Genomic and Proteomic Resolution of Heterochromatin and Its Restriction of Alternate Fate Genes

Abstract

SUMMARY Heterochromatin is integral to cell identity maintenance by impeding the activation of genes for alternate cell fates. Heterochromatic regions are associated with histone 3 lysine 9 trimethylation (H3K9me3) or H3K27me3, but these modifications are also found in euchromatic regions that permit transcription. We discovered that resistance to sonication is a reliable indicator of the heterochromatin state, and we developed a biophysical method (Gradient-seq) to discriminate subtypes of H3K9me3 and H3K27me3 domains in sonication-resistant heterochromatin (srHC) versus euchromatin. These classifications are more accurate than the histone marks alone in predicting transcriptional silence and resistance of alternate-fate genes to activation during direct cell conversion. Our proteomics of H3K9me3-marked srHC and functional screens revealed diverse proteins, including RBMX and RBMXL1, that impede gene induction during cellular reprogramming. Isolation of srHC with Gradient-seq provides a genome-wide map of chromatin structure, elucidating subtypes of repressed domains that are uniquely predictive of diverse other chromatin properties.

Expression and phase separation potential of heterochromatin proteins during early mouse development

“…Most of them focused on the identification of proteins that bind H3K9me3 using peptides or modified nucleosomes pulldowns or chromatin immunoprecipitation . More recently, heterochromatin proteins have been identified by mass spectrometry of the sonication‐resistant fraction of the chromatin . Functionally, however, much of our knowledge on heterochromatin stems from genetic screens in model organisms including Schizosaccharomyces pombe , C. elegans and Drosophila melanogaster .…”

“…In an effort to identify the most relevant protein components of constitutive heterochromatin—and thereby potential proteins that may promote heterochromatin phase separation—we undertook a bioinformatic analysis, initially based on 7 mass spectrometry studies performed in mammalian cells . We focused primarily on H3K9me3 as a proxy for constitutive heterochromatin, since it is its most prevalent mark across most, albeit not all, eukaryotes.…”

“…Unless otherwise stated, all analyses were performed in R studio (version 1.2.1335) with the R version (R version 3.5.2 (2018‐12‐20)). The bioinformatic analysis was based on 7 mass spectrometry studies performed in mammalian cells . Proteins predicted to be heterochromatic were selected based on their ability to bind H3K9me3, H3K9me3‐modified nucleosomes with and without DNA methylation, or to their enrichment in the sonication resistance fragment of the chromatin.…”

Heterochromatin protein 1γ deficiency decreases histone H3K27 methylation in mouse neurosphere neuronal genes

“…On the other hand, H3K27me3 mainly functions in facultative heterochromatin formation, repressing gene expression temporally, especially during the developmental process . Recently, the co‐occupation of certain euchromatic regions by H3K9me2/3 and H3K27me3 was reported in vitro and in vivo to silence gene expression in those regions . Both H3K9me2/3 and H3K27me3 are closely related to neural development and maintenance of neural cells.…”

“…[4][5][6] Recently, the co-occupation of certain euchromatic regions by H3K9me2/3 and H3K27me3 was reported in vitro and in vivo to silence gene expression in those regions. [7][8][9][10] Both H3K9me2/3 and H3K27me3 are closely related to neural development and maintenance of neural cells. For example, reduction of H3K9me3 amounts by mutation of Eset, a gene encoding one of the H3K9 methyltransferases that is highly expressed in mouse developing brains and helps maintenance of the global and localized H3K9me3 amounts, results in lethal damage to cells due to overexpression of endogenous retroviral-like sequences known as intracisternal A-particle (IAP).…”