A System for Genome-Wide Histone Variant Dynamics In ES Cells Reveals Dynamic MacroH2A2 Replacement at Promoters

Abstract

Dynamic exchange of a subset of nucleosomes in vivo plays important roles in epigenetic inheritance of chromatin states, chromatin insulator function, chromosome folding, and the maintenance of the pluripotent state of embryonic stem cells. Here, we extend a pulse-chase strategy for carrying out genome-wide measurements of histone dynamics to several histone variants in murine embryonic stem cells and somatic tissues, recapitulating expected characteristics of the well characterized H3.3 histone variant. We extended this system to the less-studied MacroH2A2 variant, commonly described as a “repressive” histone variant whose accumulation in chromatin is thought to fix the epigenetic state of differentiated cells. Unexpectedly, we found that while large intergenic blocks of MacroH2A2 were stably associated with the genome, promoter-associated peaks of MacroH2A2 exhibited relatively rapid exchange dynamics in ES cells, particularly at highly-transcribed genes. Upon differentiation to embryonic fibroblasts, MacroH2A2 was gained primarily in additional long, stably associated blocks across gene-poor regions, while overall turnover at promoters was greatly dampened. Our results reveal unanticipated dynamic behavior of the MacroH2A2 variant in pluripotent cells, and provide a resource for future studies of tissue-specific histone dynamics in vivo.

Application of Recombination -Induced Tag Exchange (RITE) to study histone dynamics in human cells

“…H3.3 accumulates in non-dividing cells [26,27], which is consistent with both H3.3's role as a replacement histone and the finding that nucleosomes are disrupted and reassembled using newly synthesized histones even in the absence of DNA replication [28,29,30] Studies on genome-wide replication-independent histone dynamics in mammalian cells indicate that new H3.3 is rapidly incorporated at active genes, particularly at enhancers and promoters, and at regions of transcription termination [32][33][34]. Interestingly, despite the enrichment of H3.3 in transcribed regions, depletion of H3.3 or its chaperone HIRA leads to only mild gene expression changes in dividing cells [35,36].…”

Open Chromatin, Epigenetic Plasticity, and Nuclear Organization in Pluripotency

“…Intriguingly, however, subsequent genome-wide turnover studies using sequencing approaches at a single nucleosome resolution revealed a single hyperdynamic H3.3 nucleosome marking promoters of undifferentiated 2i-grown ESCs (Schlesinger et al, 2017). This resembles the behavior of the core histone variant MacroH2A2, which was also studied using a similar approach, in serum-grown ESCs: MacroH2A2 was stably associated with large genomic blocks, which further extended upon ESC differentiation, but in promoter regions, it displayed a high turnover rate in undifferentiated ESCs (Yildirim et al, 2014). These results suggest that the global hyperdynamic behavior observed for several structural components of chromatin is not a general feature of all chromatin proteins in pluripotent cells and that at least some variants display promoter-specific dynamic turnover in ESCs (Table 1).…”

Understanding nucleosome dynamics and their links to gene expression and DNA replication

“…Exchange is even more rapid when transcription is occurring in these regions. Similar turnover studies have been conducted in flies, mice and humans, including expanded studies to monitor turnover of histone variants 62–65 , as well as the turnover rates of PTMs 66 . Together, these studies have revealed that genomic regions with the highest rates of nucleosome (and histone) turnover are biochemically active.…”