REGULATION OF THE MAMMALIAN RETINOBLASTOMA PATHWAY BY THE UBIQUITIN - PROTEASOME SYSTEM By Satyaki Sengupta A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of Physiology - Doctor of Philosophy 2015 ABSTRACT REGULATION OF THE MAMMALIAN RETINOBLASTOMA PATHWAY BY THE UBIQUITIN - PROTEASOME SYSTEM By Satyaki Sengupta The Retinoblastoma (RB) family of proteins play critical roles in normal development through their governance of genes involved in cell fate. During normal growth RB family activity is tightly regulated through Cdk - dependent phosphorylation, resulting in t heir dissociation from E2F family transcription factors. In addition, the RB pathway is also governed through the ubiquitin - proteasome system, with deregulated degradation of RB proteins frequently associated with human cancer. Recent studies from our labs have shown in D rosophila that the Retinoblastoma family (Rbf) proteins are subjected to proteasome mediated turnover during embryonic development and this process enhances Rbf engagement in transcriptional repression . This positive link between Rbf1 activity and its destruction indicates that repressor function is governed in a manner similar to that described by the degron theory of transcriptional activation. To understand the relationship between RB family stability and their repress or function during early mammalian development, we initiated studies in mouse embryonic stem (ES) cells. Our studies suggest that differentiation of mouse ES cells is associated with the establishment of a functional RB pathway and simultaneous changes in stability of RB family members. As pluripotent ES cells are characterized by unrestrained cdk activity which plummets at the onset of differentiation, we speculated that the observed changes in protein stability upon ES cell differentiation reflects an int imate relationship between RB phosphorylation and stability. Indeed, we show that phosphorylation dependent turnover of RB, p107 and p130 is mediated by an evolutionarily conserved and autonomous instability element (IE) located in their C - terminal regulat ory domain. Moreover, stabilizing mutations within the IE elements also debilitate them for transcriptional repression. We conclude that the overlap of degron sequences and repression modules is a conserved feature shared among the RB homologues, and repre sents a novel mode of transcriptional repression. Together, these findings implicate the Retinoblastoma family IE region as a regulatory nexus linking repressor potency to the ubiquitin - proteasome sy stem in development and disease. iv I dedicate this thesis to my parents, Mr. Lokendra Sankar Sengupta and Mrs. Soma Sengupta for teaching me the values of education , and for their unconditional love and support. v ACKNOWLEDGEMENTS First and foremost, I would like to thank my mentor Dr. Bill Henry for guiding me through all the se years with utmost patience and encouragement . His infectious enthusiasm for science continues to inspire me in the most fragile time s . Through our conversations, I have acquired countless pearls - of - wisdom that will be priceless in guiding me through the rest of my professional and personal life! I want to thank Dr. David Arnosti, my doctoral committe e member and an investigator of the Retinoblastoma project, for all his valuable insights t hat helped me immensely to achieve clarity . I extend my sincere gratitude to Dr. Jason Knott, my doctoral committe e member and collaborator , for his unparalleled generosity in supporting our experiment s with embryonic stem cells that have been of unmatched importance in shaping this project to its current form. I am also grateful to my other committee members Dr. Kathy Gallo , Dr. Richard Miksicek, and Dr. Hua Xiao for the ir collective wisdom that helped me in achieving my goals. I also want to thank all my co lleagues in the lab, Stacy, Sandhya, Yiliang, Rima, Rewatee, Kurtulus, Alison , Nitin, Pankaj and Liang for making it a stimulating and fun place to work. A big Monica, Raj, Haris and Z ach, the four extra - ordinary undergraduates who helped me with several projects . I am also very thankful to the members of the Knott lab, Katie and Tim, for all their support with the ES cell experiments. I would like to thank my parents, my sister Saloka, my extended family and my friends , for their unyielding support and for always being there for me. Finally , I thank the Lord Almighty for always showing me the path of reason and hope. vi TABLE OF CONTENTS viii ix CHAPTER 1: INTRODUCTION ...................................................................... .................. Abstract ........................................... Retinoblastoma Tumor Suppressor Protein .................................... ........................................ Regulation of RB - E2F pathway: general mechanisms................. ............................... ........... Ubiquitin - Regulation of RB protein stability by viral oncogenes . Regulation of E 2F1 stability by the ubiquitin - proteasome system Regulation of the ubiquitin - Roles for ubiquitin - proteasome in RB - Summary REFERENCES 1 2 3 5 8 12 13 15 16 21 22 CHAPTER 2: THE EVOLUTIONARILY CONSERVED C - TERMINAL DOMAINS IN THE MAMMALIAN RETINOBLASTOMA FAMILY SERVE AS DUAL REGULATORS OF PROTEIN STABILITY AND TRANSCRIPTIONAL Abstract. ....... ................................. Materials and Methods ............................... ...... ....................................................................... Expression Constructs ..................................................... ............................................. ES cell culture, differentiation, an d immunofluorescence ....................................... .... RNA extraction and gene expression analys Human cell culture, transfection, an Luciferase Reporte Structural Homology Results ............................................................. ................. ...................................................... . Regulation of RB, p107, and p130 localization an The RB family C - terminal regulatory domains in 34 35 3 6 38 38 3 8 39 41 41 43 43 43 43 51 72 77 vii CHAPTER 3: SUMMARY .............................................................. .................................... 84 94 APPENDIX .................................................... ........................................................................ REFERENCES 97 104 viii LIST OF FIGURES Figure 1 - 1. Regulation of RB - 6 Figure 1 - 2. Regulation of RB - E2F dependent transcription by the ubiquitin - proteasome ... 18 Figure 2 - 1. Cellular localization of RB, p107, and p130 during mouse embryonic stem (ES) cell . 45 Figure 2 - 2. RB family protein abundance decreases during differentiation concomitant with ... 48 Figure 2 - 3. The C - terminal instabil ity element is co nserved within the mammalian RB family....................................................................................................................... .................... . 53 Figure 2 - 4. The canonical instability elements in human p107 and p130 function as ... 57 Figure 2 - 5. The non - canonical instability element in human RB functions as an autonomous 61 Figure 2 - 6. RB and p107 destabilization during CDK4 inhibition is dependent upon instability ... 64 Figure 2 - 7. p130 IE activity in regulating repressor potency and stabilit y are biochemically separable ... 69 Figure 3 - 1. Parallel regulation of the RB - E2F pathway through reversible phosphorylation and the ubiquitin - proteasome system 86 Figure 3 - 2. Participation o f the RB family C - terminal instability element (IE) in dual regulation of protein stability and transcriptio Figure AP - 1. COP9 signalosome is not involved in regulation of RB family protein stability in differentiated human c Figure AP - 2. COP9 signalosome regulates p53 abundance upon doxorubicin induced DNA .102 ix KEY TO ABBREVIATIONS APC/C - Anaphase Promoting Complex/Cyclosome ATP - Adenosine Triphosphate BRG1 - BRM - related Gene product 1 CC - MB - Coiled - Coil - Marked - Box CCNA2 - Cyclin A2 CDK - Cyclin Dependent Kinase ChIP - Chromatin Immunoprecipitation CMV - Cytomegalovirus COP9 - Constitutive Photomorphogenic 9 CSN - COP9 Signalosome CtBP - C - terminal Binding Protein DP - E2F Dimerization Partner DREAM - D P, RB - like, E2F and MuvB complex E1A - Early region 1A E3 - Ubiquitin activating enzyme 3 E2F - Adenovirus E2 promoter binding factor GFP - Green Fluorescent Protein HCV - Hepatitis C Virus HDAC1/2 - Histone Deacetylase 1/2 HGP - Hutchison - Gilford - Progeria HPV E7 - Human Papilloma Virus Early 7 IE - Instability Element x - LIF - Leukemia Inhibitory Factor LOH - Loss of Heterozygosity MCM10 - Mini Chromosome Maintenance 10 MDM2 - Murine double minute 2 MEFs - Mouse Embryonic Fibroblasts Ned d4/8 - Neural precursor cell - expressed developmentally down regulated gene 4/8 NSCLC - Non Small Cell Lung Cancer PCNA - Proliferating Cell Nuclear Antigen PIP - PCNA Interacting Protein RB - Retinoblastoma Protein RBL1 - Retinoblastoma Like 1 RBL2 - Retinoblastoma Like 2 RBP1 - retinoblastoma Binding Protein 1 Rbf1/2 - Retinoblastoma Factor 1/2 SCF - Skp/Cullin/F - box complex SUMO - Small Ubiquitin Like Modification SWI/SNF - Switch/Sucrose nonfermenting complex TAD - Transactivation Domain TKO Triple Knock Out TSS Transcriptional Start Site 1 CHAPTER 1 : INTRODUCTION 1 1 Part of the work described in this chapter is used in the following manuscript: Satyaki Sengupta and R. William Henry (2015) Regulation of the RB - E2F pathway by the ubiquitin - proteasome system, Invited review from Biochemical Biophysical Acta. (BBA) Gene Regulatory Mechanisms 2 Abstract The Retinoblastoma tumor suppressor (RB) and its related family members p107 and p130 regulate cell proliferation through the transcriptional repression of genes involved in cellular G1 to S phase transition. However, RB proteins are functionally versatile , and numerous genetic and biochemical studies point to expansive roles in cellular growth control, pluripotency, apoptotic response, genomic stability, metastasis, and senescence. For the vast majority of genes, RB family members target the E2F family of transcriptional activators as an integral component of its gene regulatory mechanism. These interactions are regulated via reversible phosphorylation by Cyclins/Cyclin - dependent kinase (Cdk) complexes, a major molecular mechanism that regulates transcript ional output of RB/E2F target genes. Recent studies indicate an additional level of regulation involving the ubiquitin - proteasome system that renders pervasive control over each component of RB pathway. Disruption of the genetic circuitry for proteasome - me diated targeting of the RB pathway has serious consequences on development and cellular transformation during cancer progression, and is associated with several forms of human cancer. In this review, I discuss the role of the ubiquitin - proteasome system in proteolytic control of RB - E2F pathway components, and recent data that points to surprising non - proteolytic roles for the ubiquitin - proteasome system in novel transcriptional repression mechanisms. 3 Retinoblastoma tumor suppressor protein Precise regulation of cell cycle is key to normal development and maintenance of physiological homeostasis. Disruption of these regulatory mechanisms leads to deregulated cellular proliferatio n associated with human cancers (1, 2) . Retinoblastoma (RB) prot ein actively engages in transcriptional repression of cell cycle genes and thereby coordinates from G1 - to - S phase transition during cell cycle to limit aberrant cellular proliferation (3) . Historically, RB was attributed as a tumor suppressor protein through studies that implicated mutation in the RB1 gene as a causal event in the development of Retinoblastoma, a devastating cancer of the juvenile retina ( 4 ) . We now know that mutations in RB is a common theme in cancer initiation (3) . Besides i ts anti - proliferative function, RB employs a repertoire of others tumor suppressive mechanism s pertaining to regulation of apoptosis, genomic stability senescence, pluripotency, tumor metabolism, angiogenesis and metastasis ( 3 ) . In mammals, the RB family is composed of RB, and the RB - like proteins p107 (RBL1) and p130 (RBL2). These transcriptional repressor pro teins are primarily related through the conservation of the cyclin fold pocket domains that provides a docking interface for co - regulatory factors required for target gene regulation ( 5 ). The pocket domain is evolutionarily conserved and can be easily recognized in RB proteins from diverse metazoan species, including two members, Rbf1 and Rbf2, found in Drosophila species ( 6 ). In the canonical repression mechanism, RB interacts with and actively blocks the function of a heterodimeric transcription facto r complex composed of E2F and DP proteins bound to gene promoters ( 7, 8 ). The E2F family is composed of multiple members, but only some of these (E2F1 - 3) function as transcriptional activators in humans, while others (E2F4 - 8) lack activator capacity per se , but instead can function as co - repressors when tested in gene expression assays ( 7, 8 ). RB associates preferentially with E2F1 - 4 3 complexes, while p107 and p130 tend to associate with the co - repressor class of E2F complexes ( 7, 8 ). Similar E2F specializa tion has been observed in Drosophila with dE2F1 providing activator functionality while the only other member, dE2F2, functions as transcriptional repressor ( 6 ). RB interacts with E2F1 in a highly modular fashion to influence differential gene regulation . RB is organized in to three structured re gions that includes the N - terminus (RB - N) , the Pocket domain (RB - pocket) and the C - terminus (RB - C) ( 5 ). RB - pocket binds to E2F1 - transactivation domain ( E2F1 - TAD) to regulate transcription of cell cycle genes ( 9, 10, 11, 12) whereas RB - C interacts with the E2F1 - Coiled - Coil - Marked Box domain ( E2F1 - CC - MB) to regulate transcription of apoptotic genes ( 11, 13, 14 ) . Each interaction is regulated by distinct phosphorylation events that induce significant allosteric changes to impede the function of one domain while potentially leaving other functionality intact ( 15 ). In vivo, t argeted disruption of the interaction between RB - pocket and E2F - TAD leads to severe impairment of RB - medi at ed repression of cell cycle genes , although surprisingly these animals undergo normal development and show no evidence of tumor formation ( 12 ) . Together these studies suggest that RB - pocket dependent interaction with E2F - TAD is necessary for RB mediated transcriptional repression, but is dispensable for its tumor suppressor function. Another interesting and conserved feature of the RB family pocket domain is the presence of a conserved hydrophobic cleft that provides a binding surface for Leu - X - Cys - X - Glu (LXCXE) containing peptides. This is particularly intriguing because LXCXE serves as a signature motif in several RB - interacting proteins such as chromatin modifying enzymes an d chromatin remodelers ( 16 ) . In vivo a n allele of RB that lacks functional LXCXE binding cleft (RB ) is impaired for interaction with several chromatin modifying enzymes such as RBP1, Sin3, CtBP, HDAC1, HDAC2 while retain ing intact binding with E2F1 (17 ) . Consistent with the role of repressive 5 chromatin in RB mediated gene regulation , several cell cyc le genes are derepressed in RB1 cells during quies cence and oncogenic senescence , in part through deficiencies in establishing heterochromatin mark s on target genes ( 17, 18 ) . Together these studies suggest that L - X - C - X - E dependent interaction s with the transcr iptional machinery is critical for establishing an epigenetic landscape suited for RB mediated transcriptional control (16 , 17, 18). Regulation of RB - E2F pathway : g eneral mechanisms Attesting to its central significance in growth control, the RB/E2F pathway is subjected to tight regulation . In the canonical pathway, sequential RB phosphorylation by the Cyclin - D/Cdk4 and Cyclin - E/C dk2 complexes during G1 - S phase progression, mitigates the two general sets of interactions that are essential for RB - mediated repression, namely E2F/DP association and co - factor binding (Figure 1 - 1 ) ( 19 - 24 ). An emerging, but surprising model of RB regulatory tactics suggests that RB is phosphorylated at only one of its many potential sites by Cyclin - D/Cdk4 during early G1, whereas RB becomes hyper - phosphorylated by Cyclin - E/Cdk2 in late G1 allowing wholesa le activation of E2F driven transcription of cell cycle genes required for S phase function ( 25 ). Rather than blocking global RB function, early but limited phosphorylation may create a suite of RB molecules with specialized functions depending upon parti cular phosphorylation events. Such restrained phosphorylation may provide for more refined regulation of target genes with selective association of some co - regulatory factors, such as histone deacetylases or ATP - dependent chromatin remodeling factors at so me target genes. In addition to control by reversible phosphorylation, outpu t of the RB pathway can also be influenced by modulating the abundance of RB t hrough transcriptional and post - transcriptional mechanisms ( 26 ) . In vivo RB transcription is cell cycle independent, howe ver steady state abundance of RB - mRNA is governed through autoregulatory feedback circuits in a highly cell and tissue specific manner (27). 6 Figure 1 - 1. Regulation of RB - E2F pathway during cell cycle progre ssion 7 Figure 1 - During G1, RB is actively engaged in repression of cell cycle genes (OFF) by binding to , and inhibiting E2F - DP heterodimer. RB mediated transcriptional repression also depends upon interaction with chromatin remodelers (e.g., Brg1) and chromatin modifying enzymes (e.g., HDAC). During S - phase, multisite phosphorylation mediated by Cyclin D/Cdk 4 and Cyclin E/Cdk2 disengages RB from E2F - DP and promotes transcription of cell cycle genes (ON) . Cyclin D/Cdk4 and Cyclin E/Cdk2 activity is inhibited by Cyclin - dependent - Kinase Inhibitors (CKI s ) p16 and p27 respectively. 8 In contrast, p107 transcription is cell cycle dependent and also subjected to cell type specific regulatory networks that dictates transcriptional output ( 28, 29 ). Oncogenic mutations resulting in diminished RB transcription have been found in many cancer cell types ( 30, 31 ) . RB family abundance is also dictated post - transcriptionally through regula tion of protein stability by the ubiquitin - proteasome system and contributes to cell cycle coupled fluctuations RB levels ( 32 ). Inappropriate degradation of RB proteins through the proteasome is also associated with cellular transformation driven by viral oncogenes (33 ). Ubiquitin - Proteasome mediated regulation of RB family proteins Polyu biquitination of intracellular proteins directs them for proteasome mediated degradation (126) . U nhindered progression through cell cycle relies on timely degradation of mitotic and G1 cyclins by the ubiquitin - proteasome system (35 ). In addition, the proteasome is also implicated in degradation of RB and E2F family members. While unified by their ability to repress E2F dependent transcription, RB, p107 and p130 exhibit distinct expression patterns during cell cycle progression indicating that these factors are regulated through additional mechanisms governing their steady state levels. Data from cell cycle block and release studies demonstrated that p130 is abundantly expressed in quiescent cells, while both RB and p107 are maintained at lower stead y state levels ( 32, 35, 36 ). As cells progress through G1 to S phase, p107 and RB are expressed at higher levels, as p13 0 steady state levels plummet. The best experimental support for the ubiquitin - proteasome system in these processes was initially noted for p130 turnover involving the SCFSkp2 E3 ubiquitin ligase complex ( 32, 36 ). In this process, p130 regulation is sensitive to at least two conditions. First, early in cell cycle progression Skp2 activity is rate - limiting for p130 degradation, but upon s erum stimulation and progression into S phase, Skp2 levels accumulate ( 37 - 39 ). Secondly, Cdk4 phosphorylates p130 to trigger subsequent ubiquitination and 9 proteasome - mediated destruction ( 32 ). The timely destruction of p130 during G1 and S phase has important consequences for activation of gene expression programs during cell cycle progression. Firstly, destabilization of p130 during G1 - S transition ensures transcriptional activation of activator E2Fs (E2F1 - E2F3), whose expression is repressed by p130 - E2F4 complexes ( 40, 41 ). Secondly, as p130 binds to and inactivates Cdk2 (42 ), its degradation also ensures activation of Cdk2 that allows progression to S phase. It is interesting that during G1 - S transition p130 turnover is mediated by the same E3 ligas e complex em ployed for turnover of the p27 C dk 2 inhibitor, reinforcing the timely activation of Cdk2 for coordinated cell cycle progression (43 ). As noted for p130, p107 also exhibits a strong linkage between cell cycle progression and turnover, but in a pattern that was inversely correlated with p130 behavior ( 32, 35, 36 ) . In this case, p107 turnover occurs during the point wherein it is engaged in gene repression, suggesting that p107 potency is linked to its modification and destruction by the ubiquitin - proteasome system. Consistent with this notion, studies using the Cdk4 - specific inhibitor PD - 0332991, a cytostatic drug that causes a robust G1 ar rest demonstrated that the hypo - phosphorylated and active form of p107 is degraded by the proteasome (44 ). Earlier studies had reported that p107 levels are unaffected by treatment with proteasome inhibitors. However, in those studies, the effect of prote asome inhibition may have been obscured because MG132 treatment was carried out in cells that have entered into S - phase, a period wherein p107 is phosphorylated and refractory to proteasome - mediated turnover (45 ). Our studies suggest that dual control of p107 stability and activity is rendered by a C - terminal instability motif which functions as a phosphorylation sensitive degron to influence protein turnover and a repression domain that mediates molecular contacts with E2F to mediate gene repression ( 46 ). The instability element is evolutionarily conserved and related sequences are clearly found in Drosophila Rbf1 and human p130 ( 46, 47 ). While divergent at the amino acid 10 level, functionally related features linking activity and proteasome - mediated turno ver are also observed in regions of the human RB C - terminal domain previously demonstrated to mediate E2F/DP contacts ( 46 ). This correlation led us to propose a model for the RB family in which signaling pathways converge on common motifs to simultaneously regulate protein longevity and activity. In contrast with both p107 and p130, which exhibit robust changes in steady state levels during cell cycle progression, RB fluctuations are muted, and the connection between RB degradation during cell cycle contro l is less well understood (32 ). Nonetheless, in some disease contexts, RB stability is intimately connected to p16 mediated cell cycle arrest, but in somewhat unexpected ways. RB is anchored to the nuclear matrix by a trimeric complex formed by Lamin - A/C a 48 ). Disruptions in RB anchoring either in cells lacking Lamin - A function or via 49, 50 ). Moreover, Lamin - A deficient cells are also insensitive to p14 arf and p16 ar f mediated arrest potentially due to inappropriate RB destruction ( 50 ). It has been speculated that diminution of RB may also have implications for progressive muscular dystrophy in a mouse models and in patients with laminopathies. Deletion mutations in the Lamin - A gene are widely implicated in Hutchison - Gilford - Progeria (H GP ) syndrome (51) . Recent study suggests that levels of RB protein decrease in fibroblasts from H G P patients, but whether this is due altered stability remains to be determined ( 52 ). A dditional evidence supports a role for the ubiquitin - proteasome system in RB control in response to DNA damage. Firstly, several lines of evidence suggest that the Mdm2 E3 - ligase physically interacts with RB to promote its degradation ( 53, 54, 55 ). Human M dm2 is frequently amplified in many cancers ( 56 ) , suggesting that deregulated RB turnover may contribute to 11 tumorigenesis . In support of Mdm2 - mediated RB degradation as a putative oncogenic mechanism, an inverse correlation between Mdm2 and RB protein expr ession has been found in non - small cell lung cancer (NSCLC) tumors that do not exhibit loss - of - heterozygosity (LOH) for RB ( 55 ). The physiological significance of this regulation is further underscored by the observation that Mdm2 overexpression disrupts t he formation of RB - E2F complexes ( 53 ). Secondly, in cells that harbor genomic amplification of Mdm2, depletion of Mdm2 inhibits DNA replication in a RB - dependent manner ( 54 ). This is consistent with previous studies showing that oncogenicity of Mdm2 relies in part on its S - phase promoting function that is independent of p53. E vidence to date further suggest s that RB degradation may involve two distinct mechanisms. In one model, Mdm2 interacts with endogenous RB, and promotes its ubiquitination in a process that is dependent on the E3 ligase activity of the Ring finger domain of Mdm2 ( 55 ). In this study, RB - Md m2 interactions and RB ubiquitination were also dependent on the C terminal domain of RB harboring the instability element ( 46 ) . A second model suggests Mdm2 - mediated degradation of RB involves ubiquitin independent degradation by the 20S proteasome ( 54 ). In this scenario, Mdm2 bound to RB and promoted an interaction between RB and C8 - efficient tethering of RB to the 20S complex thereby facilitating its degradation. Even though all three pocket domain protei ns can interact with this E3 ligase, Mdm2 driven ubiquitination appears specific for RB ( 55 ). Nonetheless, Mdm2 mediated control of RB family stability and function may also extend to p107 as Mdm2 overexpression enables p53 null cells to overcome p107 medi ated G1 arrest ( 57 ), although the molecular details are currently unknown. Recent studies suggest that RB stability is also regulated by MDMX, a structural homolog of MDM2 that is deficient for E3 ligase activity (58 ). MdmX enhances RB - Mdm2 interac tion to efficiently target RB for proteasome mediated degradation and inhibits RB mediated transcriptional repression ( 59 ). 12 In vivo ablation of MdmX in highly tumorigenic p53 - null cells results in an RB - dependent reduction of tumor growth in mouse xenograft assays, suggesting that MdmX mediated RB degradation is potentially oncogenic ( 59 ) . Dual regulation by MdmX and Mdm2 have also bee n implicated in regulation of p53 stability and transactivation ( 60 ) . Collectively these studies suggest that the Mdm2 - MdmX axis functions as negative regulators of RB an d p53 tumor suppressor pathways. Regulation of RB protein stability by viral oncogenes Inactivation of tumor suppressor pathways is a hallmark of viral oncogene induced transformation. Replication of viral genome is heavily reliant on sustained availability of host cell replication factors which are abundant only during S phase , and remain transcriptionally repressed by RB family proteins during the remainder of the cell cycle . Thus disruption of RB function by viral oncogenes provide s a milieu amenable for viral replication. Indeed, early works from the Harlow ( 61 ) and Nevins lab ( 62 ) showed that oncoproteins derived from DNA tumor virus es such as Adenoviral E1A and Human Papilloma virus E7 (HPV - E7) binds to RB, sequestering it away from E2F and thereby unleashing E2F transactivation potential needed for cellular transfo rmation. I nterestingly, several E7 mutants deficient for RB sequestration retained transformation potential, suggesting that an additional E7 function is needed to harness oncogenic potential ( 63 - 66 ). This additional function of E7 required for cellular tr ansformation was defined as its ability to trigger ubiquitin - mediated degradation of RB ( 67 ). Structural ( 9 ) and biochemical studies ( 33 ) suggest that the LXCXE motif in E7 interacts with the RB - pocket , and these contacts are crucial for E7 directed RB degradation. Mechanistically, RB bound E7 forms a complex with Cullin2 - Rb x1 - Elongin B/C to constitute an active E3 ligase that directs proteasome dependent RB degradation ( 68 ). ZER1, a Cullin - 2 substrate specificity factor mediates the interaction between E7 and the 13 Cullin - 2 E3 - complex, and this bridging function of ZER1 is necessary for HPV - E7 mediated RB destabilization ( 69 ). In addition to RB, p107 and p130 are also targeted by E7 for proteasome mediated degradation ( 33 ), with consequent effects on cell proliferation. The maintenance of cellular quiescence during G0 is achieved by repression of cell cycle genes by the DREAM complex, comprised of p130, E2F4, DP1 and the MuvB core ( 70, 71 ). E7 mediated degradation of p130 prevents the formation of DREAM re pressor, leading to sustained activation of S phase genes that allows cell to escape growth arrest and sustain proliferation ( 72 ). A similar of strategy of degrading cellular RB in order to gain control over host cell machinery is employed by RNA viruses s uch as hepatitis C virus (HCV). In HC V infected cells, a LXCXE - containing viral RNA - polymerase binds to RB in the cytoplasm and recruits E6 - associated - protein (E6AP) E3 - ligase to promote RB degradation ( 73, 74 ). Together these studies suggest that invoking proteasome mediated degradation of RB family proteins is a commonly employed mechanism for oncogenesis . Regulation of E2F1 stability by the ubiquitin - proteasome system Similar to RB family proteins, the levels of E2F transcription factors are also tightly regulated through transcription dependent ( 41, 75, 76 ) and independent mechanisms ( 77, 78 ). Post - transcriptional regulation of E2F abundance through proteasome mediated turnover is an essential feature of eukaryotic cell cycle control during development and adulthood ( 79, 80, 81 ). Most of our understanding pertaining to E2F turnover is derived from studies involving E2F1, the major activator E2F which serves as a dual re gulator of cell proliferation and apoptosis ( 7 , 82 ). E2F1 protein is most abundant during late - G1 consistent with its role in activation of S phase genes, however at the onset of S/G2 phase E2F1 levels decline rapidly, as it gets richly ubiquitinated and t argeted to the proteasome ( 79, 83, 84 ). E2F1 protein is extremely unstable due to the presence of 14 an autonomously acting C - terminal degron ( 85, 86 ). Interestingly the E2F1 degron sequence overlaps with the RB binding motif located within its transactivatio n domain, suggesting that RB binding could potentially occlude the usage of the degron , thereby preventing E2F1 degradation ( 85, 86, 87 ). Indeed overexpression of RB stabilizes E2F1 by preventing its proteosomal degradation, and mutant forms of RB that are unable to bind E2F1 also fail to stabilize E2F1. Together these studies suggest that during G1, target gene repression is achieved by stable RB - E2F1 complexes, whereas during S - phase as RB - E2F complexes disengage, free E2F molecules are rapidly degraded. Cell cycle coupled E2F1 turnover is achieved through temporal recruitment of di stinct E3 ligases. For example, during S phase , E2F1 is degraded by the SCF skp2 E3 - ligase complex ( 83 ) whereas during early mitosis its degra dation is mediated by Anaphase Promoting C omplex/C (APC/C cdc20 ) E3 - ligase (84 ). Insight into the physiological sig nificance of cell - cycle associated changes in E2F1 stability is obtained from genetic analysis of S - phase coupled degradation of Drosophila E2F1 (dE2F1). dE2F1 harbors a degron sequence known as PCNA Interacting Protein (PIP) box, which in replicating cell s enables E2F1 degradation through recruitment of Cul4 Cdt2 - E3 ligase ( 88 ). Transgenic overexpression of a stable allele of d E2F1 that is refractory to degradation during S - phase, results in accelerated progression through cell cycle and induces severe apop tosis through transcription dependent and independent mechanisms (88 , 89 ). Interestingly , in a genetic background muted for apoptotic response, stabilized E2F1 results in hyperproliferation, suggesting that apoptotic clearance of cells expressing aberrant levels of E2F1 enables a check on inappropriate cellular proliferation ( 89 ). Together, th ese observations suggests that in vivo , S - phase coupled E2F1 degradation limits inappropriate gene expression and is critical for maintenance of tissue homeostasis. It is important to note that regulation of E2F1 stability is highly 15 context specific. Where as E2F1 is degraded during S - phase, its levels rapidly increase following DNA damage as a result of altered protein stability ( 82, 90, 91 ). In this context stabilized E2F1 is critical for efficient induction of proapoptotic genes. Regulation of the ubiquit in - proteasome system by RB proteins As discussed in the previous sections, components of the RB axis are highly regulated by the ubiquitin - proteasome system. Interestingly, RB family members in turn regulate components of the ubiquitin machinery through tr anscriptional and posttranscriptional mechanisms to affect cellular proliferation and tumor suppression. In particular, most studies have focused on RB - dependent regulation of Skp2, the F - box component of the SCF Skp2 - E3 ligase complex that regulates the st ability of p130 ( 32 ) and the Cdk - inhibitor p27 (43 ). Skp2 abundance is cell cycle dependent ( 37 - 39 ). G rowth arrest leads to diminished Skp2 levels, whereas its expression is induced upon active proliferation , enabling the formation of catalytically active SCF Skp2 complexes. Consistently, p27 is stabilized during quiescence, resulting in RB dephosphorylation and growth arrest, whereas during S - phase p27 is destabilized through SCF Skp2 ( 92 ) , resulting in RB inactivation and cellular proliferation . RB binds to the N - terminus of Skp2 and disrupts Skp2 - p27 interaction, resulting in p27 degradation and G1 arrest ( 93 ). In addition, RB also interacts with the catalytically active Anap hase promoting complex/cyclosome (APC/C), a multisubunit E3 ligase involved in targeted destruction of proteins a t the G1/S boundary and mitosis ( 94 ). Interestingly, RB - stability , but rather provides a scaffold to recruit Skp2 and facilitate it s degradation. RB interacts with Skp2 and APC through distinct surfaces and it is highly likely that a trimeric complex formed by RB - Skp2 - APC is required for effective stabilization of p27 during cell cycle arrest. In addition to regulating Skp2 stability a nd activity , RB is also involved in direct repression of Skp2 transcription in growth arrested cells ( 95, 16 96 ). RB heterozygous (RB +/ - ) mice develops pituitary tumors with elevated Skp2 and diminished p27 levels ( 97 ). However, pituitary specific loss of Skp2 , or introduction a stable p27 allele, completely abolishes pituitary tumors in RB+/ - mice , thereby attesting to the notion that RB mediated regulation of Skp2 abundance and activity serves a tumor suppressive function ( 97 - 99 ) . Furthermore, RB depleted human cone precursor cells and retinoblastoma cell lines exhibits similar reliance on RB - Skp2 - p27 axis for survival and proliferation ( 97, 100 ). Like RB, p107 promotes Skp2 turnover resulting in p27 stabilization and cell cycle arrest , however using mechanisms that are distinct from RB induced Skp2 destabilization ( 101 ). The p107 - Skp2 - p27 axis is also tumor suppressive, as loss of p107 in RB null mice increases Skp2 abundance, re sulting in diminished p27 and heightened CDK2 activity that leads to the formation of retinoblastoma ( 102 ). Together these studies suggest that RB mediated regulation of SCF Skp2 - E3 ligase complex is an important regulatory feature of mammalian cell cycle control and supports the idea of crosstalk between these diverse regulato ry modules. Roles for ubiquitin - proteasome in RB - E2F mediated gene regulation In addition to its function in controlling timely degradation of several transcription factors including RB and E2F , the ubiquitin - proteasome system is also directly involv ed in transcription al regulation ( 103 ) . U biquitinati on of several tr anscriptional activators promotes their activation potency ( 104, 105 ) . Interestingly , these activator proteins show an intimate sequence overlap between domains involved in transactivation and degradation ( 104 ) . Together t hese observations suggests a model where ubiquitination within this overlapping region may additional ly function in transactivation by promoting recruitment of transcriptional cofactors ( 103 , 106 ). The foll owing lines of evidence suggest that the ubiquitin - proteasome system may similarly be involved in regulating transcriptional output from the RB - E2F pathway. Firstly, in a 17 series of recent work we showed that domains involved in regulating RB family stability through ubiquitin - proteasome , are also involved in mediating transcriptional re pression ( 46, 47 ). Consistently, d eletion of this region both stabilizes the repressor (RB) and renders it ineffective for transcriptional repression, suggesting that ubiquitin mediated proteolysis may positively contribute towards gene repression. In support o f this idea, ubiquitination of D ros ophila Rbf1 potentiates repression of target genes (Figure 1 - 2 B) , although the exact role for ubiquitin in this process remains unknown ( 107 ). Secondly , the RB - E2F pathway is subject ed to regulation by COP9 signalosome (CSN), an evolutionarily conserved octameric protein complex that regulates the assembly of Skp1 - Cullin1 - Fbox (SCF) - E3 ligase ( 108 ). D uring embryonic development D rosophila RB homologs Rbf1 and Rbf2 are stabilized through interaction with COP9 signalosome (CSN) ( 109 ) . Interestingly, Rbf1 - CSN interaction is evident on chromatin and sugge sts that CSN may function a s a corepressor by stabilizing chromatin - bound Rbf1 (Figure 1 - 2 A) . Attesting to CSN corepressor function , there is a substantial overlap between gen omic regions bound by Rbf1 and CSN7 subunit of the signalosome ( 110 ) . Furthermore , CSN regulate s transcription of E2F dependent cell cycle genes (110, 111 ) probably by facilitating E2F1 degradation by the Culin4 - E3 ligase ( 112 ). In deed in the absence of CSN , E2F1 is stabilized ( 112 ) but surprisingly rendered inert for transcriptional activation of its target genes ( 110, 111 ) , suggesting that ubiquitin mediated proteolysis may positivel y contribute to transactivation. Consistent with this notion, CSN5 mediated ubiquitination and proteolysis of Myc activator protein positively regulates its transcriptional potency (113). Yet another involvement of CSN in gene regu lation is reflected in its role as a specificity factor for E2F dependent transcription of apoptotic genes (114 - 117). 18 Figure 1 - 2. Regulation of RB - E2F dependent transcription by the ubiquitin - proteasome system. 19 Figure 1 - (A) C hromatin bound D rosophila Rbf1 is stabil ized by COP9 signalosome (CSN) and may enable appropriate gene expression program during embryonic development . (B) A proposed model depicting that ubiquitin (Ub) attached to D rosophila Rbf1 may engage in both targe ting Rbf1 for proteosomal degradation (broken green arrow) , and in enhancing Rbf1 repression by promoting Rbf1 interaction with co - repressors (solid green arrow) . (C) Hypophosphorylated RB is SUMOylated and the SUMO tag may promote RB interaction with transcriptional co - repressors to facilitate repression . 20 Consistently, CSN5 binds to E2F1 target genes involved in apoptosis and is absent at promoters of replication genes (117 ). Intriguingly , in this context the transcriptional function of CSN5 is independent of its deneddylase activity, suggesting that CSN contributes to transcr iptional activation through non - proteolytic mechanisms ( 115 ). One such possible mechanism may involve CSN5 mediated disruption of th e interaction between RB - C terminus and E2F1 marked box domain that specifically inhibit s transcription of apoptotic genes ( 11, 14 ) . Alternatively CSN5 may be involved in recruiting coactivators needed for transcriptional activation. Thirdly , RB - E2F mediated gene regulation is reliant on chromatin remodelers that affect histone H2B ubiquitination , a chromatin mark associated with active transcription ( 121 ) . RB and E2F interact with two distinct remodelling complexes, Brg - BAF250a containing SW I - SNF - A , and Brm - BAF250b containing SWI - SNF - B ( 118, 119 ). During quiescence , both complexes are required to repress E2F target genes, whereas o nly BAF250b containing complex remain bound during proliferation, suggesting an activator function for this compl ex ( 120 ). BAF250b via its BC - box interacts with ElonginC - Cullin2 - Roc1 E3 - ligase to promote H2B ubiquitination and activate transcription. Consistently, cells devoid of BAF250b exhibit delayed induction of cell cycle genes upon exit from quiescence ( 120 ). Together , these studies suggest that appropriate induction of E2F target genes may rely on chrom atin modifications driven by E3 - ubiquitin ligases. Finally , in addition to ubiquitination, RB is also posttranslationally modified by conjugation of Small - Ubiquitin - Like - Modification (SUMOylation) catalyzed by various classes of SUMO - E3 ligases (122, 123 ). Hypophosphorylated RB is SUMOylated in the pocket domain that enhances its potency for transcriptional repression ( 122 ). In this context, the SUMO tag on RB may promote its interaction with transcriptional co - repressors to facilitate repression (Figure 1 - 2 C) (125). Moreover, overexpression of SUMO - E3 ligases induce a senescent phenotype that 21 is reliant on a functional RB pathway ( 123 , 124 ). Taken together, these studies suggest that the ubiquitin - proteasome system plays diverse roles in regulation RB - E2F dependent transcriptional control. Summary I n addition to regulation by reversible phosphorylation, RB - E2F pathway is governed by the ubiquitin - proteasome system , and this mode of governance signi ficantly contributes towards RB - E2F function s during development and disease . RB regulation through both of these m echanisms involve hierarchical control by upstream regulators, and there is substantial crosstalk between these regulatory schemes . Continuing work from our lab has characterized the molecular pathway involved in ubiquitin - proteasome mediated regulation of D rosophila Rbf1, and its implication for Rbf function in development and gene regulation . However such detailed under standing of mammalian RB regulation by the ubiquitin - proteasome system is missing. In chapter II I present and discuss my findings pertaining to molecular mechanisms involved in regulating mammalian RB family stability and their functional significance . 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Nature 458, 422 - 429 34 CHAPTER 2: THE EVOLUTIONARILY CONSERVED C - TERMINAL DOMAINS IN THE MAMMALIAN RETINOBLASTOMA FAMILY SERVE AS DUAL REGULATORS OF PROTEIN STABILITY AND TRANSCRIPTIONAL POTENCY 1 1 T he work described in this chapter was published as the following manuscript: Satyaki Sengupta , Raj Lingnurkar, Timothy S. Carey, Monica Pomaville, Parimal Kar, Michael Feig, Catherine A. Wilson, Jason G. Knott, David N. Arnosti and R. William Henry (2015) The evolutionarily conserved C - terminal domains in the mammalian retinoblastoma tumor suppressor family serve as dual regulators of protein stabil ity and transcriptional potency , Journal of Biological Chemistry , In Press 35 Abstract The Retinoblastoma (RB) tumor suppressor and related family of proteins play critical roles in development through their regulation of genes involved in cell fate. Multiple regulatory pathways impact RB function, including the ubiquitin - proteasome system with deregulated RB destruction frequently associated with pathogenesis. With the current study, we explored the mechanisms connecting proteasome - mediated turnover of the RB family to the regulation of repressor activity. We find that steady state levels of all RB family members, RB, p107, and p130 were diminished during embryonic stem (ES) cell differentiation concomitant with their target gene acquisition. Proteasome - dependent turnover of the RB family is mediated by distinct and autonomously acting instability elements (IE) located in their C - terminal regulatory domains in a pr ocess that is sensitive to Cyclin - dependent kinase (CDK4) perturbation. The IE regions include motifs that contribute to E2F/DP transcription factor interaction, and consistently, p107 and p130 repressor potency was reduced by IE deletion. The juxtaposit ion of degron sequences and E2F interaction motifs appears to be a conserved feature across the RB family, suggesting the potential for repressor ubiquitination and specific target gene regulation. These findings establish a mechanistic link between regul ation of RB family repressor potency and the ubiquitin - proteasome system. 36 Introduction The Retinoblastoma (RB) tumor suppressor regulates cell fate through its governance of distinct gene sets that promote cell division, differentiation, and apoptosis ( 1 , 2 ) . RB is related to two other family members, p107 and p130 that share substantial structural conservation along with some overlapping function in target gene regulation ( 3 - 6 ) . While not as tightly linked to tumor suppression as RB, tumor suppressive capacity has been assigned to these other family members in some contexts. In mouse studies, RB deficient mice were prone to pituitary tumor formation ( 7 ) , whereas mice deficient for RB and p107 or p130 developed retinoblastoma ( 8 , 9 ) , suggesting that p107 and p130 can influence tissue specific predisposition towards tumor development. Similarly, conditional loss of p130 in adult lung epithelial cells in a RB - / - /p53 - / - null background enhanced development of small cell lung carcinoma ( 10 ) . Thus, all three RB family members can act as tumor suppressors in specific contexts. In their roles as tumor suppressors, RB family members predominately function as transcriptional repressors of target genes through their antagonism of the E2F/DP family of transcription factors ( 11 , 12 ) . Recent evidence suggests that RB also plays a positive role in transcriptional activation of some pro - apoptotic response genes, again in a mechanism requiring E2F activity ( 13 ) , although RB may also induce apoptosis through a mechanism that is independent of transcription ( 14 ) . In this scenario, RB - mediated tumor suppression is enabled through blockade of gene products necessary for cell growth with concomitant invocation of cell death pathways. As key regula tors of cell fate, the RB family is tightly controlled by CDK - mediated phosphorylation in response to environmental conditions ( 15 - 17 ) . Hypo - phosphorylated RB interacts with E2F1/DP1 ( 18 ) , blocking activated transcrip tion of cell cycle genes involved in DNA replication and S - phase progression ( 11 , 12 ) . In response to mitogenic signals, serial 37 phosphorylation via cyclin D - CDK 4/6 and cyclin E - CDK2 renders RB family members inactive by disengaging their association with E2F complexes ( 19 - 22 ) . Cyclin - CDK activity is also critically regulated during early steps in normal embryonic development ( 23 ) . Rapidly dividing pluripotent embryonic stem (ES) cells of the early developing embryo employ constitutive CDK - mediated inhibition of RB proteins as a mechanism to maintain rapid cell division d uring blastocyst formation ( 24 - 26 ) . As ES cells differentiate, CDK activity plummets, allowing RB family proteins to regulate E2F activity in a cell cycle dependent manner ( 26 , 27 ) . Despite this unifying model for cyclin - CDK regulation, there are significant differences in the coordination of RB family member activities and steady state levels. For example, RB and p107 are active in cycling cells, while p130 functions predominately i n quiescent cells that have exited from the cell cycle. Experiments performed with staged cells showed that steady state p130 levels peak in G0 in contrast to RB and p107 that increase as cells progress through G1 ( 28 , 29 ) . Consistently, CDK4 activity has opposite effects on p107 and p130 steady state levels; inhibition of the enzyme leads to diminished levels of p107 and higher levels of p130 ( 30 ) . Thus, RB family member activity and stability clearly respond differently to cyclin - CDK signaling during cell cycle progression. However, the mechanisms that link regulation of RB family activity to their turnover are poorly understood. Previous studies from our lab showed that the Drosophila melanogaster RB homologue Rbf1 is subjected to proteasome mediated turnover during embryonic development ( 31 , 32 ) . We within its C - terminal regulatory domain. Importantly, the IE region is also critical for full repressor potency for some cell cycle regulated genes, but not for non - canonical targets whose expression is not usually integrated with the cell cycle ( 31 , 33 ) . Interestingly, Rbf1 ubiquitination also enhanced 38 specif ic activity at select cell cycle target genes ( 33 ) , suggesting that the potency of the repressor at specific genes and overall Rbf1 stability are coordinated. The IE region is well conserved within the mammalian p107 and p130 factors, and we hypothesized that the activity of mammalian RB family members may also be coordinated via integration of the cyclin - CDK signaling pathway with the ubiquitin - proteasome system. We demonstrate here that this regulato ry mechanism is indeed shared among the human RB family proteins. The IE regions within the RB, p107, and p130 C - terminal domains negatively regulate repressor stability through a cyclin - CDK responsive proteasome dependent pathway and contribute to effect ive gene rep ression. These findings indicate that an evolutionarily conserved regulatory pathway links stability and potency for the mammalian RB family. Materials and Methods Expression Constructs - Expression plasmids encoding mutant forms of human RB, p107 and p130 were obtained by site - directed mutagenesis of the pCMV - GFP - RB, pCMV - GFP - p107, pCMV - GFP - p130 parental plasmids ( 34 ) . To generate GFP fusion proteins, PCR amplified instability elements from RB (residues 786 - 864), p107 (residues 964 - 1024), and p130 (residues 1035 - 1095) were fused in frame between the HindIII and KpnI sites of pEGFP - C3 (Clontech). All pla smids were verified by sequencing for the desired mutation. ES cell culture, differentiation, and immunofluorescence - Mouse R1 ES cells were obtained from American Type Culture Collection (ATCC, Manassas, VA), and were cultured on mitomycin - treated mouse embryonic fibroblasts (MEFs) in medium containing high - glucose DMEM supplemented with fetal calf serum (FCS), LIF, L - glutamine, nonessential amino acids, and - mercaptoethanol. J1 - ES cells and the RB - / - , p107 - / - , p130 - / - triple knock out (TKO) ES cells we re a kind gift from Julien Sage ( 35 ) . For ES cell differentiation, cells were plated on 39 gelatin - coated plates to eliminate contaminating MEFs. Differentiation was induced by growing cells in presence of 10 µ M retinoic acid (R2625, Sigma) for 72h. Control cells were treated with DMSO for similar time. For immunofluorescence analysis , ES cells were g rown on lab - tek II chamber slides (Nalge Nunc International, Naperville, IL) under similar conditions and differentiation was induced as discussed above. Cells were fixed in 3.7% freshly made paraformaldehyde for 20 min, washed three times in wash buffer (phosphate buffered saline (PBS) pH 7.4, 0.1% BSA, and 0.01% Tween - 20). Cells were permeabilized in PBS containing 0.1% Triton X - 100 for 15 min, washed, and blocked for one hour at RT in blocking solution (PBS pH 7.4, 1% BSA and 0.01% Tween 20). Cells we re incubated in primary antibody against anti - RB (G3245, mouse monoclonal, 1:100, BD Pharmingen), anti - p107 (SC - 318, rabbit polyclonal, 1:100, Santa Cruz Biotechnology), or anti - p130 (SC - 317, rabbit polyclonal, 1:100, Santa Cruz Biotechnology) in blocking buffer either overnight at 4 o C (Fig 1A - C) or fo r 2 h at room temperature (Fig 2 - 1 D - F). Following three washes in wash buffer, cells were incubated in secondary antibody (Alexa Fluor 488 Goat anti - rabbit - A11008, Alexa Fluor 488 Goat anti - mouse - A11001, Life technologies, Carlsbad, CA) for one hour. Following three washes in wash buffer, slides were mounted with Vectashield mounting media containing DAPI (Vector Laboratories Inc., Burlingame, CA). Images were obtained using an Olympus Fluoview 1000 Filt er - based laser scanning confocal microscope. Chromatin Immunoprecipitation - ES cells were grown on T - 125 flasks and treated with either DMSO or 10 µ M RA ( R2625, Sigma) for 3 days. Cells were washed in PBS, trypsinized, suspended in DMEM and cross - linked with 1% formaldehyde for 18h at 4°C. Cells were then pelleted, washed with PBS and flash frozen in liquid nitrogen. Soluble chromatin was prepared as previously described ( 36 ) . Chromatin bound protein complexes were immunoprecipitated in low 40 salt buffer (20mM Tris HCl, pH 8 .1, 2mM EDTA, 150 mM NaCl, 0.1% SDS, 1% Triton X 100) using 5 µ g of anti - RB (G3245, BD) or anti - p107 (SC - 318) or anti - p130 (SC - 317) or 5 µ g of rabbit non - specific IgG (Millipore). Chromatin - antibody complexes were isolated using 50 µ l protein G Dynabeads ( Life technologies, Carlsbad, CA). Beads were washed once each in low salt buffer, high salt buffer (20 mM Tris HCl, pH 8.1, 2 mM EDTA, 500 mM NaCl, 0.1% SDS, 1% Triton X 100), LiCl buffer (10 mM Tris HCl pH 8.1, 1 mM EDTA, 250 mM LiCl, 1% deoxycholate, 1% IGEPAL630), and twice with TE buffer (10mM Tris HCl, pH 8.1, 1mM EDTA). Chromatin - antibody complexes were eluted from the beads in 200 µ l of elution buffer (100 mM NaHCO 3 , 1% SDS) at 65°C for 30 min with occasional vortexing. Crosslinking was reversed b y addition of 8 µ l of 5 M NaCl and incubation overnight at 65°C. Extracts were then treated with 1 µ l of RNase A (10 mg/ml) for 1h. Subsequently, 13 µ l of Proteinase K buffer (8 µ l 1M Tris pH 6.8, 4 µl 0.5M EDTA and 1 µ l Proteinase K (10 mg/ml) was added and samples were incubated for an additional 1.5 h at 45°C. Associated DNA was purified using QIAquick PCR purification kit (Qiagen, Valencia, CA). Quantitative real - time PCR was performed on input DNA, and antibody spe cific ChIP DNA using SYBR Green Master Mix reagents with an ABI Step one plus thermocycler (Applied Biosystems, Foster City, CA) detection system. Enrichment of RB family members at target gene promoters was examined using primers spanning known E2F bindi ng sites at the murine CCNA2 and MCM10 loci. An intergenic region on mouse chromosome 6 was used as a negative control. Primer sequences are as follows: CCNA2 - F: AATAG TCGCGGGCTACTTGA; CCNA2 - R: GAGCG TAGAGCCCAGGAG; MCM10 - F: AGCGTC CTCCACAAATGAAC; MCM10 - R : ACCCCG TGACGCTTACCTA; Intergenic mouse chr6F: TTTTCAGTT CACACATATAAAGCAGA; Intergenic mouse chr6R: TGTT GTTGTTGTT GCTTCACTG. 41 RNA extraction and gene expression analysis using quantitative real time PCR Pluripotent or differentiated ES cells were harve sted, snap frozen and stored at - 80°C. RNA was extracted using synthesized using SuperScript II Reverse Transcriptase (Invitrogen, Carlsbad, CA). Quantitative real - ti me PCR for RB, p107, and p130 was performed using gene specific primers ( 10 ) ) and SYBR Green Master Mix reagents with an ABI Step one plus thermocycler (Applied Biosystems, Foste r City, CA) detection system. Primers sequences are as follows: RB - F : GCTTGGCTAACTTGGGAG; RB - R : CAACTGCTGCGATAAAGATG; p107 - F : CCGAAGCCCTGGATGACTT; p107 - R : GCATGCCAGCCAGTGTATAACTT; p130 - F : AAGGCACATGCTAACCAATGAA; p130 - R : GAGCAGTTACCGCAGCATGA. Transcript l evels were measured using Taqman probes (Applied Biosystems, Foster City, CA) for Pou5f1 (Mm03053917_g1), Nanog (Mm02019550_s1), and the Eukaryotic elongation factor 1a1 (Mm01966122_u1) as an endogenous control. Relative gene expression was measured by th e 2 - C T method ( 37 ) . Human cell culture, transfection, and drug treatments - To determine the effect of CDK4 inhibition on endogenous RB family stability, approximately 5 x 10 5 U2OS cells were grown for 48h in DMEM containing 10% fetal bovine serum and penicillin - streptomycin. Cells were then treated with 1 µ M PD0332991 (Selleck Chemicals, Houston, TX), and were cultured for an additional 24 hours with or without 1 µ M MG132 for the last 6h. Cells were harvested and the pellet was snap frozen in liquid nitrogen. Cell extracts were prepared in lysis buffer (50mM Tris HCl pH 8.0, 150mM NaCl, 1% Triton X - 100) and the total protein concentration determined using Bradford assay. Equal amounts of whole cell extracts (50 µg) were separated by 12.5% SDS - PAGE and transferred to nitrocellulose membranes for Western analys es. Endogenous proteins 42 were detected by using the following antibodies: anti - RB (G3245, mouse monoclonal, 1:1,000, BD Pharmingen), anti - p107 (SC - 318, rabbit polyclonal, 1:1,000, Santa Cruz Biotechnology), anti - p130 (610261, mouse monoclonal, 1:1000, BD Bi oscience), anti - tubulin (mouse monoclonal, 1:20,000, Iowa Hybridoma Bank), and anti - actin (A5441, mouse monoclonal, 1:10,000, Sigma). In Figure 2 - 2B, p130 was detected using the rabbit polyclonal antibody (SC - 317, rabbit polyclonal, Santa Cruz Biotechnolog y). All antibody incubations were performed in 5% milk in TBST (20 mM Tris HCl, pH 7.5, 120 mM NaCl, 0.1% Tween - 20). Blots were developed using peroxidase conjugated goat anti - rabbit or goat anti - mouse secondary antibodies, as appropriate (1:5000, Thermo Scientific, Waltham, MA) and SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific, Waltham, MA). To measure the effect of CDK4 inhibition on recombinant RB family proteins, approximately 5 x 10 5 U2OS cells were cultured for 24 h prior to tr ansfection with GFP - tagged full - length or IE mutant constructs using Nanojuice transfection reagent (Novagen, EMD Chemicals, San Diego, CA). 24 hours after transfection, cells were treated with 1 µ M PD0332991 for an additional 24 hours. Western analysis w as performed, as above, using anti - GFP antibodies (SC - 9996, mouse monoclonal, 1:1,000, Santa Cruz Biotechnology). Stability assays The steady state abundance of GFP - tagged full length, IE, and 4KRA proteins (Figure 3) was determined by Western blot anal yses, as described above. To determine the relative stability of GFP and GFP fused to the RB family instability elements, transfected cells were treated with 100 µ M cycloheximide 40 hour after transfection, and samples were harvested at 3h, 6h, and 9h post treatment. For proteasome inhibitor treatments in Figures 4B and 5A, cells were treated with DMSO or 1 µ M MG132 for 24h. 43 Luciferase Reporter Assay - U2OS cells were transfected using with Nanojuice transfection reagent, as described above. Typically 5 x 10 5 cells were transfected with 100 ng of a human cyclin A promoter - driven luciferase reporter ( human Cyclin A promoter ( - 89 to +11, ( 38 ) ), 50 ng of pRL - CMV Renilla luciferase reporter (Promega), and 500 ng of plasmid expressing the GFP - tagged effector proteins. After 48 h, cells were harvested and luciferase activity was measured using the Dual - Glo Luciferase assay system (Promega) and Veritas m icroplate luminometer (Turner Biosystems). Firefly luciferase activity was normalized to Renilla luciferase reading. Luciferase measurements were made in triplicate and at least three biological experiments were performed. Structural Homology Modeling - Structure homology modeling of the p130 IE in complex with the E2F4/DP1 was performed using SWISS - MODEL ( 39 ) . The crystal structure of the RB C - terminal domain bound to an E2F1 - DP1 heterodimer (PDB code: 2AZE) ( 40 ) was used to generate the homology model. Results Regulation of RB, p107, and p130 localization and stability in mouse ES cells Previous studies in Drosophila suggested that Rbf1 turnover and function are linked during embryonic development ( 31 , 32 ) . We therefore examined the behavior of mammalian RB fami ly members in pluripotent self - renewing mouse embryonic stem (ES) cells before and after differentiation. RB function is limited in ES cells due to elevated cyclin - CDK activity, but is established at the onset of differentiation, in part due to down regul ation of cyclin - CDK kinase complexes ( 23 , 27 ) . Thus, these cells offer a useful system to examine regulation of the RB family as members are mobilized to gene promoters in response to dynamic CDK activity during de velopment. In these 44 experiments, ES cells were cultured on mitotically inactive embryonic fibroblasts in the presence of leukemia inhibitory factor (LIF) to sustain their self - renewing potential or in the presence of retinoic acid (RA) to induce different iation, and the effect on RB, p107, and p130 was first examined by immunofluorescence analyses. In undifferentiated R1 - ES cells, RB and p107 exhibited predominate cytoplasmic staining, which shifted to a stronger nuclear pattern after RA treatment (Figure 2 - 1 A, B). In contrast, p130 was detected in the nuclear compartment both before and after RA - induced differentiation (Figure 2 - 1 C). To control for antibody specificity, RB family staining in WT J1 - ES were compared to triple knock out (TKO) ES cells under identical imaging conditions, showing that RB and p107 were preferentially detected in the WT - ES cells (Figures 2 - 1, D - E). The pa ttern for RB subcellular localization was similar for both R1 - ES and J1 - ES cells with increased nuclear retention observed after RA challenge (see also Figure 2 - 1, H). However, the strong cytoplasmic staining for p107 was muted in J1 - ES cells, and nuclear staining was observed both before and after RA treatment. Unexpectedly, p130 was equivalently detected in the nuclei of both WT and TKO ES cells (Figure 2 - 1, F). In Western blot analyses, we also detected anti - p130 reactive species in both WT and TKO cell s (Figure 2 - 1, G) and using multiple antibodies that recognize distinct epitopes (not shown), whereas RB and p107 were detected only in WT but not TKO ES cells, confirming specificity for these antibodies. Based on these findings, we conclude that RB and p 107 can exhibit differential subcellular localization in response to induced differentiation. During the execution of these experiments, we frequently observed that the staining intensity of endogenous RB family members was reduced after RA treatment. The se differences are not obvious in Figures 2 - 1, A - C because the relative luminosity of the images before and after differentiation was normalized. 45 Figure 2 - 1. Cellular localization of RB, p107, and p130 during mouse embryonic stem (ES) cell differentiation. 46 Figure 2 - (A - C) Confocal imaging showing localization of RB, p107, and p130 in a single section of pluripotent mouse R1 - ES cell colonies before and after differentiation with retinoic acid (RA), as indicated. Cells were counterstained with DAPI to detect nuclear DNA. Thre e biological replicates were performed and at least ten ES cell colonies were imaged for each experiment. Representative examples are presented. (D - F) Maximal intensity projection confocal images comparing WT J1 - ES and TKO cells before and after RA treatm ent. Samples were processed in parallel and data collected using identical imaging parameters (G) Western blots were performed on whole cell extracts prepared from wild type (lane 1) and triple knockout (TKO) ES (lane 2) cells using the indicated antibodie s. Comparable amounts of p107 - / - MEFs (lane 3) and U2OS ( lane 4) extracts were included for comparison. Different exposures are shown for the p130 Western blot to permit visualization of the differently migrating species (labeled a - d). (H) RB exhibits nuc lear localization in response to RA treatment. Sections of the data presented in panel D (white boxes) were enlarged to demonstrate the predominately nuclear staining pattern for RB in WT ES cells, which was not observed for the TKO cells. Total RB stainin g was significantly reduced after RA treatment with a broad range of intensity noted among cells. 47 However, reduced RB expression after RA treatment is noticeable for the experiment presented in Figure 2 - 1 , D wherein the images were acquired using identical parameters, suggesting that ES cell differentiation is correlated with re duced steady state expression. Variation in RB family abundance has been observed during cell cycle progression ( 24 , 28 , 29 , 41 ) , therefore, we considered that RB family levels during differentiation might be associated with the differing cell cycle profiles for pluripotent ES cells compared to cel ls undergoing differentiation. As shown in Figure 2 - 2 A, the proportion of cells in G1 indeed increased concomitant with a diminished S phase population in differentiated ES cells, consistent with tight relationship between RB family levels and cell cycle progression. Our previous studies in Drosophila further demonstrated that Rbf1 is less stable in its active state ( 31 , 42 ) , and we surmised that mammalian RB family proteins likewise become destabilized as they engage target genes in differentiating cells during G1 phase. To test this hypothesis, the levels of RB family proteins before and after RA treatment were compared to their genomic binding at endogenous target genes. Levels in ES cells were also compared to those in MCF - 7 breast adenocarcinoma cells, as a positive control for RB family members and as a negative control for the stem cell marker Oct - 4. As shown in Figu re 2 - 2 B, Oct - 4 expression was significantly reduced by RA treatment, in line with the expected changes for this molecular marker of pluripotency. Consistent with the reduced RB family staining that we previously noted using confocal microscopy, the stead y state abundances of all three family members were clearly reduced after RA - induced differentiation as observed by direct Western blot assay. In two replicates, RB levels were reduced by 69 percent while levels of p107 and p130 were reduced by 64 and 46 percent, respectively. The decrease in RB family protein levels were not due to change in transcription or 48 Figure 2 - 2. RB family protein abundance decreases during differentiation concomitant with increased engagement at target gene promoters . 49 Figure 2 - (A) FACS analysis showing increased G1 and reduced S phase population in differentiated ES cells ( - LIF, +RA) as compared to pluripotent ES cells (+LIF, - RA). (B) Western blot analysis of RB, p107 and p130 in whole cell extract derived from pl uripotent ( - RA, lane 1) and differentiated (+RA, lane 2) mouse ES cells. RB, p107, and p130 levels in differentiated ES cells were decreased by 69, 64, and 46 percent, respectively, as compared to pluripotent ES cells (n=2). Oct4 was analyzed as a positiv e control of differentiation and was substantially diminished in RA treated ES cells (lane 2). Actin and tubulin were analyzed as loading controls. Whole cell extracts from MCF7 breast adenocarcinoma cells (lane 3) were analyzed as a negative control for Oct4 and as a positive control for RB, p107, and p130 detection. (C) Quantitative real time PCR showing relative changes in abundance of RB, p107, and p130 mRNA transcripts upon differentiation (+RA/ - RA). RB and p107 mRNA levels increased modestly after differentiation whereas p130 levels were modestly reduced. Transcript levels of Nanog and Pou5f1 (Oct4) were reduced after RA treatment, as expected. (D) p107 and p130 association at target promoters is stimulated during RA - induced differentiation. Chro matin immunoprecipitation assays were performed with the indicated antibodies to determine enrichment of RB, p107, and p130 on the CCNA2 and MCM10 promoters before and after RA treatment. After differentiation, CCNA2 start site (TSS) DNA was significantly enriched in both the p107 and p130 immunoprecipitated samples (n=6, p<0.05), whereas enrichment of the MCM10 promoter was observed only during p107 immunoprecipitation (n=6, p<0.05). Under these conditions, no significant enrichment of any loci was obser ved for the anti - RB immunoprecipitated samples, nor was enrichment observed with species matched IgG control antibodies . Amplification of an intergenic region on mouse chromosome 6 was performed as an additional negative control. 50 or RNA stability because steady state mRNA levels were either unaffected by RA treatment, such as for p130, or were modestly stimulated, such as for p107 and RB (Figure 2 - 2 C). Nanog and Pou5f1 (Oct - 4) expression were significantly reduced during differen tiation, as expected. These results point to a post - translational mechanism for RB family regulation, such as through proteas ome mediated turnover pathway. To examine this possibility, we treated pluripotent ES cells and RA - differentiated cells with t he p roteasome inhibitor MG132. However, proteasome inhibition induced substantial cell death for both pluripotent and differentiated ES cells (not shown), precluding direct assessment of proteasome involvement for RB family turnover in these cells. Next, we d etermined whether the observed changes in cell cycle arrest and RB family localization during differentiation could be correlated with repressor binding at target gene promoters as one measure of function. To this end, we measured RB family occupancy on a set of well - characterized E2F - dependent promoters that were demonstrated to be RB family target genes ( 5 , 6 ) and whose expression was affected by RB family loss in ES cells ( 43 ) . As shown in Figure 2 - 2 D increased promoter binding by p107 and p130 was correlated with RA - induced differen tiation and cell cycle arrest. Interestingly, p107 and p130 exhibited distinct gene association, as both were bound to the CCNA2, TK1, and E2F1 loci (Figure 2 - 2 D and data not shown), while only p107 but not p130 was as sociated with the MCM10 locus. Finally, our data provide some evidence for differential binding by RB family members depending upon cell type. Specifically, p130 was not associated at the MCM10 locus in ES cells, even t hough it had been detected at this locus in other cell types ( 6 ) . We also did not observe RB association at any of these target genes, although we have routinely used this antibody to detect RB binding in other cell types ( 44 ) , and we cannot conclude whether the lack of RB binding in these experiments is biologically 51 relevant. The lack of RB signal at these cell cycle loci, otherwise bound by p107 and/or p130 is consistent with data previously published using T98G cells ( 5 ) . Interestingly, the current observations indicate that p107 levels drop during differentiation even as there is increa sed residency at target genes. Unlike p107, increased p130 presence at target promoters and its diminished expression upon differentiation are independent of any changes in subcellular localization, suggesting that turnover regulation is probably not coupled to nuclear transport processes. The RB family C - terminal regulatory domains influence repressor stability We next considered a model that signaling mechanisms governing mammalian RB family activity are involved in regulation of repressor turnover. In Drosophila, the Rbf1 homolog harbors an instability element (IE) within its C - terminal domain that contribute s to both repressor activity and destruction ( 31 , 33 ) . As indicated by the alignme nts shown in Figures 2 - 3 A and 2 - 3 B, just such a sequence is clearly identifiable within the C - terminal regions of both p107 and p130. RB exhibits substantial sequence differences throughout this region. Nonetheless, previous studies demonstrated that the RB C - terminal region is structurally related to p107 and thus, this region might likewise participate in both repression and turnover. Within RB, the IE can be subdivided into two regions previously called the RBC NTer and RBC Core , which are important for specific interactions with the marked box domains of the E2F1/DP1 complex (see Figure 2 - 3 A, and ( 40 ) ). The RBC NTer region can also interact with the MDM2 E3 ubiquitin ligase ( 45 ) , suggesting that corresponding IE region with in RB similarly coordinates repressor stability. To test whether the IE regions are important for regulation of RB family turnover, we deleted these regions from RB, p107, and p130 and examined the effect on steady state expression during transient transf ection in U2OS cells. These cells were chosen because we could achieve more efficient and reliable transfection in this system than in ES 52 cells. Moreover, U2OS cells do not express the p16 CDK inhibitor ( 46 , 47 ) ; hence these cells exhibit unrestrained cyclin - CDK activity, analogously to ES cells ( 25 ) . As shown in Figure 2 - 3C, deletion of the RBC NTer r egion ( 786 - 800) alone, harboring the putative MDM2 binding site, did not affect RB steady state levels. In contrast, mutant RB lacking both the RBC NTer plus RBC Core regions (RB 786 - 864) exhibited a significant elevat ion in steady state abundance. Thus, the IE region within RB negatively influences repressor abundance. The C - terminal regulatory regions from p107 and p130 are less well characterized than for RB, and yet these proteins clearly exhibit the highest homology to the Drosophila Rbf1 IE region, a s noted previously. Therefore, a more detailed analysis of these family members was undertaken. As shown in Figure 2 - 3 D, IE deletion from GFP - tagged p107 incr eased steady state expression. Similarly, GFP - p107 abundance was increased by alanine substituti on of four conserved lysine and arginine residues within the p107 IE region (GFP - p107 4KRA ) that were previously shown to influence Rbf1 half - life. As observed for p107, deletion of the IE region in GFP - p130 resulted in an even more profound fold - increase in abundance, a result that was mirrored by the corresponding 4KR to A substitution (Figure 2 - 3 E). Ablating E2F/DP interaction by deletion of the entire p130 A/B pocket domain did not affect steady state expression, suggesting that E2F/DP association per se doe s not modulate p130 abundance. Moreover, IE deletion did not affect the nuclear accumulation of either p107 or p130 as observed by immunofluorescence assay (data not shown). The quantification of the effects of IE deletion on RB family abundance is summarized in Figure 2 - 3 F. The effects of IE deletion were similar in magnitude for RB and p107 showing an increase of 2.4 fold (n=5, p<0.05) and 2.8 fold (n=7, p<0.05), respectively, while p130 abundance was increased 8.8 fold (n=3, p<0.05). Together, these data demonstrate that all three mammalian RB family members harbor C - terminal regulatory domains that contribute to their reduced steady state expression. 53 Figure 2 - 3. Th e C - terminal instability element is conserved within the mammalian RB family . 54 Figure 2 - (A) Schematic representation of the human and Drosophila RB family. The canonical instability element (magenta box) first discovered in Rbf1 (residues 728 - 786) is also present in the C - terminus of human p107 (residues 964 - 1024) and p130 (residues 1035 - 1095) . The corresponding C - terminal region in human RB that functions in E2F1/DP1 interactions, shown in red, contains two discrete regions called RBC NTer (residues 786 - 800) and RBC Core (residues 829 - 864) ( 40 ) . Drosophila Rbf2 does not appear to harbor a C - terminal IE. The A and B cyclin fold domains within the central pocket domains are shown as grey boxes. Potential cyclin fold domains within the N - terminal regions are shown as blue boxes. (B) Sequence alignment of the C - terminal regions from RB, p107, p130, and Rbf1. Residues that are identical in at least two members are highlighted in yellow. The position of the experimentally determined inst ability element within Rbf1 is boxed in blue. The RBC NTer and RBC Core regions are schematically represented above the alignment. Asterisks indicate the position of CDK phosphorylation sites within RB that modulate intermolecular interactions with E2F1/DP 1 and intramolecular interactions with the B domain ( 40 ) . The positions of positively charged lysine and arginine residues that increase Rbf1 stability when mutated are indicated as black triangles. The position of a lysine residue within Rbf1 (K774) that induces profound developmental phenotypes when mutated ( 31 ) is indicated as an open triangle. (C) Western blot analysis of w hole cell extracts derived from U2OS osteosarcoma cells transfected with GFP - RB WT (lane 1), GFP - RB NTer (lane 2) and GFP - RB NTer+Core (lane 3). (D) Western blot analysis of whole cell extracts derived from U2OS osteosarcoma cells transfected with wild type GFP - p107 (lane 2) or mutant GFP - 107 harboring a deletion of the C - terminal instability element (GFP - p107 , lane 3) or alanine substitutions at f our conserved positively charged residues (K970, R977, R978 and K991) within the instability element (GFP - p107 4KR - A , 55 Figure 2 - lane 4). (E) Western blot analysis of mutant p130 harboring a deletion of the C - terminal instability element (GFP - p13 0 , lane 2) or bearing alanine substitutions at four conserved positively charged residues (R1041, R1046, R1047, and K1062) within the instability element (GFP - p130 4KR - A , lane 4). Total p130 levels were unaffected by deletion of the A/B pocket domain GFP - p130 A/B (lane 5). Actin was used as a loading control for all experiments. (F) Quantification of the Western data presented in 3C - E for IE - deletion mutants. Deletion of the IE within RB (n=5), p107 (n=7), and p130 (n=3) resulted in 2.4 - fold, 2.8 - fo ld, and 8.8 - fold increase in abundance, respectively (*, p<0.05). 56 RB family members lacking their IE regions exhibited increased steady state abundance; therefore, we hypothesized that the IE - containing regions function as degradation signals or degrons to direct destruction of their cognate proteins. To test whether th e Rbf1 - related IE regions within p107 and p130 are sufficient for autonomous recognition and target protein degradation by the ubiquitin proteasome system, these regions were appended to GFP, and the effect on chimera protein abundance was examined. As sh own by the immunofluorescence data presented in Figure 2 - 4 A, the GFP - IE p107 chimera was expressed at reduced levels in most, but not all cells when directly compared to GFP. The total numbers of transfected cells were similar for both constructs, as assessed by scoring the number of GFP - positive cells independently of fluorescen ce intensity. These analyses indicate that the major difference in steady state abundance is likely due to reduced accumulation rather than differe ntial transfection efficiency. The GFP - IE chimeras containing either the p107 or p130 IE regions were also m arkedly diminished compared to GFP alone in Western blot analyses (Figure 2 - 4 B). MG132 treatment increased the steady state abundance of both GFP - IE p107 and GFP - IE p130 , whereas GFP levels were unaffected, consistent with the model that the p107 and p130 IE regions facilitate degradation by the proteasome (Figure 2 - 4 B) . Significantly, alanine substitution of key lysine and arginine residues that affected p107 and p130 expression in the context of the full - length proteins also lead to elevated steady stat e levels of the GFP - IE chimeras (Figure 2 - 4 C). These data also indicate that the cellular degradation machinery does not require additional interactions with other domains for turnover activity. Nonetheless, neither proteasome inhibition by MG132 nor IE mutation in this context restored expression to levels observed for GFP alone, suggesting that other unidentified features contribute to regulation of degron activity. We next treated cells with cycloheximide to test whether the reduced levels of GFP - IE fusion proteins were indeed a function of accelerated turnover. The measured stabilities 57 Figure 2 - 4. The canonical instability elements in human p107 and p130 function as autonomously acting degrons. 58 Figure 2 - (A) The p107 IE contributes to red uced steady expression of GFP. GFP fluorescence was measured in U2OS cells transfected with GFP alone or the GFP - IE p107 chimera containing GFP fused to residues 964 - 1024, corresponding to the p107 instability element. Cells were counterstained with DAPI to detect cellular DNA, and overlays (OL) of the GFP and DAPI signals are shown. GFP - IE p107 showed much reduced expression as compared to GFP in most, but not all cells. (B) GFP - IE p107 and GFP - IE p130 steady state expression is enhanced by proteasome inhibition. Anti - GFP western blot analysis was performed on whole cell extracts derived from U2OS cells transfected with GFP (lanes 1, 2), GFP - IE p107 (lane 3, 4), or GFP - IE p130 containing GFP fused to residues 1035 - 1095 correspon ding to the p130 instability element (lane 5, 6), in the absence or presence of MG132, as indicated. Whereas GFP remained insensitive to the proteasome inhibition, the GFP - IE fusion proteins accumulated to higher levels during MG132 treatment, suggesting the involvement of the instability element in proteasome mediated turnover. Actin was detected as a loading control and was refractory to proteasome inhibition. (C) Conserved positively charged amino acids contribute to autonomous IE function. Anti - GFP western blot analysis was performed on cells extracts transfected with either wild type GFP - IE p107 (lane 2) or mutant GFP - IE p107 containing alanine substitutions of four positively charged resides within the IE (lane 3). Similar analysis was performed for wild type GFP - IE p130 (lanes 5, 8) and mutant GFP - IE p130 (lanes 6, 9). In all cases, the GFP - chimeras containing the wild type IE sequences were expressed at lower levels than GFP alone (lanes 1, 4, 7), while alanine substitution within the IE resulted in increased steady state expression. The effect of alanine substitutions on GFP - IE p130 was more evident at a higher exposure of the same blot (lane 8 vs. lane 9). Actin was detected as a loading control. (D, E) The p107 and p130 instability elements con tribute to enhanced protein turnover. Anti - GFP 59 Figure 2 - Western analyses was performed on cells expressing either GFP or the wild type GFP - IE chimeras incubated in the presence of the translation inhibitor cycloheximide for 0, 3, 6, or 9 h, as indicated. GFP exhibited a half - life greater than 9 hours, whereas the half - lives of the GFP - IE chimeras were less than 3 h. Actin was detected as a loading control and its levels remained unperturbed by cycloheximide treatment. 60 of the GFP - IE chimeras were substantially lower, demonstrating that these effects are directed towards protein turnover (Figures 2 - 4 D, 2 - 4 E). Together, these studies define these canonical IE regions as independently acting degrons that are capable of d irecting substrate degradation by the proteasome. The canonical IE regions from p107 and p130 differ from RB at the primary sequence level. However, structure prediction analysis suggested that these regions may be conserved at the tertiary level, and th us we next tested the ability of the non - canonical RB - IE r egion to function as a degron. As shown in Figure 2 - 5 A, GFP appended with the RB - IE was expressed at a substantially lower level than GFP, and levels were increased by MG132 treatment, suggesting t hat this region is a bona fide degron. Cycloheximide treatment demonstrated that the RB - derived IE was also destabilizing (Figure 2 - 5 B), as previously noted for the canonical p107 and p130 IE constructs. We conclude that the RBC NTer and RBC Core regions together constitute a functional degron. RB family members are differentially expressed during cell cycle progression with low levels of RB and p107 during G0 or early G1, and increasing levels as cells progress towards late G1/S (29,48). In cont rast, p130 is typically expressed at its highest levels during G0 but at reduced levels at other stages. These differences suggest that cyclin/CDK activity may be key for regulation of RB family protein levels. At least for p130, proteasome - mediated turnov er is known to contribute to cell cycle associated changes (28,29). To assess the potential role of the IE in this process, we first measured the effect of CDK4 inhibition by PD0332991 on endogenous RB family members in asynchronously dividing U2OS cells. As shown in Figure 2 - 6 A, p107, and RB levels 61 Figure 2 - 5. The non - canonical instability element in human RB functions as an autonomous degron. 62 Figure 2 - (A) The RB IE contributes to reduced steady expression. Anti - GFP Western blot analysis was performed on whole cell extracts derived from U2OS cells that were transfected with GFP (lanes 1, 2) or GFP - IE RB containing GFP fused to residues 786 - 864 from human RB (lanes 3, 4) in the absence or presence of the proteasome inhibitor MG132, as indicated. Actin was detected as a loading control. In these experiments, the GFP - IE RB chimera was expressed at much low er levels than GFP alone. GFP - IE RB expression was also enhanced during proteasome inhibition. (B) The RB IE region contributes to enhanced substrate turnover . Cycloheximide experiments were performed as described previously, demonstrating that the GFP - IE RB chimera exhibited diminished stability (t 1/2 < 3hr), as compared to GFP (t 1/2 > 9hr). Acti n was used as a loading control. 63 were markedly diminished after CDK4 inhibition, whil e endogenous p130 levels were si gnificantly increased. These data are consistent with the diver gent regulation of the RB family m embers during cell cycle progression in staged cells (28, 29), with experiments testing the effect of PD0332991 in asynchronously dividing hepatocellular carcinoma cells (30). Moreover, levels of p107 and p130 were significantly increased during a short duration (6 h) of MG132 treatment, while RB levels were modestly diminished. These data indicate that endogenous p107 and p130 are subjected to proteasome - mediated turnover unde r these growth conditions. Secondly, RB is either not subjected to proteasome turnover or MG132 induced RB turnover via a proteasome - independent pathway. Interestingly, levels of p107, but not RB, were rescued by proteasome blockade during Cdk4 inhibition (PD+MG132), suggesting that Cdk4 - mediated phosphorylation of p107 prevents its proteasome - mediated turnover. In contrast, concomitant proteasome and Cdk4 inhibition did not affect p130 levels compared to that observed for PD alone, suggesting that hypo - p hosphorylated p130 is not a substrate for ubiquitin mediated degradation, as described previously ( 29 ) . We next tested whether the IE is essential for the observed destabilization of p107 by examining the effect of CDK4 inhibition on wild type and mutant p107 lacking the IE in transiently transfected cells. Indeed, PD0332991 treatment destabilized the wild protein, indicating that the IE is required for CDK - sensitive proteasome - mediated turnover of p107 (Figure 2 - 6 B). Wild type RB was also destabilized during Cdk4 inhibition, while RB lacking both the RBC NTer plus RBC Core (GFP - RB I E ) regions was refractory to PD0332991 influence (Figure 2 - 6 C), suggesting that the IE plays a similar role for CDK4 regulation of RB. In contrast to endogenous p130, recombinant GFP - p130 was destabilized, not stabilized during CDK4 inhibition, and this destabilization was observed regardless of IE status (Figure 2 - 6 D). This 64 Figure 2 - 6. RB and p107 destabilization during CDK4 inhibition is dependent upon instability element function. 65 Figure 2 - 6 (A) CDK4 inhibition differentially affects steady state expression of endogenous RB, p107, and p130. Western blot analysis was performed to detect endogenous RB, p107 and p130 in whole cell extracts derived from U2OS cells treated with DMSO (lanes 1), MG132 (lane 2), the CDK4 - specific inhibitor PD0332991 (lane 3), or PD0332991 plus MG132 (lane 4). Proteasome inhibition by MG132 (6h) increased steady state abundance of p107 and p130 by 90% and 30%, respectively as compared to the DMSO treated samples (n=3, p< 0.05), whereas RB abundance decreased by 15% (n=2, p<0.05). The steady state expression of both RB and p107 were reduced by 80% during PD treatment as compared to the DMSO treated samples (n=5, p<0.05), whereas p130 expression was increased by 49% (n=7, p <0.05). The PD - induced down - regulation of p107, but not RB, could be restored by MG132 to levels comparable to the DMSO control. Actin as used as a loading control and its levels remained unperturbed by PD or MG132 treatment. (B) p107 destabilization du ring CDK4 inhibition requires IE function. Western blots analysis was performed on U2OS cells that expressed wild type GFP - p107 (lanes 1 - 3) or mutant GFP - p107 IE (lanes 4 - 6) in the absence or presence of PD0332991, as indicated. In response to CDK4 inhibition, wild - type GFP - p107 levels were reduced by 68% as compared to the DMSO control (n=3, p<0.05), whereas the levels of p107 lacking the instability element w ere modestly reduced (18%) as compared to the DMSO control (n=3, p<0.05). (C) RB destabilization during CDK4 inhibition requires IE function. Western blot analyses were performed as above, showing that wild - type GFP - RB levels were reduced by 66% in respo nse to PD as compared to the DMSO control (n=3, p<0.05), whereas steady state expression of GFP - RB IE was increased by 82% compared to the DMSO control samples (n=3, p<0.05). (D) p130 destabilization during CDK4 inhibition does not require IE function. We stern blot an alyses were performed as above, showing that both wild type GFP - p130 66 Figure 2 - and GFP - p130 IE were destabilized to similar extents during PD0332991 treatment. On average, the levels of both proteins were decreased by 92% in respons e to PD as compared to DMSO control (n=3, p<0.05). Actin was used as a loading control in panels B - D. 67 outcome suggests that an additional IE - independent pathway can contribute to p130 turnover. The differences in response to CDK4 inhibition for the endogenous and overexpressed p130 proteins also suggests that some turnover pathways are active only in one setting, perhaps dictated by additional regulatory phosphorylation events that affected endogenous p130 in this context. Previous biochemical and structural studies of the C - terminal domains of the human RB family showed that these regions are important f or interactions with the marked box domains of E2F/DP complexes ( 40 , 49 - 51 ) . A model of the p130 IE region in a complex with the marked box domains of E2F4 and DP1 was generated by homology modeling using the RB C - terminal domain bound to a heterodimer of E2F1 and DP1 (Figure 2 - 7 A). In this model, the C - terminal portion of the IE harbors a sheet - turn - helix motif, which contacts the E2F4/DP1 complex, consistent with a potential role for the IE in repression. Interestingly, we noted that p107 ( RBL1 ) and p130 ( RBL2 ) harbor low frequency somatic mutations in cancer patients that map within the IE regions, as documented in the TCGA and COSMIC databases ( http://cancergenome.nih.gov/ ) ( 52 ) . These missense and nonsense mutations are found in carcinomas of the ovary, large intesti ne, endometrium, and pancreas. An additional independent study focusing on RBL2 found that mutations within the p130 IE were frequently observed in a cohort of lung adenocarcinomas ( 53 ) , including missense mutations affecting lysine 1083. It is notable that comparable lesions have strong biological effects in Drosophila wherein Rbf1 bearing a homologous substitution at K1083 (K7 74R) caused severe developmental defects ( 31 , 54 ) , suggesting that some mutations may play important roles in vivo. As also shown in Figure 2 - 7 A, the locations of cancer - associated RBL2 mutations map to different regions of the p130 structure, suggesting that these mutations may generate different effects, including modulation of E2F/DP interactions or potential E3 ligase association. We first t ested whether cancer - associated point mutations can affect p130 stability by 68 expression of the proteins in U2OS cells. Substitutions within the C - terminal portion of the alpha helix (S1090I, I1092M) significantly enhanced p130 levels, comparable to the ef fect of mutating four conserved lysine/arginine residues in the adjacent but unstructured region of the IE. Other point mutations tested, including R1070G, N1079F, K1083R, and K1083T that map more proximal to the E2F4/DP1 dimer interface had no effect on p130 steady state levels (Figure 2 - 7 B). Thus, some IE - associated cancer mutations result in enhanced expression of p130 potentially due to disrupted E3 ligase binding. We next tested whether deletion of the entire IE or amino acid substitutions within t he IE regions from p107 and p130 can impact repression potency (Figures 2 - 7 C, 2 - 7 D). Both wild type p107 and p130 repressed transcription driven by the CCNA2 reporter to levels approximately 50 - 63% of that observed for the control. This magnitude of rep ression is consistent with previously reported activities for p107 and p130 ( 34 ) , but is not as profound as that reported for Drosophila Rbf1 ( 31 , 33 ) . Removal of the IE, or substitution of four conserved lysines/arginines with alanine significantly impaired repression activity for both p107 and p130, indicating that the IE in these mammalian homologs contribute to full repression potential. Consiste ntly, truncation of the entire C - terminus (p130 - - terminus in nuclear localization and in mediating contacts with E2F4/DP ( 55 ) . In contrast, none of the missense mutations within the p130 - IE, as reported in human cancer samples, were significantly altered for repression of either the cell cycle regulated CCNA2 gene or the apoptotic TP73 reporter (not shown). We conclude that these particular point mutations are unlikely to critical ly affect transcriptional regulation of canonical E2F/DP target genes. 69 Figure 2 - 7. p130 IE activity in regulating repressor potency and stability are biochemically separable functions . 70 Figure 2 - 7 (A) Model of the human p130 IE in a complex with E2F4/DP1. Homology model of the p130 C - terminus (residues 1035 - 1113, red) in complex with the coiled coil - marked box domains from E2F4 (residues 94 - 198, light green) and DP1 (residues 199 - 350, dark green) was generated using the crystal structure of the RB C - terminal domain bound to an E2F1 - DP1 heterodimer as an template (PDB code: 2AZE, ( 40 ) ). The N - terminal portion of the p130 IE is unstructured in this model (dashed red line), whereas the C - terminal portion of the IE harbors a sheet (residues 1071 - 1077) - turn - helix (1083 - 1093) motif . The positions of some amino acid residues altered in human cancer patients are indicated (documented in COSMIC - S1090I: TCGA - 23 - 1118 - 01; I1092M: TCGA - AA - 3864 - 01; R1093H: TCGA - AA - A01Q - 01; R1093C: TCGA - D1 - A15W - 01A - 11D - A12 2 - mutants (R1070G, 2/14 cases; N1079F, 2/14 cases; K1083R, 4/14 cases; K1083T, 1/14 cases) are based on the study presented in reference 53. Residues highlighted in y ellow (N1079, K1083) are located towards the N - terminal region of the - helix while residues highlighted in blue (S1090, I1092, R1093) are located within the C - terminal region of this helix . (B) Cancer associated IE mutations have variable effects on p130 levels. Anti - GFP Western blot analysis was performed on U2OS cells transfected with plasmids expressing either wild type GFP - p130 or mutant versions harboring single poin t substitutions, as indicated. In two replicates, GFP - 130 containing the S1090I and I1092M substitutions was expressed 2.2 - and 1.7 - fold greater than the wild type (n=2). Vertical dashed lines indicate positions where image had been cut to rearrange the order of lanes. (C) The p130 IE is required for full repression potency. Luciferase reporter assays were performed in the presence of wild type or mutant versions of GFP - 130, as indicated, testing repression of the human CCNA2 luciferase reporter. Wild type GFP - p130 repressed transcripti on by 63%, as 71 Figure 2 - compared to GFP alone. GFP - p130 4KR - A , GFP - p130 , and GFP - p130 were significantly less effective than wild type GFP - p130 (n=8, p<0.05, *). None of the IE mutations reported in human cancers statistically altered the re pression of CCNA2 reporter by GFP - p130. (D) The p107 IE is required for full repression potency. Luciferase reporter assays were performed in the presence of wild type or mutant versions of GFP - 107, as indicated, testing repression of the human CCNA2 luciferase reporter. Wild GFP - p107 repressed tra nscription by 50%, as compared to GFP alone. Both GFP - p107 4KR - A and GFP - p107 were significantly less effective than wild type GFP - p107 (n=5, p<0.05, *). 72 Discussion The RB tumor suppressor family governs key steps in cellular proliferation through the transcriptional regulation of distinct genes associated with growth control ( 3 , 4 ) . Phosphorylation of RB proteins by cyclin/CDK complexes in mouse and human ES cells was previously demonstrated to inhibit RB/E2F interaction with consequent effects on gene expression and cell proliferation ( 25 , 26 , 43 , 56 ) . In this study, we report that early st eps during developmental regulation of the RB family in ES cells are additionally rendered through governance of subcellular localization. Specifically, RB and p107 accumulated in the nucleus during mouse ES cell differentiation mediated by concomitant LI F withdrawal and retinoic acid addition. This transition was concurrent with enhanced p107 and p130 association at select target genes. Promoter association by p107 and p130, but not RB in this developmental context is similar to that observed in tissue c ulture experiments performed using T98G glioblastoma cells ( 5 ) . It is also interesting that p107 and p130 exhibited different behavior for promoter binding with some genes harboring only p107 and others harboring both p107 and p130. However, the functional significance of differential promoter association during ES differentiation remains to be determined. It should be noted that ES cells can undergo differen tiation independently of RB family control ( 43 ) , and thus consequenc es for promoter - specific binding may depend upon additional cues, such as cell type and developmental context. These data point to two mechanisms governing activity of some RB family members during early development, one involving post - translational regula tion by the cyclin/CDK system and another involving control of subcellular localization. Our data further indicate that RB family function in transcriptional repression is linked to increased repressor turnover. This connection was first suggested by observations that steady state levels of RB family members in ES cells were diminished during RA - induced differentiation and 73 cell cycle attenuation. Our observations are also consistent with previous studies in mouse ES cells where total RB levels dropped in early G1 after release from nocodazole blockade ( 24 ) . Taken together these observations suggest an intim ate connection between cell cycle progression and RB protein levels in development. In response to RA, cellular mRNA levels encoding RB, p107, and p130 were either unaffected or were slightly increased, suggesting that changes in protein abundance during differentiation are influenced at a post - transcriptional level, potentially involvi ng regulated protein turnover. To understand the mechanism underlying RB family abundance, we tested the effect of proteasome inhibition on repressor levels in ES cells. Ho wever, these cells were extremely sensitive to MG132 treatment precluding direct assessment of RB family member half - lives. We therefore performed a biochemical structure - function study in a human osteosarcoma cell line that also maintains elevated CDK ac tivity, analogously to that observed in ES cells. In this context, p107, and p130 turnover were indeed directed via a proteasome - mediated pathway that involved the evolutionarily conserved instability elements located within their C - terminal regulatory do mains. The primary sequences of the mammalian p107 and p130 IE regions are most similar, and both of these are clearly related to the prototypical IE initially identified within Drosophila Rbf1 ( 31 ) . However, RB differs substantially in primary sequence throughout the IE. Nonetheless, structural studies have indicated that pocket proteins maintain secondary and tertiary conservation throughout this region ( 40 ) . We show herein that the corresponding region within RB also functions as an autonomously acting degron, suggesting that regulation of repressor stability via these C - terminal degrons represent s an important and evolutionarily conserved component of global RB family control. Mammalian RB family members are differentially expressed during cell cycle progression ( 28 , 29 , 48 ) with rapid degradation of hyper - phosphorylated p130 correlated with G0 exit and cell 74 cycle reentry. In contrast, p107 and RB levels tend to increase as cells progress towards S phase. Our studies showed that both wild type p107 and RB but not mutant ver sions lacking the IE are diminished by CDK4 inhibition, and are consistent with a role of IE - mediated degron function in these cell cycle fluctuations. These findings are consistent with earlier observations wherein mouse ES cells ablated for the CDK2 inh ibitor Cdk2AP1 exhibited enhanced RB phosphorylation concomitant with increased RB abundance ( 57 ) . Together, these studies demonstrate a clear linkage between the onset of CDK regulatory activity in ES cells and inversely correlated changes in RB family activity and abundance. Although the IE regions of RB, p107, and p130 share similar function in turnover control, the notable primary sequence divergence within these regions suggests that different E3 ligases participate in RB family turnov er. We propose that these distinct IE regions provide regulatory flexibility for RB family responses to distinct cell signaling events. Such events may include differential responses to DNA damage or during regulated cell cycle progression. For example, the RBC NTer region can bind to the Mdm2 E3 ligase for targeted destruction of hypo - phosphorylated RB ( 45 , 58 , 59 ) . As noted, corresp onding regions in p107 and p130 are only minimally conserved with RB, and these proteins are refractory to Mdm2 expression ( 59 ) . In contrast, proteolysis of p130, but not that of RB or p107 is dependent on the ubiquitin ligase activity of SCF Skp2 ( 28 , 29 ) , which is minimally expressed during G0 but peaks during S phase ( 60 - 62 ) , suggesting that E3 availability also plays a significant role in turnover of specific family members. Although SCF Skp2 and Mdm2 have been suggested as E3 ligases for cell cycle and DNA damage - associated degradation of RB family proteins, the involvement of these ligases in turnover during ES cell differentiation remains to be established. It is interesting that the RB C - terminal domain is also sufficient for F - box protein Skp2 association, but in this case, the RB - Skp2 75 interaction is mainly implicated in the regulation of p27 turnover ( 63 ) , suggesting a role for the RB - IE in non - autonomous protein turnover. We consider it likely that multiple E3 lig ases participate in RB family regulation through differential contacts with the IE regions of the different RB family members. Our study has additionally uncovered an intriguing aspect of mammalian RB regulation, namely that the sequences guiding represso r instability physically overlap with regions that are important for transcriptional repression. The inability of mutant p107 and p130 lacking the IE to fully engage in transcriptional repression is consistent with biochemical studies that have demonstrat ed a role for the IE in intermolecular contacts with the coiled coil - marked box (CC - MB) regions of E2F1 - DP1 complexes ( ( 40 ) , see also Figure 7). In this regard, our observations with the RB family of repressor proteins are similar to the intimate association of degrons within the activation domains of potent trans - activator proteins, such as E2F1 and c - Myc ( 64 ) , regulatory factors that control critical st eps in cellular proliferation. A common theme emerges from these studies that key activators and repressors governi ng cell fate outcomes are inherently engineered with limitations on their life span through turnover by the ubiquitin - proteasome system. Depending upon how the RB family interacts with distinct E2F/DP complexes, the interesting possibility arises that IE - E2F/DP engagement may reciprocally influence interactions with E3 ubiquitin ligases. In one model, E2F/DP complexes compete with E3 ligase for access to IE surfaces. In an alternative cooperative model, IE interactions with E2F complexes may portend enga gement with E3 ubiquitin ligases. This latter model is supported, in part, by our data showing that the steady state levels of p107 and RB are diminished by Cyclin D/Cdk4 inhibition, a process that also licenses these pocket proteins for E2F/DP interactio ns and target gene engagement. Moreover, some cancer - associated p130 mutants tested in the current study showed increased 76 steady state expression without significant effects on CCNA2 repression in vitro, suggesting that E3 binding and E2F/DP engagemen t ar e biochemically separable. While ineffectual for perturbation of p130 - mediated repression in this context, it remains possible that in vivo , these mutations are associated with deregulation of other, as yet uncharacterized, classes of target genes with significant effects on cellular physiology ( 6 , 65 ) . We note that the in the developing Drosophila embryo, Rbf1 associates with many genes involved in cell signaling and metabolism ( 66 ) and similar categories of genes may likewise become deregulated during human cancer progression in cells lacking IE fun ction. In flies, expression of Rbf1 lacking the IE enhanced DNA replication in vitro ( 67 ) and drove increased organ size when expressed in a tissue specific manner during development (54), suggesting a critical role for IE function in developmental and proliferative pathways . 77 REFERENCES 78 REFERENCES 1. Burkhart, D. L., and Sage, J. (2008) Cellular mechanisms of tumour suppression by the retinoblastoma gene. Nat. Rev. Cancer 8 , 671 - 682 2. Dick, F. A., and Rubin, S. M. (2013) Molecular mechanisms underlying RB protein function. Nat. R ev. Mol. Cell. Biol . 14 , 297 - 306 3. Hurford, R. K., Jr., Cobrinik, D., Lee, M. H., and Dyson, N. 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Multisite p hosphorylation on RB by Cyclin s/Cyclin - dependent kinase (Cdk) complexes, a bolish es interaction with E2Fs and increases transcriptional output of RB/E2F target genes ( 2, 3 ) . As discu ssed in Chapter 1, a n additional level of regulation on RB and E2F is implemented by the ubiquitin - proteasome system. Regulation of RB function through both these mechanisms involve hierarchical control by upstream regulators, and there is a substantial crosstalk between these regulons (Figure 3 - 1 ) . Disruption of the genetic circuitry involved in phosphorylation and proteasome - medi ated targeting of the RB and E2F molecules results in severe deficiencies in embryonic development ( 4 - 9 ) , and is a lso an early event in cellular transformation during cancer progres sion ( 10 ). Recent st udies from our labs have shown that in D rosophila the Retinoblastoma family (Rbf) proteins are subject to proteasome mediated turnover during embryonic development , and this process enhances Rbf engagement in transcriptional repression ( 4,5, 6,11 ) . This positive linkage between Rbf1 activity and its destruction indicates that repressor function is governed in a manner similar to that described by the degron theory of transcriptional activation ( 12 ) . To understand the relationship between RB family s tability and their repressor function during early mammalian development, I initiated studies in mouse embryonic stem (ES) cells. These studies revealed that differentiation of mouse ES cells is associated with the establishment of a functional RB pathway and simultaneous destabilization of RB family members ( 13 ) . 86 Figure 3 - 1. Parallel regulation of the RB - E2F pathway through reversible phosphorylation and the ubiquitin - proteasome system. 87 Figure 3 - RB is inactivated by upstream Cyclin/Cdk complexes, which in turn are negatively regulated by CDK inhibitors. In parallel RB is inactivated by E3 - ligases whose activity is modulated by upstream E3 - ligase inhibitors. Regulation of RB function through both these mechanisms involve hierar chical control by upstream regulators, and there is substantial crosstalk between these regulons. 88 As pluripotent ES cells are characterized by unrestrained C dk activity which plummets at the onset of differentiation ( 14 ) , we speculated that the observed changes in protein stability upon ES cell differentiation reflects an intimate relationship between RB phosphory lation and stability. Indeed, a C - terminal instability element (IE) in RB, p107 and p130 mediates their proteasome dependent turno ver in response to changes in phosphorylation status ( 13 ) . The IE sequence is evolutionarily conserved and functions autonomous to direct degradation of heterologous proteins ( 13 ) . This study has additionally uncovered another intriguing aspect of mammalia n RB regulation, namely that the sequences involved in regulating repressor instability physically overlaps with regions that are important for transcriptional repression. This idea is supported by the observation that m utant p107 and p130 lacking the IE a lthough very stable , were unable to fully engage in transcriptional repression. This dependence on IE for transcriptional repression is consistent with its role in mediating intermolecular contacts with the coiled coil - marked box (CC - MB ) regions of E2F1 - DP 1 and E2F4 - DP1 (Figure 3 - 2A) ( 13, 15 ) . To this end, it is intriguing to speculate that IE - E2F/DP interaction may reciprocally influence interacti ons with E3 ubiquitin ligases. The observation that the steady state levels of RB and p107 wer e diminished by C yclin D/Cdk4 inhibition, a process that also licenses these pocket proteins for E2F/DP interacti on and gene regulation , supports a model where IE interactions with E2F complexes may allow interaction association with E3 - ubiquitin ligases (Figure 3 - 2B) . Thi s overlap of degron sequences and repression domains is a conserved feature shared among the RB homologues ( 5, 13 ) , and represents a novel mod e of regulated transcriptional repression, whereby repressors governing critical cell fate outcomes are inherently engineered with limitations on their longevity through turnover by t he ubiquitin - proteasome system. Together, these findings implicate Retinoblastoma family IE region as a regulatory nexus linking repressor potency to the ubiquitin - proteasome system in development and 89 disease. The instability element (IE) mediated regulation of RB family abundance and transcriptional potency is evolutionarily conserved (5, 13). As part of ongoing and future experiments, I am most keen on un derstanding the differential use of RB family degron for protein turnover in response to diverse physiological perturbations, and the mechanisms of cross - talk between CDK - directed phosphorylation control of RB proteins and the cellular degradation machiner y. Firstly , as deregulated proteosomal degradation of RB, p107 and p130 contributes to cellular transformation (10), it will be very intriguing to determine whether the instability element is involved in viral oncogene (HPV - E7) induced RB degradation through the pro teasome. Secondly , based on our recent observation that UV induced DNA damage results in proteosomal degradation of p107 and p130 (Figure 3 - 3A, B), it will be interesting to study the role of IE in DNA damage induced regulation of p107 and p130 stability. Thirdly , we also found that the Mdm2 inhibitor Nutlin - 3 stabilizes Mdm2, and induces proteasome mediated degradation of RB and p107 (Figure 3 - 3C). Given the involvement of Mdm2 in RB turnover, we hypothesize that Nutlin - 3 promotes IE - MDM2 interaction that ultimately leads to RB/p107 degradation. Based on our previous findings that the instability element (IE) binds to E2F and directs protein turnover in an ubiquitin dependent manner, we propose a model where E2F and Mdm2 cooperatively binds to the hypo - phos phorylated p107 via the instability element, thereby ensuring degradation of active p107. Finally, our studies suggested that Cdk4 inhibition by PD0332991 leads to destabilization of wild - type RB and p107 via proteasome dependent degradation, and mutant fo rm of these proteins lacking the instability element were refractory to PD mediated destabilization. This led us to propose a model whereby, dephosphorylation of specific Cdk4 S - T/P sites within the IE may lead to direct binding of E3 ligases, resulting in protein turnover. We are currently testing this model using a battery of phospho - resistant and phospho mimetic mutations in RB and p107. 90 Figure 3 - 2 . Participation of the RB family C - terminal instability element (IE) in dual regulation of protein stabili ty and transcriptional potency. 91 Figure 3 - 2 (A) IE mediates intermolecular contacts with the coiled coil - marked box (CC - MB) regions of E2F1 - DP1 and E2F4 - DP 1 , and thereby contributes to transcriptional repression. ( B) RB and p107 is destabilized upon Cyclin D/Cdk4 inhibition, a process that also licenses these pocket proteins for E2F/DP interaction and gene regulation . This supports a model where by IE interactions with E2F complexes may allow simultaneous interaction with E3 - ubiquitin ligases to limit the life span of active repressor molecules. 92 93 Figure 3 - 3 94 REFERENCES 95 REFERENCES 1. Burkhart, D. L., and Sage, J. (2008) Cellular mechanisms of tumour suppression by the retinoblastoma gene. Nat. Rev. Cancer 8, 671 - 682. 2. Dick, F. A., and Rubin, S. M. (2013) Molecular mechanisms underlying RB protein function. Nat. R ev. Mol. Cell. Biol 14, 297 - 306 3. Rubin S. M. (2013). Deciphering the retinoblastoma protein phosphorylation code. Trends Biochem. Sci. 38 12 19 4. Ullah, Z., Buckley, M. S., Arnosti, D. N., and Henry, R. W. (2007) Retinoblastoma protein regulation by the COP9 signalosome. Mol. Biol. Cell 18, 1179 1186 5. Acharya, P., Raj, N., Buckley, M. S., Zhang, L., Duperon, S., Williams, G., Henry, R. W., and Arnosti, D. N. 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The SCF core , comprised of Cullin1 and the Ring - finger protein Roc1, provides a molecular scaffold that via the adaptor protein Skp1 allows modular recruitment of distinct F - box receptors, which in turn recruits specific substrates, thereby generating a repertoire of substrate specific E3 ligases. Covalent conjugation of N edd8 to Culin1 ( Nedd ylation) promotes the assembly of active SCF by recruitment of the adaptor and substrate specific F - box proteins to the SCF core. Similarly, removal of Nedd8 from Cul1 catalyzed by CSN5 (de Nedd ylation), disassembles and inactivates the SCF complex. This dynamic neddylation and deneddylation of Culin1 mediated by COP9 is the major regulatory control that ensures optimal function of SCF E3 - ligases. Studies from our lab found that during embryogenesis, D rosophila RB homologs Rbf1 and Rbf2 a re stabilized through interaction with COP9 signalosome (CSN) ( 4 ). Interestingly, Rbf1 - CSN interaction was evident on chromatin and suggested that CSN may function as a corepressor by stabilizing chromatin - bound Rbf1 ( 4 ). Both RB and COP9 being evolutiona rily conserved proteins , we hypothesized that a similar mechanism governing RB stability should exist in human cells . To test this hypothesis, we knock ed down CSN subunits in MCF7 breast adenocarcinoma cell lines, and examined the steady state abundance of RB, p107 and p130 through western blot analysis . In parallel, we also studied the effect of CSN knockdown on cell cycle progression, because CSN has been implicated in transcriptional control of cell cycle genes ( 5 ) . As shown in Figures AP - 1.1 A and AP - 1. 1B , siRNA s against CSN1 and CSN5 lead to significant decrease in CSN1 and CSN5 protein levels, without any significant effect on the steady 99 state abundance of RB family proteins. Similar experiments were also performed in three other RB - positive cell lines namely mammary MD A - MB - 231 , mammary 184B5, and colorectal HCT116 cells, and n o significant changes in RB pr otein levels were observed (data not shown) in any of the cell types . In addition, we also examined the effect of CSN5 knockdown on cell cycle distribution (8 - 11) and in regulating p53 stability (7) as positive controls for CSN5 deficiency. As shown in Figure AP - 1.1D , CSN5 knockdown resulted in a G1 arrest, consistent with previous reports that CSN5 deficiency influences cellular proliferation as a result of reduced CDK2 activity and impaired RB phosphorylation ( 6 ) . Moreover, CSN5 knockdown resulted in increased p53 abundance upon Doxorubicin induced DNA damage (AP - 1.2) , consistent with the notion that CSN5 plays a role in MDM2 - mediated p53 ubiquitination and degradation ( 7 ). In agreement with our observation, two independent groups recently re ported that unlike Drosophila Rb f1, mammalian RB proteins do not interact with CSN and, its knockdown is ineffectual for mammalian RB family abundance ( 5, 6 ). Taken together, these studies suggests that in mammals, CSN is not directly involved in regulation of RB family stability in differentiated cells , but affects the transcriptional output of the RB - E2F pathway by modulating RB phosphorylation by CDKs . Based on our studies with D rosophila Rbf1, an alternative possibility is that COP9 mediated protection of RB occurs only in the context of early embryonic development. Interestingly, knockdown of CSN2 in mouse embryonic stem (ES) cells caused a modest reduction in RB levels (data not shown). Future studies should aim at understanding context dependent function of CSN in regulating RB biology. 100 Figure AP - 1. COP9 signalosome is not involved in regulation of RB family protein stability in differentiated human cells. 101 Figure AP - (A, B) Western blot analysis of MCF - 7 cell extracts for RB, p107 and p130 after siRNA mediated knockdown of CSN1 and CSN5. Two specific siRNAs against CSN5 were used. CSN1 and CSN5 levels were substantially reduced using respective siRNAs, but none of the siRNAs affected RB, p107, and p130 levels. Actin and tubulin were used as loading controls. (C) Endogenous p130 interacts with E2F4, but not CSN5. (D) CSN5 knockdown causes a G1 cell cycle arrest in MCF7 cells. 102 Figure AP - 2. COP9 signalosome regulates p53 abundance upon doxorubicin induced DNA damage 103 Figure AP - 2 (A) Western blot analysis of MCF - 7 cell extracts for p53, after doxorubicin induced DNA damage in cells that were transfected with either control - siRNA or CSN5 - siRNA ( B ) Validation of CSN5 knockdown in these samples using western blot analysis. 104 REFERENCES 105 REFERENCES 1. 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