., Brech, A., Latz, E. and Espevik, T. (2006). Endocytic pathways regulate Toll-like receptor 4 signaling and link innate and adaptive immunity. EMBO J , 25, 683—692. Ishiura, N., Nakashima, H., Watanabe, R., Kuwano, Y., Adachi, T., Takahashi, Y., Tsubata, T., Okochi, H., Tamaki, K., Tedder, T. F. and Fujimoto, M. (2010). Differen- tial phosphorylation of functional tyrosines in CD19 modulates B-lymphocyte activation. European Journal of Immunology , . Ivan, M., Harris, A. L., Martelli, F. and Kulshreshtha, R. (2008). Hypoxia response and microRNAs: no longer two separate worlds. Journal of Cellular and Molecular Medicine , 12, 1426—1431. Kantor, A. B. and Herzenberg, L. A. (1993). Origin of Murine B Cell Lineages. Annual Review of Immunology , 11, 501-538. Kato, M., Miura, A., Bender, J ., Jacobsen, S. E. and Kakutani, T. (2003). Role of CG and Non-CG Methylation in Immobilization of Transposons in Arabidopsis. Current Biology , 13, 421—426. Kawai, T. and Akira, S. (2006). TLR signaling. Cell Death Differ , 13, 816—825. Kee, B. L. (2009). E and ID proteins branch out. Nat Rev Immunol , 9, 175—184. Kerkvliet, N. I. (2002). Recent advances in understanding the mechanisms of TCDD immunotoxicity. Int Immunopharmacol., 2, 277—91. Kim, Y.-H., Shim, Y.-J., Shin, Y.-J., Sul, D., Lee, E. and Min, B.-H. (2009). 2,3,7,8- Tetrachlorodibenzo-p-Dioxin (TCDD) Induces Calcium Influx Through T-type Calcium Channel and Enhances Lysosomal Exocytosis and Insulin Secretion in IN S-l Cells. Inter- national Journal of Toxicology , 28, 151—161. Klein, U. and Dalla-Favera, R. (2008). Germinal centres: role in B-cell physiology and malignancy. Nat Rev Immunol , 8, 22—33. Kristensen, L. S. and Hansen, L. L. (2009). PCR-Based Methods for Detecting Single- Locus DNA Methylation Biomarkers in Cancer Diagnostics, Prognostics, and Response to Treatment. Clin Chem , 55, 1471—1483. Kulshreshtha, R., Davuluri, R. V., Calin, G. A. and Ivan, M. (2008). A microRNA compo- nent of the hypoxic response. Cell Death Differ , 15, 667—67 1. 119 Kurkjian, C., Kummar, S. and Murgo, A. J. (2008). DNA methylation: its role in cancer development and therapy. Current Problems in Cancer , 32, 185—186. Kurosaki, T. (1999). Genetic Analysis of B Cell Antigen Receptor Signaling. Annual Review of Immunology , 17, 555-592. Lanzavecchia, A. and Sallusto, F. (2007). Toll-like receptors and innate immunity in B- cell activation and antibody responses. Current Opinion in Immunology , 19, 268—274. Li, B., Desai, S. A., MacCorkle-Chosnek, R. A., Fan, L. and Spencer, D. M. (2002). A novel conditional Akt ’survival switch’ reversibly protects cells from apoptosis. Gene Ther., 9, 233—44. Li, W., Khor, T. O., Xu, C., Shen, G., Jeong, W.-S., Yu, S. and Kong, A.-N. (2008). Activa- tion of Nrf2-antioxidant signaling attenuates NFKB-inflammatory response and elicits apoptosis. Biochemical Pharmacology , 76, 1485-1489. Liu, G., Park, Y.-J., Tsuruta, Y., Lorne, E. and Abraham, E. (2009a). p53 Attenuates Lipopolysaccharide-Induced NF-kappaB Activation and Acute Lung Injury. J Immunol , 182, 5063-5071. Liu, W. L., Coleman, R. A., Ma, E., Grob, P., Yang, J. L., Zhang, Y., Dailey, G., Nogales, E. and Tjian, R. (2009b). Structures of three distinct activator-TFIID complexes. Genes Dev., 23, 1510—21. Ma, Q., Kinneer, K., Bi, Y., Chan, J. Y. and Kan, Y. W. (2004). Induction of murine NAD(P)H:quinone oxidoreductase by 2,3,7,8-tetrachlorodibenzo-p-dioxin requires the CNC (cap ’n’ collar) basic leucine zipper transcription factor Nrf2 (nuclear factor erythroid 2-related factor 2): cross-interaction between AhR (aryl hydrocarbon receptor) and Nrf2 signal transduction. Biochem. J ., 377, 205—213. MacDonald, J. L. and Roskams, A. J. (2009). Epigenetic regulation of nervous system development by DNA methylation and histone deacetylation. Progress in Neurobiology , 88, 170—183. MacPherson, L., Lo, R., Ahmed, S., Pansoy, A. and Matthews, J. (2009). Activation function 2 mediates dioxin-induced recruitment of estrogen receptor alpha to CYP1A1 and CYPlBl. Biochemical and Biophysical Research Communications , 385, 263—268. Mares, J., Krz, V., Weinhusel, A., Vodickov, S., Kodet, R., Haas, O. A. and Zdenek (2001). Methylation changes in promoter and enhancer regions of the WT] gene in Wilms’ tumours. Cancer Letters , 166, 165 - 171. Martinowich, K., Hattori, D., Wu, H., Fouse, S., He, F., Hu, Y., Fan, G. and Sun, Y. E. (2003). DNA methylation-related chromatin remodeling in activity-dependent BDNF gene regulation. Science, 302, 890—3. Masciarelli, S., Fra, A. M., Pengo, N., Bertolotti, M., Cenci, S., Fagioli, C., Ron, D., Hendershot, L. M. and Sitia, R. (2010). CHOP-independent apoptosis and pathway- selective induction of the UPR in developing plasma cells. Molecular Immunology , 47, 1356—1365. Matsumura, F. (2009). The significance of the nongenomic pathway in mediating inflam- matory signaling of the dioxin-activated Ah receptor to cause toxic effects. Biochemical Pharmacology , 77, 608-626. 120 Matthews, J. and Gstafsson, J .-. (2006). Estrogen receptor and aryl hydrocarbon receptor signaling pathways. Nuclear Receptor Signaling , 4. . McClure, E., North, C., Kaminski, N. and Goodman, J. (2010). DNA methylation: a potential source of crosstalk occurring in Lipopolysaccharide (LPS)-challenged and 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD)-exposed murine splenocytes. Toxicol. Sci., in Preparation. Medema, R. H., Kops, G. J. P. L., Bos, J. L. and Burgering, B. M. T. (2000). AFX- like Forkhead transcription factors mediate cell—cycle regulation by Ras and PKB through p27kipl. Nature , 404, 782—787. Middendorp, S., Dingjan, G. M., Maas, A., Dahlenborg, K. and Hendriks, R. W. (2003). Function of Bruton’s Tyrosine Kinase during B Cell Development Is Partially Independent of Its Catalytic Activity. J Immunol , I 71, 5988—5996. Minguet, S., Dopfer, E. P., Pollmer, C., Freudenberg, M. A., Galanos, C., Reth, M., Huber, M. and Schamel, W. W. (2008). Enhanced B-cell activation mediated by TLR4 and BCR crosstalk. European Journal of Immunology , 38, 2475—2487. Mitoma, Y., Tasaka, N., Takase, M., Masuda, T., Tashiro, H., Egashira, N. and Oki, T. (2006). Calcium-promoted catalytic degradation of PCDDs, PCDFs, and coplanar PCBs under a mild wet process. Environ Sci Technol., 40, 1849-54. Monteiro, P., Gilot, D., Langouet, S. and Fardel, O. (2008a). Activation of the Aryl Hydro- carbon Receptor by the Calcium/Calmodulin-Dependent Protein Kinase Kinase Inhibitor 7-Oxo—7H-benzimidazo[2,1-a]benz[de]isoquinoline-3-carboxylic Acid (STD-609). Drug Metabolism and Disposition , 36, 2556—2563. Monteiro, P., Gilot, D., Le Ferrec, E., Rauch, C., Lagadic-Gossmann, D. and Fardel, O. (2008b). Dioxin-Mediated Up-Regulation of Aryl Hydrocarbon Receptor Target Genes Is Dependent on the Calcium/Calmodulin/CaMKI Pathway. Molecular Pharmacology , 73, 769—777. Mora-Lopez, F., Reales, E., Brieva, J. A. and Campos-Caro, A. (2007). Human BSAP and BLIMPl conform an autoregulatory feedback loop. Blood , 110, 3150—3157. Mukherjee, S., Chen, L. Y., Papadimos, T. J., Huang, S., Zuraw, B. L. and Pan, Z. K. (2009). Lipopolysaccharide-driven Th2 cytokine production in macrophages is regulated by both MyD88 and TRAM. J Biol Chem , 284, 29391—8. Naveh-Many, T. and Cedar, H. (1981a). Active gene sequences are undermethylated. Proceedings of the National Academy of Sciences of the United States of America , 78, 4246—4250. Naveh-Many, T. and Cedar, H. (1981b). Active gene sequences are undermethylated. Proceedings of the National Academy of Sciences of the United States of America , 78, 4246—4250. Nebert, D. W., Roe, A. L., Dieter, M. Z., Solis, W. A., Yang, Y. and Dalton, T. P. (2000). Role of the aromatic hydrocarbon receptor and [Ah] gene battery in the oxidative stress response, cell cycle control, and apoptosis. Biochem Pharmacol., 59, 65—85. Nohara, K., Fujimaki, H., Tsukumo, S., Inouye, K., Sone, H. and Tohyama, C. (2002). Effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on T cell-derived cytokine production in ovalbumin (OVA)-immunized C57B1/6 mice. Toxicology , 172, 49—58. 121 North, C. M., Crawford, R. B., Lu, H. and Kaminski, N. (2009). Simultaneous In vivo Time Course and Dose Response Evaluation for TCDD-Induced Impairment of the LPS- stimulated Primary IgM Response. Toxicol. Sci., 112, 123—32. Nutt, S. L. and Kee, B. L. (2007). The Transcriptional Regulation of B Cell Lineage Commitment. Immunity , 26, 715—725. Okino, S. T., Pookot, D., Li, L.-C., Zhao, H., Urakami, S., Shiina, H., Igawa, M. and Dahiya, R. (2006). Epigenetic Inactivation of the Dioxin-Responsive Cytochrome P4501Al Gene in Human Prostate Cancer. Cancer Res , 66, 7420—7428. Paajarvi, G., Viluksela, M., Pohjanvirta, R., Stenius, U. and Hogberg, J. (2005). TCDD activates Mdm2 and attenuates the p53 response to DNA damaging agents. Carcinogen- esis , 26, 201—208. ' Park, S., Zhao, D., Hatanpaa, K. J., Mickey, B. E., Saha, D., Boothman, D. A., Story, M. D., Wong, E. T., Burma, S., Georgescu, M.-M., Rangnekar, V. M., Chauncey, S. S. and Habib, A. A. (2009). RIP] Activates PI3K-Akt via a Dual Mechanism Involving NF-kappaB-Mediated Inhibition of the mTOR-SéK-IRSI Negative Feedback Loop and Down-regulation of PTEN. Cancer Res , 69, 4107—4111. Patke, A., Mecklenbrauker, I., Erdjument-Bromage, H., Tempst, P. and Tarakhovsky, A. (2006). BAFF controls B cell metabolic fitness through a PKCbeta- and Akt-dependent mechanism. J. Exp. Med., 203, 2551—2562. Peng, S. L. (2005). Signaling in B cells via Toll-like receptors. Current Opinion in Immunology , 17, 230—236. . Phillips, J. M., Burgoon, L. D. and Goodman, J. I. (2009). Phenobarbital Elicits Unique, Early Changes in the Expression of Hepatic Genes that Affect Critical Pathways in Tumor- Prone B6C3Fl Mice. Toxicol. Sci., 109, 193—205. Phillips, J. M. and Goodman, J. I. (2009). Inhalation of cigarette smoke induces regions of altered DNA methylation (RAMs) in SENCAR mouse lung. Toxicology , 260, 7-15. Pillai, S. and Cariappa, A. (2009). The follicular versus marginal zone B lymphocyte cell fate decision. Nat Rev Immunol , 9, 767—777. Prawan, A., Keum, Y.-S., Khor, T., Yu, S., Nair, S., Li, W., Hu, L. and Kong, A.-N. (2008). Structural Influence of Isothiocyanates on the Antioxidant Response Element (ARE)-Mediated Heme Oxygenase-l (HO-1) Expression. Pharmaceutical Research , 25, 836—844. Puga, A., Hoffer, A., Zhou, S., Bohm, J. M., Leikauf, G. D. and Shertzer, H. G. (1997). Sustained Increase in Intracellular Free Calcium and Activation of Cyclooxygenase-2 Expression in Mouse Hepatoma Cells Treated with Dioxin. Biochemical Pharmacology , 54, 1287 — 1296. Puga, A., Ma, C. and Marlowe, J. L. (2009). The aryl hydrocarbon receptor cross-talks with multiple signal transduction pathways. Biochemical Pharmacology , 77, 713—722. Ramsahoye, B. H., Biniszkiewicz, D., Lyko, F., Clark, V., Bird, A. P. and Jaenisch, R. (2000). Non-CpG methylation is prevalent in embryonic stem cells and may be mediated by DNA methyltransferase 3a. Proc Natl Acad Sci U S A., 97, 5237—42. Roth, S. Y., Denu, J. M. and Allis, C. D. (2001). HISTONE ACETYLTRANSFERASES. Annual Review of Biochemistry , 70, 81—120. 122 Russo, V., Martienssen, R. and Riggs, A., eds (1996). Epigenetic Mechanisms of Gene Regulation. Cold Spring Harbor Laboratory Press, Woodbury. Saarikettu, J., Sveshnikova, N. and Grundstrm, T. (2004). Calcium/Calmodulin Inhibition of Transcriptional Activity of E-proteins by Prevention of Their Binding to DNA. Journal of Biological Chemistry 279, 41004—41011. Sadakata, T., Washida, M., Morita, N. and Furuichi, T. (2007). Tissue Distribution of Ca2+-dependent Activator Protein for Secretion Family Members CAPS] and CAPS2 in Mice. J. Histochem. Cytochem. 55, 301—311. Savitsky, D. and Calame, K. (2006). B-l B lymphocytes require Blimp-l for immuno- globulin secretion. J. Exp. Med. 203, 2305—2314. Schebesta, A., McManus, S., Salvagiotto, G., Delogu, A., Busslinger, G. A. and Busslinger, M. (2007). Transcription Factor Pax5 Activates the Chromatin of Key Genes Involved in B Cell Signaling, Adhesion, Migration, and Immune Function. Immunity 27, 49—63. Schneider, D., Manzan, M. A., Yoo, B. S., Crawford, R. B. and Kaminski, N. (2009). Involvement of Blimp-1 and AP-l Dysregulation in the 2,3,7,8-Tetrachlorodibenzo-p- dioxin-mediated Suppression of the IgM Response by B Cells. Toxicol. Sci. 108, 377- 388. Seguin-Devaux, C., Devaux, Y., Latger-Cannard, V., Grosjean, S., Rochette-Egly, C., Zannad, F., Meistelman, C., Mertes, P.-M. and Longrois, D. (2002). Enhancement of the inducible NO synthase activation by retinoic acid is mimicked by RARalpha agonist in vivo. Am J Physio] Endocrinol Metab 283, E525—535. Seguin-Devaux, C., Hanriot, D., Dailloux, M., Latger—Cannard, V., Zannad, F., Mertes, P.-M., Longrois, D. and Devaux, Y. (2005). Retinoic acid amplifies the host immune response to LPS through increased T lymphocytes number and LPS binding protein expression. Molecular and Cellular Endocrinology 245, 67—76. Shaffer, A. L., Lin, K.-I., Kuo, T. C., Yu, X., Hurt, E. M., Rosenwald, A., Giltnane, J. M., Yang, L., Zhao, H., Calame, K. and Staudt, L. M. (2002). Blimp-1 Orches- trates Plasma Cell Differentiation by Extinguishing the Mature B Cell Gene Expression Program. Immunity 17, 51—62. Shapiro-Shelef, M. and Calame, K. (2005). Regulation of plasma-cell development. Nat Rev Immunol 5, 230—242. Shen, E. S. and Whitlock, J. P. (1989). The potential role of DNA methylation in the response to 2,3,7,8-tetrachlorodibenzo-p-dioxin. Journal of Biological Chemistry 264, 17754-17758. Sutter, C. H., Yin, H., Li, Y., Mammen, J. S., Bodreddigari, S., Stevens, G., Cole, J. A. and Sutter, T. R. (2009). EGF receptor signaling blocks aryl hydrocarbon receptor-mediated transcription and cell differentiation in human epidermal keratinocytes. Proc Natl Acad Sci U S A. 106, 4266—71. Szutorisz, H., Canzonetta, C., Georgiou, A., Chow, C. M., Tora, L. and Dillon, N. (2005). Formation of an active tissue-specific chromatin domain initiated by epigenetic marking at the embryonic stem cell stage. Mol Cell Biol. 25, 1804-20. 123 Takahashi, K., Sugi, Y., Hosono, A. and Kaminogawa, S. (2009). Epigenetic Regulation of TLR4 Gene Expression in Intestinal Epithelial Cells for the Maintenance of Intestinal Homeostasis. J Immunol 183, 6522—6529. Takeda, K. and Akira, S. (2004). TLR signaling pathways. Seminars in Immunology 16, 3—9. Tatemichi, M., Hata, H., Tazawa, H. and Nakadate, T. (2008). Lipopolysaccharide induces aberrant hypermethylation of Hie-l in mouse embryonic fibroblasts lacking p53 gene. Anticancer Res. 28, 2101—8. Thimmulappa, R. K., Scollick, C., Traore, K., Yates, M., Trush, M. A., Liby, K. T., Spom, M. B., Yamamoto, M., Kensler, T. W. and Biswal, S. (2006). Nrf2-dependent protec- tion from LPS induced inflammatory response and mortality by CDDO-Imidazolide. Biochemical and Biophysical Research Communications 351, 883 - 889. Tobian, A. A. R., Canaday, D. H. and Harding, C. V. (2004). Bacterial Heat Shock Proteins Enhance Class II MHC Antigen Processing and Presentation of Chaperoned Peptides to CD4+ T Cells. J Immunol 173, 5130—5137. Tran, H., Brunet, A., Griffith, E. C. and Greenberg, M. E. (2003). The Many Forks in FOXO’s Road. Sci. STKE 2003. Ulrey, C. L., Liu, L., Andrews, L. G. and Tollefsbol, T. O. (2005). The impact of metabolism on DNA methylation. Hum. Mol. Genet. 14, R139—147. van Anken, F... Romijn, E. P., Maggioni, C., Mezghrani, A., Sitia, R., Braakman, I. and Heck, A. J. R. (2003). Sequential Waves of Functionally Related Proteins Are Expressed When B Cells Prepare for Antibody Secretion. Immunity 18, 243—253. Vasanwala, F. H., Kusam, S., Toney, L. M. and Dent, A. L. (2002). Repression of AP—l Function: A Mechanism for the Regulation of Blimp-l Expression and B Lymphocyte Differentiation by the B Cell Lymphoma-6 Protooncogene. J Immunol 169, 1922—1929. Vecchi, A., Mantovani, A., Sironi, M., Luini, W., Cairo, M. and Garattini, S. (1980). Effect of acute exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin on humoral antibody production in mice. Chem Biol Interact 30, 337—42. Vivarelli, M. S., McDonald, D., Miller, M., Cusson, N., Kelliher, M. and Geha, R. S. (2004). RIP Links TLR4 to Akt and Is Essential for Cell Survival in Response to LPS Stimulation. J. Exp. Med. 200, 399—404. Vogel, C. F. and Matsumura, F. (2009). A new cross-talk between the aryl hydrocarbon receptor and RelB, a member of the NF-kappaB family. Biochem Pharrnacol. 7 7, 734—45. Vorderstrasse, B. A., Steppan, L. B., Silverstone, A. E. and Kerkvliet, N. I. (200]). Aryl Hydrocarbon Receptor-Deficient Mice Generate Normal Immune Responses to Model Antigens and Are Resistant to TCDD-Induced Immune Suppression. Toxicology and Applied Pharmacology 171, 157—164. Waddington, C. H. (1957). The Strategy of the Genes. Allen & Unwin, London. Walsh, C. P., Chaillet, J. R. and Bestor, T. H. (1998). Transcription of IAP endogenous retroviruses is constrained by cytosine methylation. Nat Genet. 20, 116—7. 124 Wang, L., Liu, L. and Berger, S. L. (1998). Critical residues for histone acetylation by Gcn5, functioning in Ada and SAGA complexes, are also required for transcriptional function invivo. Genes & Development 12, 640—653. Wang, Y., Zhang, R, Li, W., Hou, L., Wang, J., Liang, Y. and Han, H. (2006). Mouse follicular and marginal zone B cells show differential expression of Dnmt3a and sensi- tivity to 5’-azacytidine. Immunology Letters 105, 174—179. Weake, V. M. and Workman, J. L. (2008). Histone Ubiquitination: Triggering Gene Activity. 29, 653—663. Woodland, R. T., Fox, C. J., Schmidt, M. R., Hammerman, P. S., Opferrnan, J. T., Korsmeyer, S. J., Hilbert, D. M. and Thompson, C. B. (2008). Multiple signaling pathways promote B lymphocyte stimulator dependent B-cell growth and survival. Blood 1 I l , 750—760. Womer, W. and Schrenk, D. (1996). Influence of Liver Tumor Promoters on Apoptosis in Rat Hepatocytes Induced by 2-Acetylaminofluorene, Ultraviolet Light, or Transforming Growth Factor betal. Cancer Res 56, 1272—1278. Wu, H. and Sun, Y. E. (2006). Epigenetic regulation of stem cell differentiation. Pediatr Res. 59, 2lR-5R. Wu, Q., Ohsako, S., Ishimura, R., Suzuki, J. S. and Tohyama, C. (2004). Exposure of Mouse Preimplantation Embryos to 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) Alters the Methylation Status of Imprinted Genes H19 and Igf2. Biology of Reproduction 70, 1790—1797. Yang, W. and Desiderio, S. (1997). BAP-135, a target for Bruton’s tyrosine kinase in response to B cell receptor engagement. Proc Natl Acad Sci U S A. 94, 604—9. Yeager, R. L., Reisman, S. A., Aleksunes, L. M. and Klaassen, C. D. (2009). Introducing the ‘TCDD Inducible AhR-Nrf2 Gene Battery’. Toxicol. Sci. 111, 238—46. Zhang, C., Walker, L. M., Hinson, J. A. and Mayeux, P. R. (2000). Oxidant Stress in Rat Liver after Lipopolysaccharide Administration: Effect of Inducible Nitric-Oxide Synthase Inhibition. Jouma] of Pharmacology and Experimental Therapeutics 293, 968—972. Zhang, X. K., Moussa, O., LaRue, A., Bradshaw, S., Molano, I., Spyropoulos, D. D., Gilkeson, G. S. and WatsOn, D. K. (2008). The Transcription Factor Fli-l Modulates Marginal Zone and Follicular B Cell Development in Mice. J Immunol 181, 1644—1654. Zhang, Y. and Reinberg, D. (2001). Transcription regulation by histone methylation: interplay between different covalent modifications of the core histone tails. Genes & Development 15, 2343—2360. Zhu, Y., Wloch, A., Wu, Q., Peters, G, Pagenstecher, A., Bertalanffy, H. and Sure, U. (2009). Involvement of PTEN Promoter Methylation in Cerebral Cavernous Malforma- tions. Stroke 40, 820—826. 125 M'11111111119111iiiiiiljiiiiflliii“