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Epigenetic inheritance of silent chromatin domains is fundamental to cellular memory during embryogenesis, but it must overcome the dilution of repressive histone modifications during DNA replication 1 . One such modification, histone H2A lysine 119 monoubiquitination (H2AK119Ub), needs to be re-established by the Polycomb repressive complex 1 (PRC1) E3 ligase to restore the silent Polycomb domain 2,3 . However, the exact mechanism behind this restoration remains unknown. Here, combining cryo-electron microscopy (cryo-EM) and functional approaches, we characterize the read-write mechanism of the non-canonical PRC1-containing RYBP (ncPRC1 RYBP ). This mechanism, which functions as a positive-feedback loop in epigenetic regulation 4,5 , emphasizes the pivotal role of ncPRC1 RYBP in restoring H2AK119Ub. We observe an asymmetrical binding of ncPRC1 RYBP to H2AK119Ub nucleosomes, guided in part by the N-terminal zinc-finger domain of RYBP binding to residual H2AK119Ub on nascent chromatin. This recognition positions the RING domains of RING1B and BMI1 on the unmodified nucleosome side, enabling recruitment of the E2 enzyme to ubiquitinate H2AK119 within the same nucleosome (intra-nucleosome read-write) or across nucleosomes (inter-nucleosome read-write). Collectively, our findings provide key structural and mechanistic insights into the dynamic interplay of epigenetic regulation, highlighting the significance of ncPRC1 RYBP in H2AK119Ub restoration to sustain repressive chromatin domains., Competing Interests: Competing interests: The authors declare no competing interests., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

During tumor development, promoter CpG islands that are normally silenced by Polycomb repressive complexes (PRCs) become DNA-hypermethylated. The molecular mechanism by which de novo DNA methyltransferase(s) [DNMT(s)] catalyze CpG methylation at PRC-regulated regions remains unclear. Here, we report a cryo-electron microscopy structure of the DNMT3A long isoform (DNMT3A1) amino-terminal region in complex with a nucleosome carrying PRC1-mediated histone H2A lysine-119 monoubiquitination (H2AK119Ub). We identify regions within the DNMT3A1 amino terminus that bind H2AK119Ub and the nucleosome acidic patch. This bidentate interaction is required for effective DNMT3A1 engagement with H2AK119Ub-modified chromatin in cells. Further, aberrant redistribution of DNMT3A1 to Polycomb target genes recapitulates the cancer-associated DNA hypermethylation signature and inhibits their transcriptional activation during cell differentiation. This effect is rescued by disruption of the DNMT3A1-acidic patch interaction. Together, our analyses reveal a binding interface critical for mediating promoter CpG island DNA hypermethylation, a major molecular hallmark of cancer.

SUV420H1 di- and tri-methylates histone H4 lysine 20 (H4K20me2/H4K20me3) and plays crucial roles in DNA replication, repair, and heterochromatin formation. It is dysregulated in several cancers. Many of these processes were linked to its catalytic activity. However, deletion and inhibition of SUV420H1 have shown distinct phenotypes, suggesting that the enzyme likely has uncharacterized non-catalytic activities. Our cryoelectron microscopy (cryo-EM), biochemical, biophysical, and cellular analyses reveal how SUV420H1 recognizes its nucleosome substrates, and how histone variant H2A.Z stimulates its catalytic activity. SUV420H1 binding to nucleosomes causes a dramatic detachment of nucleosomal DNA from the histone octamer, which is a non-catalytic activity. We hypothesize that this regulates the accessibility of large macromolecular complexes to chromatin. We show that SUV420H1 can promote chromatin condensation, another non-catalytic activity that we speculate is needed for its heterochromatin functions. Together, our studies uncover and characterize the catalytic and non-catalytic mechanisms of SUV420H1, a key histone methyltransferase that plays an essential role in genomic stability., Competing Interests: Declaration of interests G.N. is a member of the Molecular Cell advisory board., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Histone H2A lysine 119 (H2AK119Ub) is monoubiquitinated by Polycomb repressive complex 1 and deubiquitinated by Polycomb repressive deubiquitinase complex (PR-DUB). PR-DUB cleaves H2AK119Ub to restrict focal H2AK119Ub at Polycomb target sites and to protect active genes from aberrant silencing. The PR-DUB subunits (BAP1 and ASXL1) are among the most frequently mutated epigenetic factors in human cancers. How PR-DUB establishes specificity for H2AK119Ub over other nucleosomal ubiquitination sites and how disease-associated mutations of the enzyme affect activity are unclear. Here, we determine a cryo-EM structure of human BAP1 and the ASXL1 DEUBAD in complex with a H2AK119Ub nucleosome. Our structural, biochemical, and cellular data reveal the molecular interactions of BAP1 and ASXL1 with histones and DNA that are critical for restructuring the nucleosome and thus establishing specificity for H2AK119Ub. These results further provide a molecular explanation for how >50 mutations in BAP1 and ASXL1 found in cancer can dysregulate H2AK119Ub deubiquitination, providing insight into understanding cancer etiology.

Little is understood about how the two major types of heterochromatin domains (HP1 and Polycomb) are kept separate. In the yeast Cryptococcus neoformans, the Polycomb-like protein Ccc1 prevents deposition of H3K27me3 at HP1 domains. Here we show that phase separation propensity underpins Ccc1 function. Mutations of the two basic clusters in the intrinsically disordered region or deletion of the coiled-coil dimerization domain alter phase separation behavior of Ccc1 in vitro and have commensurate effects on formation of Ccc1 condensates in vivo, which are enriched for PRC2. Notably, mutations that alter phase separation trigger ectopic H3K27me3 at HP1 domains. Supporting a direct condensate-driven mechanism for fidelity, Ccc1 droplets efficiently concentrate recombinant C. neoformans PRC2 in vitro whereas HP1 droplets do so only weakly. These studies establish a biochemical basis for chromatin regulation in which mesoscale biophysical properties play a key functional role., (© 2023. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Certain large DNA viruses, including those in the Marseilleviridae family, encode histones. Here we show that fused histone pairs Hβ-Hα and Hδ-Hγ from Marseillevirus are structurally analogous to the eukaryotic histone pairs H2B-H2A and H4-H3. These viral histones form 'forced' heterodimers, and a heterotetramer of four such heterodimers assembles DNA to form structures virtually identical to canonical eukaryotic nucleosomes.

Dormant spores of Bacillus species lack ATP and NADH and contain notable levels of only a few other common low mol wt energy reserves, including 3-phosphoglyceric acid (3PGA), and glutamic acid. Recently, Bacillus subtilis spores were reported to contain ~ 30 μmol of L-malate/g dry wt, which also could serve as an energy reserve. In present work, L-malate levels were determined in the core of dormant spores of B. subtilis, Bacillus cereus, Bacillus megaterium and Clostridium difficile, using both an enzymatic assay and 13C-NMR on extracts prepared by several different methods. These assays found that levels of L-malate in B. cereus and B. megaterium spores were ≤ 0.5 μmol/g dry wt, and ≤ 1 μmol/g dry wt in B. subtilis spores, and levels of L-lactate and pyruvate in B. megaterium and B. subtilis spores were < 0.5 μmol/g dry wt. Levels of L-malate in C. difficile spores were ≤ 1 μmol/g dry wt, while levels of 3PGA were ~ 7 μmol/g; the latter value was determined by 31P-NMR, and is in between the 3PGA levels in B. megaterium and B. subtilis spores determined previously. 13C-NMR analysis of spore extracts further showed that B. megaterium, B. subtilis and C. difficile contained significant levels of carbonate/bicarbonate in the spore core. Low mol wt carbon-containing small molecules present at > 3 μmol/g dry spores are: i) dipicolinic acid, carbonate/bicarbonate and 3PGA in B. megaterium, B. subtilis and C. difficile; ii) glutamate in B. megaterium and B. subtilis; iii) arginine in B. subtilis; and iv) at least one unidentified compound in all three species.