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Bivalent genes are ready for activation upon the arrival of developmental cues. Here, we report that BEND3 is a CpG island (CGI)–binding protein that is enriched at regulatory elements. The cocrystal structure of BEND3 in complex with its target DNA reveals the structural basis for its DNA methylation–sensitive binding property. Mouse embryos ablated of Bend3 died at the pregastrulation stage. Bend3 null embryonic stem cells (ESCs) exhibited severe defects in differentiation, during which hundreds of CGI-containing bivalent genes were prematurely activated. BEND3 is required for the stable association of polycomb repressive complex 2 (PRC2) at bivalent genes that are highly occupied by BEND3, which suggests a reining function of BEND3 in maintaining high levels of H3K27me3 at these bivalent genes in ESCs to prevent their premature activation in the forthcoming developmental stage.

A pot study was conducted to elucidate morphological and physiological responses of Spatholobus suberectus Dunn accessions to four nitrogen application rates (0, 10, 20, and 30 mmol L-1) and two watering regimes (daily irrigation and irrigation every 10 d for a month). The results showed that water deficit led to marked decreases in leaf water relations and photosynthetic parameters regardless of the N treatments. Under drought conditions, the reductions in net photosynthesis occurred mainly due to stomatal factors. Although the leaf N content and stem mass ratio were positively correlated with N application, other morphological and physiological characteristics were negatively correlated or nonsignificantly correlated with N application under limited water supply. As the root nodules of S. suberectus can provide sufficient N supply, and high N applications made S. suberectus plants more vulnerable to drought stress, we suggest that no N supplement is required for S. suberectus seedlings.

Stella is a maternal gene required for oogenesis and early embryogenesis. Stella overexpression in somatic cells causes global demethylation. As we have recently shown, Stella sequesters nuclear ubiquitin-like with PHD and RING finger domains 1 (UHRF1), a RING finger–type E3 ubiquitin ligase essential for DNA methylation mediated by DNA methyltransferase 1 and triggers global demethylation. Here, we report an overexpressed mutant Stella protein without nuclear export activity surprisingly retained its ability to cause global demethylation. By combining biochemical interaction assays, isothermal titration calorimetry, immunostaining, and live-cell imaging with fluorescence recovery after photobleaching, we found that Stella disrupts UHRF1's association with chromatin by directly binding to the plant homeodomain of UHRF1 and competing for the interaction between UHRF1 and the histone H3 tail. Consistently, overexpression of Stella mutants that do not directly interact with UHRF1 fails to cause genome-wide demethylation. In the presence of nuclear Stella, UHRF1 could not bind to chromatin and exhibited increased dynamics in the nucleus. Our results indicate that Stella employs a multilayered mechanism to achieve robust UHRF1 inhibition, which involves the dissociation from chromatin and cytoplasmic sequestration of UHRF1.

IL-32 is a cytokine involved in proinflammatory immune responses to bacterial and viral infections. However, the role of epigenetic events in the regulation of IL-32 gene expression is understudied. Here we show that IL-32 is repressed by DNA methylation in HEK293 cells. Using ChIP sequencing, locus-specific methylation analysis, CRISPR/Cas9-mediated genome editing, and RT-qPCR (quantitative RT-PCR) and immunoblot assays, we found that short-term treatment (a few hours) with the proinflammatory cytokine tumor necrosis factor α (TNFα) activates IL-32 in a DNA demethylation–independent manner. In contrast, prolonged TNFα treatment (several days) induced DNA demethylation at the promoter and a CpG island in the IL-32 gene in a TET (ten-eleven translocation) family enzyme– and NF-κB–dependent manner. Notably, the hypomethylation status of transcriptional regulatory elements in IL-32 was maintained for a long time (several weeks), causing elevated IL-32 expression even in the absence of TNFα. Considering that IL-32 can, in turn, induce TNFα expression, we speculate that such feedforward events may contribute to the transition from an acute inflammatory response to chronic inflammation.

Summary The multisubunit chromatin remodeler SWR1/SRCAP/p400 replaces the nucleosomal H2A-H2B dimer with the free-form H2A.Z-H2B dimer, but the mechanism governing the unidirectional H2A-to-H2A.Z exchange remains elusive. Here, we perform single-molecule force spectroscopy to dissect the disassembly/reassembly processes of the H2A nucleosome and H2A.Z nucleosome. We find that the N-terminal 1–135 residues of yeast SWR1 complex protein 2 (previously termed Swc2-Z) facilitate the disassembly of nucleosomes containing H2A but not H2A.Z. The Swc2-mediated nucleosome disassembly/reassembly requires the inherently unstable H2A nucleosome, whose instability is conferred by three H2A α2-helical residues, Gly47, Pro49, and Ile63, as they selectively weaken the structural rigidity of the H2A-H2B dimer. It also requires Swc2-ZN (residues 1–37) that directly anchors to the H2A nucleosome and functions in the SWR1-catalyzed H2A.Z replacement in vitro and yeast H2A.Z deposition in vivo. Our findings provide mechanistic insights into how the SWR1 complex discriminates between the H2A nucleosome and H2A.Z nucleosome, establishing a simple paradigm for the governance of unidirectional H2A.Z exchange.

Transcriptional memory allows certain genes to respond to previously experienced signals more robustly. However, whether and how the key proinflammatory cytokine TNF-α mediates transcriptional memory are poorly understood. Using HEK293F cells as a model system, we report that sustained TNF-α stimulation induces transcriptional memory dependent on TET enzymes. The hypomethylated status of transcriptional regulatory regions can be inherited, facilitating NF-κB binding and more robust subsequent activation. A high initial methylation level and CpG density around κB sites are correlated with the functional potential of transcriptional memory modules. Interestingly, the CALCB gene, encoding the proven migraine therapeutic target CGRP, exhibits the best transcriptional memory. A neighboring primate-specific endogenous retrovirus stimulates more rapid, more strong, and at least 100-fold more sensitive CALCB induction in subsequent TNF-α stimulation. Our study reveals that TNF-α-mediated transcriptional memory is governed by active DNA demethylation and greatly sensitizes memory genes to much lower doses of inflammatory cues., eLife digest Genes are the instruction manuals of life and contain the information needed to build the building blocks that keep cells alive. To read these instructions, cells use specific signals that activate genes. The process, known as gene expression, is tightly controlled and for the most part, fairly stable. But gene expression can be modified in various ways. Epigenetics is a broad term for describing reversible changes made to genes to switch them on and off. Sometimes, certain genes even develop a kind of ‘transcriptional memory’ where over time, their expression is enhanced and speeds up with repeated activation signals. But this may also have harmful effects. For example, the signalling molecule called tumour necrosis factor α (TNF-α) is an essential part of the immune system. But it is also implicated in chronic inflammatory diseases, such as rheumatoid arthritis. In these conditions, cell signalling pathways triggering inflammation are overactive. One possibility is that TNF-α could be inducing the transcriptional memory of certain genes, amplifying their expression. But little is known about which fraction of genes exhibits transcriptional memory, and what differentiates memory genes from genes with stable expression. Here, Zhao et al. treated cells grown in the laboratory with TNF-α to investigate its role in transcriptional memory and find out what epigenetic features might govern the process. The experiments showed that mimicking a sustained inflammation by stimulating TNF-α, triggered a transcriptional memory in some genes, and enabled them to respond to much lower levels of TNF-α on subsequent exposure. Zhao et al. also discovered that genes tagged with methyl groups are more likely to show transcriptional memory when stimulated by TNF-α. However, they also found that these groups must be removed to consolidate any transcriptional memory. This work shows how TNF-α influences can alter the expression of certain genes. It also suggests that transcriptional memory, stimulated by TNF-α, may be a possible mechanism underlying chronic inflammatory conditions. This could help future research in identifying more genes with transcriptional memory.

The stable maintenance of DNA methylation patterns during mitotic cell division is crucial for cell identity. Precisely determining the maintenance kinetics and dissecting the exact contributions of relevant regulators requires a method to accurately measure parent and daughter strand DNA methylation at the same time, ideally at the single-molecule level. Recently, we developed a method referred to as Hammer-seq (hairpin-assisted mapping of methylation of replicated DNA) that fulfils the above criteria. This method integrates 5-ethynyl-2'-deoxyuridine (EdU) labeling of replicating DNA, biotin conjugation and streptavidin-based affinity purification, and whole-genome hairpin bisulfite sequencing technologies. Hammer-seq offers the unique advantage of simultaneously measuring the methylation status of parent and daughter strands within a single DNA molecule, which makes it possible to determine maintenance kinetics across various genomic regions without averaging effects from bulk measurements and to assess de novo methylation events that accompany methylation maintenance. Importantly, when combined with mutant cell lines in which mechanisms of interest are disrupted, Hammer-seq can be applied to determine the functional contributions of potential regulators to methylation maintenance, with accurate kinetics information that cannot be acquired with other currently available methods. Hammer-seq library preparation requires ~100 ug EdU-labeled genomic DNA as input (~15 million mammalian cells). The whole protocol, from pulse labeling to library construction, can be completed within 2-3 d, depending on the chasing time.

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.

Table S1. Sequencing depth and mapping efficiency of ChIP-seq and ATAC-seq experiments. Table S2. Peak numbers called in ATAC-seq and ChIP-seq experiments. Table S3. Stats for mRNA-seq experiments. Table S4 Sequences of DNA oligos used in this study. (PDF 459 kb)

Figure S1. Strategy for CRISPR-Cas9-mediated gene editing at H3f3a and H3f3b loci. Figure S2. H3.3K27R mutation does not affect the H3.3 protein level and pluripotent state of ESCs. Figure S3. Genome-wide decrease in H3K27ac levels in individual H3.3K27R mutant mouse ESC lines. Figure S4. Averaged profiles of H3.3 at active enhancers and H3K27-occupied promoters in mouse ESCs. Figure S5. H3.3K27R mutation does not affect the genome-wide distribution of H3K27me3 in mouse ESCs. Figure S6. Heatmap clustering of differentially expressed genes in WT and H3.3K27R mutant mouse ESC lines. Figure S7. H3.3K27R mutation does not affect the transcription of genes associated with super enhancers in mouse ESCs. Figure S8. H3K4me1 distribution at enhancers and H3K27ac-occupied promoters in WT and H3.3K27R mutant ESC lines. Figure S9. H3K4me3 distribution at H3K27ac-occupied promoters in WT and H3.3K27R mutant ESC lines. Figure S10. Averaged profiles of H3K9ac, H3K18ac, H3K122ac, H4K5ac, and H4K8ac at enhancers and H3K27ac-occupied promoters in WT and H3.3K27R mutant ESC lines. Figure S11. Barplots show Pearson correlation coefficients between ChIP-seq signal of histone acetylation and chromatin accessibility (ATAC-seq signal). (PDF 2893 kb)

Methods and supplementary references. (PDF 121 kb)

Review history.

Ash1 is a Trithorax group protein that possesses H3K36-specific histone methyltransferase activity, which antagonizes Polycomb silencing. Here we report the identification of two Ash1 complex subunits, Mrg15 and Nurf55. In vitro, Mrg15 stimulates the enzymatic activity of Ash1. In vivo, Mrg15 is recruited by Ash1 to their common targets, and Mrg15 reinforces Ash1 chromatin association and facilitates the proper deposition of H3K36me2. To dissect the functional role of Mrg15 in the context of the Ash1 complex, we identify an Ash1 point mutation (Ash1-R1288A) that displays a greatly attenuated interaction with Mrg15. Knock-in flies bearing this mutation display multiple homeotic transformation phenotypes, and these phenotypes are partially rescued by overexpressing the Mrg15-Nurf55 fusion protein, which stabilizes the association of Mrg15 with Ash1. In summary, Mrg15 is a subunit of the Ash1 complex, a stimulator of Ash1 enzymatic activity and a critical regulator of the TrxG protein function of Ash1 in Drosophila., Ash1 is an H3K36 methyltransferase, Trithorax group protein and is critical in antagonizing Polycomb silencing. Here, the authors purify the Drosophila Ash1 complex and identify Mrg15 and Nurf55 as subunits, finding that Mrg15 is recruited by Ash1 and reinforces Ash1 activity.

We carried out a field experiment in order to study effects of fertilization in juvenile plants of three coffee (Coffea arabica) cultivars in Yunnan, SW China. Fertilization treatments included a control without fertilizer (CK), combinations of three NPK fertilization rates [high fertilization (FH), medium fertilization (FM), and low fertilization (FL) with 135, 90, and 45 g per plant per year, respectively], and at two N:P2O5:K2O ratios (R1, 1:0.5:0.8; R2, 1:0.8:0.5). The growth in juvenile plants was not altered by fertilization, with two clear growth peaks being observed in both the height and stem growth rates (RGRs) throughout a year. Both FM and FH resulted in significantly higher RGRs in both height and stem diameter compared to FL and CK in all three cultivars. At the same fertilization rate, the leaf area, branch number, longest branch length, internode number, and biomass of R2 were higher than those of R1, and P significantly affected the root biomass and root to shoot ratio. Compared to the FL treatment, both FM and FH treatments resulted in higher net photosynthetic rates and stomatal conductance across seasons, and in higher intrinsic water-use efficiency during the dry season and at the middle of the wet season. Photosynthetic nitrogen-use efficiency at R2 was higher than that at R1, but no significant differences were observed between the different fertilization rates. Among the three coffee cultivars, Caturra exhibited the highest height, stem diameter, longest branch length, and internode number. Our results indicated that the optimal N:P2O5:K2O ratio was 1:0.8:0.5 for the juvenile growth of coffee plants. Both FM and FH could help optimize the growth and photosynthetic rate of coffee plants, but FM is suitable for the ecological friendly agriculture and economic sustainability at coffee plantations.

Histone variant H2A.Z, a universal mark of dynamic nucleosomes flanking gene promoters and enhancers, is incorporated into chromatin by SRCAP (SWR1), an ATP-dependent, multicomponent chromatin-remodeling complex. The YL1 (Swc2) subunit of SRCAP (SWR1) plays an essential role in H2A.Z recognition, but how it achieves this has been unclear. Here, we report the crystal structure of the H2A.Z-binding domain of Drosophila melanogaster YL1 (dYL1-Z) in complex with an H2A.Z-H2B dimer at 1.9-Å resolution. The dYL1-Z domain adopts a new whip-like structure that wraps over H2A.Z-H2B, and preferential recognition is largely conferred by three residues in loop 2, the hyperacidic patch and the extended αC helix of H2A.Z. Importantly, this domain is essential for deposition of budding yeast H2A.Z in vivo and SRCAP (SWR1)-catalyzed histone H2A.Z replacement in vitro. Our studies distinguish YL1-Z from known H2A.Z chaperones and suggest a hierarchical mechanism based on increasing binding affinity facilitating H2A.Z transfer from SRCAP (SWR1) to the nucleosome.

For decades, researchers have made a tremendous effort to characterize the chromosome organization in the nucleus. With the development of chromosome conformation capture (1) and related techniques, researchers have begun to gain insight at the molecular level into how inter- and intrachromosome contacts are formed. Quantitative measurement of the contact frequencies in metazoans suggested the existence of topologically associating domains (TADs) as the building blocks of chromosome organization (1⇓–3). These domains are often demarcated by distinct patterns of histone modifications. For example, compartments that are enriched with H3K27me3 and have depleted H3K36me3 are signatures of facultative heterochromatin in the human genome. Although the features of chromosome organization have been characterized in several organisms, the principles and mechanisms governing the formation of chromosome organization have remained a mystery. In PNAS, Klocko et al. (4) report that the Polycomb Repressive Complex 2 (PRC2) and H3K27me3 have a role in shaping genome organization in Neurospora crassa . In mammalian and Drosophila cells, chromosomes are segregated into large TADs, and boundaries between adjacent TADs are characterized by enriched insulator protein CTCF occupancy (2, 3). Plants, yeast, and N. crassa lack large local interactive domains in their genomes, which is likely a result of the loss of genes encoding the CTCF protein (5, 6). The most obvious chromosomal contacts in plants and N. crassa genomes are formed between heterochromatin regions. However, H3K9me3 and the constitutive heterochromatin machinery seemed to be dispensable for this contact (5, 6). Fission yeast does not have H3K27 methylation, and its sole H3K9 methyltransferase is needed for proper genome organization (7). This result raises the question of whether H3K27 methylation and facultative heterochromatin contribute to genome structure formation. Klocko et al. (4) first profile chromosome organization with a high-throughput chromosome conformation capture (Hi-C) technique in … [↵][1]1To whom correspondence should be addressed. Email: zhubing{at}ibp.ac.cn. [1]: #xref-corresp-1-1

Epigenetic silencing can be mediated by various mechanisms, and many regulators remain to be identified. Here, we report a genome-wide siRNA screening to identify regulators essential for maintaining gene repression of a CMV promoter silenced by DNA methylation. We identified CSE1L (chromosome segregation 1 like) as an essential factor for the silencing of the reporter gene and many endogenous methylated genes. CSE1L depletion did not cause DNA demethylation. On the other hand, the methylated genes derepressed by CSE1L depletion largely overlapped with methylated genes that were also reactivated by treatment with histone deacetylase inhibitors (HDACi). Gene silencing defects observed upon CSE1L depletion were linked to its nuclear import function for certain protein cargos because depletion of other factors involved in the same nuclear import pathway, including KPNAs and KPNB1 proteins, displayed similar derepression profiles at the genome-wide level. Therefore, CSE1L appears to be critical for the nuclear import of certain key repressive proteins. Indeed, NOVA1, HDAC1, HDAC2, and HDAC8, genes known as silencing factors, became delocalized into cytosol upon CSE1L depletion. This study suggests that the cargo specificity of the protein nuclear import system may impact the selectivity of gene silencing.

Regulation of gene expression by epigenetic modifications such as DNA methylation is crucial for developmental and disease processes, including cell differentiation and cancer development. Genes repressed by DNA methylation can be derepressed by various compounds that target DNA methyltransferases, histone deacetylases, and other regulatory factors. However, some additional, unknown mechanisms that promote DNA methylation-mediated gene silencing may exist. Chemical agents that can counteract the effects of epigenetic repression that is not regulated by DNA methyltransferases or histone deacetylases therefore may be of research interest. Here, we report the results of a high-throughput screen using a 308,251-member chemical library to identify potent small molecules that derepress an EGFP reporter gene silenced by DNA methylation. Seven hit compounds were identified that did not directly target bulk DNA methylation or histone acetylation. Analyzing the effect of these compounds on endogenous gene expression, we discovered that three of these compounds (compounds LX-3, LX-4, and LX-5) selectively activate the p38 mitogen-activated protein kinase (MAPK) pathway and derepress a subset of endogenous genes repressed by DNA methylation. Selective agonists of the p38 pathway have been lacking, and our study now provides critical compounds for studying this pathway and p38 MAPK-targeted genes repressed by DNA methylation.

B cells can present antigens to CD4

Among all tissues of the metazoa, the transcritpome of testis displays the highest diversity and specificity. However, its composition and dynamics during spermatogenesis have not been fully understood. Here, we have identified 20,639 message RNAs (mRNAs), 7,168 long non-coding RNAs (lncRNAs) and 15,101 circular RNAs (circRNAs) in mouse spermatogenic cells, and found many of them were specifically expressed in testes. lncRNAs are significantly more testis-specific than mRNAs. At all stages, mRNAs are generally more abundant than lncRNAs, and linear transcripts are more abundant than circRNAs. We showed that the productions of circRNAs and piRNAs were highly regulated instead of random processes. Based on the results of a small-scale functional screening experiment using cultured mouse spermatogonial stem cells, many evolutionarily conserved lncRNAs are likely to play roles in spermatogenesis. Typical classes of transcription factor binding sites are enriched in the promoters of testis-specific m/lncRNA genes. Target genes of CREM and RFX2, 2 key TFs for spermatogenesis, were further validated by using ChIP-chip assays and RNA-seq on RFX2-knockout spermatogenic cells. Our results contribute to the current understanding of the transcriptomic complexity of spermatogenic cells and provide a valuable resource from which many candidate genes may be selected for further functional studies.

Postnatal growth of mammalian oocytes is accompanied by a progressive gain of DNA methylation, which is predominantly mediated by DNMT3A, a de novo DNA methyltransferase1,2. Unlike the genome of sperm and most somatic cells, the oocyte genome is hypomethylated in transcriptionally inert regions2-4. However, how such a unique feature of the oocyte methylome is determined and its contribution to the developmental competence of the early embryo remains largely unknown. Here we demonstrate the importance of Stella, a factor essential for female fertility5-7, in shaping the oocyte methylome in mice. Oocytes that lack Stella acquire excessive DNA methylation at the genome-wide level, including in the promoters of inactive genes. Such aberrant hypermethylation is partially inherited by two-cell-stage embryos and impairs zygotic genome activation. Mechanistically, the loss of Stella leads to ectopic nuclear accumulation of the DNA methylation regulator UHRF18,9, which results in the mislocalization of maintenance DNA methyltransferase DNMT1 in the nucleus. Genetic analysis confirmed the primary role of UHRF1 and DNMT1 in generating the aberrant DNA methylome in Stella-deficient oocytes. Stella therefore safeguards the unique oocyte epigenome by preventing aberrant de novo DNA methylation mediated by DNMT1 and UHRF1.

The regulatory factor X (RFX) family of transcription factors is crucial for ciliogenesis throughout evolution. In mice, Rfx1-4 are highly expressed in the testis where flagellated sperm are produced, but the functions of these factors in spermatogenesis remain unknown. Here, we report the production and characterization of the Rfx2 knockout mice. The male knockout mice were sterile due to the arrest of spermatogenesis at an early round spermatid step. The Rfx2-null round spermatids detached from the seminiferous tubules, forming large multinucleated giant cells that underwent apoptosis. In the mutants, formation of the flagellum was inhibited at its earliest stage. RNA-seq analysis identified a large number of cilia-related genes and testis-specific genes that were regulated by RFX2. Many of these genes were direct targets of RFX2, as revealed by chromatin immunoprecipitation-PCR assays. These findings indicate that RFX2 is a key regulator of the post-meiotic development of mouse spermatogenic cells.

Epigenetic systems are well known for the roles they play in regulating the differential expression of the same genome in different cell types. However, epigenetic systems can also directly impact genomic integrity by protecting genetic sequences. Using an experimental evolutionary approach, we studied rates of mutation in the fission yeast Schizosaccharomyces pombe strains that lacked genes encoding several epigenetic regulators or mismatch repair components. We report that loss of a functional mismatch repair pathway in S. pombe resulted in the preferential enrichment of mutations in euchromatin, indicating that the mismatch repair machinery preferentially protected genetic fidelity in euchromatin. This preference is probably determined by differences in the accessibility of chromatin at distinct chromatin regions, which is supported by our observations that chromatin accessibility positively correlated with mutation rates in S. pombe or human cancer samples with deficiencies in mismatch repair. Importantly, such positive correlation was not observed in S. pombe strains or human cancer samples with functional mismatch repair machinery.

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.

Maintaining Repression The Polycomb Repressive Complex 2 (PRC2) plays a critical role in gene silencing in metazoans, methylating histone H3 on lysine 27 (H3K27) to generate a repressive chromatin mark. The catalytic subunit E(z)/Ezh2 requires the presence of two other subunits—ESC/EED and Su(z)12—for enzyme activity. Yuan et al. (p. 971 ; see the Perspective by Pirrotta ) show that both a fragment of the histone H3 N-terminal tail, and histone H1 stimulated PRC2 enzyme activity on poor, low-density chromatin substrates, indicating that that PRC2 is regulated by the density and compaction states of chromatin. The histone H3 fragment binds to the Su(z)12 subunit of PRC2 to stimulate E(z)/Ezh2. Local chromatin compaction preceded establishment of histone H3K27 methylation indicating how PRC2 might maintain the repressed state.

Nodal and Smad2/3 signals play pivotal roles in mesendoderm induction and axis determination during late blastulation and early gastrulation in vertebrate embryos. However, Smad2/3 direct target genes during those critical developmental stages have not been systematically identified. Here, through ChIP-chip assay, we show that the promoter/enhancer regions of 679 genes are bound by Smad2 in the zebrafish early gastrulas. Expression analyses confirm that a significant proportion of Smad2 targets are indeed subjected to Nodal/Smad2 regulation at the onset of gastrulation. The co-existence of DNA-binding sites of other transcription factors in the Smad2-bound regions allows the identification of well known Smad2-binding partners, such as FoxH1 and Lef1/β-catenin, as well as many previously unknown Smad2 partners, including Oct1 and Gata6, during embryogenesis. We demonstrate that Oct1 physically associates with and enhances the transcription and mesendodermal induction activity of Smad2, whereas Gata6 exerts an inhibitory role in Smad2 signaling and mesendodermal induction. Thus, our study systemically uncovers a large number of Smad2 targets in early gastrulas and suggests cooperative roles of Smad2 and other transcription factors in controlling target gene transcription, which will be valuable for studying regulatory cascades during germ layer formation and patterning of vertebrate embryos.

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 3′ 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.

Summary B cells can present antigens to CD4+ T cells, but it is thought that dendritic cells (DCs) are the primary initiators of naive CD4+ T cell responses. Nanoparticles, including virus-like particles (VLPs), are attractive candidates as carriers for vaccines and drug delivery. Using RNA phage Qβ-derived VLP (Qβ-VLP) as a model antigen, we found that antigen-specific B cells were the dominant antigen-presenting cells that initiated naive CD4+ T cell activation. B cells were sufficient to induce T follicular helper cell development in the absence of DCs. Qβ-specific B cells promoted CD4+ T cell proliferation and differentiation via cognate interactions and through Toll-like receptor signaling-mediated cytokine production. Antigen-specific B cells were also involved in initiating CD4+ T cell responses during immunization with inactivated influenza virus. These findings have implications for the rational design of nanoparticles as vaccine candidates, particularly for therapeutic vaccines that aim to break immune tolerance.

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.

Mouse RING finger protein Rnf133 is a testis-specific endoplasmic reticulum-associated E3 ubiquitin ligase

Histone H3 lysine 27 (H3K27) methylation and H2A monoubiquitination (ubH2A) are two closely related histone modifications that regulate Polycomb silencing. Previous studies reported that H3K27 trimethylation (H3K27me3) rarely coexists with H3K36 di- or tri-methylation (H3K36me2/3) on the same histone H3 tails, which is partially controlled by the direct inhibition of the enzymatic activity of H3K27-specific methyltransferase PRC2. By contrast, H3K27 methylation does not affect the catalytic activity of H3K36-specific methyltransferases, suggesting other Polycomb mechanism(s) may negatively regulate the H3K36-specific methyltransferase(s). In this study, we established a simple protocol to purify milligram quantities of ubH2A from mammalian cells, which were used to reconstitute nucleosome substrates with fully ubiquitinated H2A. A number of histone methyltransferases were then tested on these nucleosome substrates. Notably, all of the H3K36-specific methyltransferases, including ASH1L, HYPB, NSD1, and NSD2 were inhibited by ubH2A, whereas the other histone methyltransferases, including PRC2, G9a, and Pr-Set7 were not affected by ubH2A. Together with previous reports, these findings collectively explain the mutual repulsion of H3K36me2/3 and Polycomb modifications.

Previously, we reported that little canonical (H3.1–H4)2 tetramers split to form “hybrid” tetramers consisted of old and new H3.1–H4 dimers, but approximately 10% of (H3.3–H4)2 tetramers split during each cell cycle. In this report, we mapped the H3.3 nucleosome occupancy, the H3.3 nucleosome turnover rate and H3.3 nucleosome splitting events at the genome-wide level. Interestingly, H3.3 nucleosome turnover rate at the transcription starting sites (TSS) of genes with different expression levels display a bimodal distribution rather than a linear correlation towards the transcriptional activity, suggesting genes are either active with high H3.3 nucleosome turnover or inactive with low H3.3 nucleosome turnover. H3.3 nucleosome splitting events are enriched at active genes, which are in fact better markers for active transcription than H3.3 nucleosome occupancy itself. Although both H3.3 nucleosome turnover and splitting events are enriched at active genes, these events only display a moderate positive correlation, suggesting H3.3 nucleosome splitting events are not the mere consequence of H3.3 nucleosome turnover. Surprisingly, H3.3 nucleosomes with high splitting index are remarkably enriched at enhancers in a cell-type specific manner. We propose that the H3.3 nucleosomes at enhancers may be split by an active mechanism to regulate cell-type specific transcription., Author Summary In our previous study, we unexpectedly discovered that nucleosomes containing the variant H3.3 histones experience substantial splitting events, resulting hybrid nucleosomes containing both “old” and “new” H3.3–H4 dimers. Here, we mapped the genomic distribution of these splitting events at the genome-wide level and studied the connections among gene transcriptional activity, H3.3 nucleosome occupancy, H3.3 nucleosome turnover and H3.3 nucleosome splitting events. We found that H3.3 nucleosome splitting events are better markers that reflect the transcriptional activity. Moreover, we discovered that H3.3 nucleosome splitting events feature the cell-type specific enhancers, which do not appear to the mere consequence of H3.3 nucleosome turnover. These findings may suggest an active mechanism regulating the H3.3 nucleosome splitting events at the enhancers.