This is to certify that the thesis entitled IDENTIFICATION AND CHARACTERIZATION OF PLANT HOMOLOGS OF YEAST ELONGATOR AND OTHER TRANSCRIPTON ELONGATION FACTORS IN ARABIDOPSIS presented by Ying Yan has been accepted towards fulfillment of the requirements for the MS. degree in Cell and Molecular Biology _ <23: .4, LL a. Major Profe§éb’r’s Signature 7/17 /o¢-/ Date MSU is an Affirmative Action/Equal Opportunity Institution LIBRARY Michigan State University PLACE IN RETURN BOX to remove this checkout from your record. TO AVOID FINES return on or before date due. MAY BE RECALLED with earlier due date if requested. DATE DUE DATE DUE DATE DUE MAR 21 2007 lib—31:) .V 6/01 cJClRC/DateDuepBS-pls IDENTIFICATION AND CHARACTERIZATION OF PLANT HOMOLOGS OF YEAST ELONGATOR AND OTHER TRANSCRIPTION ELONGATION FACTORS IN ARABIDOPSIS By Ying Yan A THESIS Submitted to Michigan State University In partial fulfillment of the requirements For the degree of MASTER OF SCIENCE Cell and Molecular Biology Program 2004 ABSTRACT IDENTIFICATION AND CHARACTERIZATION OF PLANT HOMOLOGS OF YEAST ELONGATOR AND OTHER TRANSCRIPTION ELONGATION FACTORS IN ARABIDOPSIS By Yin g Yan Whereas much attention has been placed on characterizing the multitude of protein factors and sequence of events involved in recruitment of RNA polymerase II (Pol II) to eukaryotic gene promoters, relatively little is known about subsequent events associated with promoter clearance and transcriptional elongation. In cells of budding yeast, the hyperphosphorylated, elongation-competent form of PolII is associated with several factors that may facilitate transcription through chromatin. These include Elongator, a multisubunit histone acetyltransferase (HAT) that may act to disrupt chromatin packaging. The three subunits of 'core' Elongator (Elpl, Elp2 and Elp3) are highly conserved among eukaryotes. In humans, mutation in the Elpl homolog IKAP is associated with a severe neurodegenerative disorder termed familial dysautonomia. We found that in Arabidopsis, each of the core subunits is represented by a single clear homolog. Mutations in either AtELPI and AtELPZ lead to identical defects in vegetative and floral development. Double mutants of AtelpI/AtelpZ were phenotypically similar to either single mutant, suggesting AtELPI and AtELPZ have identical function. AtELPI and AtELPZ functionally interact with genes encoding homologs of elongation factors TFIIS and Pafl. Transcriptional profiling experiments revealed that AtELPI is involved in regulation of a limited and diverse subset of genes, including the MADS-box flowering time regulator FLC. To My family iii ACKNOWLEDGEMENTS My sincerest thanks and appreciation to my mentor Dr. Steven van Nocker for his guidance, support and inspiration throughout the course of this research. \Vithout his valuable, kind consideration, understanding, firm belief in my capabilities and stimulation of my mind this thesis would not be an object of my pride. A special thanks to the Director Peter Briggs of International Center, Dean George Leroi, Associate Dean Doug Estry, and Associate Dean Estelle McGroarty of College of Natural Science for supporting me financially and offering me help when I was stranded in unexpected situation. I also appreciate the valuable insights, ideas, and comments from my committee members: Dr. Steven Triezenberg, Dr. Zachary Burton, and Dr. Robert Larkin. Sincere gratitude is extended to every lab mate: Julissa, Hua, Lingxia, Philip, Sookyung, and Nobuko. Thank you all for your friendship and support. Thanks to my 128 students for offering awesome evaluation of my teaching. I would not be who I am today without the unconditional love, support and encouragement of my parents. They have shaped me into the individual I am and inspired me to pursue my dreams in life. My sincere thanks and gratitude to my boy friend Weiguo Liang for his patience, love affection, support, encouragement and sacrifice during every step that I made towards the completion of study at MSU. iv TABLE OF CONTENTS LIST OF TABLES ............................................................................................................. vi LIST OF FIGURES .......................................................................................................... vii LITERATURE REVIEW ................................................................................................... 1 INTRODUCTION .............................................................................................................. 9 RESULTS AND DISCUSSION ....................................................................................... 10 METHODS ....................................................................................................................... 29 LITERATURE CITED ..................................................................................................... 36 LIST OF TABLES Table 1.Arabidopsis homologs of elongation factors other than Paf 1C subunits .......... 11 Table 2. Oligonucleotides used as PCR primers for the identification and characterization of T-DNA disruption mutants of AtELPI , AtELPZ, AtTFIIS, AtSOHI and AtCHDIgenes.34 Table 3. RT-PCR primers and cycle numbers .................................................... 35 vi LIST OF FIGURES Figure 1.Position of T—DNA insertions in the Arabidopsis AtELPI, AtEPLZ, AtTFIIS, AtSOHI and AtCHDI genes ....................................................................... 13 Figure 2. Phenotypes of the AtelpI—004690, Atelp2-106485, and AtelpI/AtelpZ double mutants ................................................................................................ 16 Figure 3. Phenotypes of Atelp/Attflls and Atelp/vip double mutants ......................... 19 Figure 4. Characteristics of microarray data derived from wild—type Col-O and the AtelpI mutant ................................................................................................ 23 Figure 5. RNA gel-blot analysis of COR and HSP70 gene expression in Atelpl and AtelpZ mutant plants ......................................................................................... 26 vii LITERATURE REVIEW Transcription initiation and elongation Eukaryotic transcription by RNA polymerase H (Pol H), a ~600-kDa, l2-subunit enzyme highly conserved from yeast to mammals, is a remarkably intricate biochemical and multistage process that is tightly regulated at many levels. This process proceeds via five stages: preinitiation, initiation, promoter clearance, elongation, and termination. An enormous body of work generated over the past decades suggests that during initiation, Pol II assembles at promoters together with general transcription factors IIA, IID, IIB, IIF, HE, and HH into a pre-initiation complex (PIC) (Roeder, 1996). Apart from possessing intrinsic capabilities for basal transcription, these proteins are also believed to represent the ultimate targets of various gene specific DNA-binding activators or repressors. For example, TFIID, a multisubunit complex that consists Of the TATA box-binding protein TBP and a number of TBP associated factors (TAFs), is regarded as the major, if not exclusive, target of transcriptional activator proteins (Muller and Tora, 2004). In Dmsophila, some of these TAFs have been shown to directly bind activation domains of activators, and in turn activators can recruit TFIID by interacting with TFIID through TAFs (Dikstein, 2001). This interaction is essential for activator-dependent transcription in vitro. In addition, biochemical and genetic evidence showed that a protein complex termed Mediator (MedC) serves as an adaptor between activator proteins and the basal transcription machinery (Myers and Kornberg, 2000). Subunits of Mediator interact with the C-terminal domain of Pol II in vivo. The mammalian counterparts of Mediator also interact with transcriptional activator proteins and have an important role in modulating Pol II activity in promoter-dependent transcription (Myers and Kornberg, 2000). Recently, a novel form of Pol H has been purified from yeast cells, which contains Pafl (RNA polymerase H associated factor 1) and Cdc73, together with other proteins implicated in transcription initiation such as Galll, TFIIB, and TFIIF, but lacks TBP, TFIIH, transcription elongation factor TFIIS, and the Srbs subunits of MedC (Shi et al., 1997). It is unclear why cells contain both the Srb/MedC-containing and the Pafl/Cdc73-containing RNA polymerase II complexes at the initiation stage. Transcription elongation factors Although the initiation stage of transcription has received much attention during the past decades, little is known about events associated with promoter clearance and transcriptional elongation. Transcription elongation by RNA polymerase II in eukaryotes has blossomed into a broadly active area of investigation (Conaway et al., 2000; Orphanides and Reinberg, 2000). A major advance in the understanding of elongation has come with the recent determination of the high-resolution crystal structures of free and elongating forms of Pol H (Cramer et al., 2001; Gnatt et al., 2001). These structures revealed many of the key catalytic properties of the polymerase, leading to a better understanding of the enzyme’s catalytic mechanism, the nature of the enzyme’s interactions with its DNA template and nascent transcript, and the mechanisms underlying the enzyme’s propensity to pause and arrest (Shilatifard, 2003). More importantly, a diverse collection of nuclear proteins that regulate the activity of Pol H during the elongation phase of messenger RNA synthesis have been identified and biochemically characterized (Conaway et al., 2000). These so-called transcription elongation factors typically display some common features: First, these proteins can be crosslinked with both the promoters and coding regions. Second, deletion of the genes encoding such proteins typically render cells sensitive to the drug 6-azauracil (6-AU), a transcription elongation inhibitor. This compound is believed to cause the depletion of intracellular GTP or UTP level by inhibiting their biosynthetic enzymes. Third, such proteins have copurified with the hyperphosphorylated, elongating form of Pol II. Finally, genes have been implicated in transcription elongation by genetic or physical interaction with other known elongation factors. Transcription elongation factors have been classified into three distinct groups (Shilatifard, 1998): Class 1, those involved in drug-induced arrest or sequence-dependent arrest of transcription, such as DSIF (Spt4, SptS) and TFIIS; Class II, those that function to suppress transient pausing and increase the catalytic rate of elongation by altering the Km and /or the Vmax of the polymerase, such as ELL, TFIIF and Elongin; and Class III, those that facilitate passage of polymerase through chromatin, such as FACT (Sptl6, Pob3) and Elongator. Transcription elongation by Pol H is a dynamic process that does not occur at a constant rate. Throughout its elongation phase, Pol II can encounter constraints causing pause, arrest, and even termination (Uptain et al., 1997). Pausing results from reversible backtracking of the enzyme by 2 to 4 nucleotides. This leaves the mRNA transcript’s misaligned 3’-OH terminus unable to serve as an acceptor for the incoming ribonucleoside triphosphate in synthesis of the next phosphodiester. In contrast, arrest is caused by irreversible backsliding of the enzyme by 7-14 nucleotides. An arrested elongation complex is unable to efficiently resume transcript synthesis without the aid of accessory factors (Uptain et al., 1997). The first class of transcriptional elongation factors plays an indispensable role to overcome arrest. For example, at some T-rich sites scattered along the DNA template, arrested polymerase can be reactivated only by the action of TFIIS (Vde and Reines, 2000). Elegant biochemical studies have revealed that TFHS allows the passage of arrested Pol II by directly interacting with the catalytic site of the enzyme and creating a new 3’-OH terminus that can be reextended by the polymerase (Reines and Mote, 1993). Rather than rescue polymerase from arrested state, Class II elongation factors stimulate the rate of elongation by interacting directly with elongating polymerase and suppressing transient pausing (Uptain et al., 1997). Although the exact mechanism of suppressing RNA polymerase II pausing is unknown, some evidence suggested that these proteins help to maintain the 3’-OH terminus of the nascent RNA chain in its proper position in the polymerase catalytic site, thereby preventing backtracking of the enzyme (Takagi et al., 1995). Class III elongation factors do not affect polymerase activity directly, but instead modify the chromatin template and enhance movement through chromatin. In eukaryotes, DNA typically exists in vivo as a repeating array of nucleosomes, in which 146 bp of DNA are wound around a histone octamer (consisting of two each of histone proteins HZA, HZB, H3 and H4), which can be subject to higher-order packaging (Kornberg and Lorch, 1999). In this way, genomic DNA is compacted some 10,000-fold. Such condensation of DNA provides a considerable obstacle to transcription. A direct connection between chromatin alteration and transcriptional elongation has been demonstrated in recent years by the finding that Class III elongation factors have chromatin modification activity. For example, FACT binds to the nucleosomes and promotes removal of nucleosomal histones H2A and H2B, hence facilitating elongation by RNA Pol II (Shilatifard et al., 2003). Chdl is a member of a large class of ATP-dependent chromatin remodeling proteins. These proteins alter the distribution of nucleosomes along DNA (BjOrklund et al., 1999). Other chromatin-altering elongation factors act by covalently modifying histone proteins. Each core histone consists a structured domain and an unstructured amino-terminal ‘tail’ of 25-40 residues extending through the DNA gyres and into the space surrounding the nucleosomes. These histone tails provide sites for a variety of posttranscriptional modifications, including acetylation, methylation, and phosphorylation (He and Lehming, 2004). It is becoming increasingly apparent that such modifications of histone tails determine the interactions of histones with other proteins, which may in turn regulate chromatin structure. For example, the Pafl complex, which consists of seven subunits: Pafl, Cdc73 (Shi, 1997), Leol, Rtf 1, Ctr9 (Mueller and Jaehning, 2002), Ccr4 and Hprl (Wade et al., 1996), is required for the recruitment of Setl, Set2 and Dotl methylases, which catalyze histone H3 methylation at lysine 4, 36 and 79 (Santos-Rosa et al., 2002). The evidence that hypermethylated H3-K4 is exclusively associated with active transcription, and that the association of Paf 1C with elongating Pol 11, suggests that this modification provides a molecular memory of recent transcriptional activity (Ng et al., 2003). In contrast, Elongator functions in histone acetylation. The Elp3 subunit of Elongator is a member of GN AT (GcnS-related N acetyltransferase) superfamily (Sterner and Berger, 2000). Histone acetyltransferases (HATS) transfer an acetyl group from acetyl-coenzyme A (acetyl-CoA) to the e-amino group of certain lysine side chains (Loidl, 1994). In particular, the highly conserved histone H3 lysines at amino acid positions 9, 14, 18 and 23, and H4 lysines at 5, 8, 12, and 16 are target sites for acetylation by HATS (Roth et al., 2001). It is theorized that Elongator may perform a function during elongation that is similar to the function of the related HAT GcnS during initiation. In yeast, GcnS is the catalytical subunit of two multi-subunit protein complexes, SAGA and ADA (Grant et al., 1997). Recruitment of SAGA to the nucleosomes by some transcriptional activators causes histone acetylation and concomitant activation of transcription in vitro (Utley et al., 1998). Acetylation of histones can be reversed by histone deacetylases (HDACs) (Khochbin et al., 2001). Acetylated histones are usually associated with transcriptionally active chromatin and deacetylated histones with inactive chromatin (Sterner and Berger, 2000). The interplay between HDACs and HATS results in dynamic transitions in chromatin structure, which may affect the efficiency of transcription elongation. The Elongator complex was isolated by its ability to interact with the hyperphosphorylated CTD of the elongating form of Pol II in yeast (Otero et al., 1999). Elongator is composed of a tightly bound core of Elpl-Elp3, and an additional trimer of Elp4-Elp6. In vitro work using recombinant Elp3 demonstrated that Elp3 possesses intrinsic HAT activity (Wittschieben et al., 1999). Further investigation has been done to show Elp3 could physically interact with both naked and nucleosomal DNA, and predominantly acetylates lysine-l4 of histone H3 and lysine-8 of histone H4 (Winkler et al., 2002). Chromatin immunoprecipitation experiments Show that Elongator is important to keep the normal histone H3 and H4 acetylation levels in chromatin in vivo (Winkler et al., 2002). The ELP2 gene encodes a protein with eight WD repeats (Fellows et al., 2000). WD repeats are thought to be a platform for protein-protein interaction (Smith et al., 1999), and thus, Elp2 might have a role in the assembly of the Elongator complex. Mutant cells lacking any of the six ELP genes showed similar phenotypes, such as slow adaptation to new growth conditions and hypersensitivity to salt and temperature stress (Otero et al., 1999; Fellows et al., 2000; Wittschieben et al., 1999). These phenotypes might result from a delay in gene expression under rapidly changing conditions. Microarray experiments demonstrated that the gene expression profiles of strains carrying deletions of genes encoding any of Elpl, Elp2, E1p4 and Elp6 subunits are very similar (Krogan and Greenblatt, 2001). Different combination of elp double mutants, even triple mutants, do not enhance the phenotype (Otero et al., 1999, Wittschieben et al., 1999, Fellows et al., 2000), suggesting that these proteins function exclusively as part of Elongator. Although Elp4, Elp5 and Elp6 do not have HAT activity by themselves, and do not exhibit homology with known HATS, they are required for HAT activity of Elp3 in the core Elongator complex (Winkler et al., 2002). The observation that Elongator subunits are not essential in yeast suggests that Elongator does not provide a crucial function, or that other HATS may replace the role of Elongator in the absence of Elongator function (Wittschieben et al., 1999). Elongator subunits are highly conserved in humans. Human Elongator complex has been purified and was found to consist of six subunits (hElpl-hElp6) presenting as a Six-subunit holo-Elongator complex (Hawkes, 2002). A mutation in IKAP, the human homolog of yeast Elpl, results in familial dysautonomia, a fatal disorder affecting the nervous system (Anderson et al., 2001), and causes bronchial asthma in children (Takeoka et al., 2001). As in yeast, the human holo—Elongator complex has HAT activity targeted to histone H3 and H4 in vitro, and associates with the elongating form of Pol II (Hawkes, 2002). This suggests that Elongator in higher eukaryotes may also have a role in transcriptional elongation. However, researchers failed to detect an interaction between human Elongator and other transcription elongation factors (Kim et al., 2002). A role for Elongator in elongation has recently been put in doubt by the finding that the majority of Elongator Elpl-Elp3 subunits in yeast are excluded from the nucleus (Pokholok et al., 2002). In addition, it has been noted that in contrast to other elongation factors, Elongator subunits have not been found crosslinked with ORFs (Krogan et al., 2002). Thus, Elongator may play a unique and unanticipated role in transcription elongation. INTRODUCTION Whereas much progress has been made to discover transcription elongation mechanisms in yeast and humans, very little is known about transcription elongation in plants. Our interest in the role of transcriptional elongation in plant development came from the finding that mutations in the plant homologs of the yeast Paf 1C subunits Leo] , Rtfl and Ctr9 led to developmental pleiotropy, including reduced plant size, reduced apical dominance, male sterility, defects in floral organs, and early flowering (Zhang and van Nocker, 2002; Zhang et al., 2003; Oh et al., unpublished). Inniguingly, we found that the early flowering phenotype was associated with silencing of the MADS-box flowering inhibitor FLC (FLOWERING LOCUS C) and MAF genes (Bastow et al., 2004; Oh et al., unpublished). In natural, winter-annual ecotypes of Arabidopsis represses flowering until after an appreciable period of cold, a phenomenon called vemalization. Cold results in epigenetic silencing of FLC, associated with hypoacetylation and repressive methylation of FLC chromatin (Bastow et al., 2004). In light of the strong effect of mutations in plant Paf 1C genes on development, we explored the significance of other transcriptional elongation factors in plant development. RESULTS AND DISCUSSION Identification of potential components of the transcriptional elongation machinery in Arabidopsis. As a first step to explore the Significance of transcriptional elongation in plant development, we evaluated the Arabidopsis predicted proteome for proteins closely related to yeast transcription elongation factors (Table 1), because highly conserved proteins might have similar functions. We found that most, but not all, of the characterized yeast transcription elongation factors were represented in Arabidopsis, suggesting that mechanisms of elongation may be well conserved in plants. The Arabidopsis genome encodes for more than one homolog of Spt4/Spt5/DSIF and P-TEFB subunits, suggesting that their function might have evolved to mediate different aspects of elongation. However the Arabidopsis genome does not encode for obvious homologs of ngl (a subunit of TFIIF), or the Elp4-Elp6 subunits of Elongator. In yeast, Elp4-Elp6 are not catalytic subunits, but they interact with the core subunits and have a regulatory role (Li et al., 2001). This suggested that plant Elongator might have a different structural composition. We also found that each of the core subunits of Elongator, Sptl6, Pob3/FACT, Dstl/TFIIS, Sohl or Chdl, is represented by a single clear homolog in Arabidopsis. We designated these AtElpl, AtElp2, AtE1p3, AtSptl6, AtPob3, AtSohl and AtChdl , respectively. To investigate the functions of these plant homologs of yeast transcriptional elongation factors in Arabidopsis, we obtained mutants for several of these genes from a large sequenced-indexed, T-DNA mutagenized population (Httpzllsignalsalkedu) (Table l and Fig.1). Because of the potential for paralogous genes to have redundant function, 10 Table l. Arabidopsis homolgogs of elongation factors other than PaflC subunits Protein name in yeast Arabidgsis homolog(s) T-DNA lines E value* Elongator Elpl At5g13680 Salk-004690 1.3e-67 Salk-011529 Elp2 Atl g49540 Salk-106485 3. 1e-66 Elp3 At5g50320 4.4e-204 Elp4 None ElpS None Elp6 None Spt4, Spt5/DSIF Spt4 At5g63670 1.5e-74 At5g08565 3.1e-13 Spt5 At2g34210 1. 1e-43 At4g08350 3.2e-42 Spt6 Atlg63210 1.5e-74 Atl g65440 1.5e-7l stl Atlg32130 8.3e-23 At4g19000 9.9e-15 Sptl6,Pob3/FACT Spt16 At4g10710 5.1e-107 Pob3 At3g28730 Salk-058731 2.7e-46 P-TEFB CycT ~8 genes, overlapping with Cch Cdk9 > 100 Cch ~8 genes, overlapping with CycT Dstl/TFIIS At2g3 8560 Salk-056755 7. le-3O Salk-064316 TFIIF ng1 None ng2 Atlg75510, At3g52270 1.7e-14 3.6e-O9 Taf14/I‘fg3 At2gl8000 5.6e-10 Sohl At5gl9910 Salk-OS 1025 1.2e-13 Salk-035522 Chdl At2gl3370 Salk-087283 3.8e-l76 Salk-020296 * E value: false positive expectation value 11 we focused our efforts on those genes with a single, clear homolog in Arabidopsis. No mutants were available for ATELP3, ATSPT16, or A'ITFG3. We obtained two alleles for the AtELPI gene, designated Atelp1-004690 and AtelpI-011529. Both of these contained a T-DNA insertion in the third of total five exons. For the AtELPZ gene, the Atelp2-106485 allele contains a T—DNA insertion in the third of total ten exons. The AtTFIIS gene had two alleles; AttflIs-0643I6 contains a T-DNA in the 5’ UTR, and AttflIs-056755 contains a T-DNA insertion in the second of total two exons. AtSOHI had two alleles; AtsohI-051025 contains a T-DNA insertion in the second of total six exons, and AtsohI-035522 in the 3’UTR. Atchd-020296 has a T-DNA insertion in the Sixth of a total of thirty exons. Based on the location of the T-DNA insertion in these alleles, all of these mutations have the potential to significantly affect gene expression. We isolated homozygous mutants for all of these genes, and analyzed the phenotype of these mutants under Standard growth conditions. However, we did not observe obvious defects in development for Attflls, AtsohI, and Atchdl mutants. In yeast, none of these three proteins are essential (Giaever, et al., 2002), and mutations in any of these genes do not obviously affect growth. Therefore, if these proteins have conserved functions in plants, the observed normal development of the mutant plants would not be surprising. In addition, because the VIP4, VIP5 and VIP6 genes, which encode homologs of Paf 1C subunits, are required for the expression of FLC/MAF genes and proper tinting of flowering, we were curious to see if AtTFIIS, AtSOHI, and AtCHDI influence flowering. FLC is not strongly expressed in the genetic background in which the T-DNA lines were isolated, due to a mutation in its activator FRIGIDA. Therefore, to assess the effect of 12 .ConEac o_m__w 05953950 9: new .mcoEmmE (20.... .6989 x028 mEb ADE. xoflnv 95:5 .3982 no. 62mm. 83mg: ”moxon xomB .mEbv 9.96 .o 9.258 m>=m_m._ 9: 2m 3506:. .350 “Q20; can “tow; flip; Nani; .hmama £30233 05 5 «BED»... 53.9 .0 :oEmom .w 2:9“. .m g .m Fute< «Sustain. .m .m “10%; MI: TIM“ 338133 323.3: .m _ \fl want; 8839.2.» «5.8.53 1’! 348333 .. IIIIMNI a an; 0mmvcclv: (m mNmF FOIXJdm l3 these mutations on FLC expression, we introduced these mutations into a synthetic introgression line containing a functional FRI allele (see Methods). Although we did not evaluate F LC expression directly, none of the resulting plants flowered abnormally early, suggesting that F LC was expressed to wild-type levels (not shown). Phenotypic analysis of Atelpl, Atelpz and AtelpI/Atelpz double mutants In contrast, mutations in ATELPI and ATELPZ conferred moderate defects in development. Both Atelp] and Atelp2 mutant plants showed indistinguishable pleiotropic phenotypes at multiple stages of development (Fig. 2). When the second pair of rosette leaves expanded, the leaf lamina of the mutant plants was narrower than that of wild type plants and exhibited obvious serrated edges (Fig. 2B and C). In addition, the leaves were curled downward, suggesting that the adaxial cells had divided or expanded faster than the abaxial cells. The leaf color was yellowish compared to that of the wild type (Fig. 2B, C and D). Moreover, AtelpI and Atelp2 mutant plants showed significantly higher trichome density on the leaf surface, inflorescence stems and sepals (Fig. 2B, C, D and E). In addition, the Atelpl or AtelpZ mutant plants grew much more slowly than the wild type plants (not shown), suggesting defects in cell division and/or cell expansion. Floral development was also affected in AteIpI and AtelpZmutant plants. Flowers exhibited abnormally small petals and large sepals, and the stamens were significantly shorter than the carpels (Fig. 2E and not shown). The flowers of both AtelpIand AtelpZ showed a phenotypic gradient (not shown); whereas early-formed flowers were severely affected and sterile, later-formed flowers were less affected and produced shorter but thicker siliques compared to the wild type (Fig. 2F and not shown). In addition, seed size was 14 larger than that of the wild type (Fig. 2G). All of the seeds from heterozygous plants were wild type Size, suggesting that seed size resulted from maternal affects. We also found that AtELPI and AtELPZ mutations did not obviously affect flowering time when introduced into a genetic background containing an active FRI allele, suggesting that these genes have little role in FLC expression (not shown). However, we later discovered through transcriptional profiling that FLC in fact was down-regulated in an Atelpl mutant (see below). The implications of this are discussed below. To confirm that the mutant phenotypes were caused by T-DNA insertion in the AtELPZ gene, we expressed antisense RNA for AtELPZ in wild-type Col-0 plants. A subset of primary transformants showed all of the phenotypic defects seen in the Atelp2 mutant plants (Fig. 2B), including narrower, yellowish and serrated leaves that curled downwards; high trichome density on the leaf surface, inflorescence stems and sepals; flowers with small petals covered by large sepals; short stamens, reduced fertility, and slow growth rate (not shown). RT-PCR analysis using primers spanning the insertion sites did not detect the AtELPI transcripts in homozygous AtelpI-004690 mutant plants or AtELPZ transcripts in homozygous Atelp2-106485 mutant plants (Fig. 2A). This suggests that these are null mutations, and these phenotypes (discussed below) reflect the absence of gene activity. The apparently indistinguishable phenotypes exhibited by AtelpI and AtelpZ mutant plants suggested that AtELPI and AIELPZ have similar function. To explore this idea further, we constructed a AtelpI/AtelpZ double mutant. If AtELPI and AtELPZ have completely overlapping function, then we predicted that the AtelpI/AtelpZ double mutant 15 .acma 9.8 EE 82-23, Em £555 22.8 333353 News. .333 9: E momma A9 new 39:» 055% $5950: Amy awe. ozomo. R: ”enema. 6v ”$.53 22; av .acma RS 25, Ba .acma magma: 852:3 .Emse 288 wq§§q§< .mEmSE NED? new BEE Co mambo—Coca 5-9 .888 m an mote» ZRO< ”mecca Nam; 5 Kim? 9: .8 050me 9955 9:3 avenues. E 82:88 mm moan—t 2 omaomfism new mogfioom 20-3: Eo: Doc—mama was <21 .m0d..E E 953 EmSE NOE? Em Bees. .Eé 83-2? 5 8:8. «Ema. Em Esme. 9: a mazmcm coameaxm E 64:83:. 29.8 «32:33»? 25 $32.33... $88333 9: .o 8322.. .u 2:2". l6 .CS cozmSE BEE D is I :89qu mam? U N303: Sm; N30; meme: Ema. ts BEE News. O m<.Nl.~.£< .CS NSS<>S$< N32< “32.x 3483an 8963383 Es 2E0< l7 would be phenotypically similar to the AtelpI or AtelpZ Single mutants. This was indeed the case; the AtelpI/AtelpZ double mutant exhibited all the phenotypic defects of AteIpI or AtelpZ single mutants (Fig. ZB, C, D and E). RT-PCR analysis (Fig. 2A) showed that neither the expression level of AtELP] in the Atelp2-106485 mutant, nor the expression level of AtELP2 in the AtelpI -004690 mutant, was affected. This suggests that AtELP] and AtELP2 are not regulated by each other. These findings reveal that the AtELP] and AtELP2 genes are essential for proper development of Arabidopsis, and likely carry out an overlapping function, potentially as components of a protein complex. AtELPl and AtELP2 genetically interact with AtTFIIS and VIPs In yeast, TFIIS promotes transcription elongation and rescues arrested polymerase by creating a new 3’-OH group that can be extended by the polymerase. The moderate sensitivity to the transcription elongation inhibitor 6-AU exhibited by tflls mutants was greatly enchanced when combined with an eIpI mutant (Otero et al., 1999). This suggested a role for Elongator in transcription elongation in vivo. As a transcription elongation factor, Elongator in plants might interact with other hypothetical elongation factors. To test this possibility, we evaluated genetic interaction between AtELP and AtTFIIS by combining Atelpl and Atelp2 mutations with the AtlflIs-056755 allele. Although the AttflIs mutant plants developed normally (see above), both AteIpI/AttflIs-056755 and AtelpZ/Atq‘IIs-056755 double mutant plants exhibited more severe phenotypic defects than seen in the Atelp single mutants: leaves were narrow and serrated, growth was extremely slow (not shown), inflorescence development was delayed, formation of sepal, petal, and stamen was defective, and fertility was further reduced (Fig. 3A). The enhancement of the 18 ©8>\NS$< VASNQEE MQSNQEE N30; .3: 3 9m: 02:39am. 683058 was cashew? Raises. E§§e< mains? “so? $2.52 o>mc was van was .3: .mASdEmSE mason message: can engages. engages. engage? a woabococm .a SENSE 29:8 8::an Em washes? XEEDE .35:an Co mambococmd .mEmSE assoc geese. a 8382; EC .339“. .o .532“. .n .Ema 205$ .m.£:m§E mason aE$mq§< Em m=§3q§< seaaew Rea afiefim‘ofi 9%.: «$5 .6 woaaocoig . flazx Imminlx $.wa \5 4. .mEmSE Enact x. , ease; use as: Semi Co moabocosm .m 2:9". 19 Atelp phenotypes by loss of TFIIS activity suggests that AtELP] and AtELP2 may carry out a function that is identical with that of AtTFIIS, such that when the AtTFIIS is absent, the plants developed normally. AtELP and AtTFIIS might be in parallel pathways that perform a non-essential function. The absence of this function gives rise to an enhanced phenotype. In addition, if AtTFIIS functions in transcription elongation, the genetic interaction between AtELP and AtTFIIS suggests that the AtELPl and AtELP2 in plants have a role in transcription elongation. In a similar manner, we tested genetic interaction between AtELP and plant homologs of subunits of Paf 1C. The previously identified VIP4, VIP5, and VIP6 genes in Arabidopsis encode the counterparts of Leo], Rtf 1, and Ctr9 subunits of the yeast Pafl complex, respectively (Zhang and van Nocker, 2002, Zhang et al., 2003, Oh et al., unpublished). Mutation in AtELP] or AtELP2 and in VIP4-VIP6 showed similar defects in petal development, suggesting that Elongator and VIPs have some overlapping genetic targets. Therefore, we postulated that there might be functional interaction between the plant homolog of Elongator and Pafl proteins. To test our hypothesis, we combined the Atelpl and Atelp2 mutations with vip3-vip6 mutations, and carried out a phenotypic analysis of the resulting double mutants. Although the VIP3 protein does not show homology to any subunit of yeast Pafl complex, this gene is functionally identical to VIP4, VIP5, and VIP6 (Oh et al., unpublished). We found that all of these double mutants were similar and showed a remarkably enhanced phenotype as compared with either Atelp or vip single mutants: the plants were tiny and eventually died after producing only four to six rosette leaves (Fig. 3B). This piece of evidence revealed that plant homologs of Elongator and Pafl proteins functionally interact. They may not encode the same biochemical 20 function, but act in parallel pathways (Guarente, 1993). If their functions are essential, then loss of both pathways will result in synthetic lethality. Gene expression profiling of Atelpl mutants The pleiotropic phenotypes of AtelpI and AtelpZ mutants suggested that AtELP] and AtELP2 function in multiple aspects of development through regulation of a variety of genes. To identify such genes, we used transcriptional profiling to detect differences in gene expression between the Atelpl mutant and its wild-type Col-0 genetic background (Fig.5A). We used the Arabidopsis ATHl Genome Array representing 22,747 different Arabidopsis genes (Affymetrix, Santa Clara, CA). For this study, we considered only flowers and inflorescence apices, because of the striking effect of Atelpl on floral morphology, and because transcriptional and genetic networks contributing to floral development are widely studied (Buzgo et al., 2004). We employed two independent biological replicates, with each replicate composed of three independent samples, and with each sample composed of 10 inflorescences. Mld-type samples were harvested when the first flower was fully opened and stigmatic papillae were fully developed (stage 12). Because AtelpI has defects in petal development, we use stigmatic papillae development as a benchmark to coordinate developmental stages between mutant and wild type flowers. Using both replicates of AtelpI and wild-type, four pair-wise comparisons were created. Genes were considered to be down- or up-regulated if differences in hybridization signals were threefold or more in at least three of the four comparisons (see Methods). Based on these criteria, 196 genes were affected (159 were down-regulated, and 37 were up-regulated). 21 To confirm the Affymetrix array data, we performed relatively quantitative RT-PCR analysis for a subset of the affected genes (Fig. 4B). We chose these genes for further study, because they encode putative transcription factors, and they may represent more direct targets of AtELP] and play a role in the developmental defects that we observed in Atelpl mutants. The Atlg71770 and Ar1g22760 genes encode PABS and PAB3, respectively, two members of the polyadenylate-binding proteins (PABPS) family. Poly(A)-binding proteins are multifunctional proteins that play important roles in mRNA stability and protein translation (Wang and Grumet, 2004). Although PAB3 and PABS are not essential for viability, they are important for post-transcriptional regulation in plant sexual reproduction. Atlg71770 (PABS) expression in Arabidopsis is restricted to pollen and ovule development and early embryogenesis, and PAB5 protein is capable of rescuing a PABP-deficient yeast strain by partially restoring both poly(A) shortening and translational initiation functions of PABP (Belostotsky and Meagher, 1996). At1g22760 (PAB3) expression is restricted to late pollen development in immature flowers, and PAB3 is capable of promoting partial Shortening of poly (A) tails in yeast (Belostotsky and Shaw, 1997). We confirmed that the PAB3 and/or PAB5 gene expression was significantly repressed in AtelpI mutant flowers. Potentially, the striking defects that we observed on floral development could be due to loss of activity of these genes. Alternatively, because PAB3 and PAB5 are expressed in stamens, under-representation of PAB3 and PAB5 in AtelpI mutant samples might merely be explained by failure of stamen development. 22 A 10 V1 '1 l I I V ‘0‘ E E 103 - .: o. 102:“ ‘: 6 = ‘ O 101 r 3 0 10 .- ~ E i -1 10 I I “.1 I l 10 ’ 10" 10’ 10’ 103 10‘ 10’ B Polyadenylate-binding proteins (At1g71770 At1922760) DNA-binding family protein (At2942940) NAM family protein (At1gs1110) AC TIN Figure 4. Characteristics of microarray data derived from wild-type Col-0 and the Atelp1 mutant. (A) Signal intensity was plotted to compare single replicates of WT with Atelp1. (B) Genes identified by microarray analysis as down-regulated in Ate/p1 mutants relative to WT plants were monitored by RT—PCR; the constitutively expressed gene ACT/N served as a control. 23 The At2g42940 gene encodes a potential DNA-binding protein containing AT-hook motifs, which preferentially bind to AT-rich regions in double-stranded DNA. Although its function is not well known, the study of other members of this family of proteins, such as HMG (High Mobility Group) proteins, revealed that AT-hook-containing proteins may regulate nucleosome phasing and 3' end processing of mRNA transcripts, and transcriptional regulation of genes containing AT—rich regions (Reeves and Nissen, 1990). The At1g61110 gene encodes a member of the NAM (No Apical Meristem) protein family. In Petunia, NAM mRNA accumulates in cells at the boundaries of meristems and primordia, implicating a role for NAM in determining positions of meristems and primordia (Souer et al., 1996). Another member of this family in Arabidopsis, NAP, is a target gene of APETAlA3/PISTIL1ATA (AP3/PI; Sablowski and Meyerowitz, 1998). AP3 and P1 are homeotic proteins belonging to the MADS-box family of transcription factors, and are involved in petal and stamen formation in the Arabidopsis flower (Jack et a1, 1992). If Atlg61110 has a function similar to that of NAP, then down-regulation of Atlg61110 could help explain the defects in petal and stamen formation in Atelp1 mutant plants. We also found that FLC expression was down-regulated about 4-fold in Atelp1 mutant flowers compared to the wild-type Col-0 flowers, revealing that AtELP] directly or indirectly activates FLC. This was surprising, because we did not observe early flowering after we transferred the homozygous Atelp1 and Atelp2 mutations into ColeRI‘SF2 background, and because antisense AtELP2 plants in the Colzli‘RISFz background did not flower early (not shown). Potentially because FRI is epistatic to AtELP], or AtELP] regulates F LC only in floral phase. 24 Induction of cold-response genes and heat shock genes is not affected by Atelp1 and Atelpz mutations. In Arabidopsis and other plants, a set of genes referred to as COR genes are induced upon cold (Uemura et al., 1996). Previous studies demonstrated that in Arabidopsis, low-temperature induction of COR genes was delayed, and the final levels of transcripts were reduced, in gcn5 mutants (Vlachonasios et al., 2003). The ELP3 subunit of Elongator and Gcn5 subunit of SAGA and ADA complexes both belong to the GNAT class of HATS, and in yeast these genes have an overlapping function (Wittschieben et al., 2000). Therefore, we tested whether induction of COR genes was affected in Atelp1 and AtelpZ mutants. Using RNA gel-blot analysis, we found that COR6.6 and COR47 transcripts became detectable about 4h after cold treatment in Atelp1 and AtelpZ mutants, and reached a plateau by 24 hours (Fig. 5A). This response was not obviously different from the wild type, suggesting that AtELP] and AtELPZ do not have a crucial role in COR gene induction. We also evaluated expression of the heat response pathway gene HSP70 in Atelp1 and AtelpZ mutant plants. The heat Shock (HS) response is a conserved cellular defense mechanism, which is activated by elevated temperature and a number of chemical Stresses. 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