bims-crepig Biomed News
on Chromatin regulation and epigenetics in cell fate and cancer
Issue of 2020‒12‒06
thirty-six papers selected by
Connor Rogerson
University of Cambridge, MRC Cancer Unit

  1. Cell Rep. 2020 Dec 01. pii: S2211-1247(20)31449-2. [Epub ahead of print]33(9): 108460
      Technologies to reprogram cell-type specification have revolutionized the fields of regenerative medicine and disease modeling. Currently, the selection of fate-determining factors for cell reprogramming applications is typically a laborious and low-throughput process. Therefore, we use high-throughput pooled CRISPR activation (CRISPRa) screens to systematically map human neuronal cell fate regulators. We utilize deactivated Cas9 (dCas9)-based gene activation to target 1,496 putative transcription factors (TFs) in the human genome. Using a reporter of neuronal commitment, we profile the neurogenic activity of these factors in human pluripotent stem cells (PSCs), leading to a curated set of pro-neuronal factors. Activation of pairs of TFs reveals neuronal cofactors, including E2F7, RUNX3, and LHX8, that improve conversion efficiency, subtype specificity, and maturation of neuronal cell types. Finally, using multiplexed gene regulation with orthogonal CRISPR systems, we demonstrate improved neuronal differentiation with concurrent activation and repression of target genes, underscoring the power of CRISPR-based gene regulation for programming complex cellular phenotypes.
    Keywords:  CRISPR; CRISPR activation; CRISPR screening; cell fate; gene regulation; neuronal differentiation; transcription factor
  2. Nucleic Acids Res. 2020 Dec 03. pii: gkaa1129. [Epub ahead of print]
      The Polycomb repressive complex 2 (PRC2) catalyzes histone H3 Lys27 trimethylation (H3K27me3) to repress gene transcription in multicellular eukaryotes. Despite its importance in gene silencing and cellular differentiation, how PRC2 is recruited to target loci is still not fully understood. Here, we report genome-wide evidence for the recruitment of PRC2 by the transcriptional repressors VIVIPAROUS1/ABI3-LIKE1 (VAL1) and VAL2 in Arabidopsis thaliana. We show that the val1 val2 double mutant possesses somatic embryonic phenotypes and a transcriptome strikingly similar to those of the swn clf double mutant, which lacks the PRC2 catalytic subunits SWINGER (SWN) and CURLY LEAF (CLF). We further show that VAL1 and VAL2 physically interact with SWN and CLF in vivo. Genome-wide binding profiling demonstrated that they colocalize with SWN and CLF at PRC2 target loci. Loss of VAL1/2 significantly reduces SWN and CLF enrichment at PRC2 target loci and leads to a genome-wide redistribution of H3K27me3 that strongly affects transcription. Finally, we provide evidence that the VAL1/VAL2-RY regulatory system is largely independent of previously identified modules for Polycomb silencing in plants. Together, our work demonstrates an extensive genome-wide interaction between VAL1/2 and PRC2 and provides mechanistic insights into the establishment of Polycomb silencing in plants.
  3. Nat Commun. 2020 Dec 04. 11(1): 6222
      Using Hi-C, promoter-capture Hi-C (pCHi-C), and other genome-wide approaches in skeletal muscle progenitors that inducibly express a master transcription factor, Pax7, we systematically characterize at high-resolution the spatio-temporal re-organization of compartments and promoter-anchored interactions as a consequence of myogenic commitment and differentiation. We identify key promoter-enhancer interaction motifs, namely, cliques and networks, and interactions that are dependent on Pax7 binding. Remarkably, Pax7 binds to a majority of super-enhancers, and together with a cadre of interacting transcription factors, assembles feed-forward regulatory loops. During differentiation, epigenetic memory and persistent looping are maintained at a subset of Pax7 enhancers in the absence of Pax7. We also identify and functionally validate a previously uncharacterized Pax7-bound enhancer hub that regulates the essential myosin heavy chain cluster during skeletal muscle cell differentiation. Our studies lay the groundwork for understanding the role of Pax7 in orchestrating changes in the three-dimensional chromatin conformation in muscle progenitors.
  4. Nat Commun. 2020 12 02. 11(1): 6161
      DNA 5-hydroxymethylcytosine (5hmC) modification is known to be associated with gene transcription and frequently used as a mark to investigate dynamic DNA methylation conversion during mammalian development and in human diseases. However, the lack of genome-wide 5hmC profiles in different human tissue types impedes drawing generalized conclusions about how 5hmC is implicated in transcription activity and tissue specificity. To meet this need, we describe the development of a 5hmC tissue map by characterizing the genomic distributions of 5hmC in 19 human tissues derived from ten organ systems. Subsequent sequencing results enabled the identification of genome-wide 5hmC distributions that uniquely separates samples by tissue type. Further comparison of the 5hmC profiles with transcriptomes and histone modifications revealed that 5hmC is preferentially enriched on tissue-specific gene bodies and enhancers. Taken together, the results provide an extensive 5hmC map across diverse human tissue types that suggests a potential role of 5hmC in tissue-specific development; as well as a resource to facilitate future studies of DNA demethylation in pathogenesis and the development of 5hmC as biomarkers.
  5. Elife. 2020 Dec 02. pii: e59404. [Epub ahead of print]9
      Our understanding of the beads-on-a-string arrangement of nucleosomes has been built largely on high-resolution sequence-agnostic imaging methods and sequence-resolved bulk biochemical techniques. To bridge the divide between these approaches, we present the single-molecule adenine methylated oligonucleosome sequencing assay (SAMOSA). SAMOSA is a high-throughput single-molecule sequencing method that combines adenine methyltransferase footprinting and single-molecule real-time DNA sequencing to natively and nondestructively measure nucleosome positions on individual chromatin fibres. SAMOSA data allows unbiased classification of single-molecular 'states' of nucleosome occupancy on individual chromatin fibres. We leverage this to estimate nucleosome regularity and spacing on single chromatin fibres genome-wide, at predicted transcription factor binding motifs, and across both active and silent human epigenomic domains. Our analyses suggest that chromatin is comprised of a diverse array of both regular and irregular single-molecular oligonucleosome patterns that differ subtly in their relative abundance across epigenomic domains. This irregularity is particularly striking in constitutive heterochromatin, which has typically been viewed as a conformationally static entity. Our proof-of-concept study provides a powerful new methodology for studying nucleosome organization at a previously intractable resolution, and offers up new avenues for modeling and visualizing higher-order chromatin structure.
    Keywords:  chromosomes; gene expression; genetics; genomics; human
  6. Sci Rep. 2020 Dec 03. 10(1): 21072
      Bone homeostasis is maintained by a balance between osteoblast-mediated bone formation and osteoclast-driven bone resorption. Hypoxia modulates this relationship partially via direct and indirect effects of the hypoxia-inducible factor-1 alpha (HIF-1α) transcription factor on osteoclast formation and bone resorption. Little data is available on the role(s) of the HIF-2α isoform of HIF in osteoclast biology. Here we describe induction of HIF-1α and HIF-2α during the differentiation of human CD14+ monocytes into osteoclasts. Knockdown of HIF-1α did not affect osteoclast differentiation but prevented the increase in bone resorption that occurs under hypoxic conditions. HIF-2α knockdown did not affect bone resorption but moderately inhibited osteoclast formation. Growth of osteoclasts in 3D gels reversed the effect of HIF-2α knockdown; HIF-2α siRNA increasing osteoclast formation in 3D. Glycolysis is the main HIF-regulated pathway that drives bone resorption. HIF knockdown only affected glucose uptake and bone resorption in hypoxic conditions. Inhibition of glycolysis with 2-deoxy-D-glucose (2-DG) reduced osteoclast formation and activity under both basal and hypoxic conditions, emphasising the importance of glycolytic metabolism in osteoclast biology. In summary, HIF-1α and HIF-2α play different but overlapping roles in osteoclast biology, highlighting the importance of the HIF pathway as a potential therapeutic target in osteolytic disease.
  7. Biology (Basel). 2020 Nov 27. pii: E428. [Epub ahead of print]9(12):
      Emerging evidence suggests that chromodomain-helicase-DNA-binding (CHD) proteins are involved in stem cell maintenance and differentiation via the coordination of chromatin structure and gene expression. However, the molecular function of some CHD proteins in stem cell regulation is still poorly understood. Herein, we show that Chd9 knockdown (KD) in mouse embryonic stem cells (ESCs) cultured in normal serum media, not in 2i-leukemia inhibitory factor (LIF) media, causes rapid cell proliferation. This is caused by transcriptional regulation related to the cell cycle and the response to growth factors. Our analysis showed that, unlike the serum cultured-Chd9 KD ESCs, the 2i-LIF-cultured-Chd9 KO ESCs displayed elevated levels of critical G1 phase regulators such as p21 and p27. Consistently, the DNA binding sites of CHD9 overlap with some transcription factor DNA motifs that are associated with genes regulating the cell cycle and growth pathways. These transcription factors include the cycle gene homology region (CHR), Arid5a, and LIN54. Collectively, our results provide new insights into CHD9-mediated gene transcription for controlling the cell cycle of ESCs.
    Keywords:  CHD9; ES cell; cell cycle; chromatin structure; transcription
  8. Nat Commun. 2020 Dec 04. 11(1): 6216
      Histone H3 lysine 27 (H3K27M) mutations represent the canonical oncohistone, occurring frequently in midline gliomas but also identified in haematopoietic malignancies and carcinomas. H3K27M functions, at least in part, through widespread changes in H3K27 trimethylation but its role in tumour initiation remains obscure. To address this, we created a transgenic mouse expressing H3.3K27M in diverse progenitor cell populations. H3.3K27M expression drives tumorigenesis in multiple tissues, which is further enhanced by Trp53 deletion. We find that H3.3K27M epigenetically activates a transcriptome, enriched for PRC2 and SOX10 targets, that overrides developmental and tissue specificity and is conserved between H3.3K27M-mutant mouse and human tumours. A key feature of the H3K27M transcriptome is activation of a RAS/MYC axis, which we find can be targeted therapeutically in isogenic and primary DIPG cell lines with H3.3K27M mutations, providing an explanation for the common co-occurrence of alterations in these pathways in human H3.3K27M-driven cancer. Taken together, these results show how H3.3K27M-driven transcriptome remodelling promotes tumorigenesis and will be critical for targeting cancers with these mutations.
  9. Cell. 2020 Nov 20. pii: S0092-8674(20)31529-4. [Epub ahead of print]
      Histone H3.3 glycine 34 to arginine/valine (G34R/V) mutations drive deadly gliomas and show exquisite regional and temporal specificity, suggesting a developmental context permissive to their effects. Here we show that 50% of G34R/V tumors (n = 95) bear activating PDGFRA mutations that display strong selection pressure at recurrence. Although considered gliomas, G34R/V tumors actually arise in GSX2/DLX-expressing interneuron progenitors, where G34R/V mutations impair neuronal differentiation. The lineage of origin may facilitate PDGFRA co-option through a chromatin loop connecting PDGFRA to GSX2 regulatory elements, promoting PDGFRA overexpression and mutation. At the single-cell level, G34R/V tumors harbor dual neuronal/astroglial identity and lack oligodendroglial programs, actively repressed by GSX2/DLX-mediated cell fate specification. G34R/V may become dispensable for tumor maintenance, whereas mutant-PDGFRA is potently oncogenic. Collectively, our results open novel research avenues in deadly tumors. G34R/V gliomas are neuronal malignancies where interneuron progenitors are stalled in differentiation by G34R/V mutations and malignant gliogenesis is promoted by co-option of a potentially targetable pathway, PDGFRA signaling.
    Keywords:  GSX2; H3.3 G34R/V; PDGFRA; cell-of-origin; chromatin conformation; gliomas; interneuron progenitors; oncohistones; pediatric cancer; single-cell transcriptome
  10. Nat Metab. 2020 Nov 30.
      The in vitro differentiation of insulin-producing beta-like cells can model aspects of human pancreatic development. Here, we generate 95,308 single-cell transcriptomes and reconstruct a lineage tree of the entire differentiation process from human embryonic stem cells to beta-like cells to study temporally regulated genes during differentiation. We identify so-called 'switch genes' at the branch point of endocrine/non-endocrine cell fate choice, revealing insights into the mechanisms of differentiation-promoting reagents, such as NOTCH and ROCKII inhibitors, and providing improved differentiation protocols. Over 20% of all detectable genes are activated multiple times during differentiation, even though their enhancer activation is usually unimodal, indicating extensive gene reuse driven by different enhancers. We also identify a stage-specific enhancer at the TCF7L2 locus for diabetes, uncovered by genome-wide association studies, that drives a transient wave of gene expression in pancreatic progenitors. Finally, we develop a web app to visualize gene expression on the lineage tree, providing a comprehensive single-cell data resource for researchers studying islet biology and diabetes.
  11. Proc Natl Acad Sci U S A. 2020 Nov 30. pii: 202017094. [Epub ahead of print]
      The evolutionary significance of epigenetic inheritance is controversial. While epigenetic marks such as DNA methylation can affect gene function and change in response to environmental conditions, their role as carriers of heritable information is often considered anecdotal. Indeed, near-complete DNA methylation reprogramming, as occurs during mammalian embryogenesis, is a major hindrance for the transmission of nongenetic information between generations. Yet it remains unclear how general DNA methylation reprogramming is across the tree of life. Here we investigate the existence of epigenetic inheritance in the honey bee. We studied whether fathers can transfer epigenetic information to their daughters through DNA methylation. We performed instrumental inseminations of queens, each with four different males, retaining half of each male's semen for whole genome bisulfite sequencing. We then compared the methylation profile of each father's somatic tissue and semen with the methylation profile of his daughters. We found that DNA methylation patterns were highly conserved between tissues and generations. There was a much greater similarity of methylomes within patrilines (i.e., father-daughter subfamilies) than between patrilines in each colony. Indeed, the samples' methylomes consistently clustered by patriline within colony. Samples from the same patriline had twice as many shared methylated sites and four times fewer differentially methylated regions compared to samples from different patrilines. Our findings indicate that there is no DNA methylation reprogramming in bees and, consequently, that DNA methylation marks are stably transferred between generations. This points to a greater evolutionary potential of the epigenome in invertebrates than there is in mammals.
    Keywords:  Apis mellifera; DNA methylation; epigenetic inheritance; epigenetic remodeling
  12. J Biol Chem. 2020 Dec 02. pii: jbc.RA120.015896. [Epub ahead of print]
      The Yes-associated protein YAP, one of the major effectors of the Hippo pathway together with its related protein TAZ, mediates a range of cellular processes from proliferation and death to morphogenesis. YAP and TAZ regulate a large number of target genes, acting as co-activators of DNA-binding transcription factors or as negative regulators of transcription by interacting with the nucleosome remodeling and histone deacetylase complexes. YAP is expressed in self-renewing embryonic stem cells (ESCs), although it is still debated whether it plays any crucial roles in the control of either stemness or differentiation. Here we show that the transient downregulation of YAP in mouse ESCs perturbs cellular homeostasis, leading to the inability to differentiate properly. Bisulfite genomic sequencing revealed that this transient knockdown caused a genome-wide alteration of the DNA methylation remodeling that takes place during the early steps of differentiation, suggesting that the phenotype we observed might be due to the dysregulation of some of the mechanisms involved in regulation of ESC exit from pluripotency. By gene expression analysis we identified two molecules which could have a role in the altered genome-wide methylation profile: the long non-coding RNA Ephemeron, whose rapid upregulation is crucial for ESCs transition into epiblast, and the methyltransferase-like protein Dnmt3l, which, during the embryo development, cooperates with Dnmt3a and Dnmt3b to contribute to the de novo DNA methylation that governs early steps of ESC differentiation. These data suggest a new role for YAP in the governance of the epigenetic dynamics of exit from pluripotency.
    Keywords:  DNA methylation; Dnmt3l; Ephemeron; cell differentiation; embryonic stem cell; pluripotency; yes-associated protein (YAP)
  13. Nat Commun. 2020 12 02. 11(1): 6168
      Annotations of evolutionary sequence constraint based on multi-species genome alignments and genome-wide maps of epigenomic marks and transcription factor binding provide important complementary information for understanding the human genome and genetic variation. Here we developed the Constrained Non-Exonic Predictor (CNEP) to quantify the evidence of each base in the genome being in an evolutionarily constrained non-exonic element from an input of over 60,000 epigenomic and transcription factor binding features. We find that the CNEP score outperforms baseline and related existing scores at predicting evolutionarily constrained non-exonic bases from such data. However, a subset of them are still not well predicted by CNEP. We developed a complementary Conservation Signature Score by CNEP (CSS-CNEP) that is predictive of those bases. We further characterize the nature of constrained non-exonic bases with low CNEP scores using additional types of information. CNEP and CSS-CNEP are resources for analyzing constrained non-exonic bases in the genome.
  14. Nat Biotechnol. 2020 Nov 30.
      Human pluripotent stem cells (hPSCs) offer an unprecedented opportunity to model diverse cell types and tissues. To enable systematic exploration of the programming landscape mediated by transcription factors (TFs), we present the Human TFome, a comprehensive library containing 1,564 TF genes and 1,732 TF splice isoforms. By screening the library in three hPSC lines, we discovered 290 TFs, including 241 that were previously unreported, that induce differentiation in 4 days without alteration of external soluble or biomechanical cues. We used four of the hits to program hPSCs into neurons, fibroblasts, oligodendrocytes and vascular endothelial-like cells that have molecular and functional similarity to primary cells. Our cell-autonomous approach enabled parallel programming of hPSCs into multiple cell types simultaneously. We also demonstrated orthogonal programming by including oligodendrocyte-inducible hPSCs with unmodified hPSCs to generate cerebral organoids, which expedited in situ myelination. Large-scale combinatorial screening of the Human TFome will complement other strategies for cell engineering based on developmental biology and computational systems biology.
  15. Nat Commun. 2020 12 03. 11(1): 6196
      The Transforming Growth Factor-β (TGFβ) signaling pathway controls transcription by regulating enhancer activity. How TGFβ-regulated enhancers are selected and what chromatin changes are associated with TGFβ-dependent enhancers regulation are still unclear. Here we report that TGFβ treatment triggers fast and widespread increase in chromatin accessibility in about 80% of the enhancers of normal mouse mammary epithelial-gland cells, irrespective of whether they are activated, repressed or not regulated by TGFβ. This enhancer opening depends on both the canonical and non-canonical TGFβ pathways. Most TGFβ-regulated genes are located around enhancers regulated in the same way, often creating domains of several co-regulated genes that we term TGFβ regulatory domains (TRD). CRISPR-mediated inactivation of enhancers within TRDs impairs TGFβ-dependent regulation of all co-regulated genes, demonstrating that enhancer targeting is more promiscuous than previously anticipated. The area of TRD influence is restricted by topologically associating domains (TADs) borders, causing a bias towards co-regulation within TADs.
  16. Cell Rep. 2020 Dec 01. pii: S2211-1247(20)31439-X. [Epub ahead of print]33(9): 108450
      The nucleosome remodeling and deacetylase (NuRD) complex is essential for metazoan development but has been refractory to biochemical analysis. We present an integrated analysis of the native mammalian NuRD complex, combining quantitative mass spectrometry, cross-linking, protein biochemistry, and electron microscopy to define the architecture of the complex. NuRD is built from a 2:2:4 (MTA, HDAC, and RBBP) deacetylase module and a 1:1:1 (MBD, GATAD2, and Chromodomain-Helicase-DNA-binding [CHD]) remodeling module, and the complex displays considerable structural dynamics. The enigmatic GATAD2 controls the asymmetry of the complex and directly recruits the CHD remodeler. The MTA-MBD interaction acts as a point of functional switching, with the transcriptional regulator PWWP2A competing with MBD for binding to the MTA-HDAC-RBBP subcomplex. Overall, our data address the long-running controversy over NuRD stoichiometry, provide imaging of the mammalian NuRD complex, and establish the biochemical mechanism by which PWWP2A can regulate NuRD composition.
    Keywords:  DIA-MS; EM; NuRD; PWWP2A; cross-linking MS; electron microscopy; gene regulation; nucleosome remodeling
  17. PLoS Biol. 2020 Nov 30. 18(11): e3000749
      Clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR-associated (Cas) 9 has been widely used far beyond genome editing. Fusions of deactivated Cas9 (dCas9) to transcription effectors enable interrogation of the epigenome and controlling of gene expression. However, the large transgene size of dCas9-fusion hinders its applications especially in somatic tissues. Here, we develop a robust CRISPR interference (CRISPRi) system by transgenic expression of doxycycline (Dox) inducible dCas9-KRAB in mouse embryonic stem cells (iKRAB ESC). After introduction of specific single-guide RNAs (sgRNAs), the induced dCas9-KRAB efficiently maintains gene inactivation, although it modestly down-regulates the expression of active genes. The proper timing of Dox addition during cell differentiation or reprogramming allows us to study or screen spatiotemporally activated promoters or enhancers and thereby the gene functions. Furthermore, taking the ESC for blastocyst injection, we generate an iKRAB knock-in (KI) mouse model that enables the shutdown of gene expression and loss-of-function (LOF) studies ex vivo and in vivo by a simple transduction of gRNAs. Thus, our inducible CRISPRi ESC line and KI mouse provide versatile and convenient platforms for functional interrogation and high-throughput screens of specific genes and potential regulatory elements in the setting of development or diseases.
  18. Sci Adv. 2020 Dec;pii: eabc8696. [Epub ahead of print]6(49):
      While a genetic component of preterm birth (PTB) has long been recognized and recently mapped by genome-wide association studies (GWASs), the molecular determinants underlying PTB remain elusive. This stems in part from an incomplete availability of functional genomic annotations in human cell types relevant to pregnancy and PTB. We generated transcriptome (RNA-seq), epigenome (ChIP-seq of H3K27ac, H3K4me1, and H3K4me3 histone modifications), open chromatin (ATAC-seq), and chromatin interaction (promoter capture Hi-C) annotations of cultured primary decidua-derived mesenchymal stromal/stem cells and in vitro differentiated decidual stromal cells and developed a computational framework to integrate these functional annotations with results from a GWAS of gestational duration in 56,384 women. Using these resources, we uncovered additional loci associated with gestational duration and target genes of associated loci. Our strategy illustrates how functional annotations in pregnancy-relevant cell types aid in the experimental follow-up of GWAS for PTB and, likely, other pregnancy-related conditions.
  19. Blood. 2020 Nov 30. pii: blood.2020007075. [Epub ahead of print]
      Isocitrate dehydrogenase (IDH) mutations are common genetic alterations in myeloid disorders, including acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). Epigenetic changes, including abnormal histone and DNA methylation, have been implicated in the pathogenic build-up of hematopoietic progenitors, but it is still unclear whether and how IDH mutations themselves affect hematopoiesis. Here, we show that IDH1-mutant mice develop myeloid dysplasia in that these animals exhibit anemia, ineffective erythropoiesis, increased immature progenitor and erythroblast. In erythroid cells of these mice, D-2-hydroxyglutarate (D-2HG), an aberrant metabolite produced by the mutant IDH1 enzyme, inhibits oxoglutarate dehydrogenase (OGDH) activity and diminishes succinyl-CoA production. This succinyl-CoA deficiency attenuates heme biosynthesis in IDH1-mutant hematopoietic cells, thus blocking erythroid differentiation at the late erythroblast stage and the erythroid commitment of hematopoietic stem cells (HSC), while the exogenous succinyl-CoA or 5-ALA rescues erythropoiesis in IDH1-mutant erythroid cells. Heme deficiency also impairs heme oxygenase-1 (HO-1) expression, which reduces levels of important heme catabolites such as biliverdin and bilirubin. These deficits result in accumulation of excessive reactive oxygen species (ROS) that induce the cell death of IDH1-mutant erythroid cells. Our results clearly demonstrate the essential role of IDH1 in normal erythropoiesis and show how its mutation leads to myeloid disorders. Our data thus have important implications for the devising of new treatments for IDH-mutant tumors.
  20. Commun Biol. 2020 Nov 30. 3(1): 725
      Temporal and spatial colinear expression of the Hox genes determines the specification of positional identities during vertebrate development. Post-translational modifications of histones contribute to transcriptional regulation. Lysine demethylase 7A (Kdm7a) demethylates lysine 9 or 27 di-methylation of histone H3 (H3K9me2, H3K27me2) and participates in the transcriptional activation of developmental genes. However, the role of Kdm7a during mouse embryonic development remains to be elucidated. Herein, we show that Kdm7a-/- mouse exhibits an anterior homeotic transformation of the axial skeleton, including an increased number of presacral elements. Importantly, posterior Hox genes (caudally from Hox9) are specifically downregulated in the Kdm7a-/- embryo, which correlates with increased levels of H3K9me2, not H3K27me2. These observations suggest that Kdm7a controls the transcription of posterior Hox genes, likely via its demethylating activity, and thereby regulating the murine anterior-posterior development. Such epigenetic regulatory mechanisms may be harnessed for proper control of coordinate body patterning in vertebrates.
  21. Nat Commun. 2020 12 01. 11(1): 6146
      Cohesin plays an essential role in chromatin loop extrusion, but its impact on a compartmentalized nuclear architecture, linked to nuclear functions, is less well understood. Using live-cell and super-resolved 3D microscopy, here we find that cohesin depletion in a human colon cancer derived cell line results in endomitosis and a single multilobulated nucleus with chromosome territories pervaded by interchromatin channels. Chromosome territories contain chromatin domain clusters with a zonal organization of repressed chromatin domains in the interior and transcriptionally competent domains located at the periphery. These clusters form microscopically defined, active and inactive compartments, which likely correspond to A/B compartments, which are detected with ensemble Hi-C. Splicing speckles are observed nearby within the lining channel system. We further observe that the multilobulated nuclei, despite continuous absence of cohesin, pass through S-phase with typical spatio-temporal patterns of replication domains. Evidence for structural changes of these domains compared to controls suggests that cohesin is required for their full integrity.
  22. J Neurosci. 2020 Nov 30. pii: JN-RM-1821-20. [Epub ahead of print]
      The dynamic regulation of DNA methylation in post-mitotic neurons is necessary for memory formation and other adaptive behaviors. Ten-eleven translocation 1 (TET1) plays a part in these processes by oxidizing 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), thereby initiating active DNA demethylation. However, attempts to pinpoint its exact role in the nervous system have been hindered by contradictory findings, perhaps due in part, to a recent discovery that two isoforms of the Tet1 gene are differentially expressed from early development into adulthood. Here, we demonstrate that both the shorter transcript (Tet1S ) encoding an N-terminally truncated TET1 protein and a full-length Tet1 (Tet1FL ) transcript encoding canonical TET1 are co-expressed in the adult mouse brain. We show that Tet1S is the predominantly expressed isoform, and is highly enriched in neurons, whereas Tet1FL is generally expressed at lower levels and more abundant in glia, suggesting their roles are at least partially cell type-specific. Using viral-mediated, isoform- and neuron-specific molecular tools, we find that the individual disruption of each isoform leads to the dysregulation of unique gene ensembles and contrasting changes in basal synaptic transmission. In addition, Tet1S repression enhances, while Tet1FL impairs, hippocampal-dependent memory in male mice. Together, our findings demonstrate that each Tet1 isoform serves a distinct role in the mammalian brain.SIGNIFICANCE STATEMENTIn the brain, activity-dependent changes in gene expression are required for the formation of long-term memories. DNA methylation plays an essential role in orchestrating these learning-induced transcriptional programs by influencing chromatin accessibility and transcription factor binding. Once thought of as a stable epigenetic mark, DNA methylation is now known to be impermanent and dynamically regulated, driving neuroplasticity in the brain. We found that Tet1-a member of the Ten-eleven translocation family of enzymes that mediates removal of DNA methyl marks-is expressed as a two separate isoforms in the adult mouse brain and that each differentially regulates gene expression, synaptic transmission and memory formation. Together, our findings demonstrate that each Tet1 isoform serves a distinct role in the central nervous system.
  23. Mol Cell Biol. 2020 Nov 30. pii: MCB.00210-20. [Epub ahead of print]
      Copper homeostasis is crucial for cellular processes. The balance between nutritional and toxic level is maintained through the regulation of uptake, distribution and detoxification via antagonistic actions of two transcription factors AceI and Mac1. AceI responds to toxic copper levels by transcriptional regulation of detoxification genes CUP1 and CRS5 Cup1 metallothionein (MT) confers protection against toxic copper levels. CUP1 gene regulation is a multifactorial event requiring AceI, TBP (TATA-binding protein), chromatin remodeler, acetyltransferase (Spt10) and histones. However, the role of histone H3 residues has not been fully elucidated. To investigate the role of H3 tail in CUP1 transcriptional regulation, we screened the library of histone mutants in copper stress. We identified mutations in H3 (K23Q, K27R, K36Q, Δ5-16, Δ13-16, Δ13-28, Δ25-28, Δ28-31, Δ29-32) that reduce CUP1 expression. We detected reduced AceI occupancy across CUP1 promoter in K23Q, K36Q, Δ5-16, Δ13-28, Δ25-28 and Δ28-31 correlating with the reduced CUP1 transcription. Majority of these mutations affect TBP occupancy at CUP1 promoter augmenting the CUP1 transcription defect. Additionally, some mutants display cytosolic protein aggregation upon copper stress. Altogether, our data establish previously unidentified residues of H3 N-terminal tail and their modifications in CUP1 regulation.
  24. Nat Genet. 2020 Dec;52(12): 1271-1281
      Histone-modifying enzymes are implicated in the control of diverse DNA-templated processes including gene expression. Here, we outline historical and current thinking regarding the functions of histone modifications and their associated enzymes. One current viewpoint, based largely on correlative evidence, posits that histone modifications are instructive for transcriptional regulation and represent an epigenetic 'code'. Recent studies have challenged this model and suggest that histone marks previously associated with active genes do not directly cause transcriptional activation. Additionally, many histone-modifying proteins possess non-catalytic functions that overshadow their enzymatic activities. Given that much remains unknown regarding the functions of these proteins, the field should be cautious in interpreting loss-of-function phenotypes and must consider both cellular and developmental context. In this Perspective, we focus on recent progress relating to the catalytic and non-catalytic functions of the Trithorax-COMPASS complexes, Polycomb repressive complexes and Clr4/Suv39 histone-modifying machineries.
  25. Sci Rep. 2020 Dec 04. 10(1): 21228
      Ascorbic acid (AA, vitamin C) serves as a cofactor for ten-eleven translocation (TET) enzymes and induces DNA demethylation in vitro. However, its role in DNA demethylation in vivo remains unclear. We previously reported that DNA demethylation in the mouse liver was enhanced during the suckling period. Therefore, we hypothesized that DNA demethylation is enhanced in an AA-dependent manner during the suckling period. To examine our hypothesis, we employed wild-type (WT) mice, which synthesize AA, and senescence marker protein-30/gluconolactonase (SMP30/GNL) knockout (KO) mice, which cannot synthesize AA, and analyzed the DNA methylation status in the livers of offspring in both the suckling period and adulthood. SMP30/GNL KO offspring showed DNA hypermethylation in the liver possibly due to low plasma and hepatic AA levels during the suckling period despite the administration of rescue-dose AA to dams. Furthermore, DNA hypermethylation of the fibroblast growth factor 21 gene (Fgf21), a PPARα target gene, persisted into adulthood. In contrast, a high-dose AA administration to SMP30/GNL KO dams during the lactation period restored DNA demethylation in the livers of offspring. Even though a slight increase was observed in plasma AA levels with the administration of rescue-dose AA to WT dams during the gestation and lactation periods, DNA demethylation in the livers of offspring was minimally enhanced. The present results demonstrate that AA intake during the suckling period is required for proper DNA demethylation in the liver.
  26. Nat Commun. 2020 11 30. 11(1): 6114
      DNA methylation differences in Alzheimer's disease (AD) have been reported. Here, we conducted a meta-analysis of more than 1000 prefrontal cortex brain samples to prioritize the most consistent methylation differences in multiple cohorts. Using a uniform analysis pipeline, we identified 3751 CpGs and 119 differentially methylated regions (DMRs) significantly associated with Braak stage. Our analysis identified differentially methylated genes such as MAMSTR, AGAP2, and AZU1. The most significant DMR identified is located on the MAMSTR gene, which encodes a cofactor that stimulates MEF2C. Notably, MEF2C cooperates with another transcription factor, PU.1, a central hub in the AD gene network. Our enrichment analysis highlighted the potential roles of the immune system and polycomb repressive complex 2 in pathological AD. These results may help facilitate future mechanistic and biomarker discovery studies in AD.
  27. Cell Rep. 2020 Dec 01. pii: S2211-1247(20)31441-8. [Epub ahead of print]33(9): 108452
      Plasma cells provide high-affinity antibodies against invading pathogens. Although transcriptional and epigenetic mechanisms have been extensively studied for plasma cell differentiation, how these mechanisms respond to environmental cues remains largely unexplored. In this study, we show that ascorbic acid (vitamin C), an essential nutrient, is able to promote plasma cell differentiation and humoral immune response by enhancing TET2/3-mediated DNA demethylation. Ascorbic acid treatment during B cell activation has persistent effects on later plasma cell differentiation by predisposing germinal center B cells toward the plasma cell lineage. Conversely, ascorbic acid deficiency in vivo blocks plasma cell differentiation and attenuates the humoral immune response following antigen immunization. We further demonstrate that such effects of ascorbic acid on plasma cell differentiation require DNA methylcytosine oxidases TET2 and TET3. Our study thus reveals a previously uncharacterized link between essential nutrients and epigenetic regulation of plasma cell differentiation and humoral immune response.
    Keywords:  Ascorbic acid; B cell in vitro differentiation; Blimp-1; Epigenetic regulation; Germinal center B cell; Immune respones; Plasma B cell; Ten-eleven translocation (TET) enzymes
  28. Nucleic Acids Res. 2020 Dec 03. pii: gkaa1104. [Epub ahead of print]
      DNA methylation is an important epigenetic regulator in gene expression and has several roles in cancer and disease progression. MethHC version 2.0 (MethHC 2.0) is an integrated and web-based resource focusing on the aberrant methylomes of human diseases, specifically cancer. This paper presents an updated implementation of MethHC 2.0 by incorporating additional DNA methylomes and transcriptomes from several public repositories, including 33 human cancers, over 50 118 microarray and RNA sequencing data from TCGA and GEO, and accumulating up to 3586 manually curated data from >7000 collected published literature with experimental evidence. MethHC 2.0 has also been equipped with enhanced data annotation functionality and a user-friendly web interface for data presentation, search, and visualization. Provided features include clinical-pathological data, mutation and copy number variation, multiplicity of information (gene regions, enhancer regions, and CGI regions), and circulating tumor DNA methylation profiles, available for research such as biomarker panel design, cancer comparison, diagnosis, prognosis, therapy study and identifying potential epigenetic biomarkers. MethHC 2.0 is now available at∼MethHC.
  29. Nat Commun. 2020 11 30. 11(1): 6077
      Single-cell RNA sequencing (scRNA-seq) has become an empowering technology to profile the transcriptomes of individual cells on a large scale. Early analyses of differential expression have aimed at identifying differences between subpopulations to identify subpopulation markers. More generally, such methods compare expression levels across sets of cells, thus leading to cross-condition analyses. Given the emergence of replicated multi-condition scRNA-seq datasets, an area of increasing focus is making sample-level inferences, termed here as differential state analysis; however, it is not clear which statistical framework best handles this situation. Here, we surveyed methods to perform cross-condition differential state analyses, including cell-level mixed models and methods based on aggregated pseudobulk data. To evaluate method performance, we developed a flexible simulation that mimics multi-sample scRNA-seq data. We analyzed scRNA-seq data from mouse cortex cells to uncover subpopulation-specific responses to lipopolysaccharide treatment, and provide robust tools for multi-condition analysis within the muscat R package.
  30. Sci Adv. 2020 Dec;pii: eabe2955. [Epub ahead of print]6(49):
      Developmental enhancers control the expression of genes prefiguring morphological patterns. The activity of an enhancer varies among cells of a tissue, but collectively, expression levels in individual cells constitute a spatial pattern of gene expression. How the spatial and quantitative regulatory information is encoded in an enhancer sequence is elusive. To link spatial pattern and activity levels of an enhancer, we used systematic mutations of the yellow spot enhancer, active in developing Drosophila wings, and tested their effect in a reporter assay. Moreover, we developed an analytic framework based on the comprehensive quantification of spatial reporter activity. We show that the quantitative enhancer activity results from densely packed regulatory information along the sequence, and that a complex interplay between activators and multiple tiers of repressors carves the spatial pattern. Our results shed light on how an enhancer reads and integrates trans-regulatory landscape information to encode a spatial quantitative pattern.
  31. Nat Plants. 2020 Nov 30.
      DNA methylation is important for silencing transposable elements (TEs) in diverse eukaryotes, including plants. In plant genomes, TEs are silenced by methylation of histone H3 lysine 9 (H3K9) and cytosines in both CG and non-CG contexts. The role of RNA interference (RNAi) in establishing TE-specific silent marks has been extensively studied, but the importance of RNAi-independent pathways remains largely unexplored. Here, we directly investigated transgenerational de novo DNA methylation of TEs after the loss of silent marks. Our analyses uncovered potent and precise RNAi-independent pathways for recovering non-CG methylation and H3K9 methylation in most TE genes (that is, coding regions within TEs). Characterization of a subset of TE genes without the recovery revealed the effects of H3K9 demethylation, replacement of histone H2A variants and their interaction with CG methylation, together with feedback from transcription. These chromatin components are conserved among eukaryotes and may contribute to chromatin reprogramming in a conserved manner.
  32. Sci Adv. 2020 Dec;pii: eabc9207. [Epub ahead of print]6(49):
      Although somatic mutations in Histone 3.3 (H3.3) are well-studied drivers of oncogenesis, the role of germline mutations remains unreported. We analyze 46 patients bearing de novo germline mutations in histone 3 family 3A (H3F3A) or H3F3B with progressive neurologic dysfunction and congenital anomalies without malignancies. Molecular modeling of all 37 variants demonstrated clear disruptions in interactions with DNA, other histones, and histone chaperone proteins. Patient histone posttranslational modifications (PTMs) analysis revealed notably aberrant local PTM patterns distinct from the somatic lysine mutations that cause global PTM dysregulation. RNA sequencing on patient cells demonstrated up-regulated gene expression related to mitosis and cell division, and cellular assays confirmed an increased proliferative capacity. A zebrafish model showed craniofacial anomalies and a defect in Foxd3-derived glia. These data suggest that the mechanism of germline mutations are distinct from cancer-associated somatic histone mutations but may converge on control of cell proliferation.
  33. Nucleic Acids Res. 2020 Dec 02. pii: gkaa1134. [Epub ahead of print]
      We report an approach to predict DNA specificity of the tetracycline repressor (TetR) family transcription regulators (TFRs). First, a genome sequence-based method was streamlined with quantitative P-values defined to filter out reliable predictions. Then, a framework was introduced to incorporate structural data and to train a statistical energy function to score the pairing between TFR and TFR binding site (TFBS) based on sequences. The predictions benchmarked against experiments, TFBSs for 29 out of 30 TFRs were correctly predicted by either the genome sequence-based or the statistical energy-based method. Using P-values or Z-scores as indicators, we estimate that 59.6% of TFRs are covered with relatively reliable predictions by at least one of the two methods, while only 28.7% are covered by the genome sequence-based method alone. Our approach predicts a large number of new TFBs which cannot be correctly retrieved from public databases such as FootprintDB. High-throughput experimental assays suggest that the statistical energy can model the TFBSs of a significant number of TFRs reliably. Thus the energy function may be applied to explore for new TFBSs in respective genomes. It is possible to extend our approach to other transcriptional factor families with sufficient structural information.
  34. Nature. 2020 Dec;588(7836): 124-129
      Ageing is a degenerative process that leads to tissue dysfunction and death. A proposed cause of ageing is the accumulation of epigenetic noise that disrupts gene expression patterns, leading to decreases in tissue function and regenerative capacity1-3. Changes to DNA methylation patterns over time form the basis of ageing clocks4, but whether older individuals retain the information needed to restore these patterns-and, if so, whether this could improve tissue function-is not known. Over time, the central nervous system (CNS) loses function and regenerative capacity5-7. Using the eye as a model CNS tissue, here we show that ectopic expression of Oct4 (also known as Pou5f1), Sox2 and Klf4 genes (OSK) in mouse retinal ganglion cells restores youthful DNA methylation patterns and transcriptomes, promotes axon regeneration after injury, and reverses vision loss in a mouse model of glaucoma and in aged mice. The beneficial effects of OSK-induced reprogramming in axon regeneration and vision require the DNA demethylases TET1 and TET2. These data indicate that mammalian tissues retain a record of youthful epigenetic information-encoded in part by DNA methylation-that can be accessed to improve tissue function and promote regeneration in vivo.
  35. Nucleic Acids Res. 2020 Dec 04. pii: gkaa1138. [Epub ahead of print]
      Recent advances in single-cell open-chromatin and transcriptome profiling have created a challenge of exploring novel applications with a meaningful transformation of read-counts, which often have high variability in noise and drop-out among cells. Here, we introduce UniPath, for representing single-cells using pathway and gene-set enrichment scores by a transformation of their open-chromatin or gene-expression profiles. The robust statistical approach of UniPath provides high accuracy, consistency and scalability in estimating gene-set enrichment scores for every cell. Its framework provides an easy solution for handling variability in drop-out rate, which can sometimes create artefact due to systematic patterns. UniPath provides an alternative approach of dimension reduction of single-cell open-chromatin profiles. UniPath's approach of predicting temporal-order of single-cells using their pathway enrichment scores enables suppression of covariates to achieve correct order of cells. Analysis of mouse cell atlas using our approach yielded surprising, albeit biologically-meaningful co-clustering of cell-types from distant organs. By enabling an unconventional method of exploiting pathway co-occurrence to compare two groups of cells, our approach also proves to be useful in inferring context-specific regulations in cancer cells. Available at
  36. Sci Signal. 2020 Dec 01. pii: eabc4537. [Epub ahead of print]13(660):
      We previously reported that an ortholog of STING regulates infection by picorna-like viruses in Drosophila In mammals, STING is activated by the cyclic dinucleotide 2'3'-cGAMP produced by cGAS, which acts as a receptor for cytosolic DNA. Here, we showed that injection of flies with 2'3'-cGAMP induced the expression of dSTING-regulated genes. Coinjection of 2'3'-cGAMP with a panel of RNA or DNA viruses resulted in substantially reduced viral replication. This 2'3'-cGAMP-mediated protection was still observed in flies with mutations in Atg7 and AGO2, genes that encode key components of the autophagy and small interfering RNA pathways, respectively. By contrast, this protection was abrogated in flies with mutations in the gene encoding the NF-κB transcription factor Relish. Transcriptomic analysis of 2'3'-cGAMP-injected flies revealed a complex response pattern in which genes were rapidly induced, induced after a delay, or induced in a sustained manner. Our results reveal that dSTING regulates an NF-κB-dependent antiviral program that predates the emergence of interferons in vertebrates.