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Chromatin dynamics play a critical role in cell fate determination and maintenance by regulating the expression of genes essential for development and differentiation. In mouse embryonic stem cells (mESCs), maintenance of pluripotency coincides with a poised chromatin state containing active and repressive histone modifications. However, the structural features of poised chromatin are largely uncharacterized. By adopting mild time-course MNase-seq with computational analysis, the low-compact chromatin in mESCs is featured in two groups: one in more open regions, corresponding to an active state, and the other enriched with bivalent histone modifications, considered the poised state. A parameter called the chromatin opening potential index (COPI) is also devised to quantify the transcription potential based on the dynamic changes of MNase-seq signals at promoter regions. Use of COPI provides effective prediction of gene activation potential and, more importantly, reveals a few developmental factors essential for mouse neural progenitor cell (NPC) differentiation., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: The hierarchical organization of eukaryotic chromatin plays a central role in gene regulation, by controlling the extent to which the transcription machinery can access DNA. The histone variants H3.3 and H2A.Z have recently been identified as key regulatory players in this process, but the underlying molecular mechanisms by which they permit or restrict gene expression remain unclear. Here, we investigated the regulatory function of H3.3 and H2A.Z on chromatin dynamics and Polycomb-mediated gene silencing., Results: Our ChIP-seq analysis reveals that in mouse embryonic stem (mES) cells, H3K27me3 enrichment correlates strongly with H2A.Z. We further demonstrate that H2A.Z promotes PRC2 activity on H3K27 methylation through facilitating chromatin compaction both in vitro and in mES cells. In contrast, PRC2 activity is counteracted by H3.3 through impairing chromatin compaction. However, a subset of H3.3 may positively regulate PRC2-dependent H3K27 methylation via coordinating depositions of H2A.Z to developmental and signaling genes in mES cells. Using all-trans retinoic acid (tRA)-induced gene as a model, we show that the dynamic deposition of H2A.Z and H3.3 coordinately regulates the PRC2-dependent H3K27 methylation by modulating local chromatin structure at the promoter region during the process of turning genes off., Conclusions: Our study provides key insights into the mechanism of how histone variants H3.3 and H2A.Z function coordinately to finely tune the PRC2 enzymatic activity during gene silencing, through promoting or impairing chromosome compaction respectively.

Genomic DNA of eukaryotic cells is hierarchically packaged into chromatin by histones. The dynamic organization of chromatin fibers plays a critical role in the regulation of gene transcription and other DNA-associated biological processes. Recently, numerous approaches have been developed to map the chromatin organization by characterizing chromatin accessibilities in genome-wide. However, reliable methods to quantitatively map chromatin accessibility are not well-established, especially not on a genome-wide scale. Here, we developed a modified MNase-seq for mouse embryonic fibroblasts, wherein chromatin was partially digested at multiple digestion times using micrococcal nuclease (MNase), allowing quantitative analysis of local yet genome-wide chromatin compaction. Our results provide strong evidence that the chromatin accessibility at promoter regions are positively correlated with gene activity. In conclusion, our assay is an ideal tool for the quantitative study of gene regulation in the perspective of chromatin accessibility., (Copyright © 2017 Elsevier Inc. All rights reserved.)

The histone variants H3.3 and H2A.Z have recently emerged as two of the most important features in transcriptional regulation, the molecular mechanism of which still remains poorly understood. In this study, we investigated the regulation of H3.3 and H2A.Z on chromatin dynamics during transcriptional activation. Our in vitro biophysical and biochemical investigation showed that H2A.Z promoted chromatin compaction and repressed transcriptional activity. Surprisingly, with only four to five amino acid differences from the canonical H3, H3.3 greatly impaired higher-ordered chromatin folding and promoted gene activation, although it has no significant effect on the stability of mononucleosomes. We further demonstrated that H3.3 actively marks enhancers and determines the transcriptional potential of retinoid acid (RA)-regulated genes via creating an open chromatin signature that enables the binding of RAR/RXR. Additionally, the H3.3-dependent recruitment of H2A.Z on promoter regions resulted in compaction of chromatin to poise transcription, while RA induction results in the incorporation of H3.3 on promoter regions to activate transcription via counteracting H2A.Z-mediated chromatin compaction. Our results provide key insights into the mechanism of how histone variants H3.3 and H2A.Z function together to regulate gene transcription via the modulation of chromatin dynamics over the enhancer and promoter regions.

Nucleosome is the basic structural unit of the genome. During processes like DNA replication and gene transcription, the conformation of nucleosomes undergoes dynamic changes, including DNA unwrapping and rewrapping, as well as histone disassembly and assembly. However, the wrapping characteristics of nucleosomes across the entire genome, including region-specificity and their correlation with higher-order chromatin organization, remains to be studied. In this study, we investigate the wrapping length of DNA on nucleosomes across the whole genome using wrapping-seq. We discover that the chromatin of mouse ES cells forms Nucleosome Wrapping Domains (NRDs), which can also be observed in yeast and fly genomes. We find that the degree of nucleosome wrapping decreases after DNA replication and is promoted by transcription. Furthermore, we observe that nucleosome wrapping domains delineate Hi-C compartments and replication timing domains. In conclusion, we have unveiled a previously unrecognized domainization principle of the chromatin, encoded by nucleosome wrapping states. Nucleosome is the basic structural unit of the genome. Here, the authors find that the genome is segregated into domains with distinct nucleosome wrapping features, which coincident with Hi-C compartments and replication timing domains. [ABSTRACT FROM AUTHOR]

Histone lysine methyltransferase SUV420H1, which is responsible for site-specific di-/tri-methylation of histone H4 lysine 20 (H4K20), has crucial roles in DNA-templated processes, including DNA replication, DNA damage repair, and chromatin compaction. Its mutations frequently occur in human cancers. Nucleosomes containing the histone variant H2A.Z enhance the catalytic activities of SUV420H1 on H4K20 di-methylation deposition, regulating early replication origins. However, the molecular mechanism by which SUV420H1 specifically recognizes and deposits H4K20 methyl marks on nucleosomes remains poorly understood. Here we report the cryo-electron microscopy structures of SUV420H1 associated with H2A-containing nucleosome core particles (NCPs), and H2A.Z-containing NCPs. We find that SUV420H1 makes extensive site-specific contacts with histone and DNA regions. SUV420H1 C-terminal domain recognizes the H2A–H2B acidic patch of NCPs through its two arginine anchors, thus enabling H4K20 insertion for catalysis specifically. We also identify important residues increasing the catalytic activities of SUV420H1 bound to H2A.Z NCPs. In vitro and in vivo functional analyses reveal that multiple disease-associated mutations at the interfaces are essential for its catalytic activity and chromatin state regulation. Together, our study provides molecular insights into the nucleosome-based recognition and methylation mechanisms of SUV420H1, and a structural basis for understanding SUV420H1-related human disease. [ABSTRACT FROM AUTHOR]

DNA replication ensures the accurate transmission of genetic information during the cell cycle. Histone variant H2A.Z is crucial for early replication origins licensing and activation in which SUV420H1 preferentially recognizes H2A.Z-nucleosome and deposits H4 lysine 20 dimethylation (H4K20me2) on replication origins. Here, we report the cryo-EM structures of SUV420H1 bound to H2A.Z-nucleosome or H2A-nucleosome and demonstrate that SUV420H1 directly interacts with H4 N-terminal tail, the DNA, and the acidic patch in the nucleosome. The H4 (1–24) forms a lasso-shaped structure that stabilizes the SUV420H1-nucleosome complex and precisely projects the H4K20 residue into the SUV420H1 catalytic center. In vitro and in vivo analyses reveal a crucial role of the SUV420H1 KR loop (residues 214–223), which lies close to the H2A.Z-specific residues D97/S98, in H2A.Z-nucleosome preferential recognition. Together, our findings elucidate how SUV420H1 recognizes nucleosomes to ensure site-specific H4K20me2 modification and provide insights into how SUV420H1 preferentially recognizes H2A.Z nucleosome. [Display omitted] • Determine the cryo-EM structures of SUV420H1 bound to H2A.Z- and H2A-nucleosome • The H4 lasso structure precisely projects H4K20 into SUV420H1 catalytic center • SUV420H1 KR loop and H2A.Z D97/S98 are essential for H2A.Z-nucleosome preference • The KR-loop mutations diminish the preference for H2A.Z and impact DNA replication H2A.Z nucleosomes establish the H4K20me2 mark at replication origin through the recruitment of SUV420H1. Huang et al. determined the structure of SUV420H1-H2A.Z/H2A-nucleosome complex, elucidating how SUV420H1 recognizes nucleosomes to ensure site-specific H4K20me2 modification. This provides insights into SUV420H1's preferential recognition of the H2A.Z nucleosome. [ABSTRACT FROM AUTHOR]