Addressing the specific roles of histone modifications in transcriptional repression [ChIP-seq and CUT&RUN]

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.