Antiviral memory B cells exhibit enhanced innate immune response facilitated by epigenetic memory.

The long-lasting humoral immunity induced by viral infections or vaccinations depends on memory B cells with greatly increased affinity to viral antigens, which are evolved from germinal center (GC) responses. However, it is unclear whether antiviral memory B cells represent a distinct subset among the highly heterogeneous memory B cell population. Here, we examined memory B cells induced by a virus-mimicking antigen at both transcriptome and epigenetic levels and found unexpectedly that antiviral memory B cells exhibit an enhanced innate immune response, which appeared to be facilitated by the epigenetic memory that is established through the memory B cell development. In addition, T-bet is associated with the altered chromatin architecture and is required for the formation of the antiviral memory B cells. Thus, antiviral memory B cells are distinct from other GC-derived memory B cells in both physiological functions and epigenetic landmarks.

[Combination of acupuncture and medication for dry eye complicated with computer vision syndrome: a randomized controlled trial].

OBJECTIVE: To compare the clinical efficacy of acupuncture, Chinese medication and combination of acupuncture and medication in the treatment of dry eye complicated with computer vision syndrome (CVS). METHODS: A total of 152 patients with dry eye complicated with CVS were randomly divided into an acupuncture-medication group (38 cases, 1 case was removed), an acupuncture group (38 cases, 1 case dropped off), a Chinese medication group (38 cases, 1 case was removed), and a western medication group (38 cases, 1 case dropped off). In the western medication group, sodium hyaluronate eye drop combined with esculin and digitalis glycosides eye drop were used. In the acupuncture group, acupuncture was applied at bilateral Taiyang (EX-HN 5)， Cuanzhu (BL 2), Fengchi (GB 20), Qimen (LR 14) , and Hegu (LI 4) etc., once a day. In the Chinese medication group, Yiqi Congming decoction formula ganule was given orally, one dose a day. In the acupuncture-medication group, acupuncture combined with Yiqi Congming decoction formula granule were used. All groups were treated for 14 d. The non-invasive first tear film break-up time (NIBUT f), non-invasive average tear film break-up time (NIBUT av), tear meniscus height (TMH), ocular surface disease index (OSDI) score, and CVS symptom score were compared between the patients of each group before and after treatment. RESULTS: After treatment, the NIBUT f, NIBUT av, and TMH were increased compared with those before treatment in the patients of the 4 groups (P<0.01); the NIBUT f and NIBUT av in the acupuncture-medication group and the acupuncture group were higher than those in the Chinese medication group and the western medication group (P<0.05), and the TMH in the acupuncture-medication group and the Chinese medication group were higher than those in the acupuncture group and the western medication group (P<0.05). After treatment, the OSDI scores, the various scores and total scores of CVS (except for head symptom score in the western medication group) were decreased compared with those before treatment in the patients of the 4 groups (P<0.01). The OSDI score, total score, eye symptom score, and body symptom score of CVS in the acupuncture-medication group were lower than those in the acupuncture group, the Chinese medication group, and the western medication group (P<0.01, P<0.05), the head symptom score of the acupuncture-medication group was lower than that in the western medication group (P<0.05), and the CVS physical symptom scores and mental cognitive symptom scores of the acupuncture-medication group and the acupuncture group were lower than those in the Chinese medication group and the western medication group (P<0.05). CONCLUSION: Acupuncture has advantages in improving NIBUT f, NIBUT av, and CVS physical symptoms and cognitive symptoms, and the Chinese medication has advantage in improving TMH. The combination of acupuncture and Chinese medication has better effects compared with monotherapy.

Structural insights into histone binding and nucleosome assembly by chromatin assembly factor-1.

Chromatin inheritance entails de novo nucleosome assembly after DNA replication by chromatin assembly factor-1 (CAF-1). Yet direct knowledge about CAF-1's histone binding mode and nucleosome assembly process is lacking. In this work, we report the crystal structure of human CAF-1 in the absence of histones and the cryo-electron microscopy structure of CAF-1 in complex with histones H3 and H4. One histone H3-H4 heterodimer is bound by one CAF-1 complex mainly through the p60 subunit and the acidic domain of the p150 subunit. We also observed a dimeric CAF-1-H3-H4 supercomplex in which two H3-H4 heterodimers are poised for tetramer assembly and discovered that CAF-1 facilitates right-handed DNA wrapping of H3-H4 tetramers. These findings signify the involvement of DNA in H3-H4 tetramer formation and suggest a right-handed nucleosome precursor in chromatin replication.

Polymerase iota (POLI) confers radioresistance of esophageal squamous cell carcinoma by regulating RAD51 stability and facilitating homologous recombination.

Radiotherapy resistance is an important and urgent challenge in the clinical management of esophageal squamous carcinoma (ESCC). However, the factors mediating the ESCC resistance to radiotherapy and its underlying molecular mechanisms are not fully clarified. Our previous studies have demonstrated the critical role of DNA polymerase iota (POLI) in ESCC development and progression, here, we aimed to investigate the involvement of POLI in ESCC radiotherapy resistance and elucidate the underlying molecular mechanism. We found that highly expressed POLI was correlated with shorter overall survival of ESCC patients received radiotherapy. Down-regulation of POLI sensitized ESCC to IR, prolonged γH2AX foci in nuclei and comet tails after IR. HR but not NHEJ repair is inhibited in POLI-deficient ESCC cells. POLI stabilizes RAD51 protein via competitively binding with and blocking the interaction between RAD51 and E3 ligase XIAP and XIAP-mediated ubiquitination. Furthermore, loss of POLI leads to the activation of GAS signaling. Our findings provide novel insight into the role of POLI in the development of radioresistance mediated by stabilizing RAD51 protein in ESCC.

The patterns and participants of parental histone recycling during DNA replication in Saccharomyces cerevisiae.

Epigenetic information carried by histone modifications not only reflects the state of gene expression, but also participates in the maintenance of chromatin states and the regulation of gene expression. Recycling of parental histones to daughter chromatin after DNA replication is vital to mitotic inheritance of epigenetic information and the maintenance of cell identity, because the locus-specific modifications of the parental histones need to be maintained. To assess the precision of parental histone recycling, we developed a synthetic local label-chasing system in budding yeast Saccharomyces cerevisiae. Using this system, we observed that parental histone H3 can be recycled to their original position, thereby recovering their position information after DNA replication at all tested loci, including heterochromatin boundary, non-transcribed region, and actively transcribed regions. Moreover, the recycling rate appears to be affected by local chromatin environment. We surveyed a number of potential regulatory factors and observed that histone H3-H4 chaperon Asf1 contributed to parental histone recycling, while the eukaryotic replisome-associated components Mcm2 and Dpb3 displayed compounding effects in this process. In addition, the FACT complex also plays a role in the recycling of parental histones and helps to stabilize the nucleosomes.

[Effect of acupuncture on dry eye and tear inflammatory factors].

OBJECTIVE: On the basis of sodium hyaluronate eye drops, to observe the clinical efficacy of acupuncture on dry eye and explore the effect mechanism of ocular surface protection. METHODS: A total of 80 patients with dry eye were randomly divided into an observation group and a control group, 40 cases in each group. The control group was treated with routine cleaning of eyelid margin, hot compress of eyes with warm towel, and external application of sodium hyaluronate eye drops for 5 weeks. On the basis of the treatment as the control group, the observation group was treated with acupuncture at Jingming (BL 1), Cuanzhu (BL 2), Chengqi (ST 1), Hegu (LI 4), Zusanli (ST 36), Sanyinjiao (SP 6), etc., once a day, 6 times a week for 5 weeks (30 times totally). Before and after treatment, SchirmerⅠtest (SⅠT), breaking up time (BUT), corneal fluorescent (FL) score, ocular surface disease index (OSDI) score, and the contents of IL-6 and TNF-α in tears were evaluated and the therapeutic effect was compared between the two groups. RESULTS: Compared with before treatment, SⅠT and BUT after treatment in the observation group were prolonged (P<0.05), the scores of FL and OSDI and the contents of IL-6 and TNF-α in tears were decreased (P<0.05). After treatment, SⅠT and BUT in the observation group were longer than the control group (P<0.05), and the scores of FL and OSDI and the contents of IL-6 and TNF-α in tears in the observation group were lower than the control group (P<0.05). The total effective rate in the observation group was 87.5% (35/40), which was higher than 45.0% (18/40) in the control group (P<0.05). CONCLUSION: On the basis of sodium hyaluronate eye drops, acupuncture could improve the clinical symptoms of dry eye, promote the secretion of tears, prolong the tear film breaking up time, and reduce corneal damage, and effect mechanism may be related to the reduction of inflammatory response.

Highly enriched BEND3 prevents the premature activation of bivalent genes during differentiation.

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.

Recognition of the inherently unstable H2A nucleosome by Swc2 is a major determinant for unidirectional H2A.Z exchange.

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.

Simultaneously measuring the methylation of parent and daughter strands of replicated DNA at the single-molecule level by Hammer-seq.

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.

Sustained TNF-α stimulation leads to transcriptional memory that greatly enhances signal sensitivity and robustness.

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.

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.

Kinetics and mechanisms of mitotic inheritance of DNA methylation and their roles in aging-associated methylome deterioration.

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.

Histone H3K27 acetylation is dispensable for enhancer activity in mouse embryonic stem cells.

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.

Stella protein facilitates DNA demethylation by disrupting the chromatin association of the RING finger-type E3 ubiquitin ligase UHRF1.

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.

The proinflammatory cytokine TNFα induces DNA demethylation-dependent and -independent activation of interleukin-32 expression.

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.

Stella safeguards the oocyte methylome by preventing de novo methylation mediated by DNMT1.

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.

B Cells Are the Dominant Antigen-Presenting Cells that Activate Naive CD4+ T Cells upon Immunization with a Virus-Derived Nanoparticle Antigen.

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 Qß-derived VLP (Qß-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. Qß-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.

Roles of the CSE1L-mediated nuclear import pathway in epigenetic silencing.

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.

Small molecules capable of activating DNA methylation-repressed genes targeted by the p38 mitogen-activated protein kinase pathway.

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.