bims-crepig Biomed News
on Chromatin regulation and epigenetics in cell fate and cancer
Issue of 2020‒07‒05
thirty-nine papers selected by
Connor Rogerson
University of Cambridge, MRC Cancer Unit


  1. Genome Biol. 2020 Jul 02. 21(1): 158
    Jiang Y, Huang J, Lun K, Li B, Zheng H, Li Y, Zhou R, Duan W, Wang C, Feng Y, Yao H, Li C, Ji X.
      BACKGROUND: The relationship between transcription and the 3D chromatin structure is debated. Multiple studies have shown that transcription affects global Cohesin binding and 3D genome structures. However, several other studies have indicated that inhibited transcription does not alter chromatin conformations.RESULTS: We provide the most comprehensive evidence to date to demonstrate that transcription plays a relatively modest role in organizing the local, small-scale chromatin structures in mammalian cells. We show degraded Pol I, Pol II, and Pol III proteins in mESCs cause few or no changes in large-scale 3D chromatin structures, selected RNA polymerases with a high abundance of binding sites or active promoter-associated interactions appear to be relatively more affected after the degradation, transcription inhibition alters local, small loop domains, as indicated by high-resolution chromatin interaction maps, and loops with bound Pol II but without Cohesin or CTCF are identified and found to be largely unchanged after transcription inhibition. Interestingly, Pol II depletion for a longer time significantly affects the chromatin accessibility and Cohesin occupancy, suggesting that RNA polymerases are capable of affecting the 3D genome indirectly. These direct and indirect effects explain the previous inconsistent findings on the influence of transcription inhibition on the 3D genome.
    CONCLUSIONS: We conclude that Pol I, Pol II, and Pol III loss alters local, small-scale chromatin interactions in mammalian cells, suggesting that the 3D chromatin structures are pre-established and relatively stable.
    Keywords:  3D chromatin organization; RNA polymerases; Transcription
    DOI:  https://doi.org/10.1186/s13059-020-02067-3
  2. Gastroenterology. 2020 Jun 30. pii: S0016-5085(20)34854-X. [Epub ahead of print]
    Jiang YY, Jiang Y, Li CQ, Zhang Y, Dakle P, Kaur H, Deng JW, Yu-Tong Lin R, Han L, Xie JJ, Yan Y, Doan N, Zheng Y, Mayakonda A, Hazawa M, Xu L, Li Y, Aswad L, Jeitany M, Kanojia D, Guan XY, Said JW, Yang W, Fullwood MJ, Lin DC, Koeffler HP.
      BACKGROUND & AIMS: We investigated the transcriptome of esophageal squamous cell carcinoma (ESCC) cells, activity of gene regulatory (enhancer and promoter regions), and the effects of blocking epigenetic regulatory proteins.METHODS: We performed chromatin immunoprecipitation sequencing with antibodies against H3K4me1, H3K4me3, and H3K27ac and an assay for transposase accessible chromatin to map the enhancer regions and accessible chromatin in 8 ESCC cell lines. We used the CRC_Mapper algorithm to identify core regulatory circuitry transcription factors in ESCC cell lines, and determined genome occupancy profiles for 3 of these factors. In ESCC cell lines, expression of transcription factors was knocked down with small hairpin RNAs, promoter and enhancer regions were disrupted by CRISPR/Cas9 genome editing, or bromodomains and extra-terminal (BET) family proteins and histone deacetylases (HDACs) were inhibited with ARV-771 and romidepsin, respectively. ESCC cell lines were then analyzed by whole-transcriptome sequencing, immunoprecipitation, immunoblots, immunohistochemistry, and viability assays. Interactions between distal enhancers and promoters were identified and verified with circular chromosome conformation capture sequencing. NOD-SCID mice were given injections of modified ESCC cells, some mice where given injections of HDAC or BET inhibitors, and growth of xenograft tumors was measured.
    RESULTS: We identified super enhancer-regulated circuits and transcription factors TP63, SOX2, and KLF5 as core regulatory factors in ESCC cells. Super-enhancer regulation of ALDH3A1 mediated by core regulatory factors was required for ESCC viability. We observed direct interactions between the promoter region of TP63 and functional enhancers, mediated by the core regulatory circuitry transcription factors. Deletion of enhancer regions from ESCC cells decreased expression of the core regulatory circuitry transcription factors and reduced cell viability; these same results were observed with knockdown of each core regulatory circuitry transcription factor. Incubation of ESCC cells with BET and HDAC disrupted the core regulatory circuitry program and the epigenetic modifications observed in these cells; mice given injections of HDAC or BET inhibitors developed smaller xenograft tumors from the ESCC cell lines. Xenograft tumors grew more slowly in mice given the combination of ARV-771 and romidepsin than mice given either agent alone.
    CONCLUSIONS: In epigenetic and transcriptional analyses of ESCC cell lines, we found the transcription factors TP63, SOX2, and KLF5 to be part of a core regulatory network that determines chromatin accessibility, epigenetic modifications, and gene expression patterns in these cells. A combination of epigenetic inhibitors slowed growth of xenograft tumors derived from ESCC cells in mice.
    Keywords:  ChIP-seq; cistrome; epigenome; esophageal cancer
    DOI:  https://doi.org/10.1053/j.gastro.2020.06.050
  3. Cell Syst. 2020 Jun 26. pii: S2405-4712(20)30196-4. [Epub ahead of print]
    Davis JE, Insigne KD, Jones EM, Hastings QA, Boldridge WC, Kosuri S.
      In eukaryotes, transcription factors (TFs) orchestrate gene expression by binding to TF-binding sites (TFBSs) and localizing transcriptional co-regulators and RNA polymerase II to cis-regulatory elements. However, we lack a basic understanding of the relationship between TFBS composition and their quantitative transcriptional responses. Here, we measured expression driven by 17,406 synthetic cis-regulatory elements with varied compositions of a model TFBS, the c-AMP response element (CRE) by using massively parallel reporter assays (MPRAs). We find CRE number, affinity, and promoter proximity largely determines expression. In addition, we observe expression modulation based on the spacing between CREs and CRE distance to the promoter, where expression follows a helical periodicity. Finally, we compare library expression between an episomal MPRA and a genomically integrated MPRA, where a single cis-regulatory element is assayed per cell at a defined locus. These assays largely recapitulate each other, although weaker, non-canonical CREs exhibit greater activity in a genomic context.
    Keywords:  functional genomics; gene regulation; massively parallel reporter assays; synthetic biology; systems biology; transcription factor
    DOI:  https://doi.org/10.1016/j.cels.2020.05.011
  4. Nucleic Acids Res. 2020 Jul 04. pii: gkaa556. [Epub ahead of print]
    Amante SM, Montibus B, Cowley M, Barkas N, Setiadi J, Saadeh H, Giemza J, Contreras-Castillo S, Fleischanderl K, Schulz R, Oakey RJ.
      Alternative splicing (AS) and alternative polyadenylation (APA) generate diverse transcripts in mammalian genomes during development and differentiation. Epigenetic marks such as trimethylation of histone H3 lysine 36 (H3K36me3) and DNA methylation play a role in generating transcriptome diversity. Intragenic CpG islands (iCGIs) and their corresponding host genes exhibit dynamic epigenetic and gene expression patterns during development and between different tissues. We hypothesise that iCGI-associated H3K36me3, DNA methylation and transcription can influence host gene AS and/or APA. We investigate H3K36me3 and find that this histone mark is not a major regulator of AS or APA in our model system. Genomewide, we identify over 4000 host genes that harbour an iCGI in the mammalian genome, including both previously annotated and novel iCGI/host gene pairs. The transcriptional activity of these iCGIs is tissue- and developmental stage-specific and, for the first time, we demonstrate that the premature termination of host gene transcripts upstream of iCGIs is closely correlated with the level of iCGI transcription in a DNA-methylation independent manner. These studies suggest that iCGI transcription, rather than H3K36me3 or DNA methylation, interfere with host gene transcription and pre-mRNA processing genomewide and contributes to the spatiotemporal diversification of both the transcriptome and proteome.
    DOI:  https://doi.org/10.1093/nar/gkaa556
  5. Mol Cell. 2020 Jun 20. pii: S1097-2765(20)30392-0. [Epub ahead of print]
    Lu L, Liu X, Huang WK, Giusti-Rodríguez P, Cui J, Zhang S, Xu W, Wen Z, Ma S, Rosen JD, Xu Z, Bartels CF, Kawaguchi R, Hu M, Scacheri PC, Rong Z, Li Y, Sullivan PF, Song H, Ming GL, Li Y, Jin F.
      Genome-wide mapping of chromatin interactions at high resolution remains experimentally and computationally challenging. Here we used a low-input "easy Hi-C" protocol to map the 3D genome architecture in human neurogenesis and brain tissues and also demonstrated that a rigorous Hi-C bias-correction pipeline (HiCorr) can significantly improve the sensitivity and robustness of Hi-C loop identification at sub-TAD level, especially the enhancer-promoter (E-P) interactions. We used HiCorr to compare the high-resolution maps of chromatin interactions from 10 tissue or cell types with a focus on neurogenesis and brain tissues. We found that dynamic chromatin loops are better hallmarks for cellular differentiation than compartment switching. HiCorr allowed direct observation of cell-type- and differentiation-specific E-P aggregates spanning large neighborhoods, suggesting a mechanism that stabilizes enhancer contacts during development. Interestingly, we concluded that Hi-C loop outperforms eQTL in explaining neurological GWAS results, revealing a unique value of high-resolution 3D genome maps in elucidating the disease etiology.
    Keywords:  GWAS; Hi-C; HiCorr; bias correction; chromatin loop; eHi-C; enhancer-promoter interaction; neurogenesis; transcription regulation
    DOI:  https://doi.org/10.1016/j.molcel.2020.06.007
  6. Epigenetics Chromatin. 2020 Jul 01. 13(1): 27
    Zhang Y, Li Z, Bian S, Zhao H, Feng D, Chen Y, Hou Y, Liu Q, Hao B.
      BACKGROUND: Chromatin physical interactions provide essential information for understanding the regulation of cis-elements like enhancers, promoters, and insulators in cell development and differentiation. The Hi-C assay is a technique detecting chromatin structures of the whole genome, but not sensitive to interactions of regulatory elements. Several methods, like HiChIP, DNase-C, and OCEAN-C, have been developed for enriching interactions of regulatory regions, but all of them have some shortcomings. New simple, efficient, and robust methods are still in need for detecting interactions of regulatory regions.RESULTS: We developed a new, simple, and robust assay called CoP (Column Purified chromatin) for profiling of open chromatin regions by directly purifying fragmentized crosslinked chromatin with a DNA purification column. The accessible chromatin regions, including active enhancers, promoters, and insulators, were significantly enriched in CoP chromatin. The CoP-seq assay can efficiently detect open chromatin regions, especially active promoters, with a high signal-to-noise ratio. We integrated the CoP-seq and Hi-C technique (HiCoP) to detect interactions of accessible chromatin regions, which represent active cis-regulatory elements in cells. We observed that the HiCoP captured the peaks in the promoters-associated enhancer regions. HiCoP detected more promoter-enhancer (P-E), promoter-promoter (P-P), and enhancer-enhancer (E-E) interactions within 20 kb-5 Mb than Hi-C. Most of the loops identified by HiCoP are associated with the expressed genes.
    CONCLUSION: CoP assay can efficiently enrich open chromatin regions. When CoP assay was integrated with Hi-C assay, it provides a simple, robust, alternative technique for profiling accessible chromatin regions and chromatin conformation simultaneously.
    Keywords:  Chromatin accessibility; Chromatin loop; Chromatin structure; Enhancer; Promoter
    DOI:  https://doi.org/10.1186/s13072-020-00348-6
  7. Int J Cancer. 2020 Jun 29.
    Zhang X, Zegar T, Weiser T, Hamdan FH, Berger BT, Lucas R, Balourdas DI, Ladigan S, Cheung PF, Liffers ST, Trajkovic-Arsic M, Scheffler B, Joerger AC, Hahn SA, Johnsen SA, Knapp S, Siveke JT.
      Pancreatic ductal adenocarcinoma (PDAC) is resistant to virtually all chemo- and targeted therapeutic approaches. Epigenetic regulators represent a novel class of drug targets. Among them, BET and HDAC proteins are central regulators of chromatin structure and transcription, and preclinical evidence suggests effectiveness of combined BET and HDAC inhibition in PDAC. Here, we describe that TW9, a newly generated adduct of the BET inhibitor (+)-JQ1 and class I HDAC inhibitor CI994, is a potent dual inhibitor simultaneously targeting BET and HDAC proteins. TW9 has a similar affinity to BRD4 bromodomains as (+)-JQ1 and shares a conserved binding mode, but is significantly more active in inhibiting HDAC1 compared to the parental HDAC inhibitor CI994. TW9 was more potent in inhibiting tumor cell proliferation compared to (+)-JQ1, CI994 alone or combined treatment of both inhibitors. Sequential administration of gemcitabine and TW9 showed additional synergistic antitumor effects. Microarray analysis revealed that dysregulation of a FOSL1-directed transcriptional program contributed to the antitumor effects of TW9. Our results demonstrate the potential of a dual chromatin-targeting strategy in the treatment of PDAC and provide a rationale for further development of multitarget inhibitors.
    Keywords:  BET inhibitor; HDAC inhibitor; combined therapy; dual BET/HDAC inhibitor; pancreatic ductal adenocarcinoma
    DOI:  https://doi.org/10.1002/ijc.33137
  8. Nat Commun. 2020 Jul 03. 11(1): 3355
    Gao L, Emperle M, Guo Y, Grimm SA, Ren W, Adam S, Uryu H, Zhang ZM, Chen D, Yin J, Dukatz M, Anteneh H, Jurkowska RZ, Lu J, Wang Y, Bashtrykov P, Wade PA, Wang GG, Jeltsch A, Song J.
      Mammalian DNA methylation patterns are established by two de novo DNA methyltransferases, DNMT3A and DNMT3B, which exhibit both redundant and distinctive methylation activities. However, the related molecular basis remains undetermined. Through comprehensive structural, enzymology and cellular characterization of DNMT3A and DNMT3B, we here report a multi-layered substrate-recognition mechanism underpinning their divergent genomic methylation activities. A hydrogen bond in the catalytic loop of DNMT3B causes a lower CpG specificity than DNMT3A, while the interplay of target recognition domain and homodimeric interface fine-tunes the distinct target selection between the two enzymes, with Lysine 777 of DNMT3B acting as a unique sensor of the +1 flanking base. The divergent substrate preference between DNMT3A and DNMT3B provides an explanation for site-specific epigenomic alterations seen in ICF syndrome with DNMT3B mutations. Together, this study reveals distinctive substrate-readout mechanisms of the two DNMT3 enzymes, implicative of their differential roles during development and pathogenesis.
    DOI:  https://doi.org/10.1038/s41467-020-17109-4
  9. Cell Rep. 2020 Jun 30. pii: S2211-1247(20)30800-7. [Epub ahead of print]31(13): 107819
    Zheng Z, Zhang L, Cui XL, Yu X, Hsu PJ, Lyu R, Tan H, Mandal M, Zhang M, Sun HL, Sanchez Castillo A, Peng J, Clark MR, He C, Huang H.
      The RNA N6-methyladenosine (m6A) methylation is installed by the METTL3-METTL14 methyltransferase complex. This modification has critical regulatory roles in various biological processes. Here, we report that deletion of Mettl14 dramatically reduces mRNA m6A methylation in developing B cells and severely blocks B cell development in mice. Deletion of Mettl14 impairs interleukin-7 (IL-7)-induced pro-B cell proliferation and the large-pre-B-to-small-pre-B transition and causes dramatic abnormalities in gene expression programs important for B cell development. Suppression of a group of transcripts by cytoplasmic m6A reader YTHDF2 is critical to the IL-7-induced pro-B cell proliferation. In contrast, the block in the large-pre-B-to-small-pre-B transition is independent of YTHDF1 or YTHDF2 but is associated with a failure to properly upregulate key transcription factors regulating this transition. Our data highlight the important regulatory roles of the RNA m6A methylation and its reader proteins in early B cell development.
    Keywords:  B cell development; METTL14; Post-transcriptional regulation of gene expression; RNA m(6)A modification; YTHDF2
    DOI:  https://doi.org/10.1016/j.celrep.2020.107819
  10. BMC Bioinformatics. 2020 Jul 02. 21(1): 281
    Zhang J, Liu J, Lee D, Lou S, Chen Z, Gürsoy G, Gerstein M.
      BACKGROUND: During transcription, numerous transcription factors (TFs) bind to targets in a highly coordinated manner to control the gene expression. Alterations in groups of TF-binding profiles (i.e. "co-binding changes") can affect the co-regulating associations between TFs (i.e. "rewiring the co-regulator network"). This, in turn, can potentially drive downstream expression changes, phenotypic variation, and even disease. However, quantification of co-regulatory network rewiring has not been comprehensively studied.RESULTS: To address this, we propose DiNeR, a computational method to directly construct a differential TF co-regulation network from paired disease-to-normal ChIP-seq data. Specifically, DiNeR uses a graphical model to capture the gained and lost edges in the co-regulation network. Then, it adopts a stability-based, sparsity-tuning criterion -- by sub-sampling the complete binding profiles to remove spurious edges -- to report only significant co-regulation alterations. Finally, DiNeR highlights hubs in the resultant differential network as key TFs associated with disease. We assembled genome-wide binding profiles of 104 TFs in the K562 and GM12878 cell lines, which loosely model the transition between normal and cancerous states in chronic myeloid leukemia (CML). In total, we identified 351 significantly altered TF co-regulation pairs. In particular, we found that the co-binding of the tumor suppressor BRCA1 and RNA polymerase II, a well-known transcriptional pair in healthy cells, was disrupted in tumors. Thus, DiNeR successfully extracted hub regulators and discovered well-known risk genes.
    CONCLUSIONS: Our method DiNeR makes it possible to quantify changes in co-regulatory networks and identify alterations to TF co-binding patterns, highlighting key disease regulators. Our method DiNeR makes it possible to quantify changes in co-regulatory networks and identify alterations to TF co-binding patterns, highlighting key disease regulators.
    Keywords:  ENCODE; Network changes; TF dysregulation; Transcription factor co-regulation network
    DOI:  https://doi.org/10.1186/s12859-020-03605-3
  11. Nucleic Acids Res. 2020 Jul 04. pii: gkaa567. [Epub ahead of print]
    Klobučar T, Kreibich E, Krueger F, Arez M, Pólvora-Brandão D, von Meyenn F, da Rocha ST, Eckersley-Maslin M.
      Genomic imprinting is an epigenetic phenomenon leading to parental allele-specific expression. Dosage of imprinted genes is crucial for normal development and its dysregulation accounts for several human disorders. This unusual expression pattern is mostly dictated by differences in DNA methylation between parental alleles at specific regulatory elements known as imprinting control regions (ICRs). Although several approaches can be used for methylation inspection, we lack an easy and cost-effective method to simultaneously measure DNA methylation at multiple imprinted regions. Here, we present IMPLICON, a high-throughput method measuring DNA methylation levels at imprinted regions with base-pair resolution and over 1000-fold coverage. We adapted amplicon bisulfite-sequencing protocols to design IMPLICON for ICRs in adult tissues of inbred mice, validating it in hybrid mice from reciprocal crosses for which we could discriminate methylation profiles in the two parental alleles. Lastly, we developed a human version of IMPLICON and detected imprinting errors in embryonic and induced pluripotent stem cells. We also provide rules and guidelines to adapt this method for investigating the DNA methylation landscape of any set of genomic regions. In summary, IMPLICON is a rapid, cost-effective and scalable method, which could become the gold standard in both imprinting research and diagnostics.
    DOI:  https://doi.org/10.1093/nar/gkaa567
  12. Nat Commun. 2020 Jul 03. 11(1): 3326
    Ishak Gabra MB, Yang Y, Li H, Senapati P, Hanse EA, Lowman XH, Tran TQ, Zhang L, Doan LT, Xu X, Schones DE, Fruman DA, Kong M.
      Tumour cells adapt to nutrient deprivation in vivo, yet strategies targeting the nutrient poor microenvironment remain unexplored. In melanoma, tumour cells often experience low glutamine levels, which promote cell dedifferentiation. Here, we show that dietary glutamine supplementation significantly inhibits melanoma tumour growth, prolongs survival in a transgenic melanoma mouse model, and increases sensitivity to a BRAF inhibitor. Metabolomic analysis reveals that dietary uptake of glutamine effectively increases the concentration of glutamine in tumours and its downstream metabolite, αKG, without increasing biosynthetic intermediates necessary for cell proliferation. Mechanistically, we find that glutamine supplementation uniformly alters the transcriptome in tumours. Our data further demonstrate that increase in intra-tumoural αKG concentration drives hypomethylation of H3K4me3, thereby suppressing epigenetically-activated oncogenic pathways in melanoma. Therefore, our findings provide evidence that glutamine supplementation can serve as a potential dietary intervention to block melanoma tumour growth and sensitize tumours to targeted therapy via epigenetic reprogramming.
    DOI:  https://doi.org/10.1038/s41467-020-17181-w
  13. Cell. 2020 Jun 24. pii: S0092-8674(20)30689-9. [Epub ahead of print]
    Markenscoff-Papadimitriou E, Whalen S, Przytycki P, Thomas R, Binyameen F, Nowakowski TJ, Kriegstein AR, Sanders SJ, State MW, Pollard KS, Rubenstein JL.
      To discover regulatory elements driving the specificity of gene expression in different cell types and regions of the developing human brain, we generated an atlas of open chromatin from nine dissected regions of the mid-gestation human telencephalon, as well as microdissected upper and deep layers of the prefrontal cortex. We identified a subset of open chromatin regions (OCRs), termed predicted regulatory elements (pREs), that are likely to function as developmental brain enhancers. pREs showed temporal, regional, and laminar differences in chromatin accessibility and were correlated with gene expression differences across regions and gestational ages. We identified two functional de novo variants in a pRE for autism risk gene SLC6A1, and using CRISPRa, demonstrated that this pRE regulates SCL6A1. Additionally, mouse transgenic experiments validated enhancer activity for pREs proximal to FEZF2 and BCL11A. Thus, this atlas serves as a resource for decoding neurodevelopmental gene regulation in health and disease.
    Keywords:  ATAC-seq; autism; chromatin; enhancers; gene regulation; machine learning; neurodevelopment; neuropsychiatric disorders
    DOI:  https://doi.org/10.1016/j.cell.2020.06.002
  14. Cell Rep. 2020 Jun 30. pii: S2211-1247(20)30813-5. [Epub ahead of print]31(13): 107832
    Liao R, Zheng Y, Liu X, Zhang Y, Seim G, Tanimura N, Wilson GM, Hematti P, Coon JJ, Fan J, Xu J, Keles S, Bresnick EH.
      Protein ensembles control genome function by establishing, maintaining, and deconstructing cell-type-specific chromosomal landscapes. A plethora of small molecules orchestrate cellular functions and therefore may link physiological processes with genome biology. The metabolic enzyme and hemoglobin cofactor heme induces proteolysis of a transcriptional repressor, Bach1, and regulates gene expression post-transcriptionally. However, whether heme controls genome function broadly or through prescriptive actions is unclear. Using assay for transposase-accessible chromatin sequencing (ATAC-seq), we establish a heme-dependent chromatin atlas in wild-type and mutant erythroblasts lacking enhancers that confer normal heme synthesis. Amalgamating chromatin landscapes and transcriptomes in cells with sub-physiological heme and post-heme rescue reveals parallel Bach1-dependent and Bach1-independent mechanisms that target heme-sensing chromosomal hotspots. The hotspots harbor a DNA motif demarcating heme-regulated chromatin and genes encoding proteins not known to be heme regulated, including metabolic enzymes. The heme-omics analysis establishes how an essential biochemical cofactor controls genome function and cellular physiology.
    Keywords:  ATAC-seq; Bach1; GATA1; chromatin; erythroblast; erythroid; heme; transcriptome
    DOI:  https://doi.org/10.1016/j.celrep.2020.107832
  15. Nature. 2020 Jul 01.
    Hill L, Ebert A, Jaritz M, Wutz G, Nagasaka K, Tagoh H, Kostanova-Poliakova D, Schindler K, Sun Q, Bönelt P, Fischer M, Peters JM, Busslinger M.
      Nuclear processes, such as V(D)J recombination, are orchestrated by the three-dimensional organization of chromosomes at multiple levels, including compartments1 and topologically associated domains (TADs)2,3 consisting of chromatin loops4. TADs are formed by chromatin-loop extrusion5-7, which depends on the loop-extrusion function of the ring-shaped cohesin complex8-12. Conversely, the cohesin-release factor Wapl13,14 restricts loop extension10,15. The generation of a diverse antibody repertoire, providing humoral immunity to pathogens, requires the participation of all V genes in V(D)J recombination16, which depends on contraction of the 2.8-Mb-long immunoglobulin heavy chain (Igh) locus by Pax517,18. However, how Pax5 controls Igh contraction in pro-B cells remains unknown. Here we demonstrate that locus contraction is caused by loop extrusion across the entire Igh locus. Notably, the expression of Wapl is repressed by Pax5 specifically in pro-B and pre-B cells, facilitating extended loop extrusion by increasing the residence time of cohesin on chromatin. Pax5 mediates the transcriptional repression of Wapl through a single Pax5-binding site by recruiting the polycomb repressive complex 2 to induce bivalent chromatin at the Wapl promoter. Reduced Wapl expression causes global alterations in the chromosome architecture, indicating that the potential to recombine all V genes entails structural changes of the entire genome in pro-B cells.
    DOI:  https://doi.org/10.1038/s41586-020-2454-y
  16. Genome Biol. 2020 Jul 03. 21(1): 161
    Ji Z, Zhou W, Hou W, Ji H.
      Single-cell sequencing assay for transposase-accessible chromatin (scATAC-seq) is the state-of-the-art technology for analyzing genome-wide regulatory landscapes in single cells. Single-cell ATAC-seq data are sparse and noisy, and analyzing such data is challenging. Existing computational methods cannot accurately reconstruct activities of individual cis-regulatory elements (CREs) in individual cells or rare cell subpopulations. We present a new statistical framework, SCATE, that adaptively integrates information from co-activated CREs, similar cells, and publicly available regulome data to substantially increase the accuracy for estimating activities of individual CREs. We demonstrate that SCATE can be used to better reconstruct the regulatory landscape of a heterogeneous sample.
    Keywords:  Bioinformatics; Chromatin; DNase-seq; Gene regulation; Genomics; Machine learning; Single cell; Software; Statistical modeling; scATAC-seq
    DOI:  https://doi.org/10.1186/s13059-020-02075-3
  17. Cancer Cell. 2020 Jun 11. pii: S1535-6108(20)30272-5. [Epub ahead of print]
    Yuan H, Han Y, Wang X, Li N, Liu Q, Yin Y, Wang H, Pan L, Li L, Song K, Qiu T, Pan Q, Chen Q, Zhang G, Zang Y, Tan M, Zhang J, Li Q, Wang X, Jiang J, Qin J.
      The level of SETD2-mediated H3K36me3 is inversely correlated with that of EZH2-catalyzed H3K27me3. Nevertheless, it remains unclear whether these two enzymatic activities are molecularly intertwined. Here, we report that SETD2 delays prostate cancer (PCa) metastasis via its substrate EZH2. We show that SETD2 methylates EZH2 which promotes EZH2 degradation. SETD2 deficiency induces a Polycomb-repressive chromatin state that enables cells to acquire metastatic traits. Conversely, mice harboring nonmethylated EZH2 mutant or SETD2 mutant defective in binding to EZH2 develop metastatic PCa. Furthermore, we identify that metformin-stimulated AMPK signaling converges at FOXO3 to stimulate SETD2 expression. Together, our results demonstrate that the SETD2-EZH2 axis integrates metabolic and epigenetic signaling to restrict PCa metastasis.
    Keywords:  AMPK; EZH2; SETD2; epigenetic and metabolic dysregulations; metformin; prostate cancer metastasis
    DOI:  https://doi.org/10.1016/j.ccell.2020.05.022
  18. Cancer Res. 2020 Jun 30. pii: canres.4044.2020. [Epub ahead of print]
    Huang H, Wang Y, Kandpal M, Zhao G, Cardenas H, Ji Y, Chaparala A, Tanner EJ, Chen J, Davuluri RV, Matei D.
      N6-Methyladenosine (m6A) is the most abundant modification of mammalian messenger RNAs (mRNA). RNA methylation fine tunes RNA stability and translation, altering cell fate. The fat mass- and obesity-associated protein (FTO) is an m6A demethylase with oncogenic properties in leukemia. Here we show that FTO expression is suppressed in ovarian tumors and cancer stem cells (CSC). FTO inhibited the self-renewal of ovarian CSC and suppressed tumorigenesis in vivo, both of which required FTO demethylase activity. Integrative RNA-sequencing and m6A mapping analysis revealed significant transcriptomic changes associated with FTO overexpression and m6A loss involving stem cell signaling, RNA transcription, and mRNA splicing pathways. By reducing m6A levels at the 3'UTR and the mRNA stability of two phosphodiesterase genes (PDE1C and PDE4B), FTO augmented second messenger 3', 5'-cyclic adenosine monophosphate (cAMP) signaling and suppressed stemness features of ovarian cancer cells. Our results reveal a previously unappreciated tumor suppressor function of FTO in ovarian CSC mediated through inhibition of cAMP signaling.
    DOI:  https://doi.org/10.1158/0008-5472.CAN-19-4044
  19. Nat Genet. 2020 Jun 29.
    Meir Z, Mukamel Z, Chomsky E, Lifshitz A, Tanay A.
      Propagation of clonal regulatory programs contributes to cancer development. It is poorly understood how epigenetic mechanisms interact with genetic drivers to shape this process. Here, we combine single-cell analysis of transcription and DNA methylation with a Luria-Delbrück experimental design to demonstrate the existence of clonally stable epigenetic memory in multiple types of cancer cells. Longitudinal transcriptional and genetic analysis of clonal colon cancer cell populations reveals a slowly drifting spectrum of epithelial-to-mesenchymal transcriptional identities that is seemingly independent of genetic variation. DNA methylation landscapes correlate with these identities but also reflect an independent clock-like methylation loss process. Methylation variation can be explained as an effect of global trans-acting factors in most cases. However, for a specific class of promoters-in particular, cancer-testis antigens-de-repression is correlated with and probably driven by loss of methylation in cis. This study indicates how genetic sub-clonal structure in cancer cells can be diversified by epigenetic memory.
    DOI:  https://doi.org/10.1038/s41588-020-0645-y
  20. Cell Rep. 2020 Jun 30. pii: S2211-1247(20)30824-X. [Epub ahead of print]31(13): 107843
    Srinivasan K, Friedman BA, Etxeberria A, Huntley MA, van der Brug MP, Foreman O, Paw JS, Modrusan Z, Beach TG, Serrano GE, Hansen DV.
      Damage-associated microglia (DAM) profiles observed in Alzheimer's disease (AD)-related mouse models reflect an activation state that could modulate AD risk or progression. To learn whether human AD microglia (HAM) display a similar profile, we develop a method for purifying cell types from frozen cerebrocortical tissues for RNA-seq analysis, allowing better transcriptome coverage than typical single-nucleus RNA-seq approaches. The HAM profile we observe bears little resemblance to the DAM profile. Instead, HAM display an enhanced human aging profile, in addition to other disease-related changes such as APOE upregulation. Analyses of whole-tissue RNA-seq and single-cell/nucleus RNA-seq datasets corroborate our findings and suggest that the lack of DAM response in human microglia occurs specifically in AD tissues, not other neurodegenerative settings. These results, which can be browsed at http://research-pub.gene.com/BrainMyeloidLandscape, provide a genome-wide picture of microglial activation in human AD and highlight considerable differences between mouse models and human disease.
    Keywords:  Alzheimer’s disease; aging; microglia; neurodegenerative diseases; neuroinflammation; transcriptomics
    DOI:  https://doi.org/10.1016/j.celrep.2020.107843
  21. Mol Cell. 2020 Jun 23. pii: S1097-2765(20)30399-3. [Epub ahead of print]
    Wang AS, Chen LC, Wu RA, Hao Y, McSwiggen DT, Heckert AB, Richardson CD, Gowen BG, Kazane KR, Vu JT, Wyman SK, Shin JJ, Darzacq X, Walter JC, Corn JE.
      Cas9 is a prokaryotic RNA-guided DNA endonuclease that binds substrates tightly in vitro but turns over rapidly when used to manipulate genomes in eukaryotic cells. Little is known about the factors responsible for dislodging Cas9 or how they influence genome engineering. Unbiased detection through proximity labeling of transient protein interactions in cell-free Xenopus laevis egg extract identified the dimeric histone chaperone facilitates chromatin transcription (FACT) as an interactor of substrate-bound Cas9. FACT is both necessary and sufficient to displace dCas9, and FACT immunodepletion converts Cas9's activity from multi-turnover to single turnover. In human cells, FACT depletion extends dCas9 residence times, delays genome editing, and alters the balance between indel formation and homology-directed repair. FACT knockdown also increases epigenetic marking by dCas9-based transcriptional effectors with a concomitant enhancement of transcriptional modulation. FACT thus shapes the intrinsic cellular response to Cas9-based genome manipulation most likely by determining Cas9 residence times.
    Keywords:  CRISPR; CRISPRa; CRISPRi; Cas9; FACT complex; SPT16; SSRP1; histone chaperone
    DOI:  https://doi.org/10.1016/j.molcel.2020.06.014
  22. Nucleic Acids Res. 2020 Jun 29. pii: gkaa536. [Epub ahead of print]
    Vansant G, Chen HC, Zorita E, Trejbalová K, Miklík D, Filion G, Debyser Z.
      HIV-1 persists lifelong in memory cells of the immune system as latent provirus that rebounds upon treatment interruption. Therefore, the latent reservoir is the main target for an HIV cure. Here, we studied the direct link between integration site and transcription using LEDGINs and Barcoded HIV-ensembles (B-HIVE). LEDGINs are antivirals that inhibit the interaction between HIV-1 integrase and the chromatin-tethering factor LEDGF/p75. They were used as a tool to retarget integration, while the effect on HIV expression was measured with B-HIVE. B-HIVE tracks insert-specific HIV expression by tagging a unique barcode in the HIV genome. We confirmed that LEDGINs retarget integration out of gene-dense and actively transcribed regions. The distance to H3K36me3, the marker recognized by LEDGF/p75, clearly increased. LEDGIN treatment reduced viral RNA expression and increased the proportion of silent provirus. Finally, silent proviruses obtained after LEDGIN treatment were located further away from epigenetic marks associated with active transcription. Interestingly, proximity to enhancers stimulated transcription irrespective of LEDGIN treatment, while the distance to H3K36me3 only changed after treatment with LEDGINs. The fact that proximity to these markers are associated with RNA expression support the direct link between provirus integration site and viral expression.
    DOI:  https://doi.org/10.1093/nar/gkaa536
  23. Genome Biol. 2020 Jul 01. 21(1): 156
    Scott CA, Duryea JD, MacKay H, Baker MS, Laritsky E, Gunasekara CJ, Coarfa C, Waterland RA.
      BACKGROUND: The traditional approach to studying the epigenetic mechanism CpG methylation in tissue samples is to identify regions of concordant differential methylation spanning multiple CpG sites (differentially methylated regions). Variation limited to single or small numbers of CpGs has been assumed to reflect stochastic processes. To test this, we developed software, Cluster-Based analysis of CpG methylation (CluBCpG), and explored variation in read-level CpG methylation patterns in whole genome bisulfite sequencing data.RESULTS: Analysis of both human and mouse whole genome bisulfite sequencing datasets reveals read-level signatures associated with cell type and cell type-specific biological processes. These signatures, which are mostly orthogonal to classical differentially methylated regions, are enriched at cell type-specific enhancers and allow estimation of proportional cell composition in synthetic mixtures and improved prediction of gene expression. In tandem, we developed a machine learning algorithm, Precise Read-Level Imputation of Methylation (PReLIM), to increase coverage of existing whole genome bisulfite sequencing datasets by imputing CpG methylation states on individual sequencing reads. PReLIM both improves CluBCpG coverage and performance and enables identification of novel differentially methylated regions, which we independently validate.
    CONCLUSIONS: Our data indicate that, rather than stochastic variation, read-level CpG methylation patterns in tissue whole genome bisulfite sequencing libraries reflect cell type. Accordingly, these new computational tools should lead to an improved understanding of epigenetic regulation by DNA methylation.
    Keywords:  Bisulfite-seq; DNA methylation; Deconvolution; Imputation; Machine learning; Random forests; Read-level; WGBS
    DOI:  https://doi.org/10.1186/s13059-020-02065-5
  24. Nat Cell Biol. 2020 Jun 29.
    Burton A, Brochard V, Galan C, Ruiz-Morales ER, Rovira Q, Rodriguez-Terrones D, Kruse K, Le Gras S, Udayakumar VS, Chin HG, Eid A, Liu X, Wang C, Gao S, Pradhan S, Vaquerizas JM, Beaujean N, Jenuwein T, Torres-Padilla ME.
      Following fertilization in mammals, the gametes are reprogrammed to create a totipotent zygote, a process that involves de novo establishment of chromatin domains. A major feature occurring during preimplantation development is the dramatic remodelling of constitutive heterochromatin, although the functional relevance of this is unknown. Here, we show that heterochromatin establishment relies on the stepwise expression and regulated activity of SUV39H enzymes. Enforcing precocious acquisition of constitutive heterochromatin results in compromised development and epigenetic reprogramming, which demonstrates that heterochromatin remodelling is essential for natural reprogramming at fertilization. We find that de novo H3K9 trimethylation (H3K9me3) in the paternal pronucleus after fertilization is catalysed by SUV39H2 and that pericentromeric RNAs inhibit SUV39H2 activity and reduce H3K9me3. De novo H3K9me3 is initially non-repressive for gene expression, but instead bookmarks promoters for compaction. Overall, we uncover the functional importance for the restricted transmission of constitutive heterochromatin during reprogramming and a non-repressive role for H3K9me3.
    DOI:  https://doi.org/10.1038/s41556-020-0536-6
  25. Sci Adv. 2020 Jun;6(25): eaaz6699
    Ochiai H, Hayashi T, Umeda M, Yoshimura M, Harada A, Shimizu Y, Nakano K, Saitoh N, Liu Z, Yamamoto T, Okamura T, Ohkawa Y, Kimura H, Nikaido I.
      Transcriptional bursting is the stochastic activation and inactivation of promoters, contributing to cell-to-cell heterogeneity in gene expression. However, the mechanism underlying the regulation of transcriptional bursting kinetics (burst size and frequency) in mammalian cells remains elusive. In this study, we performed single-cell RNA sequencing to analyze the intrinsic noise and mRNA levels for elucidating the transcriptional bursting kinetics in mouse embryonic stem cells. Informatics analyses and functional assays revealed that transcriptional bursting kinetics was regulated by a combination of promoter- and gene body-binding proteins, including the polycomb repressive complex 2 and transcription elongation factors. Furthermore, large-scale CRISPR-Cas9-based screening identified that the Akt/MAPK signaling pathway regulated bursting kinetics by modulating transcription elongation efficiency. These results uncovered the key molecular mechanisms underlying transcriptional bursting and cell-to-cell gene expression noise in mammalian cells.
    DOI:  https://doi.org/10.1126/sciadv.aaz6699
  26. Biology (Basel). 2020 Jun 26. pii: E140. [Epub ahead of print]9(6):
    Stransky S, Aguilan J, Lachowicz J, Madrid-Aliste C, Nieves E, Sidoli S.
      Chromatin accessibility is a major regulator of gene expression. Histone writers/erasers have a critical role in chromatin compaction, as they "flag" chromatin regions by catalyzing/removing covalent post-translational modifications on histone proteins. Anomalous chromatin decondensation is a common phenomenon in cells experiencing aging and viral infection. Moreover, about 50% of cancers have mutations in enzymes regulating chromatin state. Numerous genomics methods have evolved to characterize chromatin state, but the analysis of (in)accessible chromatin from the protein perspective is not yet in the spotlight. We present an overview of the most used approaches to generate data on chromatin accessibility and then focus on emerging methods that utilize mass spectrometry to quantify the accessibility of histones and the rest of the chromatin bound proteome. Mass spectrometry is currently the method of choice to quantify entire proteomes in an unbiased large-scale manner; accessibility on chromatin of proteins and protein modifications adds an extra quantitative layer to proteomics dataset that assist more informed data-driven hypotheses in chromatin biology. We speculate that this emerging new set of methods will enhance predictive strength on which proteins and histone modifications are critical in gene regulation, and which proteins occupy different chromatin states in health and disease.
    Keywords:  DNA methylation; chromatin; histone; mass spectrometry; post-translational modification; proteome
    DOI:  https://doi.org/10.3390/biology9060140
  27. Nucleic Acids Res. 2020 Jul 03. pii: gkaa565. [Epub ahead of print]
    Escoter-Torres L, Greulich F, Quagliarini F, Wierer M, Uhlenhaut NH.
      The glucocorticoid receptor is an important immunosuppressive drug target and metabolic regulator that acts as a ligand-gated transcription factor. Generally, GR's anti-inflammatory effects are attributed to the silencing of inflammatory genes, while its adverse effects are ascribed to the upregulation of metabolic targets. GR binding directly to DNA is proposed to activate, whereas GR tethering to pro-inflammatory transcription factors is thought to repress transcription. Using mice with a point mutation in GR's zinc finger, that still tether via protein-protein interactions while being unable to recognize DNA, we demonstrate that DNA binding is essential for both transcriptional activation and repression. Performing ChIP-Seq, RNA-Seq and proteomics under inflammatory conditions, we show that DNA recognition is required for the assembly of a functional co-regulator complex to mediate glucocorticoid responses. Our findings may contribute to the development of safer immunomodulators with fewer side effects.
    DOI:  https://doi.org/10.1093/nar/gkaa565
  28. Cell. 2020 Jun 29. pii: S0092-8674(20)30624-3. [Epub ahead of print]
    Huang L, Kebschull JM, Fürth D, Musall S, Kaufman MT, Churchland AK, Zador AM.
      Comprehensive analysis of neuronal networks requires brain-wide measurement of connectivity, activity, and gene expression. Although high-throughput methods are available for mapping brain-wide activity and transcriptomes, comparable methods for mapping region-to-region connectivity remain slow and expensive because they require averaging across hundreds of brains. Here we describe BRICseq (brain-wide individual animal connectome sequencing), which leverages DNA barcoding and sequencing to map connectivity from single individuals in a few weeks and at low cost. Applying BRICseq to the mouse neocortex, we find that region-to-region connectivity provides a simple bridge relating transcriptome to activity: the spatial expression patterns of a few genes predict region-to-region connectivity, and connectivity predicts activity correlations. We also exploited BRICseq to map the mutant BTBR mouse brain, which lacks a corpus callosum, and recapitulated its known connectopathies. BRICseq allows individual laboratories to compare how age, sex, environment, genetics, and species affect neuronal wiring and to integrate these with functional activity and gene expression.
    Keywords:  BRICseq; MAPseq; connectome; high-throughput sequencing; mesoscale
    DOI:  https://doi.org/10.1016/j.cell.2020.05.029
  29. BMC Bioinformatics. 2020 Jun 29. 21(1): 269
    Chen X, Zhang B, Wang T, Bonni A, Zhao G.
      BACKGROUND: High throughput RNA sequencing is a powerful approach to study gene expression. Due to the complex multiple-steps protocols in data acquisition, extreme deviation of a sample from samples of the same treatment group may occur due to technical variation or true biological differences. The high-dimensionality of the data with few biological replicates make it challenging to accurately detect those samples, and this issue is not well studied in the literature currently. Robust statistics is a family of theories and techniques aim to detect the outliers by first fitting the majority of the data and then flagging data points that deviate from it. Robust statistics have been widely used in multivariate data analysis for outlier detection in chemometrics and engineering. Here we apply robust statistics on RNA-seq data analysis.RESULTS: We report the use of two robust principal component analysis (rPCA) methods, PcaHubert and PcaGrid, to detect outlier samples in multiple simulated and real biological RNA-seq data sets with positive control outlier samples. PcaGrid achieved 100% sensitivity and 100% specificity in all the tests using positive control outliers with varying degrees of divergence. We applied rPCA methods and classical principal component analysis (cPCA) on an RNA-Seq data set profiling gene expression of the external granule layer in the cerebellum of control and conditional SnoN knockout mice. Both rPCA methods detected the same two outlier samples but cPCA failed to detect any. We performed differentially expressed gene detection before and after outlier removal as well as with and without batch effect modeling. We validated gene expression changes using quantitative reverse transcription PCR and used the result as reference to compare the performance of eight different data analysis strategies. Removing outliers without batch effect modeling performed the best in term of detecting biologically relevant differentially expressed genes.
    CONCLUSIONS: rPCA implemented in the PcaGrid function is an accurate and objective method to detect outlier samples. It is well suited for high-dimensional data with small sample sizes like RNA-seq data. Outlier removal can significantly improve the performance of differential gene detection and downstream functional analysis.
    Keywords:  Anomaly detection; High-dimensional data; Outlier detection; PcaGrid; PcaHubert; RNA-seq; Robust principal component analysis
    DOI:  https://doi.org/10.1186/s12859-020-03608-0
  30. Proc Natl Acad Sci U S A. 2020 Jun 29. pii: 201920621. [Epub ahead of print]
    Bloomer RH, Hutchison CE, Bäurle I, Walker J, Fang X, Perera P, Velanis CN, Gümüs S, Spanos C, Rappsilber J, Feng X, Goodrich J, Dean C.
      Molecular mechanisms enabling the switching and maintenance of epigenetic states are not fully understood. Distinct histone modifications are often associated with ON/OFF epigenetic states, but how these states are stably maintained through DNA replication, yet in certain situations switch from one to another remains unclear. Here, we address this problem through identification of Arabidopsis INCURVATA11 (ICU11) as a Polycomb Repressive Complex 2 accessory protein. ICU11 robustly immunoprecipitated in vivo with PRC2 core components and the accessory proteins, EMBRYONIC FLOWER 1 (EMF1), LIKE HETEROCHROMATIN PROTEIN1 (LHP1), and TELOMERE_REPEAT_BINDING FACTORS (TRBs). ICU11 encodes a 2-oxoglutarate-dependent dioxygenase, an activity associated with histone demethylation in other organisms, and mutant plants show defects in multiple aspects of the Arabidopsis epigenome. To investigate its primary molecular function we identified the Arabidopsis FLOWERING LOCUS C (FLC) as a direct target and found icu11 disrupted the cold-induced, Polycomb-mediated silencing underlying vernalization. icu11 prevented reduction in H3K36me3 levels normally seen during the early cold phase, supporting a role for ICU11 in H3K36me3 demethylation. This was coincident with an attenuation of H3K27me3 at the internal nucleation site in FLC, and reduction in H3K27me3 levels across the body of the gene after plants were returned to the warm. Thus, ICU11 is required for the cold-induced epigenetic switching between the mutually exclusive chromatin states at FLC, from the active H3K36me3 state to the silenced H3K27me3 state. These data support the importance of physical coupling of histone modification activities to promote epigenetic switching between opposing chromatin states.
    Keywords:  ICU11; Polycomb; chromatin; epigenetic
    DOI:  https://doi.org/10.1073/pnas.1920621117
  31. Nucleic Acids Res. 2020 Jul 03. pii: gkaa563. [Epub ahead of print]
    Wragg JW, Roos L, Vucenovic D, Cvetesic N, Lenhard B, Müller F.
      The core-promoter, a stretch of DNA surrounding the transcription start site (TSS), is a major integration-point for regulatory-signals controlling gene-transcription. Cellular differentiation is marked by divergence in transcriptional repertoire and cell-cycling behaviour between cells of different fates. The role promoter-associated gene-regulatory-networks play in development-associated transitions in cell-cycle-dynamics is poorly understood. This study demonstrates in a vertebrate embryo, how core-promoter variations define transcriptional output in cells transitioning from a proliferative to cell-lineage specifying phenotype. Assessment of cell proliferation across zebrafish embryo segmentation, using the FUCCI transgenic cell-cycle-phase marker, revealed a spatial and lineage-specific separation in cell-cycling behaviour. To investigate the role differential promoter usage plays in this process, cap-analysis-of-gene-expression (CAGE) was performed on cells segregated by cycling dynamics. This analysis revealed a dramatic increase in tissue-specific gene expression, concurrent with slowed cycling behaviour. We revealed a distinct sharpening in TSS utilization in genes upregulated in slowly cycling, differentiating tissues, associated with enhanced utilization of the TATA-box, in addition to Sp1 binding-sites. In contrast, genes upregulated in rapidly cycling cells carry broad distribution of TSS utilization, coupled with enrichment for the CCAAT-box. These promoter features appear to correspond to cell-cycle-dynamic rather than tissue/cell-lineage origin. Moreover, we observed genes with cell-cycle-dynamic-associated transitioning in TSS distribution and differential utilization of alternative promoters. These results demonstrate the regulatory role of core-promoters in cell-cycle-dependent transcription regulation, during embryo-development.
    DOI:  https://doi.org/10.1093/nar/gkaa563
  32. Sci Adv. 2020 Jun;6(25): eaay5872
    Rosikiewicz W, Chen X, Dominguez PM, Ghamlouch H, Aoufouchi S, Bernard OA, Melnick A, Li S.
      The TET2 DNA hydroxymethyltransferase is frequently disrupted by somatic mutations in diffuse large B cell lymphomas (DLBCLs), a tumor that originates from germinal center (GC) B cells. Here, we show that TET2 deficiency leads to DNA hypermethylation of regulatory elements in GC B cells, associated with silencing of the respective genes. This hypermethylation affects the binding of transcription factors including those involved in exit from the GC reaction and involves pathways such as B cell receptor, antigen presentation, CD40, and others. Normal GC B cells manifest a typical hypomethylation signature, which is caused by AID, the enzyme that mediates somatic hypermutation. However, AID-induced demethylation is markedly impaired in TET2-deficient GC B cells, suggesting that AID epigenetic effects are partially dependent on TET2. Last, we find that TET2 mutant DLBCLs also manifest the aberrant TET2-deficient GC DNA methylation signature, suggesting that this epigenetic pattern is maintained during and contributes to lymphomagenesis.
    DOI:  https://doi.org/10.1126/sciadv.aay5872
  33. Sci Rep. 2020 Jul 02. 10(1): 10952
    van der Vos KE, Vis DJ, Nevedomskaya E, Kim Y, Choi W, McConkey D, Wessels LFA, van Rhijn BWG, Zwart W, van der Heijden MS.
      Muscle-invasive bladder cancer (MIBC) is a heterogeneous disease that often recurs despite aggressive treatment with neoadjuvant chemotherapy and (radical) cystectomy. Basal and luminal molecular subtypes have been identified that are linked to clinical characteristics and have differential sensitivities to chemotherapy. While it has been suggested that epigenetic mechanisms play a role in defining these subtypes, a thorough understanding of the biological mechanisms is lacking. This report details the first genome-wide analysis of histone methylation patterns of human primary bladder tumours by chromatin immunoprecipitations and next-generation sequencing (ChIP-seq). We profiled multiple histone marks: H3K27me3, a marker for repressed genes, and H3K4me1 and H3K4me3, which are indicators of active enhancers and active promoters. Integrated analysis of ChIP-seq data and RNA sequencing revealed that H3K4 mono-methylation demarcates MIBC subtypes, while no association was found for the other two histone modifications in relation to basal and luminal subtypes. Additionally, we identified differentially methylated H3K4me1 peaks in basal and luminal tumour samples, suggesting that active enhancers play a role in defining subtypes. Our study is the first analysis of histone modifications in primary bladder cancer tissue and provides an important resource for the bladder cancer community.
    DOI:  https://doi.org/10.1038/s41598-020-67850-5
  34. BMC Bioinformatics. 2020 Jul 01. 21(1): 272
    Li FZ, Liu ZE, Li XY, Bu LM, Bu HX, Liu H, Zhang CM.
      BACKGROUND: Chromatin 3D conformation plays important roles in regulating gene or protein functions. High-throughout chromosome conformation capture (3C)-based technologies, such as Hi-C, have been exploited to acquire the contact frequencies among genomic loci at genome-scale. Various computational tools have been proposed to recover the underlying chromatin 3D structures from in situ Hi-C contact map data. As connected residuals in a polymer, neighboring genomic loci have intrinsic mutual dependencies in building a 3D conformation. However, current methods seldom take this feature into account.RESULTS: We present a method called ShNeigh, which combines the classical MDS technique with local dependence of neighboring loci modeled by a Gaussian formula, to infer the best 3D structure from noisy and incomplete contact frequency matrices. We validated ShNeigh by comparing it to two typical distance-based algorithms, ShRec3D and ChromSDE. The comparison results on simulated Hi-C dataset showed that, while keeping the high-speed nature of classical MDS, ShNeigh can recover the true structure better than ShRec3D and ChromSDE. Meanwhile, ShNeigh is more robust to data noise. On the publicly available human GM06990 Hi-C data, we demonstrated that the structures reconstructed by ShNeigh are more reproducible between different restriction enzymes than by ShRec3D and ChromSDE, especially at high resolutions manifested by sparse contact maps, which means ShNeigh is more robust to signal coverage.
    CONCLUSIONS: Our method can recover stable structures in high noise and sparse signal settings. It can also reconstruct similar structures from Hi-C data obtained using different restriction enzymes. Therefore, our method provides a new direction for enhancing the reconstruction quality of chromatin 3D structures.
    Keywords:  3D organization; Chromosome; Hi-C; MDS; Reconstruction
    DOI:  https://doi.org/10.1186/s12859-020-03612-4
  35. Genome Res. 2020 Jul 02. pii: gr.260463.119. [Epub ahead of print]
    Ramaker RC, Hardigan AA, Goh ST, Partridge EC, Wold B, Cooper SJ, Myers RM.
      DNA associated proteins (DAPs) classically regulate gene expression by binding to regulatory loci such as enhancers or promoters. As expanding catalogs of genome-wide DAP binding maps reveal thousands of loci that, unlike the majority of conventional enhancers and promoters, associate with dozens of different DAPs with apparently little regard for motif preference, an understanding of DAP association and coordination at such regulatory loci is essential to deciphering how these regions contribute to normal development and disease. In this study, we aggregated publicly available ChIP-seq data from 469 human DAPs assayed in three cell lines and integrated these data with an orthogonal dataset of 352 non-redundant, in vitro-derived motifs mapped to the genome within DNase I hypersensitivity footprints to characterize regions with high numbers of DAP associations. We establish a generalizable definition for High Occupancy Target (HOT) loci and identify putative driver DAP motifs in HepG2 cells, including HNF4A, SP1, SP5, and ETV4, that are highly prevalent and exhibit sequence conservation at HOT loci. The number of different DAPs associated with an element is positively associated with evidence of regulatory activity and, by systematically mutating 245 HOT loci with a massively parallel mutagenesis assay, we localized regulatory activity to a central core region that depends on the motif sequences of our previously nominated driver DAPs. In sum, this work leverages the increasingly large number of DAP motif and ChIP-seq data publicly available to explore how DAP associations contribute to genome-wide transcriptional regulation.
    DOI:  https://doi.org/10.1101/gr.260463.119
  36. Cell Rep. 2020 Jun 30. pii: S2211-1247(20)30807-X. [Epub ahead of print]31(13): 107826
    Renoux F, Stellato M, Haftmann C, Vogetseder A, Huang R, Subramaniam A, Becker MO, Blyszczuk P, Becher B, Distler JHW, Kania G, Boyman O, Distler O.
      Regulatory T cells (Tregs) represent a major population in the control of immune homeostasis and autoimmunity. Here we show that Fos-like 2 (Fosl2), a TCR-induced AP1 transcription factor, represses Treg development and controls autoimmunity. Mice overexpressing Fosl2 (Fosl2tg) indeed show a systemic inflammatory phenotype, with immune infiltrates in multiple organs. This phenotype is absent in Fosl2tg × Rag2-/- mice lacking T and B cells, and Fosl2 induces T cell-intrinsic reduction of Treg development that is responsible for the inflammatory phenotype. Fosl2tg T cells can transfer inflammation, which is suppressed by the co-delivery of Tregs, while Fosl2 deficiency in T cells reduces the severity of autoimmunity in the EAE model. We find that Fosl2 could affect expression of FoxP3 and other Treg development genes. Our data highlight the importance of AP1 transcription factors, in particular Fosl2, during T cell development to determine Treg differentiation and control autoimmunity.
    Keywords:  FoxP3; T cells; autoimmunity; immunology; inflammation; mouse models; regulatory T cells
    DOI:  https://doi.org/10.1016/j.celrep.2020.107826
  37. Epigenetics. 2020 Jun 30.
    Perzel Mandell KA, Price AJ, Wilton R, Collado-Torres L, Tao R, Eagles NJ, Szalay AS, Hyde TM, Weinberger DR, Kleinman JE, Jaffe AE.
      DNA methylation (DNAm) is a key epigenetic regulator of gene expression across development. The developing prenatal brain is a highly dynamic tissue, but our understanding of key drivers of epigenetic variability across development is limited. We therefore assessed genomic methylation at over 39 million sites in the prenatal cortex using whole genome bisulfite sequencing and found loci and regions in which methylation levels are dynamic across development. We saw that DNAm at these loci was associated with nearby gene expression and enriched for enhancer chromatin states in prenatal brain tissue. Additionally, these loci were enriched for genes associated with neuropsychiatric disorders and genes involved with neurogenesis. We also found autosomal differences in DNAm between the sexes during prenatal development, though these have less clear functional consequences. We lastly confirmed that the dynamic methylation at this critical period is specifically CpG methylation, with generally low levels of CpH methylation. Our findings provide detailed insight into prenatal brain development as well as clues to the pathogenesis of psychiatric traits seen later in life.
    Keywords:  DNA methylation; brain development; prenatal development; whole genome bisulfite sequencing
    DOI:  https://doi.org/10.1080/15592294.2020.1786304
  38. PLoS One. 2020 ;15(7): e0235530
    Kirchner S, Reuter S, Westphal A, Mrowka R.
      BACKGROUND: Understanding complex mechanisms of human transcriptional regulation remains a major challenge. Classical reporter studies already enabled the discovery of cis-regulatory elements within the non-coding DNA; however, the influence of genomic context and potential interactions are still largely unknown. Using a modified Cas9 activation complex we explore the complexity of renin transcription in its native genomic context.METHODS: With the help of genomic editing, we stably tagged the native renin on chromosome 1 with the firefly luciferase and stably integrated a programmable modified Cas9 based trans-activation complex (SAM-complex) by lentiviral transduction into human cells. By delivering five specific guide-RNA homologous to specific promoter regions of renin we were able to guide this SAM-complex to these regions of interest. We measured gene expression and generated and compared computational models.
    RESULTS: SAM complexes induced activation of renin in our cells after renin specific guide-RNA had been provided. All possible combinations of the five guides were subjected to model analysis in linear models. Quantifying the prediction error and the calculation of an estimator of the relative quality of the statistical models for our given set of data revealed that a model incorporating interactions in the proximal promoter is the superior model for explanation of the data.
    CONCLUSION: By applying our combined experimental and modelling approach we can show that interactions occur within the selected sequences of the proximal renin promoter region. This combined approach might potentially be useful to investigate other genomic regions. Our findings may help to better understand the transcriptional regulation of human renin.
    DOI:  https://doi.org/10.1371/journal.pone.0235530
  39. Oncogene. 2020 Jul 02.
    Chen J, Dang Y, Feng W, Qiao C, Liu D, Zhang T, Wang Y, Tian D, Fan D, Nie Y, Wu K, Xia L.
      The therapeutic strategies for advanced gastric cancer (GC) remain unsatisfying and limited. Therefore, it is still imperative to fully elucidate the mechanisms underlying GC metastasis. Here, we report a novel role of SRY-box transcription factor 18 (SOX18), a member of the SOX family, in promoting GC metastasis. The elevated expression of SOX18 was positively correlated with distant metastasis, higher AJCC stage, and poor prognosis in human GC. SOX18 expression was an independent and significant risk factor for the recurrence and survival in GC patients. Up-regulation of SOX18 promoted GC invasion and metastasis, whereas down-regulation of SOX18 decreased GC invasion and metastasis. Melanoma cell adhesion molecule (MCAM) and C-C motif chemokine ligand 7 (CCL7) are direct transcriptional targets of SOX18. Knockdown of MCAM and CCL7 significantly decreased SOX18-mediated GC invasion and metastasis, while the stable overexpression of MCAM and CCL7 reversed the decrease in cell invasion and metastasis that was induced by the inhibition of SOX18. A mechanistic investigation indicated that the upregulation of SOX18 that was mediated by the CCL7-CCR1 pathway relied on the ERK/ELK1 pathway. SOX18 knockdown significantly reduced CCL7-enhanced GC invasion and metastasis. Furthermore, BX471, a specific CCR1 inhibitor, significantly reduced the SOX18-mediated GC invasion and metastasis. In human GC tissues, SOX18 expression was positively correlated with CCL7 and MCAM expression, and patients with positive coexpression of SOX18/CCL7 or SOX18/MCAM had the worst prognosis. In conclusion, we defined a CCL7-CCR1-SOX18 positive feedback loop that played a pivotal role in GC metastasis, and targeting this pathway may be a promising therapeutic option for the clinical management of GC.
    DOI:  https://doi.org/10.1038/s41388-020-1378-1