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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. The cryo-EM structure of DNA-assembled histone pairs Hβ-Hα and Hδ-Hγ from Marseillevirus, a nucleocytoplasmic large DNA virus, reveals that these proteins form viral nucleosomes with highly conserved features when compared 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.

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. Cryo-electron microscopy and biochemical studies elucidate the read–write mechanisms of non-canonical PRC1-containing RYBP in histone H2A lysine 119 monoubiquitination and their roles in maintaining epigenetic inheritance.

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. Here, the authors show that anchoring of the PRC2 complex to its own sites requires nuclear condensation of Ccc1, a H3K27me3 reader in the yeast Cryptococcus neoformans , suggesting a functional role for condensates in ensuring heterochromatin fidelity.

The nucleosome, the basic unit of chromatin, organizes 147 bases of genomic DNA wrapped around an octameric assembly of histone proteins. Eukaryotes and prokaryotes encode proteins that contain a histone-fold; however, only eukaryotes are known to encode \"core histones\" from four distinct families that heterodimerize distinctively to form nucleosomes. Some Large DNA viruses have recently been shown to encode histones. Using cryo-EM, we show that fused histone pairs Hβ-Hα and Hδ-Hγ from Marseillevirus form nucleosomes in a similar manner to eukaryotic nucleosomes. I discuss the implications of our findings in light of other significant inquiries and discoveries within the chromatin field regarding the origin and evolution of histone proteins and their role in genome organization.Furthermore, we examine chromatin regulation, another crucial aspect of chromatin biology in eukaryotes. We investigated the mechanisms of the histone lysine methyltransferase SUV420H1. This enzyme di- and tri-methylates histone H4 lysine 20 (H4K20me2/me3), which plays crucial roles in DNA replication, repair, and heterochromatin formation and is dysregulated in several cancers. While its catalytic activity is linked to these processes, it also has non-catalytic activities that contribute to its overall function in the genome. I will present a comprehensive structural, biochemical, biophysical, and cellular characterization of catalytic and non-catalytic activities of SUV420H1 and discuss how they contribute to its chromatin. Together, our work gives insights into the evolution of genome organization and uncovers the catalytic and non-catalytic mechanisms of an essential chromatin regulator. We present new insights into how protein elements ensure genomic stability across various domains of life.

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

The intricate regulation of chromatin plays a key role in controlling genome architecture and accessibility. Histone lysine methyltransferases regulate chromatin by catalyzing the methylation of specific histone residues but are also hypothesized to have equally important non-catalytic roles. SUV420H1 di- and tri-methylates histone H4 lysine 20 (H4K20me2/me3) and plays crucial roles in DNA replication, repair, and heterochromatin formation, and 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 the enzyme likely has uncharacterized non-catalytic activities. To characterize the catalytic and non-catalytic mechanisms SUV420H1 uses to modify chromatin, we determined cryo- EM structures of SUV420H1 complexes with nucleosomes containing histone H2A or its variant H2A.Z. Our structural, biochemical, biophysical, and cellular analyses reveal how both SUV420H1 recognizes its substrate and H2A.Z stimulates its activity, and show that SUV420H1 binding to nucleosomes causes a dramatic detachment of nucleosomal DNA from histone octamer. We hypothesize that this detachment increases DNA accessibility to large macromolecular complexes, a prerequisite for DNA replication and repair. We also show that SUV420H1 can promote chromatin condensates, another non-catalytic role 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.The intricate regulation of chromatin plays a key role in controlling genome architecture and accessibility. Histone lysine methyltransferases regulate chromatin by catalyzing the methylation of specific histone residues but are also hypothesized to have equally important non-catalytic roles. SUV420H1 di- and tri-methylates histone H4 lysine 20 (H4K20me2/me3) and plays crucial roles in DNA replication, repair, and heterochromatin formation, and 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 the enzyme likely has uncharacterized non-catalytic activities. To characterize the catalytic and non-catalytic mechanisms SUV420H1 uses to modify chromatin, we determined cryo- EM structures of SUV420H1 complexes with nucleosomes containing histone H2A or its variant H2A.Z. Our structural, biochemical, biophysical, and cellular analyses reveal how both SUV420H1 recognizes its substrate and H2A.Z stimulates its activity, and show that SUV420H1 binding to nucleosomes causes a dramatic detachment of nucleosomal DNA from histone octamer. We hypothesize that this detachment increases DNA accessibility to large macromolecular complexes, a prerequisite for DNA replication and repair. We also show that SUV420H1 can promote chromatin condensates, another non-catalytic role 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.

The maintenance of gene expression patterns during metazoan development is achieved by the actions of Polycomb group (PcG) complexes. An essential modification marking silenced genes is monoubiquitination of histone H2A lysine 119 (H2AK119Ub) deposited by the E3 ubiquitin ligase activity of the non-canonical Polycomb Repressive Complex 1. The Polycomb Repressive Deubiquitinase (PR-DUB) complex cleaves monoubiquitin from histone H2A lysine 119 (H2AK119Ub) to restrict focal H2AK119Ub at Polycomb target sites and to protect active genes from aberrant silencing. BAP1 and ASXL1, subunits that form active PR-DUB, are among the most frequently mutated epigenetic factors in human cancers, underscoring their biological importance. How PR-DUB achieves specificity for H2AK119Ub to regulate Polycomb silencing is unknown, and the mechanisms of most of the mutations in BAP1 and ASXL1 found in cancer have not been established. Here we determine a cryo-EM structure of human BAP1 bound to the ASXL1 DEUBAD domain 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 remodeling 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 new insight into understanding cancer etiology. We reveal the molecular mechanism of nucleosomal H2AK119Ub deubiquitination by human BAP1/ASXL1.