Project description:Our understanding of epigenetic processes is based on the hypothesis that individual posttranslational modifications of DNA and histones, or combinations thereof, function to direct unique downstream effects on transcription. Still, histone modifications are broadly categorized as repressive or activating, raising the question of potential functional redundancy. Here, we present an approach for addressing this question by substituting the genome-wide H3K27me3 pattern with other histone modifications. By taking advantage of the modular organization of PRC2, we direct de novo recruitment of H3K9me3 and H3K36me3 to PRC2 target genes in H3K27me3 null mouse embryonic stem cells (mESCs). We show that despite accurate genome-wide re-establishment of H3K36me3 at PRC2 target genes, which leads to significant reduction in H3K4me3 levels, the remaining H3K4me3 prevents H3K36me3 from recruiting sufficient DNA methylation to substitute for H3K27me3-mediated repression. In contrast, we demonstrate that H3K9me3 is more efficient in repressing H3K27me3 regulated genes, however this repression is also contingent on H3K4me3 status. Taken together, these results highlight the unique repressive functions of H3K27me3 and suggest that the functional effects of individual posttranslational modifications are highly dependent on the interplay with the existing chromatin environment.
Project description:Our understanding of epigenetic processes is based on the hypothesis that individual posttranslational modifications of DNA and histones, or combinations thereof, function to direct unique downstream effects on transcription. Still, histone modifications are broadly categorized as repressive or activating, raising the question of potential functional redundancy. Here, we present an approach for addressing this question by substituting the genome-wide H3K27me3 pattern with other histone modifications. By taking advantage of the modular organization of PRC2, we direct de novo recruitment of H3K9me3 and H3K36me3 to PRC2 target genes in H3K27me3 null mouse embryonic stem cells (mESCs). We show that despite accurate genome-wide re-establishment of H3K36me3 at PRC2 target genes, which leads to significant reduction in H3K4me3 levels, the remaining H3K4me3 prevents H3K36me3 from recruiting sufficient DNA methylation to substitute for H3K27me3-mediated repression. In contrast, we demonstrate that H3K9me3 is more efficient in repressing H3K27me3 regulated genes, however this repression is also contingent on H3K4me3 status. Taken together, these results highlight the unique repressive functions of H3K27me3 and suggest that the functional effects of individual posttranslational modifications are highly dependent on the interplay with the existing chromatin environment.
Project description:Our understanding of epigenetic processes is based on the hypothesis that individual posttranslational modifications of DNA and histones, or combinations thereof, function to direct unique downstream effects on transcription. Still, histone modifications are broadly categorized as repressive or activating, raising the question of potential functional redundancy. Here, we present an approach for addressing this question by substituting the genome-wide H3K27me3 pattern with other histone modifications. By taking advantage of the modular organization of PRC2, we direct de novo recruitment of H3K9me3 and H3K36me3 to PRC2 target genes in H3K27me3 null mouse embryonic stem cells (mESCs). We show that despite accurate genome-wide re-establishment of H3K36me3 at PRC2 target genes, which leads to significant reduction in H3K4me3 levels, the remaining H3K4me3 prevents H3K36me3 from recruiting sufficient DNA methylation to substitute for H3K27me3-mediated repression. In contrast, we demonstrate that H3K9me3 is more efficient in repressing H3K27me3 regulated genes, however this repression is also contingent on H3K4me3 status. Taken together, these results highlight the unique repressive functions of H3K27me3 and suggest that the functional effects of individual posttranslational modifications are highly dependent on the interplay with the existing chromatin environment.
Project description:Dynamic changes in histone posttranslational modifications (PTMs) are important regulators of chromatin structure and gene transcription in both normal and disease settings. Herein, we describe a novel signaling mechanism of nitric oxide (â?¢NO) by demonstrating its ability to modulate gene expression via alteration of histone PTMs. Having established that â?¢NO exposure induced differential expression of approximately 6500 genes, we set out to determine if there was an epigenetic component to their regulation. â?¢NO exposure led to alterations in the global levels of acetyl and methyl modifications at numerous lysine residues on core histones H3 and H4. Residues H3K9me2/ac were examined further and determined to have differential distribution at various loci throughout the genome in response to â?¢NO. Changes in the enrichment levels of H3K9me2/ac at specific genes correlated with changes in the expression levels of their transcripts. Molecular mechanisms contributing to phenotypic outcomes in â?¢NO-associated cancers remain to be well understood since traditional modes of â?¢NO-signaling do not explain a large proportion of its impact on tumor cell behavior. Our results reveal that â?¢NO drives a significant amount of gene expression changes epigenetically by changing the distribution of numerous histone marks. Cultured cells were treated with 500uM DETA/NO to examine the effects of a physiologically relevant â?¢NO concentration on gene expression. A total of two untreated biological replicates and two â?¢NO-treated biological replicates were harvested. The untreated samples served as control against which comparisons were made to elucidate â?¢NO-mediated changes in the gene expression.
Project description:Dynamic changes in histone posttranslational modifications (PTMs) are important regulators of chromatin structure and gene transcription in both normal and disease settings. Herein, we describe a novel signaling mechanism of nitric oxide (â¢NO) by demonstrating its ability to modulate gene expression via alteration of histone PTMs. Having established that â¢NO exposure induced differential expression of approximately 6500 genes, we set out to determine if there was an epigenetic component to their regulation. â¢NO exposure led to alterations in the global levels of acetyl and methyl modifications at numerous lysine residues on core histones H3 and H4. Residues H3K9me2/ac were examined further and determined to have differential distribution at various loci throughout the genome in response to â¢NO. Changes in the enrichment levels of H3K9me2/ac at specific genes correlated with changes in the expression levels of their transcripts. Molecular mechanisms contributing to phenotypic outcomes in â¢NO-associated cancers remain to be well understood since traditional modes of â¢NO-signaling do not explain a large proportion of its impact on tumor cell behavior. Our results reveal that â¢NO drives a significant amount of gene expression changes epigenetically by changing the distribution of numerous histone marks. Cultured cells were treated with 500uM DETA/NO to examine the effects of a physiologically relevant â¢NO concentration on differential distribution of H3K9ac/H3K9me2. A total of two untreated biological replicates and two â¢NO-treated biological replicates were harvested. The untreated samples served as control against which comparisons were made to elucidate â¢NO-mediated changes in the histone landscape.