Nicetto, D. | bioRxiv Science

bioRxiv2024

Distinct H3K9me3 heterochromatin maintenance dynamics govern different gene programs and repeats in pluripotent cells

H3K9me3-heterochromatin, established by lysine methyltransferases (KMTs) and compacted by HP1 isoforms, represses alternative lineage genes and DNA repeats. Our understanding of H3K9me3-heterochromatin stability is presently limited to individual domains and DNA repeats. We engineered Suv39h2 KO mouse embryonic stem cells to degrade remaining two H3K9me3- KMTs within one hour and found that both passive dilution and active removal contribute to H3K9me3 decay within 12-24 hours. We discovered four different H3K9me3 decay rates across the genome and chromatin features and transcription factor binding patterns that predict the stability classes. A "binary switch" governs heterochromatin compaction, with HP1 rapidly dissociating from heterochromatin upon KMTs depletion and a particular threshold level of HP1 limiting pioneer factor binding, chromatin opening, and exit from pluripotency within 12 hr. Unexpectedly, receding H3K9me3 domains unearth residual HP1{beta} peaks enriched with heterochromatin-inducing proteins. Our findings reveal distinct H3K9me3-heterochromatin maintenance dynamics governing gene networks and repeats that together safeguard pluripotency.

cell biology↗

bioRxiv2020

Histone H4K20 methylation synchronizes cytoskeletal dynamics with cell cycle phases during epidermal differentiation

Histone tails are subject to various post-translational modifications, which play a fundamental role in altering chromatin accessibility. Although they are thought to regulate progression through development, the impact of the most abundant histone modification in vertebrates, i.e., histone H4 lysine 20 dimethylation (H4K20m2), has remained largely elusive. H4K20m2 arises from sequential methylation of new, unmodified histone H4 proteins, incorporated into chromatin during DNA replication, by the mono-methylating enzyme PR-SET7/KMT5A during G2/M phases, followed by conversion to the dimethylated state by SUV4-20H1 enzymes in the following G1/G0 phase. To address its function, we have blocked the deposition of this mark by depleting Xenopus embryos of SUV4-20H1/H2 methyltransferases, which convert H4K20 monomethylated to di- and tri-methylated states, respectively In the frog larval epidermis this results in a severe loss of cilia in multiciliated cells (MCC), a key component of all mucociliary epithelia. MCC precursor cells are correctly specified and amplify centrioles, but ultimately fail in ciliogenesis due to perturbation of cytoplasmic processes. Genome wide transcriptome profiling reveals that SUV4-20H1/H2 depleted ectodermal Animal Cap explants preferentially down-regulate the expression of several hundred cytoskeleton and cilium related genes as a consequence of persistent H4K20 monomethyl marks on postmitotic chromatin. Further analysis demonstrated that knockdown of SUV4-20H1 alone is sufficient to generate the MCC phenotype and that overexpression of the H4K20m1-specific histone demethylase PHF8 rescues the ciliogenic defect in significant, although partial, manner. Taken together, this indicates that the conversion of H4K20m1 to H4K20m2 by SUV4-20H1 is critical to synchronize cytoskeletal dynamics in concert with the cell cycle.

developmental biology↗