Epigenetic regulation and cancer
Matthieu GERARD
CEA Saclay/Bât. 142
Tél: 01 69 08 94 29
matthieu.gerard@cea.fr
CEA Saclay/Bât. 142
Tél: 01 69 08 94 29
matthieu.gerard@cea.fr
iBiTec-S / SBIGeM / LREGE
Human resources
Matthieu GERARD, Group Leader
Sylvie JOUNIER, Research Technician
Hélène HUMBERTCLAUDE, Research Technician
Isabelle HMITOU, Postdoctoral Fellow
Research Programs
Chromatin remodeling factors in mouse embryonic stem cells
The group is working on the function of histone chaperones and chromatin remodeling enzymes. Our current work is devoted to the study of the genome-wide occupancy of chromatin remodeling enzymes in mouse ES cells, in order to discover the regulatory mechanisms associated with this large family of enzymes.
Chromatin plays many roles in the regulation of gene expression in mammals. Enzymes that can modify nucleosomes are key actors in this process. Such factors can be divided in two large families: one is able to covalently modify histone residues, and the other hydrolyzes ATP to alter protein-DNA interactions within the nucleosome. This second category defines the chromatin remodeling factors of the SNF2 superfamily. These proteins all contain a combination of two conserved ATPase and helicase domains. Most of these proteins belong to large multiprotein complexes in the cell, and can stimulate the sliding of nucleosomes on DNA in vitro.
In mammals, there are about 25 genes that belong to the SNF2 superfamily. On the basis of structural features, the SNF2 family can be divided into four main groups: the SWI/SNF, ISWI, INO80/SWR and CHD families. The SWI/SNF family includes in mammals the BRM and BRG1 factors, which contain a bromodomain, a strutural motif that recognizes acetylated histones. The ISWI family (SNF2H and SNF2L in mammals) is characterized by the presence of a histone binding module, the SANT domain. The INO80/SWR subfamily is identified by the presence of a split ATPase domain. Finally, the CHD family, which contains 9 genes in mammals, is defined by the presence of a double chromodomain in the N-terminal region of the protein. Chromodomains from several proteins were shown to interact with methylated histones. Structural studies suggest that the chromodomains of the different CHD factors have divergent properties, and therefore that their target chromatin regions might be different.
Why do mammalian cells contain so many different members of the SNF2 family ? The aim of our current project is to provide a comprehensive view of the role of the chromatin remodeling factor family in the regulation of gene expression in mouse embryonic stem (ES) cells.
We have chosen to use homologous recombination in ES cells to introduce a Tandem Affinity Purification (TAP) Tag, at the carboxy-terminus of each remodeling factor, together with a selection cassette, flanked by loxP sites to allow deletion with Cre-recombinase. An important implication of this strategy is that the TAP-tagged remodeling factor is expressed at normal levels from the endogenous gene, and correctly regulated during ES cell renewal and differentiation. We have successfully used a 6his-Flag-HA triple tag in pilot experiments. This combination will be used in all future experiments.
This large-scale project requires the construction of a large number of gene targeting vectors, which we generate using a high efficiency plasmid construction pipeline based on the recombineering technology (Liu et al., 2003). These large plasmids are transfected into mouse ES cells, in order to isolate ES cell clones that have undergone a homologous recombination event. We recently improved this protocol and could isolate, for two independent genes encoding chromatin remodeling factors, ES cell clones that were TAP-tagged at both alleles.
In the next step, we use chromatin immunoprecipitation (ChIP) experiments, associated with large-scale sequencing of the immunoprecipitated DNA on an Illumina/Solexa platform (ChIP-seq technology). This part of the project is realized in close collaboration with the laboratory of Ivo Gut at the CEA/Centre National de Génotypage (CNG). Software is currently being developed to process the Solexa data into lists of target sites that are directly compared to the genome. ChIP-Seq experiments will allow us to identify, for each remodeling factor, a series of binding sites within the mouse genome. When such sites are located in the vicinity of genes or regulatory elements, they will reveal the potential target genes of each factor. To further validate (or invalidate) these potential target genes, we perform a loss of function experiment using the pHYPER shRNA vector (Berlivet et al., 2007) that was developed in our laboratory. The comparison of the genes that are deregulated following depletion of a given factor, with the list of genes that are bound by the same factor, will identify genuine target genes. The ultimate goal of this project is to integrate the information in the ENSEMBL database, and make the data available publicly by web based platforms and tools.
This project is part of the International Regulome Consortium (IRC) project, which will address the regulation of genome function at a higher level by mapping the genetic regulatory nodes and networks that control the activity of ES cells.
Berlivet, S., Guiraud, V., Houlard, M. and Gerard, M. (2007) pHYPER, a shRNA vector for high-efficiency RNA interference in embryonic stem cells. Biotechniques, 42, 738, 740-733.
Liu, P., Jenkins, N.A. and Copeland, N.G. (2003) A highly efficient recombineering-based method for generating conditional knockout mutations. Genome Res, 13, 476-484.
The group is working on the function of histone chaperones and chromatin remodeling enzymes. Our current work is devoted to the study of the genome-wide occupancy of chromatin remodeling enzymes in mouse ES cells, in order to discover the regulatory mechanisms associated with this large family of enzymes.
Chromatin plays many roles in the regulation of gene expression in mammals. Enzymes that can modify nucleosomes are key actors in this process. Such factors can be divided in two large families: one is able to covalently modify histone residues, and the other hydrolyzes ATP to alter protein-DNA interactions within the nucleosome. This second category defines the chromatin remodeling factors of the SNF2 superfamily. These proteins all contain a combination of two conserved ATPase and helicase domains. Most of these proteins belong to large multiprotein complexes in the cell, and can stimulate the sliding of nucleosomes on DNA in vitro.
In mammals, there are about 25 genes that belong to the SNF2 superfamily. On the basis of structural features, the SNF2 family can be divided into four main groups: the SWI/SNF, ISWI, INO80/SWR and CHD families. The SWI/SNF family includes in mammals the BRM and BRG1 factors, which contain a bromodomain, a strutural motif that recognizes acetylated histones. The ISWI family (SNF2H and SNF2L in mammals) is characterized by the presence of a histone binding module, the SANT domain. The INO80/SWR subfamily is identified by the presence of a split ATPase domain. Finally, the CHD family, which contains 9 genes in mammals, is defined by the presence of a double chromodomain in the N-terminal region of the protein. Chromodomains from several proteins were shown to interact with methylated histones. Structural studies suggest that the chromodomains of the different CHD factors have divergent properties, and therefore that their target chromatin regions might be different.
Why do mammalian cells contain so many different members of the SNF2 family ? The aim of our current project is to provide a comprehensive view of the role of the chromatin remodeling factor family in the regulation of gene expression in mouse embryonic stem (ES) cells.
We have chosen to use homologous recombination in ES cells to introduce a Tandem Affinity Purification (TAP) Tag, at the carboxy-terminus of each remodeling factor, together with a selection cassette, flanked by loxP sites to allow deletion with Cre-recombinase. An important implication of this strategy is that the TAP-tagged remodeling factor is expressed at normal levels from the endogenous gene, and correctly regulated during ES cell renewal and differentiation. We have successfully used a 6his-Flag-HA triple tag in pilot experiments. This combination will be used in all future experiments.
This large-scale project requires the construction of a large number of gene targeting vectors, which we generate using a high efficiency plasmid construction pipeline based on the recombineering technology (Liu et al., 2003). These large plasmids are transfected into mouse ES cells, in order to isolate ES cell clones that have undergone a homologous recombination event. We recently improved this protocol and could isolate, for two independent genes encoding chromatin remodeling factors, ES cell clones that were TAP-tagged at both alleles.
In the next step, we use chromatin immunoprecipitation (ChIP) experiments, associated with large-scale sequencing of the immunoprecipitated DNA on an Illumina/Solexa platform (ChIP-seq technology). This part of the project is realized in close collaboration with the laboratory of Ivo Gut at the CEA/Centre National de Génotypage (CNG). Software is currently being developed to process the Solexa data into lists of target sites that are directly compared to the genome. ChIP-Seq experiments will allow us to identify, for each remodeling factor, a series of binding sites within the mouse genome. When such sites are located in the vicinity of genes or regulatory elements, they will reveal the potential target genes of each factor. To further validate (or invalidate) these potential target genes, we perform a loss of function experiment using the pHYPER shRNA vector (Berlivet et al., 2007) that was developed in our laboratory. The comparison of the genes that are deregulated following depletion of a given factor, with the list of genes that are bound by the same factor, will identify genuine target genes. The ultimate goal of this project is to integrate the information in the ENSEMBL database, and make the data available publicly by web based platforms and tools.
This project is part of the International Regulome Consortium (IRC) project, which will address the regulation of genome function at a higher level by mapping the genetic regulatory nodes and networks that control the activity of ES cells.
Berlivet, S., Guiraud, V., Houlard, M. and Gerard, M. (2007) pHYPER, a shRNA vector for high-efficiency RNA interference in embryonic stem cells. Biotechniques, 42, 738, 740-733.
Liu, P., Jenkins, N.A. and Copeland, N.G. (2003) A highly efficient recombineering-based method for generating conditional knockout mutations. Genome Res, 13, 476-484.
Publications
Galvan L, Lepejova N, Gaillard M C, Malgorn C, Guillermier M, Houitte D, Bonvento G, Petit F, Dufour N, Héry P, Gérard M, Elalouf J M, Deglon N, Brouillet E, de Chaldee M. (2012). Capucin does not modify the toxicity of a mutant Huntingtin fragment in vivo. Neurobiol Aging. (Sous Presse).
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.
Chantalat S, Depaux A, Hery P, Barral S, Thuret JY, Dimitrov S, Gérard M. (2011). Histone H3 trimethylation at lysine 36 is associated with constitutive and facultative heterochromatin. Genome Res. 1426-1437.
Planson A G, Palais G, Abbas K, Gérard M, Couvelard L, Delaunay A, Baulande S, Drapier J C, Toledano M. (2011). Sulfiredoxin protects mice from lipopolysaccaride-induced endotoxic shock. Antioxid Redox Signal. 14, 2071-2080.
Berlivet S, Houlard M, Gérard M. (2010). Loss-of-function studies in mouse embryonic stem cells using the pHYPER shRNA plasmid vector. Methods Mol. Biol., 650, 85-100.
Goodfellow SJ, Graham EL, Kantidakis T, Marshall L, Coppins BA, Oficjalska-Pham D, Gérard M, Lefebvre O, White RJ. (2008). Regulation of RNA Polymerase III Transcription by Maf1 in Mammalian Cells. J Mol Biol. 378, 481-91.
Berlivet S, Guiraud V, Houlard M, Gérard M. (2007). pHYPER, a shRNA vector for high-efficiency RNA interference in embryonic stem cells BioTechniques. 42, 738-743.
Houlard M, Berlivet S, Probst AV, Quivy JP, Hery P, Almouzni G, Gérard M. (2006). CAF-1 is essential for heterochromatin organization in pluripotent embryonic cells PLoS Genetics. 2, 0001-0011.
Duquet A, Polesskaya A, Cuvellier S, Ait-Si-Ali S, Hery P, Pritchard LL, Gerard M, Harel-Bellan A. (2006). Acetylation is important for MyoD function in adult mice EMBO Rep. 7, 1140-1146.
Fritsch L, Martinez LA, Sekhri R, Naguibneva I, Gérard M, Vandromme M, Schaeffer L, Harel-Bellan A. (2004). Conditional gene knock-down by CRE-dependent short interfering RNAs EMBO Rep. 5, 178-182.
Ren J, Lee S, Pagliardini S, Gerard M, Stewart CL, Greer JJ, Wevrick R. (2003). Absence of Ndn, encoding the Prader-Willi syndrome-deleted gene necdin, results in congenital deficiency of central respiratory drive in neonatal mice. J Neurosci. 23, 1569-157
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.
Chantalat S, Depaux A, Hery P, Barral S, Thuret JY, Dimitrov S, Gérard M. (2011). Histone H3 trimethylation at lysine 36 is associated with constitutive and facultative heterochromatin. Genome Res. 1426-1437.
Planson A G, Palais G, Abbas K, Gérard M, Couvelard L, Delaunay A, Baulande S, Drapier J C, Toledano M. (2011). Sulfiredoxin protects mice from lipopolysaccaride-induced endotoxic shock. Antioxid Redox Signal. 14, 2071-2080.
Berlivet S, Houlard M, Gérard M. (2010). Loss-of-function studies in mouse embryonic stem cells using the pHYPER shRNA plasmid vector. Methods Mol. Biol., 650, 85-100.
Goodfellow SJ, Graham EL, Kantidakis T, Marshall L, Coppins BA, Oficjalska-Pham D, Gérard M, Lefebvre O, White RJ. (2008). Regulation of RNA Polymerase III Transcription by Maf1 in Mammalian Cells. J Mol Biol. 378, 481-91.
Berlivet S, Guiraud V, Houlard M, Gérard M. (2007). pHYPER, a shRNA vector for high-efficiency RNA interference in embryonic stem cells BioTechniques. 42, 738-743.
Houlard M, Berlivet S, Probst AV, Quivy JP, Hery P, Almouzni G, Gérard M. (2006). CAF-1 is essential for heterochromatin organization in pluripotent embryonic cells PLoS Genetics. 2, 0001-0011.
Duquet A, Polesskaya A, Cuvellier S, Ait-Si-Ali S, Hery P, Pritchard LL, Gerard M, Harel-Bellan A. (2006). Acetylation is important for MyoD function in adult mice EMBO Rep. 7, 1140-1146.
Fritsch L, Martinez LA, Sekhri R, Naguibneva I, Gérard M, Vandromme M, Schaeffer L, Harel-Bellan A. (2004). Conditional gene knock-down by CRE-dependent short interfering RNAs EMBO Rep. 5, 178-182.
Ren J, Lee S, Pagliardini S, Gerard M, Stewart CL, Greer JJ, Wevrick R. (2003). Absence of Ndn, encoding the Prader-Willi syndrome-deleted gene necdin, results in congenital deficiency of central respiratory drive in neonatal mice. J Neurosci. 23, 1569-157