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  • Recapitulation of gametic DNA methylation and its post-fertilization maintenance with reassembled DNA elements at the mouse Igf2/H19 locus
    Epigenet. Chromatin (IF 4.185) Pub Date : 2020-01-14
    Hitomi Matsuzaki; Daichi Kuramochi; Eiichi Okamura; Katsuhiko Hirakawa; Aki Ushiki; Keiji Tanimoto

    Paternal allele-specific DNA methylation of the H19 imprinting control region (ICR) regulates imprinted expression of the Igf2/H19 genes. The molecular mechanism by which differential methylation of the H19 ICR is established during gametogenesis and maintained after fertilization, however, is not fully understood. We previously showed that a 2.9-kb H19 ICR fragment in transgenic mice was differentially methylated only after fertilization, demonstrating that two separable events, gametic and post-fertilization methylation, occur at the H19 ICR. We then determined that CTCF/Sox-Oct motifs and the 478-bp sequence of the H19 ICR are essential for maintaining its maternal hypomethylation status and for acquisition of paternal methylation, respectively, during the post-fertilization period. Using a series of 5′-truncated H19 ICR transgenes to dissect the 478-bp sequence, we identified a 118-bp region required for post-fertilization methylation activity. Deletion of the sequence from the paternal endogenous H19 ICR caused loss of methylation after fertilization, indicating that methylation activity of the sequence is required to protect endogenous H19 ICR from genome-wide reprogramming. We then reconstructed a synthetic DNA fragment in which the CTCF binding sites, Sox-Oct motifs, as well as the 118-bp sequence, were inserted into lambda DNA, and used it to replace the endogenous H19 ICR. The fragment was methylated during spermatogenesis; moreover, its allele-specific methylation status was faithfully maintained after fertilization, and imprinted expression of the both Igf2 and H19 genes was recapitulated. Our results identified a 118-bp region within the H19 ICR that is required for de novo DNA methylation of the paternally inherited H19 ICR during pre-implantation period. A lambda DNA-based artificial fragment that contains the 118-bp sequence, in addition to the previously identified cis elements, could fully replace the function of the H19 ICR in the mouse genome.

    更新日期:2020-01-14
  • Transcription-independent TFIIIC-bound sites cluster near heterochromatin boundaries within lamina-associated domains in C. elegans
    Epigenet. Chromatin (IF 4.185) Pub Date : 2020-01-09
    Alexis V. Stutzman; April S. Liang; Vera Beilinson; Kohta Ikegami

    Chromatin organization is central to precise control of gene expression. In various eukaryotic species, domains of pervasive cis-chromatin interactions demarcate functional domains of the genomes. In nematode Caenorhabditis elegans, however, pervasive chromatin contact domains are limited to the dosage-compensated sex chromosome, leaving the principle of C. elegans chromatin organization unclear. Transcription factor III C (TFIIIC) is a basal transcription factor complex for RNA polymerase III, and is implicated in chromatin organization. TFIIIC binding without RNA polymerase III co-occupancy, referred to as extra-TFIIIC binding, has been implicated in insulating active and inactive chromatin domains in yeasts, flies, and mammalian cells. Whether extra-TFIIIC sites are present and contribute to chromatin organization in C. elegans remains unknown. We identified 504 TFIIIC-bound sites absent of RNA polymerase III and TATA-binding protein co-occupancy characteristic of extra-TFIIIC sites in C. elegans embryos. Extra-TFIIIC sites constituted half of all identified TFIIIC binding sites in the genome. Extra-TFIIIC sites formed dense clusters in cis. The clusters of extra-TFIIIC sites were highly over-represented within the distal arm domains of the autosomes that presented a high level of heterochromatin-associated histone H3K9 trimethylation (H3K9me3). Furthermore, extra-TFIIIC clusters were embedded in the lamina-associated domains. Despite the heterochromatin environment of extra-TFIIIC sites, the individual clusters of extra-TFIIIC sites were devoid of and resided near the individual H3K9me3-marked regions. Clusters of extra-TFIIIC sites were pervasive in the arm domains of C. elegans autosomes, near the outer boundaries of H3K9me3-marked regions. Given the reported activity of extra-TFIIIC sites in heterochromatin insulation in yeasts, our observation raised the possibility that TFIIIC may also demarcate heterochromatin in C. elegans.

    更新日期:2020-01-09
  • MethylToSNP: identifying SNPs in Illumina DNA methylation array data
    Epigenet. Chromatin (IF 4.185) Pub Date : 2019-12-20
    Brenna A. LaBarre; Alexander Goncearenco; Hanna M. Petrykowska; Weerachai Jaratlerdsiri; M. S. Riana Bornman; Vanessa M. Hayes; Laura Elnitski

    Current array-based methods for the measurement of DNA methylation rely on the process of sodium bisulfite conversion to differentiate between methylated and unmethylated cytosine bases in DNA. In the absence of genotype data this process can lead to ambiguity in data interpretation when a sample has polymorphisms at a methylation probe site. A common way to minimize this problem is to exclude such potentially problematic sites, with some methods removing as much as 60% of array probes from consideration before data analysis. Here, we present an algorithm implemented in an R Bioconductor package, MethylToSNP, which detects a characteristic data pattern to infer sites likely to be confounded by polymorphisms. Additionally, the tool provides a stringent reliability score to allow thresholding on SNP predictions. We calibrated parameters and thresholds used by the algorithm on simulated and real methylation data sets. We illustrate findings using methylation data from YRI (Yoruba in Ibadan, Nigeria), CEPH (European descent) and KhoeSan (southern African) populations. Our polymorphism predictions made using MethylToSNP have been validated through SNP databases and bisulfite and genomic sequencing. The benefits of this method are threefold. First, it prevents extensive data loss by considering only SNPs specific to the individuals in the study. Second, it offers the possibility to identify new polymorphisms in samples for which there is little known about the genetic landscape. Third, it identifies variants as they exist in functional regions of a genome, such as in CTCF (transcriptional repressor) sites and enhancers, that may be common alleles or personal mutations with potential to deleteriously affect genomic regulatory activities. We demonstrate that MethylToSNP is applicable to the Illumina 450K and Illumina 850K EPIC array data and is also backwards compatible to the 27K methylation arrays. Going forward, this kind of nuanced approach can increase the amount of information derived from precious data sets by considering samples of the project individually to enable more informed decisions about data cleaning.

    更新日期:2019-12-20
  • Age-associated sperm DNA methylation patterns do not directly persist trans-generationally
    Epigenet. Chromatin (IF 4.185) Pub Date : 2019-12-19
    Timothy G. Jenkins; Emma R. James; Kenneth I. Aston; Albert Salas-Huetos; Alexander W. Pastuszak; Ken R. Smith; Heidi A. Hanson; James M. Hotaling; Douglas T. Carrell

    The impact of aging on the sperm methylome is well understood. However, the direct, subsequent impact on offspring and the role of altered sperm DNA methylation alterations in this process remain poorly understood. The well-defined impact of aging on sperm DNA methylation represents an excellent opportunity to trace the direct, transgenerational transmission of these signals. We utilized the Illumina MethylationEPIC array to analyze the sperm of 16 patients with older (> 40 years of age) paternal grandfathers (‘old grand paternal age’ patients; OGPA) and 16 patients with younger (< 25 years of age) grandfathers (‘young grand paternal age’ patients; YGPA) identified through the Subfertility Health Assisted Reproduction and the Environment (SHARE) cohort to investigate differences in DNA methylation. No differentially methylated regions were identified between the OGPA and YGPA groups. Further, when assessing only the sites previously shown to be altered by age, no statistically significant differences between OGPA and YGPA were identified. This was true even despite the lower bar for significance after removing multiple comparison correction in a targeted approach. Interestingly though, in an analysis of the 140 loci known to have decreased methylation with age, the majority (~ 72%) had lower methylation in OGPA compared to YGPA though the differences were extremely small (~ 1.5%). This study suggests that the robust and consistent age-associated methylation alterations seen in human sperm are ‘reset’ during large-scale epigenetic reprograming processes and are not directly inherited trans-generationally (over two generations). An extremely small trend was present between the YGPA and OGPA groups that resemble the aging pattern in older sperm. However, this trend was not significant and was so small that, if real, is almost certainly biologically inert.

    更新日期:2019-12-19
  • Nickel-induced transcriptional changes persist post exposure through epigenetic reprogramming
    Epigenet. Chromatin (IF 4.185) Pub Date : 2019-12-19
    Cynthia C. Jose; Zhenjia Wang; Vinay Singh Tanwar; Xiaoru Zhang; Chongzhi Zang; Suresh Cuddapah

    Nickel is an occupational and environmental toxicant associated with a number of diseases in humans including pulmonary fibrosis, bronchitis and lung and nasal cancers. Our earlier studies showed that the nickel-exposure-induced genome-wide transcriptional changes, which persist even after the termination of exposure may underlie nickel pathogenesis. However, the mechanisms that drive nickel-induced persistent changes to the transcriptome remain elusive. To elucidate the mechanisms that underlie nickel-induced long-term transcriptional changes, in this study, we examined the transcriptome and the epigenome of human lung epithelial cells during nickel exposure and after the termination of exposure. We identified two categories of persistently differentially expressed genes: (i) the genes that were differentially expressed during nickel exposure; and (ii) the genes that were differentially expressed only after the termination of exposure. Interestingly, > 85% of the nickel-induced gene expression changes occurred only after the termination of exposure. We also found extensive genome-wide alterations to the activating histone modification, H3K4me3, after the termination of nickel exposure, which coincided with the post-exposure gene expression changes. In addition, we found significant post-exposure alterations to the repressive histone modification, H3K27me3. Our results suggest that while modest first wave of transcriptional changes occurred during nickel exposure, extensive transcriptional changes occurred during a second wave of transcription for which removal of nickel ions was essential. By uncovering a new category of transcriptional and epigenetic changes, which occur only after the termination of exposure, this study provides a novel understanding of the long-term deleterious consequences of nickel exposure on human health.

    更新日期:2019-12-19
  • Reduced H3K27me3 leads to abnormal Hox gene expression in neural tube defects
    Epigenet. Chromatin (IF 4.185) Pub Date : 2019-12-19
    Juan Yu; Lei Wang; Pei Pei; Xue Li; Jianxin Wu; Zhiyong Qiu; Juan Zhang; Ruifang Ao; Shan Wang; Ting Zhang; Jun Xie

    Neural tube defects (NTDs) are severe, common birth defects that result from failure of normal neural tube closure during early embryogenesis. Accumulating strong evidence indicates that genetic factors contribute to NTDs etiology, among them, HOX genes play a key role in neural tube closure. Although abnormal HOX gene expression can lead to NTDs, the underlying pathological mechanisms have not fully been understood. We detected that H3K27me3 and expression of the Hox genes in a retinoic acid (RA) induced mouse NTDs model on E8.5, E9.5 and E10.5 using RNA-sequencing and chromatin immunoprecipitation sequencing assays. Furthermore, we quantified 10 Hox genes using NanoString nCounter in brain tissue of fetuses with 39 NTDs patients including anencephaly, spina bifida, hydrocephaly and encephalocele. Here, our results showed differential expression in 26 genes with a > 20-fold change in the level of expression, including 10 upregulated Hox genes. RT-qPCR revealed that these 10 Hox genes were all upregulated in RA-induced mouse NTDs as well as RA-treated embryonic stem cells (ESCs). Using ChIP-seq assays, we demonstrate that a decrease in H3K27me3 level upregulates the expression of Hox cluster A–D in RA-induced mouse NTDs model on E10.5. Interestingly, RA treatment led to attenuation of H3K27me3 due to cooperate between UTX and Suz12, affecting Hox gene regulation. Further analysis, in human anencephaly cases, upregulation of 10 HOX genes was observed, along with aberrant levels of H3K27me3. Notably, HOXB4, HOXC4 and HOXD1 expression was negatively correlated with H3K27me3 levels. Our results indicate that abnormal HOX gene expression induced by aberrant H3K27me3 levels may be a risk factor for NTDs and highlight the need for further analysis of genome-wide epigenetic modification in NTDs.

    更新日期:2019-12-19
  • Comprehensive epigenome characterization reveals diverse transcriptional regulation across human vascular endothelial cells
    Epigenet. Chromatin (IF 4.185) Pub Date : 2019-12-19
    Ryuichiro Nakato; Youichiro Wada; Ryo Nakaki; Genta Nagae; Yuki Katou; Shuichi Tsutsumi; Natsu Nakajima; Hiroshi Fukuhara; Atsushi Iguchi; Takahide Kohro; Yasuharu Kanki; Yutaka Saito; Mika Kobayashi; Akashi Izumi-Taguchi; Naoki Osato; Kenji Tatsuno; Asuka Kamio; Yoko Hayashi-Takanaka; Hiromi Wada; Shinzo Ohta; Masanori Aikawa; Hiroyuki Nakajima; Masaki Nakamura; Rebecca C. McGee; Kyle W. Heppner; Tatsuo Kawakatsu; Michiru Genno; Hiroshi Yanase; Haruki Kume; Takaaki Senbonmatsu; Yukio Homma; Shigeyuki Nishimura; Toutai Mitsuyama; Hiroyuki Aburatani; Hiroshi Kimura; Katsuhiko Shirahige

    Endothelial cells (ECs) make up the innermost layer throughout the entire vasculature. Their phenotypes and physiological functions are initially regulated by developmental signals and extracellular stimuli. The underlying molecular mechanisms responsible for the diverse phenotypes of ECs from different organs are not well understood. To characterize the transcriptomic and epigenomic landscape in the vascular system, we cataloged gene expression and active histone marks in nine types of human ECs (generating 148 genome-wide datasets) and carried out a comprehensive analysis with chromatin interaction data. We developed a robust procedure for comparative epigenome analysis that circumvents variations at the level of the individual and technical noise derived from sample preparation under various conditions. Through this approach, we identified 3765 EC-specific enhancers, some of which were associated with disease-associated genetic variations. We also identified various candidate marker genes for each EC type. We found that the nine EC types can be divided into two subgroups, corresponding to those with upper-body origins and lower-body origins, based on their epigenomic landscape. Epigenomic variations were highly correlated with gene expression patterns, but also provided unique information. Most of the deferentially expressed genes and enhancers were cooperatively enriched in more than one EC type, suggesting that the distinct combinations of multiple genes play key roles in the diverse phenotypes across EC types. Notably, many homeobox genes were differentially expressed across EC types, and their expression was correlated with the relative position of each organ in the body. This reflects the developmental origins of ECs and their roles in angiogenesis, vasculogenesis and wound healing. This comprehensive analysis of epigenome characterization of EC types reveals diverse transcriptional regulation across human vascular systems. These datasets provide a valuable resource for understanding the vascular system and associated diseases.

    更新日期:2019-12-19
  • Ubiquitin ligases HUWE1 and NEDD4 cooperatively control signal-dependent PRC2-Ezh1α/β-mediated adaptive stress response pathway in skeletal muscle cells
    Epigenet. Chromatin (IF 4.185) Pub Date : 2019-12-19
    Peng Liu; Muhammad Shuaib; Huoming Zhang; Seba Nadeef; Valerio Orlando

    While the role of Polycomb group protein-mediated “cell memory” is well established in developmental contexts, little is known about their role in adult tissues and in particular in post-mitotic cells. Emerging evidence assigns a pivotal role in cell plasticity and adaptation. PRC2-Ezh1α/β signaling pathway from cytoplasm to chromatin protects skeletal muscle cells from oxidative stress. However, detailed mechanisms controlling degradation of cytoplasmic Ezh1β and assembly of canonical PRC2-Ezh1α repressive complex remain to be clarified. Here, we report NEDD4 ubiquitin E3 ligase, as key regulator of Ezh1β. In addition, we report that ubiquitination and degradation of Ezh1β is controlled by another layer of regulation, that is, one specific phosphorylation of serine 560 located at Ezh1β-specific C terminal. Finally, we demonstrate that also Ezh1α needs to be stabilized under stress condition and this stabilization process requires decreased association pattern between another E3 ubiquitin ligase HUWE1. Together, these results shed light on key components that regulate PRC2-Ezh1α/β pathway to direct modulation of epigenome plasticity and transcriptional output in skeletal muscle cells.

    更新日期:2019-12-19
  • Maintenance of active chromatin states by HMGN2 is required for stem cell identity in a pluripotent stem cell model
    Epigenet. Chromatin (IF 4.185) Pub Date : 2019-12-12
    Sylvia Garza-Manero; Abdulmajeed Abdulghani A. Sindi; Gokula Mohan; Ohoud Rehbini; Valentine H. M. Jeantet; Mariarca Bailo; Faeezah Abdul Latif; Maureen P. West; Ross Gurden; Lauren Finlayson; Silvija Svambaryte; Adam G. West; Katherine L. West

    Members of the HMGN protein family modulate chromatin structure and influence epigenetic modifications. HMGN1 and HMGN2 are highly expressed during early development and in the neural stem/progenitor cells of the developing and adult brain. Here, we investigate whether HMGN proteins contribute to the chromatin plasticity and epigenetic regulation that is essential for maintaining pluripotency in stem cells. We show that loss of Hmgn1 or Hmgn2 in pluripotent embryonal carcinoma cells leads to increased levels of spontaneous neuronal differentiation. This is accompanied by the loss of pluripotency markers Nanog and Ssea1, and increased expression of the pro-neural transcription factors Neurog1 and Ascl1. Neural stem cells derived from these Hmgn-knockout lines also show increased spontaneous neuronal differentiation and Neurog1 expression. The loss of HMGN2 leads to a global reduction in H3K9 acetylation, and disrupts the profile of H3K4me3, H3K9ac, H3K27ac and H3K122ac at the Nanog and Oct4 loci. At endodermal/mesodermal genes, Hmgn2-knockout cells show a switch from a bivalent to a repressive chromatin configuration. However, at neuronal lineage genes whose expression is increased, no epigenetic changes are observed and their bivalent states are retained following the loss of HMGN2. We conclude that HMGN1 and HMGN2 maintain the identity of pluripotent embryonal carcinoma cells by optimising the pluripotency transcription factor network and protecting the cells from precocious differentiation. Our evidence suggests that HMGN2 regulates active and bivalent genes by promoting an epigenetic landscape of active histone modifications at promoters and enhancers.

    更新日期:2019-12-13
  • The SMART App: an interactive web application for comprehensive DNA methylation analysis and visualization
    Epigenet. Chromatin (IF 4.185) Pub Date : 2019-12-05
    Yin Li; Di Ge; Chunlai Lu

    Data mining of The Cancer Genome Atlas (TCGA) data has significantly facilitated cancer genome research and provided unprecedented opportunities for cancer researchers. However, existing web applications for DNA methylation analysis does not adequately address the need of experimental biologists, and many additional functions are often required. To facilitate DNA methylation analysis, we present the SMART (Shiny Methylation Analysis Resource Tool) App, a user-friendly and easy-to-use web application for comprehensively analyzing the DNA methylation data of TCGA project. The SMART App integrates multi-omics and clinical data with DNA methylation and provides key interactive and customized functions including CpG visualization, pan-cancer methylation profile, differential methylation analysis, correlation analysis and survival analysis for users to analyze the DNA methylation in diverse cancer types in a multi-dimensional manner. The SMART App serves as a new approach for users, especially wet-bench scientists with no programming background, to analyze the scientific big data and facilitate data mining. The SMART App is available at http://www.bioinfo-zs.com/smartapp.

    更新日期:2019-12-05
  • The distributions of protein coding genes within chromatin domains in relation to human disease
    Epigenet. Chromatin (IF 4.185) Pub Date : 2019-12-05
    Enrique M. Muro; Jonas Ibn-Salem; Miguel A. Andrade-Navarro

    Our understanding of the nuclear chromatin structure has increased hugely during the last years mainly as a consequence of the advances in chromatin conformation capture methods like Hi-C. The unprecedented resolution of genome-wide interaction maps shows functional consequences that extend the initial thought of an efficient DNA packaging mechanism: gene regulation, DNA repair, chromosomal translocations and evolutionary rearrangements seem to be only the peak of the iceberg. One key concept emerging from this research is the topologically associating domains (TADs) whose functional role in gene regulation and their association with disease is not fully untangled. We report that the lower the number of protein coding genes inside TADs, the higher the tendency of those genes to be associated with disease (p-value = 4 × $$10^{-54}$$). Moreover, housekeeping genes are less associated with disease than other genes. Accordingly, they are depleted in TADs containing less than three protein coding genes (p-value = 3.9 × $$10^{-34}$$). We observed that TADs with higher ratios of enhancers versus genes contained higher numbers of disease-associated genes. We interpret these results as an indication that sharing enhancers among genes reduces their involvement in disease. Larger TADs would have more chances to accommodate many genes and select for enhancer sharing along evolution. Genes associated with human disease do not distribute randomly over the TADs. Our observations suggest general rules that confer functional stability to TADs, adding more evidence to the role of TADs as regulatory units.

    更新日期:2019-12-05
  • Contribution of H3K4 demethylase KDM5B to nucleosome organization in embryonic stem cells revealed by micrococcal nuclease sequencing
    Epigenet. Chromatin (IF 4.185) Pub Date : 2019-04-02
    Jiji T. Kurup; Ion J. Campeanu; Benjamin L. Kidder

    Positioning of nucleosomes along DNA is an integral regulator of chromatin accessibility and gene expression in diverse cell types. However, the precise nature of how histone demethylases including the histone 3 lysine 4 (H3K4) demethylase, KDM5B, impacts nucleosome positioning around transcriptional start sites (TSS) of active genes is poorly understood. Here, we report that KDM5B is a critical regulator of nucleosome positioning in embryonic stem (ES) cells. Micrococcal nuclease sequencing (MNase-Seq) revealed increased enrichment of nucleosomes around TSS regions and DNase I hypersensitive sites in KDM5B-depleted ES cells. Moreover, depletion of KDM5B resulted in a widespread redistribution and disorganization of nucleosomes in a sequence-dependent manner. Dysregulated nucleosome phasing was also evident in KDM5B-depleted ES cells, including asynchronous nucleosome spacing surrounding TSS regions, where nucleosome variance was positively correlated with the degree of asynchronous phasing. The redistribution of nucleosomes around TSS regions in KDM5B-depleted ES cells is correlated with dysregulated gene expression, and altered H3K4me3 and RNA polymerase II occupancy. In addition, we found that DNA shape features varied significantly at regions with shifted nucleosomes. Altogether, our data support a role for KDM5B in regulating nucleosome positioning in ES cells.

    更新日期:2019-11-28
  • Abnormal level of CUL4B-mediated histone H2A ubiquitination causes disruptive HOX gene expression
    Epigenet. Chromatin (IF 4.185) Pub Date : 2019-04-16
    Ye Lin; Juan Yu; Jianxin Wu; Shan Wang; Ting Zhang

    Neural tube defects (NTDs) are common birth defects involving the central nervous system. Recent studies on the etiology of human NTDs have raised the possibility that epigenetic regulation could be involved in determining susceptibility to them. Here, we show that the H2AK119ub1 E3 ligase CUL4B is required for the activation of retinoic acid (RA)-inducible developmentally critical homeobox (HOX) genes in NT2/D1 embryonal carcinoma cells. RA treatment led to attenuation of H2AK119ub1 due to decrease in CUL4B, further affecting HOX gene regulation. Furthermore, we found that CUL4B interacted directly with RORγ and negatively regulated its transcriptional activity. Interestingly, knockdown of RORγ decreased the expression of HOX genes along with increased H2AK119ub1 occupancy levels, at HOX gene sites in N2/D1 cells. In addition, upregulation of HOX genes was observed along with lower levels of CUL4B-mediated H2AK119ub1 in both mouse and human anencephaly NTD cases. Notably, the expression of HOXA10 genes was negatively correlated with CUL4B levels in human anencephaly NTD cases. Our results indicate that abnormal HOX gene expression induced by aberrant CUL4B-mediated H2AK119ub1 levels may be a risk factor for NTDs, and highlight the need for further analysis of genome-wide epigenetic modifications in NTDs.

    更新日期:2019-11-28
  • Tat inhibition by didehydro-Cortistatin A promotes heterochromatin formation at the HIV-1 long terminal repeat
    Epigenet. Chromatin (IF 4.185) Pub Date : 2019-04-16
    Chuan Li; Guillaume Mousseau; Susana T. Valente

    Transcription from the integrated HIV-1 promoter is directly governed by its chromatin environment, and the nucleosome-1 downstream from the transcription start site directly impedes transcription from the HIV-1 promoter. The HIV-1 Tat protein regulates the passage from viral latency to active transcription by binding to the viral mRNA hairpin (TAR) and recruiting transcriptional factors to promote transcriptional elongation. The Tat inhibitor didehydro-Cortistatin A (dCA) inhibits transcription and overtime, the lack of low-grade transcriptional events, triggers epigenetic changes at the latent loci that “lock” HIV transcription in a latent state. Here we investigated those epigenetic changes using multiple cell line models of HIV-1 latency and active transcription. We demonstrated that dCA treatment does not alter the classic nucleosome positioning at the HIV-1 promoter, but promotes tighter nucleosome/DNA association correlating with increased deacetylated H3 occupancy at nucleosome-1. Recruitment of the SWI/SNF chromatin remodeling complex PBAF, necessary for Tat-mediated transactivation, is also inhibited, while recruitment of the repressive BAF complex is enhanced. These results were supported by loss of RNA polymerase II recruitment on the HIV genome, even during strong stimulation with latency-reversing agents. No epigenetic changes were detected in cell line models of latency with Tat-TAR incompetent proviruses confirming the specificity of dCA for Tat. We characterized the dCA-mediated epigenetic signature on the HIV genome, which translates into potent blocking effects on HIV expression, further strengthening the potential of Tat inhibitors in “block-and-lock” functional cure approaches.

    更新日期:2019-11-28
  • The NuA4 acetyltransferase and histone H4 acetylation promote replication recovery after topoisomerase I-poisoning
    Epigenet. Chromatin (IF 4.185) Pub Date : 2019-04-16
    Chiaki Noguchi; Tanu Singh; Melissa A. Ziegler; Jasmine D. Peake; Lyne Khair; Ana Aza; Toru M. Nakamura; Eishi Noguchi

    Histone acetylation plays an important role in DNA replication and repair because replicating chromatin is subject to dynamic changes in its structures. However, its precise mechanism remains elusive. In this report, we describe roles of the NuA4 acetyltransferase and histone H4 acetylation in replication fork protection in the fission yeast Schizosaccharomyces pombe. Downregulation of NuA4 subunits renders cells highly sensitive to camptothecin, a compound that induces replication fork breakage. Defects in NuA4 function or mutations in histone H4 acetylation sites lead to impaired recovery of collapsed replication forks and elevated levels of Rad52 DNA repair foci, indicating the role of histone H4 acetylation in DNA replication and fork repair. We also show that Vid21 interacts with the Swi1–Swi3 replication fork protection complex and that Swi1 stabilizes Vid21 and promotes efficient histone H4 acetylation. Furthermore, our genetic analysis demonstrates that loss of Swi1 further sensitizes NuA4 and histone H4 mutant cells to replication fork breakage. Considering that Swi1 plays a critical role in replication fork protection, our results indicate that NuA4 and histone H4 acetylation promote repair of broken DNA replication forks.

    更新日期:2019-11-28
  • DNMT and HDAC inhibitors modulate MMP-9-dependent H3 N-terminal tail proteolysis and osteoclastogenesis
    Epigenet. Chromatin (IF 4.185) Pub Date : 2019-04-16
    Yonghwan Shin; Nikhil B. Ghate; Byoungsan Moon; Kyungpyo Park; Wange Lu; Woojin An

    MMP-9-dependent proteolysis of histone H3 N-terminal tail (H3NT) is an important mechanism for activation of gene expression during osteoclast differentiation. Like other enzymes targeting their substrates within chromatin structure, MMP-9 enzymatic activity toward H3NT is tightly controlled by histone modifications such as H3K18 acetylation (H3K18ac) and H3K27 monomethylation (H3K27me1). Growing evidence indicates that DNA methylation is another epigenetic mechanism controlling osteoclastogenesis, but whether DNA methylation is also critical for regulating MMP-9-dependent H3NT proteolysis and gene expression remains unknown. We show here that treating RANKL-induced osteoclast progenitor (OCP) cells with the DNMT inhibitor 5-Aza-2′-deoxycytidine (5-Aza-CdR) induces CpG island hypomethylation and facilitates MMP-9 transcription. This increase in MMP-9 expression results in a significant enhancement of H3NT proteolysis and OCP cell differentiation. On the other hand, despite an increase in levels of H3K18ac, treatment with the HDAC inhibitor trichostatin A (TSA) leads to impairment of osteoclastogenic gene expression. Mechanistically, TSA treatment of OCP-induced cells stimulates H3K27ac with accompanying reduction in H3K27me1, which is a key modification to facilitate stable interaction of MMP-9 with nucleosomes for H3NT proteolysis. Moreover, hypomethylated osteoclastogenic genes in 5-Aza-CdR-treated cells remain transcriptionally inactive after TSA treatment, because H3K27 is highly acetylated and cannot be modified by G9a. These findings clearly indicate that DNA methylation and histone modification are important mechanisms in regulating osteoclastogenic gene expression and that their inhibitors can be used as potential therapeutic tools for treating bone disorders.

    更新日期:2019-11-28
  • Ezh2-dCas9 and KRAB-dCas9 enable engineering of epigenetic memory in a context-dependent manner
    Epigenet. Chromatin (IF 4.185) Pub Date : 2019-05-03
    Henriette O’Geen; Sofie L. Bates; Sakereh S. Carter; Karly A. Nisson; Julian Halmai; Kyle D. Fink; Suhn K. Rhie; Peggy J. Farnham; David J. Segal

    Rewriting of the epigenome has risen as a promising alternative to gene editing for precision medicine. In nature, epigenetic silencing can result in complete attenuation of target gene expression over multiple mitotic divisions. However, persistent repression has been difficult to achieve in a predictable manner using targeted systems. Here, we report that persistent epigenetic memory required both a DNA methyltransferase (DNMT3A-dCas9) and a histone methyltransferase (Ezh2-dCas9 or KRAB-dCas9). We demonstrate that the histone methyltransferase requirement can be locus specific. Co-targeting Ezh2-dCas9, but not KRAB-dCas9, with DNMT3A-dCas9 and DNMT3L induced long-term HER2 repression over at least 50 days (approximately 57 cell divisions) and triggered an epigenetic switch to a heterochromatic environment. An increase in H3K27 trimethylation and DNA methylation was stably maintained and accompanied by a sustained loss of H3K27 acetylation. Interestingly, substitution of Ezh2-dCas9 with KRAB-dCas9 enabled long-term repression at some target genes (e.g., SNURF) but not at HER2, at which H3K9me3 and DNA methylation were transiently acquired and subsequently lost. Off-target DNA hypermethylation occurred at many individual CpG sites but rarely at multiple CpGs in a single promoter, consistent with no detectable effect on transcription at the off-target loci tested. Conversely, robust hypermethylation was observed at HER2. We further demonstrated that Ezh2-dCas9 required full-length DNMT3L for maximal activity and that co-targeting DNMT3L was sufficient for persistent repression by Ezh2-dCas9 or KRAB-dCas9. These data demonstrate that targeting different combinations of histone and DNA methyltransferases is required to achieve maximal repression at different loci. Fine-tuning of targeting tools is a necessity to engineer epigenetic memory at any given locus in any given cell type.

    更新日期:2019-11-28
  • Profiling of chromatin accessibility and identification of general cis-regulatory mechanisms that control two ocular lens differentiation pathways
    Epigenet. Chromatin (IF 4.185) Pub Date : 2019-05-03
    Yilin Zhao; Deyou Zheng; Ales Cvekl

    Promoters and enhancers are cis-regulatory DNA sequences that control specificity and quantity of transcription. Both are rich on clusters of cis-acting sites that interact with sequence-specific DNA-binding transcription factors (TFs). At the level of chromatin, these regions display increased nuclease sensitivity, reduced nucleosome density, including nucleosome-free regions, and specific combinations of posttranslational modifications of core histone proteins. Together, “open” and “closed” chromatins represent transcriptionally active and repressed states of individual genes, respectively. Cellular differentiation is marked by changes in local chromatin structure. Lens morphogenesis, regulated by TF Pax6, includes differentiation of epithelial precursor cells into lens fibers in parallel with differentiation of epithelial precursors into the mature lens epithelium. Using ATAC-seq, we investigated dynamics of chromatin changes during mouse lens fibers and epithelium differentiation. Tissue-specific features of these processes are demonstrated via comparative studies of embryonic stem cells, forebrain, and liver chromatins. Unbiased analysis reveals cis-regulatory logic of lens differentiation through known (e.g., AP-1, Ets, Hsf4, Maf, and Pax6 sites) and novel (e.g., CTCF, Tead, and NF1) motifs. Twenty-six DNA-binding TFs, recognizing these cis-motifs, are markedly up-regulated in differentiating lens fibers. As specific examples, our ATAC-seq data uncovered both the regulatory regions and TF binding motifs in Foxe3, Prox1, and Mip loci that are consistent with previous, though incomplete, experimental data. A cross-examination of Pax6 binding with ATAC-seq data demonstrated that Pax6 bound to both open (H3K27ac and P300-enriched) and closed chromatin domains in lens and forebrain. Our study has generated the first lens chromatin accessibility maps that support a general model of stage-specific chromatin changes associated with transcriptional activities of batteries of genes required for lens fiber cell formation. Analysis of active (or open) promoters and enhancers reveals important cis-DNA motifs that establish the molecular foundation for temporally and spatially regulated gene expression in lens. Together, our data and models open new avenues for the field to conduct mechanistic studies of transcriptional control regions, reconstruction of gene regulatory networks that govern lens morphogenesis, and identification of cataract-causing mutations in noncoding sequences.

    更新日期:2019-11-28
  • Polymer coil–globule phase transition is a universal folding principle of Drosophila epigenetic domains
    Epigenet. Chromatin (IF 4.185) Pub Date : 2019-05-13
    Antony Lesage; Vincent Dahirel; Jean-Marc Victor; Maria Barbi

    Localized functional domains within chromosomes, known as topologically associating domains (TADs), have been recently highlighted. In Drosophila, TADs are biochemically defined by epigenetic marks, this suggesting that the 3D arrangement may be the “missing link” between epigenetics and gene activity. Recent observations (Boettiger et al. in Nature 529(7586):418–422, 2016) provide access to structural features of these domains with unprecedented resolution thanks to super-resolution experiments. In particular, they give access to the distribution of the radii of gyration for domains of different linear length and associated with different transcriptional activity states: active, inactive or repressed. Intriguingly, the observed scaling laws lack consistent interpretation in polymer physics. We develop a new methodology conceived to extract the best information from such super-resolution data by exploiting the whole distribution of gyration radii, and to place these experimental results on a theoretical framework. We show that the experimental data are compatible with the finite-size behavior of a self-attracting polymer. The same generic polymer model leads to quantitative differences between active, inactive and repressed domains. Active domains behave as pure polymer coils, while inactive and repressed domains both lie at the coil–globule crossover. For the first time, the “color-specificity” of both the persistence length and the mean interaction energy are estimated, leading to important differences between epigenetic states. These results point toward a crucial role of criticality to enhance the system responsivity, resulting in both energy transitions and structural rearrangements. We get strong indications that epigenetically induced changes in nucleosome–nucleosome interaction can cause chromatin to shift between different activity states.

    更新日期:2019-11-28
  • Ovarian dysfunction following prenatal exposure to an insecticide, chlordecone, associates with altered epigenetic features
    Epigenet. Chromatin (IF 4.185) Pub Date : 2019-05-13
    Louis Legoff; Ouzna Dali; Shereen Cynthia D’Cruz; Antonio Suglia; Aurore Gely-Pernot; Chloé Hémery; Pierre-Yves Kernanec; Abbassia Demmouche; Christine Kervarrec; Sergei Tevosian; Luc Multigner; Fatima Smagulova

    Chlordecone (CD) is an insecticide that was used in the French West Indies for several years to control the banana root borer pest. Given its nonsignificant degradation, it persists in the environment. CD is a carcinogenic compound with reproductive and developmental toxicity and is a recognized endocrine-disrupting chemical. In this study, we examined the effects of CD on female reproductive system of mice with the focus on epigenetic features in ovary. Our data show that gestational exposure to low dose of CD affects meiotic double-strand breaks repair in female embryos. In adult mice derived from CD-treated pregnant females, we observed delayed puberty, decreased number of primordial and increased number of atretic follicles. Gene expression analysis revealed that Rcbtb2 and Rbpms genes were not expressed in embryonic gonads. Estrogen signaling- and oocyte maturation-associated genes were downregulated in adult ovaries. The morphological changes were associated with altered epigenetic features: increased H2Aub and increased H3K27me3 and decreased H4ac and H3K4me3 in embryonic oocytes. The DNA damage-associated, γH2AX marks were detected in the follicles of treated but not control adult ovaries. We also found reduced H3K4me3 and H4ac in fully grown oocytes of the treated ovaries. The ChIP-seq analysis of H3K4me3 in adult ovaries showed that target genes of ZFP57 and TRIM28, which regulate pluripotency and imprinting, were significantly enriched in altered regions. Our study clearly demonstrates that gestational exposure to a low dose of CD impairs the function of female reproductive system and the changes are associated with altered epigenetic features.

    更新日期:2019-11-28
  • XL-DNase-seq: improved footprinting of dynamic transcription factors
    Epigenet. Chromatin (IF 4.185) Pub Date : 2019-06-04
    Kyu-Seon Oh; Jisu Ha; Songjoon Baek; Myong-Hee Sung

    As the cost of high-throughput sequencing technologies decreases, genome-wide chromatin accessibility profiling methods such as the assay of transposase-accessible chromatin using sequencing (ATAC-seq) are employed widely, with data accumulating at an unprecedented rate. However, accurate inference of protein occupancy requires higher-resolution footprinting analysis where major hurdles exist, including the sequence bias of nucleases and the short-lived chromatin binding of many transcription factors (TFs) with consequent lack of footprints. Here we introduce an assay termed cross-link (XL)-DNase-seq, designed to capture chromatin interactions of dynamic TFs. Mild cross-linking improved the detection of DNase-based footprints of dynamic TFs but interfered with ATAC-based footprinting of the same TFs. XL-DNase-seq may help extract novel gene regulatory circuits involving previously undetectable TFs. The DNase-seq and ATAC-seq data generated in our systematic comparison of various cross-linking conditions also represent an unprecedented-scale resource derived from activated mouse macrophage-like cells which share many features of inflammatory macrophages.

    更新日期:2019-11-28
  • p63 cooperates with CTCF to modulate chromatin architecture in skin keratinocytes
    Epigenet. Chromatin (IF 4.185) Pub Date : 2019-06-04
    Jieqiong Qu; Guoqiang Yi; Huiqing Zhou

    The transcription factor p63 regulates epidermal genes and the enhancer landscape in skin keratinocytes. Its molecular function in controlling the chromatin structure is, however, not yet completely understood. Here, we integrated multi-omics profiles, including the transcriptome, transcription factor DNA-binding and chromatin accessibility, in skin keratinocytes isolated from EEC syndrome patients carrying p63 mutations, to examine the role of p63 in shaping the chromatin architecture. We found decreased chromatin accessibility in p63- and CTCF-bound open chromatin regions that potentially contributed to gene deregulation in mutant keratinocytes. Cooperation of p63 and CTCF seemed to assist chromatin interactions between p63-bound enhancers and gene promoters in skin keratinocytes. Our study suggests an intriguing model where cell type-specific transcription factors such as p63 cooperate with the genome organizer CTCF in the three-dimensional chromatin space to regulate the transcription program important for the proper cell identity.

    更新日期:2019-11-28
  • Phosphorylation of TET2 by AMPK is indispensable in myogenic differentiation
    Epigenet. Chromatin (IF 4.185) Pub Date : 2019-06-04
    Ting Zhang; Xiaowen Guan; Un Lam Choi; Qiang Dong; Melody M. T. Lam; Jianming Zeng; Jun Xiong; Xianju Wang; Terence C. W. Poon; Hongjie Zhang; Xuanjun Zhang; Hailin Wang; Ruiyu Xie; Bing Zhu; Gang Li

    TET-mediated oxidation of 5-mC participates in both passive and active DNA demethylation, which exerts a significant influence on diverse biological processes. Mass spectrometry has identified multiple phosphorylation sites of TET2. However, the functions of these phosphosites and their corresponding kinases are mostly unknown. Here, we showed that AMP-activated protein kinase (AMPK) phosphorylates murine TET2 at the serine residue 97 (S97), and the phosphorylation enhances TET2 stability through promoting its binding to 14-3-3β. AMPK ablation resulted in decreased global 5-hmC levels at the myotube stages, severe differentiation defects of C2C12 cells and significantly, total loss of expression of Pax7. Genome-wide analyses revealed increased DNA methylation at genic and enhancer regions of AMPK-null myoblasts and myotubes. Using CRISPR/Cas9 technology, we showed that a novel enhancer, which is hypermethylated in AMPK-null cells, regulates Pax7 expression. The phospho-mimicking mutant, TET2-S97E, could partly rescue the differentiation defect in AMPK-ablated C2C12 cells. Together, our data demonstrated that AMPK is a critical regulator of myogenesis, partly through phosphorylating TET2.

    更新日期:2019-11-28
  • Robust hematopoietic specification requires the ubiquitous Sp1 and Sp3 transcription factors
    Epigenet. Chromatin (IF 4.185) Pub Date : 2019-06-04
    Jane Gilmour; Leigh O’Connor; Christopher P. Middleton; Peter Keane; Nynke Gillemans; Jean-Baptiste Cazier; Sjaak Philipsen; Constanze Bonifer

    Both tissue-specific and ubiquitously expressed transcription factors, such as Sp-family members, are required for correct development. However, the molecular details of how ubiquitous factors are involved in programming tissue-specific chromatin and thus participate in developmental processes are still unclear. We previously showed that embryonic stem cells lacking Sp1 DNA-binding activity (Sp1ΔDBD/ΔDBD cells) are able to differentiate into early blood progenitors despite the inability of Sp1 to bind chromatin without its DNA-binding domain. However, gene expression during differentiation becomes progressively deregulated, and terminal differentiation is severely compromised. Here, we studied the cooperation of Sp1 with its closest paralogue Sp3 in hematopoietic development and demonstrate that Sp1 and Sp3 binding sites largely overlap. The complete absence of either Sp1 or Sp3 or the presence of the Sp1 DNA-binding mutant has only a minor effect on the pattern of distal accessible chromatin sites and their transcription factor binding motif content, suggesting that these mutations do not affect tissue-specific chromatin programming. Sp3 cooperates with Sp1ΔDBD/ΔDBD to enable hematopoiesis, but is unable to do so in the complete absence of Sp1. Using single-cell gene expression analysis, we show that the lack of Sp1 DNA binding leads to a distortion of cell fate decision timing, indicating that stable chromatin binding of Sp1 is required to maintain robust differentiation trajectories. Our findings highlight the essential contribution of ubiquitous factors such as Sp1 to blood cell development. In contrast to tissue-specific transcription factors which are required to direct specific cell fates, loss of Sp1 leads to a widespread deregulation in timing and coordination of differentiation trajectories during hematopoietic specification.

    更新日期:2019-11-28
  • Human monocyte-to-macrophage differentiation involves highly localized gain and loss of DNA methylation at transcription factor binding sites
    Epigenet. Chromatin (IF 4.185) Pub Date : 2019-06-06
    Koen F. Dekkers; Annette E. Neele; J. Wouter Jukema; Bastiaan T. Heijmans; Menno P. J. de Winther

    Macrophages and their precursors monocytes play a key role in inflammation and chronic inflammatory disorders. Monocyte-to-macrophage differentiation and activation programs are accompanied by significant epigenetic remodeling where DNA methylation associates with cell identity. Here we show that DNA methylation changes characteristic for monocyte-to-macrophage differentiation occur at transcription factor binding sites, and, in contrast to what was previously described, are generally highly localized and encompass both losses and gains of DNA methylation. We compared genome-wide DNA methylation across 440,292 CpG sites between human monocytes, naïve macrophages and macrophages further activated toward a pro-inflammatory state (using LPS/IFNγ), an anti-inflammatory state (IL-4) or foam cells (oxLDL and acLDL). Moreover, we integrated these data with public whole-genome sequencing data on monocytes and macrophages to demarcate differentially methylated regions. Our analysis showed that differential DNA methylation was most pronounced during monocyte-to-macrophage differentiation, was typically restricted to single CpGs or very short regions, and co-localized with lineage-specific enhancers irrespective of whether it concerns gain or loss of methylation. Furthermore, differentially methylated CpGs were located at sites characterized by increased binding of transcription factors known to be involved in monocyte-to-macrophage differentiation including C/EBP and ETS for gain and AP-1 for loss of methylation. Our study highlights the involvement of subtle, yet highly localized remodeling of DNA methylation at regulatory regions in cell differentiation.

    更新日期:2019-11-28
  • Aberrant methylation-mediated downregulation of lncRNA SSTR5-AS1 promotes progression and metastasis of laryngeal squamous cell carcinoma
    Epigenet. Chromatin (IF 4.185) Pub Date : 2019-06-13
    Baoshan Wang; Lei Zhao; Weiwei Chi; Huan Cao; Weina Cui; Wenxia Meng

    Laryngeal squamous cell carcinoma (LSCC) is among the most common malignant tumors with poor prognosis. Accumulating evidences have identified the important roles of long noncoding RNAs (lncRNAs) in the initiation and progression of various cancer types; however, the global lncRNAs expression profile for metastatic LSCC is limited. In the present study, we screen expression profiles of lncRNAs in advanced LSCC patients with paired tumor tissues and corresponding normal tissues by microarrays. We identify numerous differentially expressed transcripts, and after the necessary verification of the transcripts expression in expanded samples, we experimentally validate the expression patterns of the remarkable low expressed gene, SSTR5, and its antisense lncRNA, SSTR5-AS1. Downregulation of SSTR5 is detected in LSCC tissues and laryngeal carcinoma cells. Aberrant DNA hypermethylation of the CpG sites clustered in the exon 1 and accumulation of inactive histone modifications at SSTR5 promoter region may be epigenetic mechanisms for its inactivation in LSCC. SSTR5-AS1 may play antitumor role in LSCC and may be regulated by the hypermethylation of the same CpG sites with SSTR5. SSTR5-AS1 inhibits laryngeal carcinoma cells proliferation, migration, and invasion. SSTR5-AS1 increases the enrichment of MLL3 and H3K4me3 at the promoter region of SSTR5 by interacting with MLL3 and further induces the transcription of SSTR5. Furthermore, SSTR5-AS1 interacts with and recruits TET1 to its target gene E-cadherin to activate its expression. These findings suggest that the identified lncRNAs and mRNAs may be potential biomarkers in metastatic LSCC, and SSTR5-AS1 may act as a tumor suppressor as well as a potential biomarker for antitumor therapy.

    更新日期:2019-11-28
  • Dam mutants provide improved sensitivity and spatial resolution for profiling transcription factor binding
    Epigenet. Chromatin (IF 4.185) Pub Date : 2019-06-13
    Tomasz Szczesnik; Joshua W. K. Ho; Richard Sherwood

    DamID, in which a protein of interest is fused to Dam methylase, enables mapping of protein-DNA binding through readout of adenine methylation in genomic DNA. DamID offers a compelling alternative to chromatin immunoprecipitation sequencing (ChIP-Seq), particularly in cases where cell number or antibody availability is limiting. This comes at a cost, however, of high non-specific signal and a lowered spatial resolution of several kb, limiting its application to transcription factor-DNA binding. Here we show that mutations in Dam, when fused to the transcription factor Tcf7l2, greatly reduce non-specific methylation. Combined with a simplified DamID sequencing protocol, we find that these Dam mutants allow for accurate detection of transcription factor binding at a sensitivity and spatial resolution closely matching that seen in ChIP-seq.

    更新日期:2019-11-28
  • The histone variant H2A.Z in gene regulation
    Epigenet. Chromatin (IF 4.185) Pub Date : 2019-06-14
    Benedetto Daniele Giaimo; Francesca Ferrante; Andreas Herchenröther; Sandra B. Hake; Tilman Borggrefe

    The histone variant H2A.Z is involved in several processes such as transcriptional control, DNA repair, regulation of centromeric heterochromatin and, not surprisingly, is implicated in diseases such as cancer. Here, we review the recent developments on H2A.Z focusing on its role in transcriptional activation and repression. H2A.Z, as a replication-independent histone, has been studied in several model organisms and inducible mammalian model systems. Its loading machinery and several modifying enzymes have been recently identified, and some of the long-standing discrepancies in transcriptional activation and/or repression are about to be resolved. The buffering functions of H2A.Z, as supported by genome-wide localization and analyzed in several dynamic systems, are an excellent example of transcriptional control. Posttranslational modifications such as acetylation and ubiquitination of H2A.Z, as well as its specific binding partners, are in our view central players in the control of gene expression. Understanding the key-mechanisms in either turnover or stabilization of H2A.Z-containing nucleosomes as well as defining the H2A.Z interactome will pave the way for therapeutic applications in the future.

    更新日期:2019-11-28
  • PRDM14 controls X-chromosomal and global epigenetic reprogramming of H3K27me3 in migrating mouse primordial germ cells
    Epigenet. Chromatin (IF 4.185) Pub Date : 2019-06-20
    Anna Mallol; Maria Guirola; Bernhard Payer

    In order to prepare the genome for gametogenesis, primordial germ cells (PGCs) undergo extensive epigenetic reprogramming during migration toward the gonads in mammalian embryos. This includes changes on a genome-wide scale and additionally in females the remodeling of the inactive X-chromosome to enable X-chromosome reactivation (XCR). However, if global remodeling and X-chromosomal remodeling are related, how they occur in PGCs in vivo in relation to their migration progress and which factors are important are unknown. Here we identify the germ cell determinant PR-domain containing protein 14 (PRDM14) as the first known factor that is instrumental for both global reprogramming and X-chromosomal reprogramming in migrating mouse PGCs. We find that global upregulation of the repressive histone H3 lysine 27 trimethylation (H3K27me3) mark is PRDM14 dosage dependent in PGCs of both sexes. When focusing on XCR, we observed that PRDM14 is required for removal of H3K27me3 from the inactive X-chromosome, which, in contrast to global upregulation, takes place progressively along the PGC migration path. Furthermore, we show that global and X-chromosomal reprogramming of H3K27me3 are functionally separable, despite their common regulation by PRDM14. In summary, here we provide new insight and spatiotemporal resolution to the progression and regulation of epigenome remodeling along mouse PGC migration in vivo and link epigenetic reprogramming to its developmental context.

    更新日期:2019-11-28
  • BRD4 bimodal binding at promoters and drug-induced displacement at Pol II pause sites associates with I-BET sensitivity
    Epigenet. Chromatin (IF 4.185) Pub Date : 2019-07-02
    P. Khoueiry; A. Ward Gahlawat; M. Petretich; A. M. Michon; D. Simola; E. Lam; E. E. Furlong; V. Benes; M. A. Dawson; R. K. Prinjha; G. Drewes; P. Grandi

    Deregulated transcription is a major driver of diseases such as cancer. Bromodomain and extra-terminal (BET) proteins (BRD2, BRD3, BRD4 and BRDT) are chromatin readers essential for maintaining proper gene transcription by specifically binding acetylated lysine residues. Targeted displacement of BET proteins from chromatin, using BET inhibitors (I-BETs), is a promising therapy, especially for acute myeloid leukemia (AML), and evaluation of resistance mechanisms is necessary to optimize the clinical efficacy of these drugs. To uncover mechanisms of intrinsic I-BET resistance, we quantified chromatin binding and displacement for BRD2, BRD3 and BRD4 after dose response treatment with I-BET151, in sensitive and resistant in vitro models of leukemia, and mapped BET proteins/I-BET interactions genome wide using antibody- and compound-affinity capture methods followed by deep sequencing. The genome-wide map of BET proteins sensitivity to I-BET revealed a bimodal pattern of binding flanking transcription start sites (TSSs), in which drug-mediated displacement from chromatin primarily affects BRD4 downstream of the TSS and prolongs the pausing of RNA Pol II. Correlation of BRD4 binding and drug-mediated displacement at RNA Pol II pause sites with gene expression revealed a differential behavior of sensitive and resistant tumor cells to I-BET and identified a BRD4 signature at promoters of sensitive coding and non-coding genes. We provide evidence that I-BET-induced shift of Pol II pausing at promoters via displacement of BRD4 is a determinant of intrinsic I-BET sensitivity. This finding may guide pharmacological treatment to enhance the clinical utility of such targeted therapies in AML and potentially other BET proteins-driven diseases.

    更新日期:2019-11-28
  • H3K4me2 functions as a repressive epigenetic mark in plants
    Epigenet. Chromatin (IF 4.185) Pub Date : 2019-07-02
    Yuhao Liu; Kunpeng Liu; Liufan Yin; Yu Yu; Ji Qi; Wen-Hui Shen; Jun Zhu; Yijing Zhang; Aiwu Dong

    In animals, H3K4me2 and H3K4me3 are enriched at the transcription start site (TSS) and function as epigenetic marks that regulate gene transcription, but their functions in plants have not been fully characterized. We used chromatin immunoprecipitation sequencing to analyze the rice genome-wide changes to H3K4me1/H3K4me2/H3K4me3 following the loss of an H3K4-specific methyltransferase, SDG701. The knockdown of SDG701 resulted in a global decrease in H3K4me2/H3K4me3 levels throughout the rice genome. An RNA-sequencing analysis revealed that many genes related to diverse developmental processes were misregulated in the SDG701 knockdown mutant. In rice, H3K4me3 and H3K36me3 are positively correlated with gene transcription; however, surprisingly, the H3K4me2 level was negatively associated with gene transcription levels. Furthermore, the H3K4me3 level at the TSS region decreased significantly in the genes that exhibited down-regulated expression in the SDG701 knockdown mutant. In contrast, the genes with up-regulated expression in the mutant were associated with a considerable decrease in H3K4me2 levels over the gene body region. A comparison of the genome-wide distributions of H3K4me2 in eukaryotes indicated that the H3K4me2 level is not correlated with the gene transcription level in yeast, but is positively and negatively correlated with gene expression in animals and plants, respectively. Our results uncovered H3K4me2 as a novel repressive mark in plants.

    更新日期:2019-11-28
  • MYC deregulates TET1 and TET2 expression to control global DNA (hydroxy)methylation and gene expression to maintain a neoplastic phenotype in T-ALL
    Epigenet. Chromatin (IF 4.185) Pub Date : 2019-07-02
    Candace J. Poole; Atul Lodh; Jeong-Hyeon Choi; Jan van Riggelen

    While aberrant DNA methylation is a characteristic feature of tumor cells, our knowledge of how these DNA methylation patterns are established and maintained is limited. DNA methyltransferases and ten-eleven translocation methylcytosine dioxygenases (TETs) function has been found altered in a variety of cancer types. Here, we report that in T cell acute lymphoblastic leukemia (T-ALL) the MYC oncogene controls the expression of TET1 and TET2 to maintain 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) patterns, which is associated with tumor cell-specific gene expression. We found that cellular senescence and tumor regression upon MYC inactivation in T-ALL was associated with genome-wide changes in 5mC and 5hmC patterns. Correlating with the changes in DNA (hydroxy)methylation, we found that T-ALL overexpress TET1, while suppressing TET2 in a MYC-dependent fashion. Consequently, MYC inactivation led to an inverse expression pattern, decreasing TET1, while increasing TET2 levels. Knockdown of TET1 or ectopic expression of TET2 in T-ALL was associated with genome-wide changes in 5mC and 5hmC enrichment and decreased cell proliferation, suggesting a tumor promoting function of TET1, and a tumor suppressing role for TET2. Among the genes and pathways controlled by TET1, we found ribosomal biogenesis and translational control of protein synthesis highly enriched. Our finding that MYC directly deregulates the expression of TET1 and TET2 in T-ALL provides novel evidence that MYC controls DNA (hydroxy)methylation in a genome-wide fashion. It reveals a coordinated interplay between the components of the DNA (de)methylating machinery that contribute to MYC-driven tumor maintenance, highlighting the potential of specific TET enzymes for therapeutic strategies.

    更新日期:2019-11-28
  • Peak calling by Sparse Enrichment Analysis for CUT&RUN chromatin profiling
    Epigenet. Chromatin (IF 4.185) Pub Date : 2019-07-12
    Michael P. Meers; Dan Tenenbaum; Steven Henikoff

    CUT&RUN is an efficient epigenome profiling method that identifies sites of DNA binding protein enrichment genome-wide with high signal to noise and low sequencing requirements. Currently, the analysis of CUT&RUN data is complicated by its exceptionally low background, which renders programs designed for analysis of ChIP-seq data vulnerable to oversensitivity in identifying sites of protein binding. Here we introduce Sparse Enrichment Analysis for CUT&RUN (SEACR), an analysis strategy that uses the global distribution of background signal to calibrate a simple threshold for peak calling. SEACR discriminates between true and false-positive peaks with near-perfect specificity from “gold standard” CUT&RUN datasets and efficiently identifies enriched regions for several different protein targets. We also introduce a web server ( http://seacr.fredhutch.org ) for plug-and-play analysis with SEACR that facilitates maximum accessibility across users of all skill levels. SEACR is a highly selective peak caller that definitively validates the accuracy of CUT&RUN for datasets with known true negatives. Its ease of use and performance in comparison with existing peak calling strategies make it an ideal choice for analyzing CUT&RUN data.

    更新日期:2019-11-28
  • Disruption of nuclear speckles reduces chromatin interactions in active compartments
    Epigenet. Chromatin (IF 4.185) Pub Date : 2019-07-17
    Shibin Hu; Pin Lv; Zixiang Yan; Bo Wen

    Nuclei of eukaryotes contain various higher-order chromatin architectures and nuclear bodies (NBs), which are critical for proper nuclear functions. Recent studies showed that active chromatin regions are associated with nuclear speckles (NSs), a type of NBs involved in RNA processing. However, the functional roles of NSs in 3D genome organization remain unclear. Using mouse hepatocytes as the model, we knocked down SRRM2, a core protein component scaffolding NSs, and performed Hi-C experiments to examine genome-wide chromatin interactions. We found that Srrm2 depletion disrupted the NSs and changed the expression of 1282 genes. The intra-chromosomal interactions were decreased in type A (active) compartments and increased in type B (repressive) compartments. Furthermore, upon Srrm2 knockdown, the insulation of TADs was decreased specifically in active compartments, and the most significant reduction occurred in A1 sub-compartments. Interestingly, the change of intra-TAD chromatin interactions upon Srrm2 depletion was not associated with the alteration of gene expression. We show that disruption of NSs by Srrm2 knockdown causes a global decrease in chromatin interactions in active compartments, indicating critical functions of NSs in the organization of the 3D genome.

    更新日期:2019-11-28
  • Hyperinsulinemia promotes aberrant histone acetylation in triple-negative breast cancer
    Epigenet. Chromatin (IF 4.185) Pub Date : 2019-07-17
    Parijat Senapati; Hiroyuki Kato; Michael Lee; Amy Leung; Christine Thai; Angelica Sanchez; Emily J. Gallagher; Derek LeRoith; Victoria L. Seewaldt; David K. Ann; Dustin E. Schones

    Hyperinsulinemia, the presence of excess insulin relative to glucose in the blood, is considered to be a poor prognostic indicator for patients with triple-negative breast cancer (TNBC). mTOR, a downstream effector of insulin, enhances mitochondrial biogenesis and activity, thereby increasing acetyl-CoA precursors. Increased acetyl-CoA can, in turn, be utilized by nuclear acetyltransferases for histone acetylation, a critical feature of genome regulation. While signaling pathways downstream of insulin have been established for sometime, the effect of insulin on chromatin remains unclear. We hypothesized that hyperinsulinemia-induced metabolic changes lead to genome-wide changes in histone acetylation in TNBC. MDA-MB-231 cells were xenografted into hyperinsulinemic and wild-type mice. Tumors in the hyperinsulinemic mice displayed elevated levels of histone acetylation compared to tumors in normal insulin conditions. We show that insulin treatment in vitro leads to global increase in chromatin-associated histone acetylation, in particular at H3K9, through the PI3K/AKT/mTOR pathway. Genome-wide analyses revealed that most promoter regions have an increase in histone acetylation upon insulin treatment. In addition, insulin induces higher levels of reactive oxygen species and DNA damage foci in cells. These results demonstrate the impact of hyperinsulinemia on altered gene regulation through chromatin and the importance of targeting hyperinsulinemia-induced processes that lead to chromatin dysfunction in TNBC.

    更新日期:2019-11-28
  • Leo1 is essential for the dynamic regulation of heterochromatin and gene expression during cellular quiescence
    Epigenet. Chromatin (IF 4.185) Pub Date : 2019-07-17
    Eriko Oya; Mickaël Durand-Dubief; Adiel Cohen; Vladimir Maksimov; Catherine Schurra; Jun-ichi Nakayama; Ronit Weisman; Benoit Arcangioli; Karl Ekwall

    Cellular quiescence is a reversible differentiation state during which cells modify their gene expression program to inhibit metabolic functions and adapt to a new cellular environment. The epigenetic changes accompanying these alterations are not well understood. We used fission yeast cells as a model to study the regulation of quiescence. When these cells are starved for nitrogen, the cell cycle is arrested in G1, and the cells enter quiescence (G0). A gene regulatory program is initiated, including downregulation of thousands of genes—for example, those related to cell proliferation—and upregulation of specific genes—for example, autophagy genes—needed to adapt to the physiological challenge. These changes in gene expression are accompanied by a marked alteration of nuclear organization and chromatin structure. Here, we investigated the role of Leo1, a subunit of the conserved RNA polymerase-associated factor 1 (Paf1) complex, in the quiescence process using fission yeast as the model organism. Heterochromatic regions became very dynamic in fission yeast in G0 during nitrogen starvation. The reduction of heterochromatin in early G0 was correlated with reduced target of rapamycin complex 2 (TORC2) signaling. We demonstrated that cells lacking Leo1 show reduced survival in G0. In these cells, heterochromatic regions, including subtelomeres, were stabilized, and the expression of many genes, including membrane transport genes, was abrogated. TOR inhibition mimics the effect of nitrogen starvation, leading to the expression of subtelomeric genes, and this effect was suppressed by genetic deletion of leo1. We identified a protein, Leo1, necessary for survival during quiescence. Leo1 is part of a conserved protein complex, Paf1C, linked to RNA polymerase II. We showed that Leo1, acting downstream of TOR, is crucial for the dynamic reorganization of chromosomes and the regulation of gene expression during cellular quiescence. Genes encoding membrane transporters are not expressed in quiescent leo1 mutant cells, and cells die after 2 weeks of nitrogen starvation. Taken together, our results suggest that Leo1 is essential for the dynamic regulation of heterochromatin and gene expression during cellular quiescence.

    更新日期:2019-11-28
  • Interplay between FACT subunit SPT16 and TRIM33 can remodel chromatin at macrophage distal regulatory elements
    Epigenet. Chromatin (IF 4.185) Pub Date : 2019-07-22
    Federica Ferri; Vanessa Petit; Vilma Barroca; Paul-Henri Romeo

    Cell type-specific use of cis-acting regulatory elements is mediated by the combinatorial activity of transcription factors involved in lineage determination and maintenance of cell identity. In macrophages, specific transcriptional programs are dictated by the transcription factor PU.1 that primes distal regulatory elements for macrophage identities and makes chromatin competent for activity of stimuli-dependent transcription factors. Although the advances in genome-wide approaches have elucidated the functions of these macrophage-specific distal regulatory elements in transcriptional responses, chromatin structures associated with PU.1 priming and the underlying mechanisms of action of these cis-acting sequences are not characterized. Here, we show that, in macrophages, FACT subunit SPT16 can bind to positioned nucleosomes directly flanking PU.1-bound sites at previously uncharacterized distal regulatory elements located near genes essential for macrophage development and functions. SPT16 can interact with the transcriptional co-regulator TRIM33 and binds to half of these sites in a TRIM33-dependent manner. Using the Atp1b3 locus as a model, we show that FACT binds to two positioned nucleosomes surrounding a TRIM33/PU.1-bound site in a region, located 35 kb upstream the Atp1b3 TSS, that interact with the Atp1b3 promoter. At this − 35 kb region, TRIM33 deficiency leads to FACT release, loss of the two positioned nucleosomes, RNA Pol II recruitment and bidirectional transcription. These modifications are associated with higher levels of FACT binding at the Atp1b3 promoter, an increase of RNA Pol II recruitment and an increased expression of Atp1b3 in Trim33−/− macrophages. Thus, sequestering of SPT16/FACT by TRIM33 at PU.1-bound distal regions might represent a new regulatory mechanism for RNA Pol II recruitment and transcription output in macrophages.

    更新日期:2019-11-28
  • Crosstalk between chromatin structure, cohesin activity and transcription
    Epigenet. Chromatin (IF 4.185) Pub Date : 2019-07-22
    Douglas Maya-Miles; Eloísa Andújar; Mónica Pérez-Alegre; Marina Murillo-Pineda; Marta Barrientos-Moreno; María J. Cabello-Lobato; Elena Gómez-Marín; Macarena Morillo-Huesca; Félix Prado

    A complex interplay between chromatin and topological machineries is critical for genome architecture and function. However, little is known about these reciprocal interactions, even for cohesin, despite its multiple roles in DNA metabolism. We have used genome-wide analyses to address how cohesins and chromatin structure impact each other in yeast. Cohesin inactivation in scc1-73 mutants during the S and G2 phases causes specific changes in chromatin structure that preferentially take place at promoters; these changes include a significant increase in the occupancy of the − 1 and + 1 nucleosomes. In addition, cohesins play a major role in transcription regulation that is associated with specific promoter chromatin architecture. In scc1-73 cells, downregulated genes are enriched in promoters with short or no nucleosome-free region (NFR) and a fragile “nucleosome − 1/RSC complex” particle. These results, together with a preferential increase in the occupancy of nucleosome − 1 of these genes, suggest that cohesins promote transcription activation by helping RSC to form the NFR. In sharp contrast, the scc1-73 upregulated genes are enriched in promoters with an “open” chromatin structure and are mostly at cohesin-enriched regions, suggesting that a local accumulation of cohesins might help to inhibit transcription. On the other hand, a dramatic loss of chromatin integrity by histone depletion during DNA replication has a moderate effect on the accumulation and distribution of cohesin peaks along the genome. Our analyses of the interplay between chromatin integrity and cohesin activity suggest that cohesins play a major role in transcription regulation, which is associated with specific chromatin architecture and cohesin-mediated nucleosome alterations of the regulated promoters. In contrast, chromatin integrity plays only a minor role in the binding and distribution of cohesins.

    更新日期:2019-11-28
  • Identification of a pituitary ERα-activated enhancer triggering the expression of Nr5a1, the earliest gonadotrope lineage-specific transcription factor
    Epigenet. Chromatin (IF 4.185) Pub Date : 2019-08-07
    Vincent Pacini; Florence Petit; Bruno Querat; Jean-Noël Laverriere; Joëlle Cohen-Tannoudji; David L’hôte

    Gonadotrope lineage differentiation is a stepwise process taking place during pituitary development. The early step of gonadotrope lineage specification is characterized by the expression of the Nr5a1 transcription factor, a crucial factor for gonadotrope cell fate determination. Abnormalities affecting Nr5a1 expression lead to hypogonadotropic hypogonadism and infertility. Although significant knowledge has been gained on the signaling and transcriptional events controlling gonadotrope differentiation, epigenetic mechanisms regulating Nr5a1 expression during early gonadotrope lineage specification are still poorly understood. Using ATAC chromatin accessibility analyses on three cell lines recapitulating gradual stages of gonadotrope differentiation and in vivo on developing pituitaries, we demonstrate that a yet undescribed enhancer is transiently recruited during gonadotrope specification. Using CRISPR/Cas9, we show that this enhancer is mandatory for the emergence of Nr5a1 during gonadotrope specification. Furthermore, we identify a highly conserved estrogen-binding element and demonstrate that the enhancer activation is dependent upon estrogen acting through ERα. Lastly, we provide evidence that binding of ERα is crucial for chromatin remodeling of Nr5a1 enhancer and promoter, leading to RNA polymerase recruitment and transcription. This study identifies the earliest regulatory sequence involved in gonadotrope lineage specification and highlights the key epigenetic role played by ERα in this differentiation process.

    更新日期:2019-11-28
  • ZFP57 regulation of transposable elements and gene expression within and beyond imprinted domains
    Epigenet. Chromatin (IF 4.185) Pub Date : 2019-08-09
    Hui Shi; Ruslan Strogantsev; Nozomi Takahashi; Anastasiya Kazachenka; Matthew C. Lorincz; Myriam Hemberger; Anne C. Ferguson-Smith

    KRAB zinc finger proteins (KZFPs) represent one of the largest families of DNA-binding proteins in vertebrate genomes and appear to have evolved to silence transposable elements (TEs) including endogenous retroviruses through sequence-specific targeting of repressive chromatin states. ZFP57 is required to maintain the post-fertilization DNA methylation memory of parental origin at genomic imprints. Here we conduct RNA-seq and ChIP-seq analyses in normal and ZFP57 mutant mouse ES cells to understand the relative importance of ZFP57 at imprints, unique and repetitive regions of the genome. Over 80% of ZFP57 targets are TEs, however, ZFP57 is not essential for their repression. The remaining targets lie within unique imprinted and non-imprinted sequences. Though the loss of ZFP57 influences imprinted genes as expected, the majority of unique gene targets lose H3K9me3 with little effect on DNA methylation and very few exhibit alterations in expression. Comparison of ZFP57 mutants with DNA methyltransferase-deleted ES cells (TKO) identifies a remarkably similar pattern of H3K9me3 loss across the genome. These data define regions where H3K9me3 is secondary to DNA methylation and we propose that ZFP57 is the principal if not sole methylation-sensitive KZFP in mouse ES cells. Finally, we examine dynamics of DNA and H3K9 methylation during pre-implantation development and show that sites bound by ZFP57 in ES cells maintain DNA methylation and H3K9me3 at imprints and at non-imprinted regions on the maternally inherited chromosome throughout preimplantation development. Our analyses suggest the evolution of a rare DNA methylation-sensitive KZFP that is not essential for repeat silencing, but whose primary function is to maintain DNA methylation and repressive histone marks at germline-derived imprinting control regions.

    更新日期:2019-11-28
  • MEK inhibition remodels the active chromatin landscape and induces SOX10 genomic recruitment in BRAF(V600E) mutant melanoma cells
    Epigenet. Chromatin (IF 4.185) Pub Date : 2019-08-09
    Temesgen D. Fufa; Laura L. Baxter; Julia C. Wedel; Derek E. Gildea; Stacie K. Loftus; William J. Pavan

    The MAPK/ERK signaling pathway is an essential regulator of numerous cell processes that are crucial for normal development as well as cancer progression. While much is known regarding MAPK/ERK signal conveyance from the cell membrane to the nucleus, the transcriptional and epigenetic mechanisms that govern gene expression downstream of MAPK signaling are not fully elucidated. This study employed an integrated epigenome analysis approach to interrogate the effects of MAPK/ERK pathway inhibition on the global transcriptome, the active chromatin landscape, and protein–DNA interactions in 501mel melanoma cells. Treatment of these cells with the small-molecule MEK inhibitor AZD6244 induces hyperpigmentation, widespread gene expression changes including alteration of genes linked to pigmentation, and extensive epigenomic reprogramming of transcriptionally distinct regulatory regions associated with the active chromatin mark H3K27ac. Regulatory regions with differentially acetylated H3K27ac regions following AZD6244 treatment are enriched in transcription factor binding motifs of ETV/ETS and ATF family members as well as the lineage-determining factors MITF and SOX10. H3K27ac-dense enhancer clusters known as super-enhancers show similar transcription factor motif enrichment, and furthermore, these super-enhancers are associated with genes encoding MITF, SOX10, and ETV/ETS proteins. Along with genome-wide resetting of the active enhancer landscape, MEK inhibition also results in widespread SOX10 recruitment throughout the genome, including increased SOX10 binding density at H3K27ac-marked enhancers. Importantly, these MEK inhibitor-responsive enhancers marked by H3K27ac and occupied by SOX10 are located near melanocyte lineage-specific and pigmentation genes and overlap numerous human SNPs associated with pigmentation and melanoma phenotypes, highlighting the variants located within these regions for prioritization in future studies. These results reveal the epigenetic reprogramming underlying the re-activation of melanocyte pigmentation and developmental transcriptional programs in 501mel cells in response to MEK inhibition and suggest extensive involvement of a MEK-SOX10 axis in the regulation of these processes. The dynamic chromatin changes identified here provide a rich genomic resource for further analyses of the molecular mechanisms governing the MAPK pathway in pigmentation- and melanocyte-associated diseases.

    更新日期:2019-11-28
  • Accurate ethnicity prediction from placental DNA methylation data
    Epigenet. Chromatin (IF 4.185) Pub Date : 2019-08-09
    Victor Yuan; E. Magda Price; Giulia Del Gobbo; Sara Mostafavi; Brian Cox; Alexandra M. Binder; Karin B. Michels; Carmen Marsit; Wendy P. Robinson

    The influence of genetics on variation in DNA methylation (DNAme) is well documented. Yet confounding from population stratification is often unaccounted for in DNAme association studies. Existing approaches to address confounding by population stratification using DNAme data may not generalize to populations or tissues outside those in which they were developed. To aid future placental DNAme studies in assessing population stratification, we developed an ethnicity classifier, PlaNET (Placental DNAme Elastic Net Ethnicity Tool), using five cohorts with Infinium Human Methylation 450k BeadChip array (HM450k) data from placental samples that is also compatible with the newer EPIC platform. Data from 509 placental samples were used to develop PlaNET and show that it accurately predicts (accuracy = 0.938, kappa = 0.823) major classes of self-reported ethnicity/race (African: n = 58, Asian: n = 53, Caucasian: n = 389), and produces ethnicity probabilities that are highly correlated with genetic ancestry inferred from genome-wide SNP arrays (> 2.5 million SNP) and ancestry informative markers (n = 50 SNPs). PlaNET’s ethnicity classification relies on 1860 HM450K microarray sites, and over half of these were linked to nearby genetic polymorphisms (n = 955). Our placental-optimized method outperforms existing approaches in assessing population stratification in placental samples from individuals of Asian, African, and Caucasian ethnicities. PlaNET provides an improved approach to address population stratification in placental DNAme association studies. The method can be applied to predict ethnicity as a discrete or continuous variable and will be especially useful when self-reported ethnicity information is missing and genotyping markers are unavailable.

    更新日期:2019-11-28
  • The DNA repair protein SHPRH is a nucleosome-stimulated ATPase and a nucleosome-E3 ubiquitin ligase
    Epigenet. Chromatin (IF 4.185) Pub Date : 2019-08-21
    Joanna Brühl; Jonathan Trautwein; Agnes Schäfer; Uwe Linne; Karim Bouazoune

    Maintenance of genome integrity during DNA replication is crucial to the perpetuation of all organisms. In eukaryotes, the bypass of DNA lesions by the replication machinery prevents prolonged stalling of the replication fork, which could otherwise lead to greater damages such as gross chromosomal rearrangements. Bypassing DNA lesions and subsequent repair are accomplished by the activation of DNA damage tolerance pathways such as the template switching (TS) pathway. In yeast, the RAD5 (Radiation-sensitive 5) protein plays a crucial role in initiating the TS pathway by catalyzing the polyubiquitination of PCNA (Proliferation Cell Nuclear Antigen). Likewise, one of the mammalian RAD5-homologs, SHPRH (SNF2, histone linker, PHD, RING, helicase) mediates PCNA polyubiquitination. To date, the study of SHPRH enzymatic functions has been limited to this modification. It is therefore unclear how SHPRH carries out its function in DNA repair. Moreover, how this protein regulates gene transcription at the enzymatic level is also unknown. Given that SHPRH harbors domains found in chromatin remodeling proteins, we investigated its biochemical properties in the presence of nucleosomal substrates. We find that SHPRH binds equally well to double-stranded (ds) DNA and to nucleosome core particles, however, like ISWI and CHD-family remodelers, SHPRH shows a strong preference for nucleosomes presenting extranucleosomal DNA. Moreover, nucleosomes but not dsDNA strongly stimulate the ATPase activity of SHPRH. Intriguingly, unlike typically observed with SNF2-family enzymes, ATPase activity does not translate into conventional nucleosome remodeling, under standard assay conditions. To test whether SHPRH can act as a ubiquitin E3 ligase for nucleosomes, we performed a screen using 26 E2-conjugating enzymes. We uncover that SHPRH is a potent nucleosome E3-ubiquitin-ligase that can function with at least 7 different E2s. Mass spectrometry analyses of products generated in the presence of the UBE2D1-conjugating enzyme reveal that SHPRH can catalyze the formation of polyubiquitin linkages that are either branched or associated with the recruitment of DNA repair factors, as well as linkages involved in proteasomal degradation. We propose that, in addition to polyubiquitinating PCNA, SHPRH promotes DNA repair or transcriptional regulation in part through chromatin ubiquitination. Our study sets a biochemical framework for studying other RAD5- and RAD16-related protein functions through the ubiquitination of nucleosomes.

    更新日期:2019-11-28
  • TH2BS11ph histone mark is enriched in the unsynapsed axes of the XY body and predominantly associates with H3K4me3-containing genomic regions in mammalian spermatocytes
    Epigenet. Chromatin (IF 4.185) Pub Date : 2019-09-07
    Iyer Aditya Mahadevan; Satyakrishna Pentakota; Raktim Roy; Utsa Bhaduri; Manchanahalli R. Satyanarayana Rao

    TH2B is a major histone variant that replaces about 80–85% of somatic H2B in mammalian spermatocytes and spermatids. The post-translational modifications (PTMs) on TH2B have been well characterised in spermatocytes and spermatids. However, the biological function(s) of these PTMs on TH2B have not been deciphered in great detail. In our attempt to decipher the unique function(s) of histone variant TH2B, we detected the modification in the N-terminal tail, Serine 11 phosphorylation on TH2B (TH2BS11ph) in spermatocytes. The current study is aimed at understanding the function of the TH2BS11ph modification in the context of processes that occur during meiotic prophase I. Immunofluorescence studies with the highly specific antibodies revealed that TH2BS11ph histone mark is enriched in the unsynapsed axes of the sex body and is associated with XY body-associated proteins like Scp3, γH2AX, pATM, ATR, etc. Genome-wide occupancy studies as determined by ChIP sequencing experiments in P20 C57BL6 mouse testicular cells revealed that TH2BS11ph is enriched in X and Y chromosomes confirming the immunofluorescence staining pattern in the pachytene spermatocytes. Apart from the localisation of this modification in the XY body, TH2BS11ph is majorly associated with H3K4me3-containing genomic regions like gene promoters, etc. These data were also found to corroborate with the ChIP sequencing data of TH2BS11ph histone mark carried out in P12 C57BL6 mouse testicular cells, wherein we found the predominant localisation of this modification at H3K4me3-containing genomic regions. Mass spectrometry analysis of proteins that associate with TH2BS11ph-containing mononucleosomes revealed key proteins linked with the functions of XY body, pericentric heterochromatin and transcription. TH2BS11ph modification is densely localised in the unsynapsed axes of the XY body of the pachytene spermatocyte. By ChIP sequencing studies in mouse P12 and P20 testicular cells, we demonstrate that TH2BS11ph is predominantly associated with H3K4me3 positive genomic regions like gene promoters, etc. We propose that TH2BS11ph modification could act alone or in concert with other histone modifications to recruit the appropriate transcription or XY body recombination protein machinery at specific genomic loci.

    更新日期:2019-11-28
  • Detailed insight into the dynamics of the initial phases of de novo RNA-directed DNA methylation in plant cells
    Epigenet. Chromatin (IF 4.185) Pub Date : 2019-09-11
    Adéla Přibylová; Vojtěch Čermák; Dimitrij Tyč; Lukáš Fischer

    Methylation of cytosines is an evolutionarily conserved epigenetic mark that is essential for the control of chromatin activity in many taxa. It acts mainly repressively, causing transcriptional gene silencing. In plants, de novo DNA methylation is established mainly by RNA-directed DNA-methylation pathway. Even though the protein machinery involved is relatively well-described, the course of the initial phases remains covert. We show the first detailed description of de novo DNA-methylation dynamics. Since prevalent plant model systems do not provide the possibility to collect homogenously responding material in time series with short intervals, we developed a convenient system based on tobacco BY-2 cell lines with inducible production of siRNAs (from an RNA hairpin) guiding the methylation machinery to the CaMV 35S promoter controlling GFP reporter. These lines responded very synchronously, and a high level of promoter-specific siRNAs triggered rapid promoter methylation with the first increase observed already 12 h after the induction. The previous presence of CG methylation in the promoter did not affect the methylation dynamics. The individual cytosine contexts reacted differently. CHH methylation peaked at about 80% in 2 days and then declined, whereas CG and CHG methylation needed more time with CHG reaching practically 100% after 10 days. Spreading of methylation was only minimal outside the target region in accordance with the absence of transitive siRNAs. The low and stable proportion of 24-nt siRNAs suggested that Pol IV was not involved in the initial phases. Our results show that de novo DNA methylation is a rapid process initiated practically immediately with the appearance of promoter-specific siRNAs and independently of the prior presence of methylcytosines at the target locus. The methylation was precisely targeted, and its dynamics varied depending on the cytosine sequence context. The progressively increasing methylation resulted in a smooth, gradual inhibition of the promoter activity, which was entirely suppressed in 2 days.

    更新日期:2019-11-28
  • Genome-wide RNAi screen in Drosophila reveals Enok as a novel trithorax group regulator
    Epigenet. Chromatin (IF 4.185) Pub Date : 2019-09-23
    Zain Umer; Jawad Akhtar; Muhammad Haider Farooq Khan; Najma Shaheen; Muhammad Abdul Haseeb; Khalida Mazhar; Aziz Mithani; Saima Anwar; Muhammad Tariq

    Polycomb group (PcG) and trithorax group (trxG) proteins contribute to the specialization of cell types by maintaining differential gene expression patterns. Initially discovered as positive regulators of HOX genes in forward genetic screens, trxG counteracts PcG-mediated repression of cell type-specific genes. Despite decades of extensive analysis, molecular understanding of trxG action and regulation are still punctuated by many unknowns. This study aimed at discovering novel factors that elicit an anti-silencing effect to facilitate trxG-mediated gene activation. We have developed a cell-based reporter system and performed a genome-wide RNAi screen to discover novel factors involved in trxG-mediated gene regulation in Drosophila. We identified more than 200 genes affecting the reporter in a manner similar to trxG genes. From the list of top candidates, we have characterized Enoki mushroom (Enok), a known histone acetyltransferase, as an important regulator of trxG in Drosophila. Mutants of enok strongly suppressed extra sex comb phenotype of Pc mutants and enhanced homeotic transformations associated with trx mutations. Enok colocalizes with both TRX and PC at chromatin. Moreover, depletion of Enok specifically resulted in an increased enrichment of PC and consequently silencing of trxG targets. This downregulation of trxG targets was also accompanied by a decreased occupancy of RNA-Pol-II in the gene body, correlating with an increased stalling at the transcription start sites of these genes. We propose that Enok facilitates trxG-mediated maintenance of gene activation by specifically counteracting PcG-mediated repression. Our ex vivo approach led to identification of new trxG candidate genes that warrant further investigation. Presence of chromatin modifiers as well as known members of trxG and their interactors in the genome-wide RNAi screen validated our reverse genetics approach. Genetic and molecular characterization of Enok revealed a hitherto unknown interplay between Enok and PcG/trxG system. We conclude that histone acetylation by Enok positively impacts the maintenance of trxG-regulated gene activation by inhibiting PRC1-mediated transcriptional repression.

    更新日期:2019-11-28
  • Histone chaperones and the Rrm3p helicase regulate flocculation in S. cerevisiae
    Epigenet. Chromatin (IF 4.185) Pub Date : 2019-09-23
    Hollie Rowlands; Kholoud Shaban; Barret Foster; Yannic Proteau; Krassimir Yankulov

    Biofilm formation or flocculation is a major phenotype in wild type budding yeasts but rarely seen in laboratory yeast strains. Here, we analysed flocculation phenotypes and the expression of FLO genes in laboratory strains with various genetic backgrounds. We show that mutations in histone chaperones, the helicase RRM3 and the Histone Deacetylase HDA1 de-repress the FLO genes and partially reconstitute flocculation. We demonstrate that the loss of repression correlates to elevated expression of several FLO genes, to increased acetylation of histones at the promoter of FLO1 and to variegated expression of FLO11. We show that these effects are related to the activity of CAF-1 at the replication forks. We also demonstrate that nitrogen starvation or inhibition of histone deacetylases do not produce flocculation in W303 and BY4742 strains but do so in strains compromised for chromatin maintenance. Finally, we correlate the de-repression of FLO genes to the loss of silencing at the subtelomeric and mating type gene loci. We conclude that the deregulation of chromatin maintenance and transmission is sufficient to reconstitute flocculation in laboratory yeast strains. Consequently, we propose that a gain in epigenetic silencing is a major contributing factor for the loss of flocculation phenotypes in these strains. We suggest that flocculation in yeasts provides an excellent model for addressing the challenging issue of how epigenetic mechanisms contribute to evolution.

    更新日期:2019-11-28
  • CGGBP1 regulates CTCF occupancy at repeats
    Epigenet. Chromatin (IF 4.185) Pub Date : 2019-09-23
    Divyesh Patel; Manthan Patel; Subhamoy Datta; Umashankar Singh

    CGGBP1 is a repeat-binding protein with diverse functions in the regulation of gene expression, cytosine methylation, repeat silencing and genomic integrity. CGGBP1 has also been identified as a cooperator of histone-modifying enzymes and as a component of CTCF-containing complexes that regulate the enhancer–promoter looping. CGGBP1–CTCF cross talk in chromatin regulation has been hitherto unknown. Here, we report that the occupancy of CTCF at repeats depends on CGGBP1. Using ChIP-sequencing for CTCF, we describe its occupancy at repetitive DNA. Our results show that endogenous level of CGGBP1 ensures CTCF occupancy preferentially on repeats over canonical CTCF motifs. By combining CTCF ChIP-sequencing results with ChIP sequencing for three different kinds of histone modifications (H3K4me3, H3K9me3 and H3K27me3), we show that the CGGBP1-dependent repeat-rich CTCF-binding sites regulate histone marks in flanking regions. CGGBP1 affects the pattern of CTCF occupancy. Our results posit CGGBP1 as a regulator of CTCF and its binding sites in interspersed repeats.

    更新日期:2019-11-28
  • Early-life DNA methylation profiles are indicative of age-related transcriptome changes
    Epigenet. Chromatin (IF 4.185) Pub Date : 2019-10-08
    Niran Hadad; Dustin R. Masser; Laura Blanco-Berdugo; David R. Stanford; Willard M. Freeman

    Alterations to cellular and molecular programs with brain aging result in cognitive impairment and susceptibility to neurodegenerative disease. Changes in DNA methylation patterns, an epigenetic modification required for various CNS functions are observed with brain aging and can be prevented by anti-aging interventions, but the relationship of altered methylation to gene expression is poorly understood. Paired analysis of the hippocampal methylome and transcriptome with aging of male and female mice demonstrates that age-related differences in methylation and gene expression are anti-correlated within gene bodies and enhancers. Altered promoter methylation with aging was found to be generally un-related to altered gene expression. A more striking relationship was found between methylation levels at young age and differential gene expression with aging. Highly methylated gene bodies and promoters in early life were associated with age-related increases in gene expression even in the absence of significant methylation changes with aging. As well, low levels of methylation in early life were correlated to decreased expression with aging. This relationship was also observed in genes altered in two mouse Alzheimer’s models. DNA methylation patterns established in youth, in combination with other epigenetic marks, were able to accurately predict changes in transcript trajectories with aging. These findings are consistent with the developmental origins of disease hypothesis and indicate that epigenetic variability in early life may explain differences in aging trajectories and age-related disease.

    更新日期:2019-11-28
  • DNA methylation modifier LSH inhibits p53 ubiquitination and transactivates p53 to promote lipid metabolism
    Epigenet. Chromatin (IF 4.185) Pub Date : 2019-10-08
    Ling Chen; Ying Shi; Na Liu; Zuli Wang; Rui Yang; Bin Yan; Xiaoli Liu; Weiwei Lai; Yating Liu; Desheng Xiao; Hu Zhou; Yan Cheng; Ya Cao; Shuang Liu; Zanxian Xia; Yongguang Tao

    The stability of p53 is mainly controlled by ubiquitin-dependent degradation, which is triggered by the E3 ubiquitin ligase MDM2. The chromatin modifier lymphoid-specific helicase (LSH) is essential for DNA methylation and cancer progression as a transcriptional repressor. The potential interplay between chromatin modifiers and transcription factors remains largely unknown. Here, we present data suggesting that LSH regulates p53 in cis through two pathways: prevention proteasomal degradation through its deubiquitination, which is achieved by reducing the lysine 11-linked, lysine 48-linked polyubiquitin chains (K11 and K48) on p53; and revival of the transcriptional activity of p53 by forming a complex with PKM2 (pyruvate kinase 2). Furthermore, we confirmed that the LSH–PKM2 interaction occurred at the intersubunit interface region of the PKM2 C-terminal region and the coiled-coil domains (CC) and ATP-binding domains of LSH, and this interaction regulated p53-mediated transactivation in cis in lipid metabolism, especially lipid catabolism. These findings suggest that LSH is a novel regulator of p53 through the proteasomal pathway, thereby providing an alternative mechanism of p53 involvement in lipid metabolism in cancer.

    更新日期:2019-11-28
  • Genome-wide allele-specific methylation is enriched at gene regulatory regions in a multi-generation pedigree from the Norfolk Island isolate
    Epigenet. Chromatin (IF 4.185) Pub Date : 2019-10-08
    Miles C. Benton; Rodney A. Lea; Donia Macartney-Coxson; Heidi G. Sutherland; Nicole White; Daniel Kennedy; Kerry Mengersen; Larisa M. Haupt; Lyn R. Griffiths

    Allele-specific methylation (ASM) occurs when DNA methylation patterns exhibit asymmetry among alleles. ASM occurs at imprinted loci, but its presence elsewhere across the human genome is indicative of wider importance in terms of gene regulation and disease risk. Here, we studied ASM by focusing on blood-based DNA collected from 24 subjects comprising a 3-generation pedigree from the Norfolk Island genetic isolate. We applied a genome-wide bisulphite sequencing approach with a genotype-independent ASM calling method to map ASM across the genome. Regions of ASM were then tested for enrichment at gene regulatory regions using Genomic Association Test (GAT) tool. In total, we identified 1.12 M CpGs of which 147,170 (13%) exhibited ASM (P ≤ 0.05). When including contiguous ASM signal spanning ≥ 2 CpGs, this condensed to 12,761 ASM regions (AMRs). These AMRs tagged 79% of known imprinting regions and most (98.1%) co-localised with known single nucleotide variants. Notably, miRNA and lncRNA showed a 3.3- and 1.8-fold enrichment of AMRs, respectively (P < 0.005). Also, the 5′ UTR and start codons each showed a 3.5-fold enrichment of AMRs (P < 0.005). There was also enrichment of AMRs observed at subtelomeric regions of many chromosomes. Five out of 11 large AMRs localised to the protocadherin cluster on chromosome 5. This study shows ASM extends far beyond genomic imprinting in humans and that gene regulatory regions are hotspots for ASM. Future studies of ASM in pedigrees should help to clarify transgenerational inheritance patterns in relation to genotype and disease phenotypes.

    更新日期:2019-11-28
  • Joint inference and alignment of genome structures enables characterization of compartment-independent reorganization across cell types
    Epigenet. Chromatin (IF 4.185) Pub Date : 2019-10-08
    Lila Rieber; Shaun Mahony

    Comparisons of Hi–C data sets between cell types and conditions have revealed differences in topologically associated domains (TADs) and A/B compartmentalization, which are correlated with differences in gene regulation. However, previous comparisons have focused on known forms of 3D organization while potentially neglecting other functionally relevant differences. We aimed to create a method to quantify all locus-specific differences between two Hi–C data sets. We developed MultiMDS to jointly infer and align 3D chromosomal structures from two Hi–C data sets, thereby enabling a new way to comprehensively quantify relocalization of genomic loci between cell types. We demonstrate this approach by comparing Hi–C data across a variety of cell types. We consistently find relocalization of loci with minimal difference in A/B compartment score. For example, we identify compartment-independent relocalizations between GM12878 and K562 cells that involve loci displaying enhancer-associated histone marks in one cell type and polycomb-associated histone marks in the other. MultiMDS is the first tool to identify all loci that relocalize between two Hi–C data sets. Our method can identify 3D localization differences that are correlated with cell-type-specific regulatory activities and which cannot be identified using other methods.

    更新日期:2019-11-28
  • DNA methylation is maintained with high fidelity in the honey bee germline and exhibits global non-functional fluctuations during somatic development
    Epigenet. Chromatin (IF 4.185) Pub Date : 2019-10-10
    Keith D. Harris; James P. B. Lloyd; Katherine Domb; Daniel Zilberman; Assaf Zemach

    DNA methylation of active genes, also known as gene body methylation, is found in many animal and plant genomes. Despite this, the transcriptional and developmental role of such methylation remains poorly understood. Here, we explore the dynamic range of DNA methylation in honey bee, a model organism for gene body methylation. Our data show that CG methylation in gene bodies globally fluctuates during honey bee development. However, these changes cause no gene expression alterations. Intriguingly, despite the global alterations, tissue-specific CG methylation patterns of complete genes or exons are rare, implying robust maintenance of genic methylation during development. Additionally, we show that CG methylation maintenance fluctuates in somatic cells, while reaching maximum fidelity in sperm cells. Finally, unlike universally present CG methylation, we discovered non-CG methylation specifically in bee heads that resembles such methylation in mammalian brain tissue. Based on these results, we propose that gene body CG methylation can oscillate during development if it is kept to a level adequate to preserve function. Additionally, our data suggest that heightened non-CG methylation is a conserved regulator of animal nervous systems.

    更新日期:2019-11-28
  • MeCP2-E1 isoform is a dynamically expressed, weakly DNA-bound protein with different protein and DNA interactions compared to MeCP2-E2
    Epigenet. Chromatin (IF 4.185) Pub Date : 2019-10-10
    Alexia Martínez de Paz; Leila Khajavi; Hélène Martin; Rafael Claveria-Gimeno; Susanne Tom Dieck; Manjinder S. Cheema; Jose V. Sanchez-Mut; Malgorzata M. Moksa; Annaick Carles; Nick I. Brodie; Taimoor I. Sheikh; Melissa E. Freeman; Evgeniy V. Petrotchenko; Christoph H. Borchers; Erin M. Schuman; Matthias Zytnicki; Adrian Velazquez-Campoy; Olga Abian; Martin Hirst; Manel Esteller; John B. Vincent; Cécile E. Malnou; Juan Ausió

    MeCP2—a chromatin-binding protein associated with Rett syndrome—has two main isoforms, MeCP2-E1 and MeCP2-E2, differing in a few N-terminal amino acid residues. Previous studies have shown brain region-specific expression of these isoforms which, in addition to their different cellular localization and differential expression during brain development, suggest that they may also have non-overlapping molecular mechanisms. However, differential functions of MeCP2-E1 and E2 remain largely unexplored. Here, we show that the N-terminal domains (NTD) of MeCP2-E1 and E2 modulate the ability of the methyl-binding domain (MBD) to interact with DNA as well as influencing the turn-over rates, binding dynamics, response to neuronal depolarization, and circadian oscillations of the two isoforms. Our proteomics data indicate that both isoforms exhibit unique interacting protein partners. Moreover, genome-wide analysis using ChIP-seq provide evidence for a shared as well as a specific regulation of different sets of genes. Our study supports the idea that Rett syndrome might arise from simultaneous impairment of cellular processes involving non-overlapping functions of MECP2 isoforms. For instance, MeCP2-E1 mutations might impact stimuli-dependent chromatin regulation, while MeCP2-E2 mutations could result in aberrant ribosomal expression. Overall, our findings provide insight into the functional complexity of MeCP2 by dissecting differential aspects of its two isoforms.

    更新日期:2019-11-28
  • Hemimethylation of CpG dyads is characteristic of secondary DMRs associated with imprinted loci and correlates with 5-hydroxymethylcytosine at paternally methylated sequences
    Epigenet. Chromatin (IF 4.185) Pub Date : 2019-10-17
    Julianna Nechin; Emma Tunstall; Naideline Raymond; Nicole Hamagami; Chris Pathmanabhan; Samantha Forestier; Tamara L. Davis

    In mammals, the regulation of imprinted genes is controlled by differential methylation at imprinting control regions which acquire parent of origin-specific methylation patterns during gametogenesis and retain differences in allelic methylation status throughout fertilization and subsequent somatic cell divisions. In addition, many imprinted genes acquire differential methylation during post-implantation development; these secondary differentially methylated regions appear necessary to maintain the imprinted expression state of individual genes. Despite the requirement for both types of differentially methylated sequence elements to achieve proper expression across imprinting clusters, methylation patterns are more labile at secondary differentially methylated regions. To understand the nature of this variability, we analyzed CpG dyad methylation patterns at both paternally and maternally methylated imprinted loci within multiple imprinting clusters. We determined that both paternally and maternally methylated secondary differentially methylated regions associated with imprinted genes display high levels of hemimethylation, 29–49%, in comparison to imprinting control regions which exhibited 8–12% hemimethylation. To explore how hemimethylation could arise, we assessed the differentially methylated regions for the presence of 5-hydroxymethylcytosine which could cause methylation to be lost via either passive and/or active demethylation mechanisms. We found enrichment of 5-hydroxymethylcytosine at paternally methylated secondary differentially methylated regions, but not at the maternally methylated sites we analyzed in this study. We found high levels of hemimethylation to be a generalizable characteristic of secondary differentially methylated regions associated with imprinted genes. We propose that 5-hydroxymethylcytosine enrichment may be responsible for the variability in methylation status at paternally methylated secondary differentially methylated regions associated with imprinted genes. We further suggest that the high incidence of hemimethylation at secondary differentially methylated regions must be counteracted by continuous methylation acquisition at these loci.

    更新日期:2019-11-28
  • The role of cigarette smoke-induced epigenetic alterations in inflammation
    Epigenet. Chromatin (IF 4.185) Pub Date : 2019-11-11
    Dandan Zong; Xiangming Liu; Jinhua Li; Ruoyun Ouyang; Ping Chen

    Exposure to cigarette smoke (CS) is a major threat to human health worldwide. It is well established that smoking increases the risk of respiratory diseases, cardiovascular diseases and different forms of cancer, including lung, liver, and colon. CS-triggered inflammation is considered to play a central role in various pathologies by a mechanism that stimulates the release of pro-inflammatory cytokines. During this process, epigenetic alterations are known to play important roles in the specificity and duration of gene transcription. Epigenetic alterations include three major modifications: DNA modifications via methylation; various posttranslational modifications of histones, namely, methylation, acetylation, phosphorylation, and ubiquitination; and non-coding RNA sequences. These modifications work in concert to regulate gene transcription in a heritable fashion. The enzymes that regulate these epigenetic modifications can be activated by smoking, which further mediates the expression of multiple inflammatory genes. In this review, we summarize the current knowledge on the epigenetic alterations triggered by CS and assess how such alterations may affect smoking-mediated inflammatory responses. The recognition of the molecular mechanisms of the epigenetic changes in abnormal inflammation is expected to contribute to the understanding of the pathophysiology of CS-related diseases such that novel epigenetic therapies may be identified in the near future.

    更新日期:2019-11-28
  • Virtual methylome dissection facilitated by single-cell analyses
    Epigenet. Chromatin (IF 4.185) Pub Date : 2019-11-11
    Liduo Yin; Yanting Luo; Xiguang Xu; Shiyu Wen; Xiaowei Wu; Xuemei Lu; Hehuang Xie

    Numerous cell types can be identified within plant tissues and animal organs, and the epigenetic modifications underlying such enormous cellular heterogeneity are just beginning to be understood. It remains a challenge to infer cellular composition using DNA methylomes generated for mixed cell populations. Here, we propose a semi-reference-free procedure to perform virtual methylome dissection using the nonnegative matrix factorization (NMF) algorithm. In the pipeline that we implemented to predict cell-subtype percentages, putative cell-type-specific methylated (pCSM) loci were first determined according to their DNA methylation patterns in bulk methylomes and clustered into groups based on their correlations in methylation profiles. A representative set of pCSM loci was then chosen to decompose target methylomes into multiple latent DNA methylation components (LMCs). To test the performance of this pipeline, we made use of single-cell brain methylomes to create synthetic methylomes of known cell composition. Compared with highly variable CpG sites, pCSM loci achieved a higher prediction accuracy in the virtual methylome dissection of synthetic methylomes. In addition, pCSM loci were shown to be good predictors of the cell type of the sorted brain cells. The software package developed in this study is available in the GitHub repository (https://github.com/Gavin-Yinld). We anticipate that the pipeline implemented in this study will be an innovative and valuable tool for the decoding of cellular heterogeneity.

    更新日期:2019-11-28
  • Chromatin accessibility and transcription dynamics during in vitro astrocyte differentiation of Huntington’s Disease Monkey pluripotent stem cells
    Epigenet. Chromatin (IF 4.185) Pub Date : 2019-11-13
    Alexandra V. Goodnight; Isaac Kremsky; Sujittra Khampang; Yoon Hee Jung; James M. Billingsley; Steven E. Bosinger; Victor G. Corces; Anthony W. S. Chan

    Huntington’s Disease (HD) is a fatal neurodegenerative disorder caused by a CAG repeat expansion, resulting in a mutant huntingtin protein. While it is now clear that astrocytes are affected by HD and significantly contribute to neuronal dysfunction and pathogenesis, the alterations in the transcriptional and epigenetic profiles in HD astrocytes have yet to be characterized. Here, we examine global transcription and chromatin accessibility dynamics during in vitro astrocyte differentiation in a transgenic non-human primate model of HD. We found global changes in accessibility and transcription across different stages of HD pluripotent stem cell differentiation, with distinct trends first observed in neural progenitor cells (NPCs), once cells have committed to a neural lineage. Transcription of p53 signaling and cell cycle pathway genes was highly impacted during differentiation, with depletion in HD NPCs and upregulation in HD astrocytes. E2F target genes also displayed this inverse expression pattern, and strong associations between E2F target gene expression and accessibility at nearby putative enhancers were observed. The results suggest that chromatin accessibility and transcription are altered throughout in vitro HD astrocyte differentiation and provide evidence that E2F dysregulation contributes to aberrant cell-cycle re-entry and apoptosis throughout the progression from NPCs to astrocytes.

    更新日期:2019-11-28
  • BRM: the core ATPase subunit of SWI/SNF chromatin-remodelling complex—a tumour suppressor or tumour-promoting factor?
    Epigenet. Chromatin (IF 4.185) Pub Date : 2019-11-13
    Iga Jancewicz; Janusz A. Siedlecki; Tomasz J. Sarnowski; Elzbieta Sarnowska

    BRM (BRAHMA) is a core, SWI2/SNF2-type ATPase subunit of SWI/SNF chromatin-remodelling complex (CRC) involved in various important regulatory processes including development. Mutations in SMARCA2, a BRM-encoding gene as well as overexpression or epigenetic silencing were found in various human diseases including cancer. Missense mutations in SMARCA2 gene were recently connected with occurrence of Nicolaides–Baraitser genetics syndrome. By contrast, SMARCA2 duplication rather than mutations is characteristic for Coffin–Siris syndrome. It is believed that BRM usually acts as a tumour suppressor or a tumour susceptibility gene. However, other studies provided evidence that BRM function may differ depending on the cancer type and the disease stage, where BRM may play a role in the disease progression. The existence of alternative splicing forms of SMARCA2 gene, leading to appearance of truncated functional, loss of function or gain-of-function forms of BRM protein suggest a far more complicated mode of BRM-containing SWI/SNF CRCs actions. Therefore, the summary of recent knowledge regarding BRM alteration in various types of cancer and highlighting of differences and commonalities between BRM and BRG1, another SWI2/SNF2 type ATPase, will lead to better understanding of SWI/SNF CRCs function in cancer development/progression. BRM has been recently proposed as an attractive target for various anticancer therapies including the use of small molecule inhibitors, synthetic lethality induction or proteolysis-targeting chimera (PROTAC). However, such attempts have some limitations and may lead to severe side effects given the homology of BRM ATPase domain to other ATPases, as well as due to the tissue-specific appearance of BRM- and BRG1-containing SWI/SNF CRC classes. Thus, a better insight into BRM-containing SWI/SNF CRCs function in human tissues and cancers is clearly required to provide a solid basis for establishment of new safe anticancer therapies.

    更新日期:2019-11-28
  • Folate deficiency induced H2A ubiquitination to lead to downregulated expression of genes involved in neural tube defects
    Epigenet. Chromatin (IF 4.185) Pub Date : 2019-11-13
    Pei Pei; Xiyue cheng; Juan Yu; Jinying Shen; Xue Li; Jianxin Wu; Shan Wang; Ting Zhang

    Neural tube defects (NTDs) are common congenital malformations resulting in failure of the neural tube closure during early embryonic development. Although it is known that maternal folate deficiency increases the risk of NTDs, the mechanism remains elusive. Herein, we report that histone H2A monoubiquitination (H2AK119ub1) plays a role in neural tube closure. We found that the folate antagonist methotrexate induced H2AK119ub1 in mouse embryonic stem cells. We demonstrated that an increase in H2AK119ub1 downregulated expression of the neural tube closure-related genes Cdx2, Nes, Pax6, and Gata4 in mouse embryonic stem cells under folate deficiency conditions. We also determined that the E3 ligase Mdm2 was responsible for the methotrexate-induced increase in H2AK119ub1 and downregulation of neural tube closure-related genes. Surprisingly, we found that Mdm2 is required for MTX-induced H2A ubiquitination and is recruited to the sites of DSB, which is dependent on DNA damage signaling kinase ATM. Furthermore, folic acid supplementation restored H2AK119ub1 binding to neural tube closure-related genes. Downregulation of these genes was also observed in both brain tissue of mouse and human NTD cases, and high levels of H2AK119ub1 were found in the corresponding NTDs samples with their maternal serum folate under low levels. Pearson correlation analysis showed a significant negative correlation between expression of the neural precursor genes and H2AK119ub1. Our results indicate that folate deficiency contributes to the onset of NTDs by altering H2AK119ub1 and subsequently affecting expression of neural tube closure-related genes. This may be a potential risk factor for NTDs in response to folate deficiency.

    更新日期:2019-11-28
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