Genome transcriptional expression
CEA Saclay/Bât. 144
Tél : 01 69 08 93 42
michel.werner@cea.fr
The team studies the regulation of DNA transcription using the yeast as a biological model, aiming at a general understanding of the mechanisms implicated in the expression of animal genomes and other eukaryotes. Eukaryotic transcription is performed by three main enzymes, RNA polymerase I, II and III, and requires a large set of general transcription factors. How these proteins work together to activate transcription is our main goal.
Human resources
Michel WERNER , Group Leader
Julie SOUTOURINA , Researcher
Claire BOSCHIERO , Research Technician
Sylvie MARIOTTE-LABARRE , Research Technician
Helen NEIL, Postdoctoral Fellow
Fanny EYBOULET, PhD Student
Research Programs
The Mediator is a multi-protein complex consisting of 25 subunits. It is essential during the activation of RNA polymerase II, to recruit the enzyme and to initiate transcription. The study of the Mediator is hence one of the keys to understand gene expression regulation. We have recently mapped the interactions between Mediator subunits using the two hybrid method (Guglielmi et al. 2004). This approach allowed us to propose a model of the organization of the complex, some aspects of which have been confirmed by structural studies. We are now interested in the role of the Mediator in transcription activation and, in particular, in preinitiation complex formation. Our studies have led us to the discovery of an essential role of the Mediator in TFIIH general transcription factor recruitment and to propose a new model in preinitiation complex formation during transcription activation (Esnault et al. 2008).
Figure: The first step of transcription activation consists in the recruitment of the Mediator complex by transcription activation (A). The complex can then recruit RNA polymerase II (B) or TFIIE and cTFIIH general transcription factors (B’). These intermediates can lead to the formation of a full preinitiation complex through various “branched” pathways.
The general transcription factor TFIIS, that plays an important role in transcription elongation, is a partner of Mediator and the SAGA coactivator, which also plays a role in activation (Wéry et al. 2004). We have shown that Mediator and TFIIS cooperate in the activation of a subset of the yeast genome (Guglielmi et al. 2007). The analysis of TFIIS distribution on the genome unexpectedly indicated its presence on RNA polymerase III-transcribed genes in addition to those that are transcribed by RNA polymerase II. We demonstrated that TFIIS plays an important role in RNA polymerase III transcription, stimulating the transcription of this class of genes (Ghavi-Helm et al. 2008). These findings have led to reconsider and to extend largely the role of TFIIS transcription factor in genome expression.
2. RNA polymerase I and III.
The comparative study of the three yeast RNA polymerases, in close collaboration with the group “Nuclear Regulations and Stress”, constitutes the second direction of our research program on the eukaryotic transcription machinery. Based on an approach developed in our laboratory, we have recently discovered a novel player in transcription mediated by RNA polymerase I, the Hmo1 protein (Gadal et al. 2002). We were able to describe the transcriptional targets of RNA polymerase III (Harismendy et al. 2003) and to better understand the role of the Fhl1 and Ifh1 transcriptional regulators (discovered in our laboratory in the 1990s) in the global control of ribosome biogenesis (Schawalder et al. 2004). We have studied the role of two specific RNA polymerase I subunits in transcriptional elongation (Beckouët et al. 2008). Regarding RNA polymerase III, we have started a project within the framework of the Regulome consortium to map the location of the RNA polymerase III transcription machinery on the mouse genome, just like we did previously for yeast (Harismendy et al. 2003) . This work is done in collaboration with the group “Epigenetic Regulation and Cancer”. The approach is based on the use of embryonic stem cells in which proteins of interest are tagged to help immunoprecipitate chromatin efficiently. We have now identified all the genes that are transcribed by RNA polymerase III in mouse embryonic stem cells.
Technically, our group is experienced at classic genetics and molecular biology techniques of the yeast Saccharomyces cerevisiae. We have also been using for several years functional genomics techniques based on DNA chips and, more recently, high throughput DNA sequencing for studying transcription and its regulation (Fauchon et al. 2002 ; Soutourina et al. 2006) and for determining the genomic location of DNA-associated proteins (Harismendy et al. 2003) ; Schawalder et al. 2004 ; Andrau et al. 2006).
Key words
Eukaryote transcription / functional genomics / transcription coupled repair/ yeast genetics
Publications
Carrière L, Graziani S, Alibert O, Ghavi-Helm Y, Boussouar F, Humbertclaude H, Jounier S, Aude J C, Keime C, Murvai J, Foglio M, Gut M, Gut I, Lathrop M, Soutourina J, Gérard M, Werner M. (2011). Genomic binding of Pol III transcription machinery and relationship with TFIIS transcription factor distribution in mouse embryonic stem cells. Nucleic Acids Res. 40, 270-283.
Garcia-Lopez M C, Pelechano V, Miron-Garcia M C, Garrido-Godino A I, Garcia A, Calvo O, Werner M, Perez-Ortin J E, Navarro F. (2011). The Conserved Foot Domain of RNA Pol II Associates with Proteins Involved in Transcriptional Initiation and/or Early Elongation. Genetics. 189, 1235-1248.
Ruprich-Robert G, Wery M, Despres D, Boulard Y, Thuriaux P. (2011). Crucial role of a dicarboxylic motif in the catalytic center of yeast RNA polymerases. Curr Genet. 57, 327-334.
Soutourina J, Wydau S, Ambroise Y, Boschiero C, Werner M. (2011). Direct Interaction of RNA Polymerase II and Mediator Required for Transcription in Vivo. Science. 331, 1451-1454.
Werner M. (2011). Transcription and its regulation in eucaryotes. Biofutur. 35-37.
Coudreuse D, van Bakel H, Dewez M, Soutourina J, Parnell T, Vandenhaute J, Cairns B, Werner M, Hermand D. (2010). A gene-specific requirement of RNA polymerase II CTD phosphorylation for sexual differentiation in S. pombe. Curr. Biol., 20, 1053-1064.
Ruprich-Robert G, Thuriaux P. (2010). Non-canonical DNA transcription enzymes and the conservation of two-barrel RNA polymerases. Nucleic Acids Res. . 38, 4559-69.
Werner M, Thuriaux P, Soutourina J. (2009). Structure-function analysis of RNA polymerases I and III. Curr. Opin. Struc. Biol., 19, 740-745.
Beckouet F, Labarre-Mariotte S, Albert B, Imazawa Y, Werner M, Gadal O, Nogi Y, Thuriaux P. (2008). Two RNA Polymerase I Subunits Control the Binding and Release of Rrn3 during Transcription. Mol Cell Biol. 28, 1596-605.
Esnault C, Ghavi-Helm Y, Brun S, Soutourina J, Van Berkum N, Boschiero C, Holstege F, Werner M. (2008). Mediator-dependent recruitment of TFIIH modules in preinitiation complex. Mol Cell. 31, 337 - 346.
Ghavi-Helm Y, Michaut M, Acker J, Aude JC, Thuriaux T, Werner M, Soutourina J.(2008). Genome-wide location analysis reveals a role of TFIIS in RNA polymerase III transcription. Genes & Dev. 22, 1934-47.
Kwapisz M, Beckouët F, Thuriaux P. (2008). Early evolution of eukaryotic DNA-dependent RNA polymerases. Trends Genet. 24, 211-5.
Kwapisz M, Wery M, Després D, Ghavi-Helm Y, Soutourina J, Thuriaux P, Lacroute F. (2008). Mutations of RNA polymerase II activate key genes of the nucleoside triphosphate biosynthetic pathways. EMBO J. 27, 2411-21.
Beckouet F, Labarre-Mariotte S, Albert B, Imazawa Y, Werner M, Gadal O, Nogi Y, Thuriaux P. (2007). Two RNA Polymerase I Subunits Control the Binding and Release of Rrn3 during Transcription. Mol Cell Biol. 28, 1596-1605
Guglielmi B, Soutourina J, Esnault C, Werner M. (2007). TFIIS elongation factor and Mediator act in conjunction during transcription initiation in vivo. Proc Natl Acad Sci U S A. 104, 16062-7.
Guiguen A, Soutourina J, Dewez M, Tafforeau L, Dieu M, Raes M, Vandenhaute J, Werner M, Hermand D. (2007). Recruitment of P-TEFb (Cdk9-Pch1) to chromatin by the cap-methyl transferase Pcm1 in fission yeast. EMBO J. 26, 1552-1559.
Loncle N, Boube M, Joulia L, Boschiero C, Werner M, Cribbs DL, Bourbon HM. (2007). Distinct roles for Mediator Cdk8 module subunits in Drosophila development. EMBO J. 26, 1045-1054.
Zaros C, Briand JF, Boulard Y, Labarre-Mariotte S, Garcia-Lopez MC, Thuriaux P, Navarro F. (2007). Functional organization of the Rpb5 subunit shared by the three yeast RNA polymerases. Nucleic Acids Res. 35, 634-647.
Soutourina J, Bordas-Le Floch V, Gendrel G, Flores A, Ducrot C, Dumay-Odelot H, Soularue P, Navarro F, Cairns BR, Lefebvre O, Werner M. (2006). Rsc4 Connects the Chromatin Remodeler RSC to RNA Polymerases. Mol Cell Biol. 26, 4920-4930.
Proshkina GM, Shematorova EK, Proshkin SA, Zaros C, Thuriaux P, Shpakovski GV. (2006). Ancient origin, functional conservation and fast evolution of DNA-dependent RNA polymerase III. Nucleic Acids Res. 34, 3615-3624.
Guffanti E, Percudani R, Harismendy O, Soutourina J, Werner M, Lacovella G, Negri R, Dieci G. (2006). Nucleosome depletion activates poised RNA polymerase III at unconventional transcription sites in Saccharomyces cerevisiae. J Biol Chem. 281, 29155-29164.
Andrau JC, van de Pasch L, Lijnzaad P, Bijma T, Koerkamp MG, van de Peppel J, Werner M, Holstege FC. (2006). Genome-wide location of the coactivator mediator: Binding without activation and transient Cdk8 interaction on DNA. Mol Cell. 22, 179-192.
Zaros C, Thuriaux P. (2005). Rpc25, a conserved RNA polymerase III subunit, is critical for transcription initiation. Mol Microbiol. 55, 104-114.
Torchet C, Badis G, Devaux F, Costanzo G, Werner M, Jacquier A. (2005). The complete set of H/ACA snoRNAs that guide rRNA pseudouridylations in Saccharomyces cerevisiae RNA. 11, 928-938.
Lenssen E, James N, Pedruzzi I, Dubouloz F, Cameroni E, Bisig R, Maillet L, Werner M, Roosen J, Petrovic K, Winderickx J, Collart MA, De Virgilio C. (2005). The Ccr4-Not complex independently controls both Msn2-dependent transcriptional activation--via a newly identified Glc7/Bud14 type i protein phosphatase module--and TFIID promoter distribution. Mol Cell Biol. 25, 488-498.
Kabani M, Michot K, Boschiero C, Werner M. (2005). Anc1 interacts with the catalytic subunits of the general transcription factors TFIID and TFIIF, the chromatin remodeling complexes RSC and INO80, and the histone acetyltransferase complex NuA3. Biochem Biophys Res Commun. 332, 398-403
Wery M, Shematorova E, Van Driessche B, Vandenhaute J, Thuriaux P, Van Mullem V. (2004). Members of the SAGA and Mediator complexes are partners of the transcription elongation factor TFIIS. EMBO J. 23, 4232-4242.
Schawalder SB, Kabani M, Howald I, Choudhury U, Werner M, Shore D. (2004). Growth-regulated recruitment of the essential yeast ribosomal protein gene activator Ifh1. Nature. 432, 1058-1061.
Kasten M, Szerlong H, Erdjument-Bromage H, Tempst P, Werner M, Cairns BR. (2004). Tandem bromodomains in the chromatin remodeler RSC recognize acetylated histone H3 Lys14. EMBO J. 23, 1348-1359.
Guglielmi B, van Berkum NL, Klapholz B, Bijma T, Boube M, Boschiero C, Bourbon HM, Holstege FC, Werner M. (2004). A high resolution protein interaction map of the yeast Mediator complex. Nucleic Acids Res. 32, 5379-5391.
Guglielmi B, Zaros C, Thuriaux P and Werner M. (2004). Investigating protein-protein interactions in multi-subunit proteins: the case of eukaryotic RNA polymerases. Genetics, Genomics, Proteomics and Bioinformatics Online.
Siaut M, Zaros C, Levivier E, Ferri ML, Court M, Werner M, Callebaut I, Thuriaux P, Sentenac A, Conesa C. (2003). An Rpb4/Rpb7-like complex in yeast RNA polymerase III contains the orthologue of mammalian CGRP-RCP. Mol Cell Biol. 23, 195-205.
Harismendy O, Gendrel CG, Soularue P, Gidrol X, Sentenac A, Werner M, Lefebvre O. (2003). Genome-wide location of yeast RNA polymerase III transcription machinery. EMBO J. 22, 4738-4747.
Wenner C, Goodlett D R, Aebersold R, Klein HG, Marschall C, Werner M, Kratz M and Cullen P (2002). Basic concepts of gene expression. In Analysing Gene Expression : A Handbook of Methods Possibilities and Pitfalls, S. Lorkowski, and P. Cullen, eds. (Weinheim, Wiley-VCH).
Van Mullem V, Wery M, Werner M, Vandenhaute J, Thuriaux P. (2002). The Rpb9 subunit of RNA polymerase II binds transcription factor TFIIE and interferes with the SAGA and elongator histone acetyltransferases. J Biol Chem. 277, 10220-10225.
Van Mullem V, Landrieux E, Vandenhaute J, Thuriaux P. (2002). Rpa12p, a conserved RNA polymerase I subunit with two functional domains. Mol Microbiol. 43, 1105-1113.
Guglielmi B, Werner M. (2002). The yeast homolog of human PinX1 is involved in rRNA and snoRNA maturation, not in telomere length regulation. J Biol Chem. 277, 35712-35719.
Gellon L, Werner M, Boiteux S. (2002). Ntg2p, a DNA N-glycolase / AP lyase involved in base excision repair of oxydative DNA damage, interacts with the DNA mismatch repair protein Mlh1p in Saccharomyces cerevisiae: Identification of a Mlh1p binding motif. J Biol Chem. 277, 29963-29972.
Gadal O, Labarre S, Boschiero C, Thuriaux P. (2002). Hmo1, an HMG box protein, belongs to the yeast ribosomal DNA transcription system. EMBO J. 21, 5498-5507.
Fauchon M, Lagniel G, Aude JC, Lombardia L, Soularue P, Petat C, Marguerie G, Sentenac A, Werner M, Labarre J. (2002). Sulfur-sparing in the yeast proteome in response to sulfur demand. Mol Cell. 9, 713-723.
Rozenfeld S, Thuriaux P. (2001). A genetic look at the active site of RNA polymerase III EMBO Rep. 21, 598-603.
Rozenfeld S, Thuriaux P. (2001). Genetic interactions within TFIIIC, the promoter-binding factor of yeast RNA polymerase III. Mol Genet Genomics. 265, 705-710.
Pierrugues O, Brutesco C, Oshiro J, Gouy M, Deveaux Y, Carman GM, Thuriaux P, Kazmaier M. (2001). Lipid phosphate phosphatases in Arabidopsis. Regulation of the AtLPP1 gene in response to stress. J Biol Chem. 276, 20300-20308.
Briand JF, Navarro F, Rematier P, Boschiero C, Labarre S, Werner M, Shpakovski GV, Thuriaux P. (2001). Partners of Rpb8p, a small subunit shared by yeast RNA polymerases I, II, and III. Mol Cell Biol. 21, 6056-6065.
Briand JF, Navarro F, Gadal O, Thuriaux P. (2001). Cross talk between tRNA and rRNA synthesis in Saccharomyces cerevisiae. Mol Cell Biol. 21, 189-195.
Andrau JC, Werner M. (2001). B"-associated factor(s) involved in RNA polymerase III preinitiation complex formation and start-site selection Eur J Biochem. 268, 5167-5175.
Andrau JC, Werner M. (2001). B"-associated factor(s) involved in RNA polymerase III preinitiation complex formation and start-site selection Eur J Biochem. 268, 5167-5175.
Main collaborations
- Gènoscope, Evry. Sequencing of SADE libraries in preparation for transcriptome analysis.
- Service de Génomique Fonctionnelle, CEA/Evry. Development of new DNA analysis methods based on DNA chips.
- Unité de Recherches en Biologie Moléculaire, Faculté Notre Dame de la Paix, Namur, Belgium. Relation between transcriptional elongation and DNA repair.
- University Medical Center, Utrecht University, Utrecht, The Netherlands. Global analysis of protein-protein interactions in transcription complexes.
- SPI/DRM/DSV, CEA Saclay. Quantitive metabolome analysis.