Polycomb repressive complex 2 (PRC2)-mediated histone H3 lysine 27 (H3K27) methylation is vital for Polycomb gene silencing, a classic epigenetic phenomenon that maintains transcriptional silencing throughout cell divisions. We report that PRC2 activity is regulated by the density of its substrate nucleosome arrays. Neighboring nucleosomes activate the PRC2 complex with a fragment of their H3 histones (Ala(31) to Arg(42)). We also identified mutations on PRC2 subunit Su(z)12, which impair its binding and response to the activating peptide and its ability in establishing H3K27 trimethylation levels in vivo. In mouse embryonic stem cells, local chromatin compaction occurs before the formation of trimethylated H3K27 upon transcription cessation of the retinoic acid-regulated gene CYP26a1. We propose that PRC2 can sense the chromatin environment to exert its role in the maintenance of transcriptional states.

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Highlights d B cells are the dominant APCs that initiate CD4 + T cell response to VLPs d The few antigen-specific B cells in WT mice are sufficient to activate CD4 + T cells d Innate signaling in B cells is required for the VLP-induced CD4 + T cell polarization d B cell-mediated antigen-presentation can break CD4 + T cell tolerance

H3K27ac is well recognized as a marker for active enhancers and a great indicator of enhancer activity. However, its functional impact on transcription has not been characterized. By substituting lysine 27 in histone variant H3.3 with arginine in mouse embryonic stem cells, we diminish the vast majority of H3K27ac at enhancers. However, the transcriptome is largely undisturbed in these mutant cells, likely because the other enhancer features remain largely unchanged, including chromatin accessibility, H3K4me1, and histone acetylation at other lysine residues. Our results clearly reveal that H3K27ac alone is not capable of functionally determining enhancer activity.

PIWI-interacting RNAs (piRNAs) are a class of small RNAs abundantly expressed in animal gonads. piRNAs that map to retrotransposons are generated by a ''ping-pong'' amplification loop to suppress the activity of retrotransposons. However, the biogenesis and function of other categories of piRNAs have yet to be investigated. In this study, we first profiled the expression of small RNAs in type A spermatogonia, pachytene spermatocytes, and round spermatids by deep sequencing. We then focused on the computational analysis of the potential piRNAs generated in the present study as well as other published sets. piRNAs mapping to retrotransposons, mRNAs, and intergenic regions had different length distributions and were differentially regulated in spermatogenesis. piRNA-generating mRNAs (PRMRs), whose expression positively correlated with their piRNA products, constituted one-third of the protein-coding genes and were evolutionarily conserved and enriched with splicing isoforms and antisense transcripts. PRMRs with piRNAs preferentially mapped to CDSs and 39 UTRs partitioned into three clusters differentially expressed during spermatogenesis and enriched with unique sets of functional annotation terms related to housekeeping activities as well as spermatogenesis-specific processes. Intergenic piRNAs were divided into 2992 clusters probably representing novel transcriptional units that have not been reported. The transcripts of a large number of genes involved in spermatogenesis are the precursors of piRNAs, and these genes are intricately regulated by alternative splicing and antisense transcripts. piRNAs, whose regulatory role in gene expression awaits to be identified, are clearly products of a novel regulatory process that needs to be defined.

H2A.Z is a highly conserved histone variant in all species. The chromatin deposition of H2A.Z is specifically catalyzed by the yeast chromatin remodeling complex SWR1 and its mammalian counterpart SRCAP. However, the mechanism by which H2A.Z is preferentially recognized by non-histone proteins remains elusive. Here we identified Anp32e, a novel higher eukaryote-specific histone chaperone for H2A.Z. Anp32e preferentially associates with H2A.Z-H2B dimers rather than H2A-H2B dimers in vitro and in vivo and dissociates non-nucleosomal aggregates formed by DNA and H2A-H2B. We determined the crystal structure of the Anp32e chaperone domain (186-232) in complex with the H2A.Z-H2B dimer. In this structure, the region containing Anp32e residues 214-224, which is absent in other Anp32 family proteins, specifically interacts with the extended H2A.Z αC helix, which exhibits an unexpected conformational change. Genome-wide profiling of Anp32e revealed a remarkable co-occupancy between Anp32e and H2A.Z. Cells overexpressing Anp32e displayed a strong global H2A.Z loss at the +1 nucleosomes, whereas cells depleted of Anp32e displayed a moderate global H2A.Z increase at the +1 nucleosomes. This suggests that Anp32e may help to resolve the non-nucleosomal H2A.Z aggregates and also facilitate the removal of H2A.Z at the +1 nucleosomes, and the latter may help RNA polymerase II to pass the first nucleosomal barrier.

TET family enzymes successively oxidize 5-methylcytosine to 5-hydroxymethylcytosine, 5-formylcytosine, and 5-carboxylcytosine, leading to eventual demethylation. 5hmC and TET enzymes occupy distinct chromatin regions, suggesting unknown mechanisms controlling the fate of 5hmC within diverse chromatin environments. Here, we report that SALL4A preferentially associates with 5hmC in vitro and occupies enhancers in mouse embryonic stem cells in a largely TET1-dependent manner. Although most 5hmC at SALL4A peaks undergoes further oxidation, this process is abrogated upon deletion of Sall4 gene, with a concomitant reduction of TET2 at these regions. Thus, SALL4A facilitates further oxidation of 5hmC at its binding sites, which requires its 5hmC-binding activity and TET2, supporting a collaborative action between SALL4A and TET proteins in regulating stepwise oxidation of 5mC at enhancers. Our study identifies SALL4A as a 5hmC binder, which facilitates 5hmC oxidation by stabilizing TET2 association, thereby fine-tuning expression profiles of developmental genes in mouse embryonic stem cells.

Mitotic inheritance of the DNA methylome is a challenging task for the maintenance of cell identity. Whether DNA methylation pattern in different genomic contexts can all be faithfully maintained is an open question. A replication-coupled DNA methylation maintenance model was proposed decades ago, but some observations suggest that a replication-uncoupled maintenance mechanism exists. However, the capacity and the underlying molecular events of replication-uncoupled maintenance are unclear. By measuring maintenance kinetics at the single-molecule level and assessing mutant cells with perturbation of various mechanisms, we found that the kinetics of replication-coupled maintenance are governed by the UHRF1-Ligase 1 and PCNA-DNMT1 interactions, whereas nucleosome occupancy and the interaction between UHRF1 and methylated H3K9 specifically regulate replication-uncoupled maintenance. Surprisingly, replication-uncoupled maintenance is sufficiently robust to largely restore the methylome when replication-coupled maintenance is severely impaired. However, solo-WCGW sites and other CpG sites displaying aging-and cancer-associated hypomethylation exhibit low maintenance efficiency, suggesting that although quite robust, mitotic inheritance of methylation is imperfect and that this imperfection may contribute to selective hypomethylation during aging and tumorigenesis.