Biomedical subjects

Raphael Margueron

Publications and source records attributed to Raphael Margueron.

7 recordsLinked to original sources

PubMed2006

Suz12 binds to silenced regions of the genome in a cell-type-specific manner.

Suz12 is a component of the Polycomb group complexes 2, 3, and 4 (PRC 2/3/4). These complexes are critical for proper embryonic development, but very few target genes have been identified in either mouse or human cells. Using a variety of ChIP-chip approaches, we have identified a large set of Suz12 target genes in five different human and mouse cell lines. Interestingly, we found that Suz12 target promoters are cell type specific, with transcription factors and homeobox proteins predominating in embryonal cells and glycoproteins and immunoglobulin-related proteins predominating in adult tumors. We have also characterized the localization of other components of the PRC complex with Suz12 and investigated the overall relationship between Suz12 binding and markers of active versus inactive chromatin, using both promoter arrays and custom tiling arrays. Surprisingly, we find that the PRC complexes can be localized to discrete binding sites or spread through large regions of the mouse and human genomes. Finally, we have shown that some Suz12 target genes are bound by OCT4 in embryonal cells and suggest that OCT4 maintains stem cell self-renewal, in part, by recruiting PRC complexes to certain genes that promote differentiation.

Animals↗

PubMed2005

EZH2 and histone 3 trimethyl lysine 27 associated with Il4 and Il13 gene silencing in Th1 cells.

Differentiation of naïve CD4 T cells toward the T helper 1 (T(H)1) and T helper 2 (T(H)2) fates involves the transcriptional repression and enhancement, respectively, of Il4 and Il13, adjacent chromosome 11 genes encoding the canonical T(H)2 cytokines interleukin-4 and interleukin-13. Proper execution of this developmental fate choice during immune responses is critical to host defense and, when misregulated, leads to susceptibility to infectious microbes and to allergic and autoimmune diseases. Here, using chromatin immunoprecipitation and real time reverse transcription PCR we identify the Polycomb family histone methyltransferase EZH2 as the enzyme responsible for methylating lysine 27 of histone H3 at the Il4-Il13 locus of T(H)1 but not T(H)2 cells, implicating EZH2 in the mechanism of Il4 and Il13 transcriptional silencing.

Animals↗

PubMed2005

Composition and histone substrates of polycomb repressive group complexes change during cellular differentiation.

Changes in the substrate specificities of factors that irreversibly modify the histone components of chromatin are expected to have a profound effect on gene expression through epigenetics. Ezh2 is a histone-lysine methyltransferase with activity dependent on its association with other components of the Polycomb Repressive Complexes 2 and 3 (PRC2/3). Ezh2 levels are increasingly elevated during prostate cancer progression. Other PRC2/3 components also are elevated in cancer cells. Overexpression of Ezh2 in tissue culture promotes formation of a previously undescribed PRC complex, PRC4, that contains the NAD+-dependent histone deacetylase SirT1 and isoform 2 of the PRC component Eed. Eed2 is expressed in cancer and undifferentiated embryonic stem (ES) cells but is undetectable in normal and differentiated ES cells. The distinct PRCs exhibit differential histone substrate specificities. These findings suggest that formation of a transformation-specific PRC complex may have a major role in resetting patterns of gene expression by regulating chromatin structure.

Animals↗

PubMed2005

The key to development: interpreting the histone code?

Developmental stages in multicellular organisms proceed according to a temporally and spatially precise pattern of gene expression. It has become evident that changes within the chromatin structure brought about by covalent modifications of histones are of crucial importance in determining many biological processes, including development. Numerous studies have provided evidence that the enzymes responsible for the modifications of histones function in a coordinated pattern to control gene expression in the short term and, through the transferral of these modifications by inheritance to their progeny, in the long term.

Animals↗

PubMed2004

Silencing of human polycomb target genes is associated with methylation of histone H3 Lys 27.

Polycomb group (PcG) complexes 2 and 3 are involved in transcriptional silencing. These complexes contain a histone lysine methyltransferase (HKMT) activity that targets different lysine residues on histones H1 or H3 in vitro. However, it is not known if these histones are methylation targets in vivo because the human PRC2/3 complexes have not been studied in the context of a natural promoter because of the lack of known target genes. Here we report the use of RNA expression arrays and CpG-island DNA arrays to identify and characterize human PRC2/3 target genes. Using oligonucleotide arrays, we first identified a cohort of genes whose expression changes upon siRNA-mediated removal of Suz12, a core component of PRC2/3, from colon cancer cells. To determine which of the putative target genes are directly bound by Suz12 and to precisely map the binding of Suz12 to those promoters, we combined a high-resolution chromatin immunoprecipitation (ChIP) analysis with custom oligonucleotide promoter arrays. We next identified additional putative Suz12 target genes by using ChIP coupled to CpG-island microarrays. We showed that HKMT-Ezh2 and Eed, two other components of the PRC2/3 complexes, colocalize to the target promoters with Suz12. Importantly, recruitment of Suz12, Ezh2 and Eed to target promoters coincides with methylation of histone H3 on Lys 27.

Carrier Proteins↗

PubMed2004

Transcriptional regulation by the repressor of estrogen receptor activity via recruitment of histone deacetylases.

Histone acetyltransferases and deacetylases are recruited by transcription factors and adapter proteins to regulate specific subsets of target genes. We were interested in identifying interaction partners of histone deacetylase 1 (HDAC1) that might be involved in conferring target or substrate specificity. Using the yeast two-hybrid system, we isolated the repressor of estrogen receptor activity (REA) as a novel HDAC1-associated protein. We demonstrated the in vivo interaction of REA with HDAC1 and characterized the respective domains required for their interaction in vitro. In addition, we found that REA also associates with the class II histone deacetylase HDAC5. In luciferase reporter assays, REA decreased transcription, and this repression was sensitive to the deacetylase inhibitor trichostatin A. Finally, we showed that REA specifically interacts with the chicken ovalbumin upstream binding transcription factors and II. The nuclear receptor chicken ovalbumin upstream binding transcription factor I was found to cooperate with REA and histone deacetylases in the repression of target genes. We, therefore, propose a novel function for REA as a mediator of transcriptional repression by nuclear hormone receptors via recruitment of histone deacetylases.

3T3 Cells↗

PubMed2003

Ligands differentially modulate the protein interactions of the human estrogen receptors alpha and beta.

The interactions of human estrogen receptor subtypes ERalpha and ERbeta with DNA and a 210 amino acid residue fragment of the coactivator protein SRC-1 bearing three nuclear receptor interaction motifs were investigated quantitatively using fluorescence anisotropy in the presence of agonist and antagonist ligands. ERalpha and ERbeta were found to bind in a similar manner to DNA, and both salt and temperature affected the affinity and/or stoichiometry of these interactions. The agonist ligands estradiol, estrone and estriol did not modify the binding of ERalpha to the fluorescein-labeled target estrogen response element. However, in the case of ERbeta, these ligands led to the formation of some higher-order protein-DNA complexes and a small decrease in affinity. The partial agonist 4-hydroxytamoxifen had little effect on either ER subtype, whereas the pure antagonist ICI 182,780 led to the cooperative formation of protein-DNA complexes of higher order than dimer, as further demonstrated by competition experiments and gel mobility-shift assays. In addition to DNA binding, the interaction of both ER subtypes with the Alexa488-labeled SRC-1 coactivator fragment was investigated by fluorescence anisotropy. The agonist ligands estrone, estradiol, estriol, genistein and ethynyl estradiol exhibited distinct capacities for inducing the recruitment of SRC-1 that were not correlated with their affinity for the receptor. Moreover, estrone and genistein exhibited subtype specificity in that they induced SRC-1 recruitment to ERbeta with much higher efficiency than in the case of ERalpha. The differential coactivator recruitment capacities of the ER agonists and their receptor subtype coactivator recruitment specificity may be linked to the molecular structure of the agonists with respect to their interactions with a specific histidine residue located at the back of the ligand-binding pocket. Altogether, these quantitative in vitro studies of ER interactions reveal the complex energetic and stoichiometric consequences of changes in the chemical structures of these proteins and their ligands.

DNA↗