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Genome-Wide Location Analysis Reveals a Role for Sub1 in RNA Polymerase III Transcription

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Genome-Wide Location Analysis Reveals a Role for Sub1 in RNA Polymerase III Transcription Genome-wide location analysis reveals a role for Sub1 in RNA polymerase III transcription Arounie Taveneta, Audrey Suleaua,1,Ge´ raldine Dubreuila, Roberto Ferrarib,2,Ce´ cile Ducrota, Magali Michauta, Jean-Christophe Audea, Giorgio Diecib, Olivier Lefebvrea, Christine Conesaa,3, and Joe¨l Ackera,3 aCommissariat a`l’Energie Atomique, Institut de Biologie et de Technologies de Saclay, F-91101 Gif sur Yvette, France; and bDipartimento di Biochimica e Biologia Molecolare, Universita`degli Studi di Parma, 43100 Parma, Italy Edited by Robert G. Roeder, The Rockefeller University, New York, NY, and approved June 30, 2009 (received for review January 8, 2009) Human PC4 and the yeast ortholog Sub1 have multiple functions in that we used in a previous work to analyze the genomic location of RNA polymerase II transcription. Genome-wide mapping revealed the Pol III transcription machinery (15). A defined number of loci that Sub1 is present on Pol III-transcribed genes. Sub1 was found were significantly enriched (991 loci with a P value Ͻ0.01) under to interact with components of the Pol III transcription system and active growth conditions. Approximately one-fourth of the en- to stimulate the initiation and reinitiation steps in a system riched loci were located within ORF; the others corresponded to reconstituted with all recombinant factors. Sub1 was required for intergenic regions and to genes encoding nontranslated RNAs. We optimal Pol III gene transcription in exponentially growing cells. noted that the ACT1, PMA1, PYK1, ADH1, and snoRNA genes previously identified as DNA targets of Sub1 by ChIP and PCR Chip chip ͉ PC4 ͉ reinitiation ͉ TFIIIB amplification (10, 16, 17) were indeed enriched in our data. The distribution of the loci corresponding to snoRNA genes shown in C4 plays an important role in various cellular processes, includ- Fig. 1A extended the binding of Sub1 to all of the H/ACA box or Ping transcription, DNA repair, and replication (1–3). First C/D box snoRNA genes. To further identify Pol II gene targets of identified as a RNA polymerase (Pol) II coactivator (2, 4), PC4 was Sub1, the enriched loci with a P value Ͻ0.01 were analyzed by using shown to interact with activators and components of the Pol II basal the GoTermFinder software. Three overrepresented GO catego- Ϫ transcription machinery (2, 5) and to enhance activator-dependent ries (P value Ͻ10 5) were identified, indicating that Sub1 was BIOCHEMISTRY transcription, stimulating both initiation and promoter escape (6). preferentially bound to a subset of Pol II-transcribed genes encod- However, in the absence of TFIIH and TFIID, PC4 represses basal ing constituents of the cell wall (30 genes), the nucleosome (H2A, transcription (5, 7). Sub1, the yeast ortholog of PC4, was charac- H2B, H3, and H4 histone genes) and the ribosome (10% of the terized biochemically as a coactivator required for activated tran- enriched loci, corresponding to genes encoding constituents of the scription in vitro (8) and genetically as a suppressor of certain TFIIB translational apparatus, including TEF1, TEF2, and 50 ribosomal mutations (9). Further investigations extended the role of PC4/Sub1 protein (RP) genes). The distribution of the loci corresponding to to transcription elongation and mRNA processing. Sub1 was shown RP genes is shown in Fig. 1A. Because RP and histone genes are to regulate enzymes modifying the CTD of the largest subunit of Pol among the most highly transcribed genes in exponentially growing II and might therefore enhance elongation (10). Functional inter- cells, we determined the relationship between transcription rates actions between not only PC4/Sub1 and Cstf-64/Rna15 (11) but also and Sub1 occupancy. Interestingly, although Sub1 was also localized Pta1 and Sub1 (12) established additional connections between on very poorly transcribed ORFs, Sub1-associated Pol II genes Sub1/PC4 and mRNA processing. PC4 has also been copurified globally tended to be highly transcribed [supporting information with human TFIIIC and was found to stimulate RNA Pol III (SI) Fig. S1]. Remarkably, one-third of Sub1 enriched loci corre- transcription in vitro (13). Recently, PC4 was found to be associated sponded to Pol III-transcribed genes present on the arrays or to the with chromatin and to be important for chromatin organization, intergenic regions and ORFs adjacent to these genes. As shown in suggesting a more general role in transcription regulation (14). Fig. 1A, the Loci Enriched by the Pol III Transcription Machinery Apart from its role in transcription, PC4 was implicated in other (LEPTM, ref. 15), comprising tRNA genes and other genes tran- cellular processes, through its capacity to bind tightly to melted scribed by Pol III, like 5S RNA gene, SNR6, RPR1, and SCR1 were DNA and to single-stranded DNA (ssDNA, ref. 7). A direct significantly overrepresented among the most enriched DNA re- interaction between PC4 and XPG, a subunit of the nucleotide gions, suggesting the association of Sub1 to all Pol III-transcribed excision factor, was correlated with the genetic interaction between genes in conditions of active growth. their yeast counterparts, Sub1 and Rad2, suggesting a role for To confirm the results obtained by microarray hybridizations, PC4/Sub1 in the repair of oxydative DNA damage (1). Further- we performed conventional ChIP assays on a set of selected Pol more, PC4 can form complexes with human HSSB protein on III-transcribed genes. Using real-time PCR on DNA fragments ssDNA and influences its replication function in vitro (3). In this study, we identified the gene targets of Sub1 in exponentially growing yeast cells. Analysis of the genome-wide Author contributions: G. Dieci, O.L., C.C., and J.A. designed research; A.T., A.S., G. Dubreuil, localization of Sub1 revealed its association to all of the genes R.F., C.D., C.C., G. Dieci, and J.A. performed research; C.C. contributed new reagents/ analytic tools; A.T., A.S., G. Dubreuil, R.F., C.D., M.M., J.-C.A., G. Dieci, C.C., and J.A. analyzed transcribed by Pol III. We focused on the role of Sub1 in Pol III data; and C.C., G. Dieci, and J.A. wrote the paper. transcription. We present evidence that Sub1 is involved in Pol The authors declare no conflict of interest. III transcription initiation and reinitiation processes in vitro and This article is a PNAS Direct Submission. is required for optimal Pol III transcription in vivo. Freely available online through the PNAS open access option. Results 1Present address: Plateforme Diagnostic C. G. Harray, Hoˆpital Henri Mondor, 94000 Cre´teil, France. Genome-Wide Analysis of Sub1 Occupancy. To define Sub1 gene 2Present address: Department of Biological Chemistry, University of California, Los Angeles, targets in vivo, we performed a genome-wide analysis of Sub1 CA 90095. occupancy in exponentially growing cells. Chromatin immunopre- 3To whom correspondence may be addressed. E-mail: [email protected] or christine. cipitation (ChIP) assays were performed on epitope-tagged Sub1– [email protected]. 3HA cross-linked chromatin and analyzed by hybridization to DNA This article contains supporting information online at www.pnas.org/cgi/content/full/ microarrays harboring ORFs and intergenic regions similar to those 0900162106/DCSupplemental. www.pnas.org͞cgi͞doi͞10.1073͞pnas.0900162106 PNAS Early Edition ͉ 1of6 Downloaded by guest on September 25, 2021 Ileu A that encompassed SCR1 and the tI(AAU)E1 (tRNA ) gene. As 40 LEPTM 40 shown in Fig. 1C, Sub1 was detected significantly all along the SCR1 gene and over the tRNAIleu gene, but not on the intergenic regions that separate these loci. A comparison of the enrichment profile of snoRNA Sub1 across the SCR1 gene with that of TFIIIC, TFIIIB and Pol III (19) suggested a colocalization of Sub1 with Pol III. The DNA arrays used in our studies had a poor coverage of 500 40 Full scale the rDNA gene locus. Nevertheless, we found a significant enrichment of all loci corresponding to that region, suggesting Number of loci RP that Sub1 associates at many locations throughout the rDNA gene. A conventional ChIP experiment on the rDNA locus (Fig. S2) confirmed that Sub1 (but not TFIIIC) bound all along the 0 50 100 0 50 100 rDNA gene locus but preferentially to the Pol I-transcribed Median rank region, an enrichment profile similar to that of TFIIS, a protein B involved in Pol II transcription that has been shown recently to 1 play a role in Pol III transcription (19). 0.8 We wondered whether Sub1 genome-wide location could be modified under conditions where a global decrease of transcrip- 0.6 tion was observed. To address this question, we performed ChIP 0.4 on chip experiments from cells grown to stationary phase where a severe shutdown of transcription by Pol II has been described 0.2 Sub1 occupancy (%) (20). The comparison of Sub1 binding in exponential or station- 0 ary phase grown cells showed that Sub1 occupancy went down GAL1 Tel15 tDNALeu SNR6 5S SCR1 substantially in stationary phase as illustrated in Fig. S3 for a C subset of its Pol II targets or for LEPTM where the decrease in 1 Sub1 binding was highly similar (Fig. S3B) to those published 0.8 (21) for TFIIIB or Pol III. The binding of Sub1 to the transcribed sequences of genes that 0.6 tend to be highly transcribed raised the possibility that part of the binding signals were because of nonspecific interactions of Sub1 0.4 with RNA transcripts (22). Although this hypothesis could not be ruled out, all in vitro data presented below strongly suggested that Sub1 occupancy (%) 0.2 ChIP binding signals resulted from Sub1 association with DNA. 0 GAL1 123456789101112 All together, our results suggested that Sub1 could be involved in all 3 transcription systems.
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