bims-crepig Biomed News
on Chromatin regulation and epigenetics in cell fate and cancer
Issue of 2020‒06‒14
thirty-nine papers selected by
Connor Rogerson
University of Cambridge, MRC Cancer Unit


  1. Mol Ther. 2020 Jun 01. pii: S1525-0016(20)30290-2. [Epub ahead of print]
    Qiao Y, Wang Z, Tan F, Chen J, Lin J, Yang J, Li H, Wang X, Sali A, Zhang L, Zhong G.
      Transcription growth factor β (TGF-β) signaling-triggered epithelial-to-mesenchymal transition (EMT) process is associated with tumor stemness, metastasis, and chemotherapy resistance. However, the epigenomic basis for TGF-β-induced EMT remains largely unknown. Here we reveal that HDAC1-mediated global histone deacetylation and the gain of specific histone H3 lysine 27 acetylation (H3K27ac)-marked enhancers are essential for the TGF-β-induced EMT process. Enhancers gained upon TGF-β treatment are linked to gene activation of EMT markers and cancer metastasis. Notably, dynamic enhancer gain or loss mainly occurs within pre-existing topologically associated domains (TADs) in epithelial cells, with minimal three-dimensional (3D) genome architecture reorganization. Through motif enrichment analysis of enhancers that are lost or gained upon TGF-β stimulation, we identify FOXA2 as a key factor to activate epithelial-specific enhancer activity, and we also find that TEAD4 forms a complex with SMAD2/3 to mediate TGF-β signaling-triggered mesenchymal enhancer reprogramming. Together, our results implicate that key transcription-factor (TF)-mediated enhancer reprogramming modulates the developmental transition in TGF-β signaling-associated cancer metastasis.
    Keywords:  EMT; FOXA2; Hi-C; TEAD2; TEAD4; TGFβ; enhancer reprogramming; epithelial-to-mesenchymal transition; metastasis
    DOI:  https://doi.org/10.1016/j.ymthe.2020.05.026
  2. Nat Cell Biol. 2020 Jun 08.
    Martínez-Zamudio RI, Roux PF, de Freitas JANLF, Robinson L, Doré G, Sun B, Belenki D, Milanovic M, Herbig U, Schmitt CA, Gil J, Bischof O.
      Senescent cells affect many physiological and pathophysiological processes. While select genetic and epigenetic elements for senescence induction have been identified, the dynamics, epigenetic mechanisms and regulatory networks defining senescence competence, induction and maintenance remain poorly understood, precluding the deliberate therapeutic targeting of senescence for health benefits. Here, we examined the possibility that the epigenetic state of enhancers determines senescent cell fate. We explored this by generating time-resolved transcriptomes and epigenome profiles during oncogenic RAS-induced senescence and validating central findings in different cell biology and disease models of senescence. Through integrative analysis and functional validation, we reveal links between enhancer chromatin, transcription factor recruitment and senescence competence. We demonstrate that activator protein 1 (AP-1) 'pioneers' the senescence enhancer landscape and defines the organizational principles of the transcription factor network that drives the transcriptional programme of senescent cells. Together, our findings enabled us to manipulate the senescence phenotype with potential therapeutic implications.
    DOI:  https://doi.org/10.1038/s41556-020-0529-5
  3. Mol Cell. 2020 Jun 04. pii: S1097-2765(20)30345-2. [Epub ahead of print]
    Louphrasitthiphol P, Siddaway R, Loffreda A, Pogenberg V, Friedrichsen H, Schepsky A, Zeng Z, Lu M, Strub T, Freter R, Lisle R, Suer E, Thomas B, Schuster-Böckler B, Filippakopoulos P, Middleton M, Lu X, Patton EE, Davidson I, Lambert JP, Wilmanns M, Steingrímsson E, Mazza D, Goding CR.
      It is widely assumed that decreasing transcription factor DNA-binding affinity reduces transcription initiation by diminishing occupancy of sequence-specific regulatory elements. However, in vivo transcription factors find their binding sites while confronted with a large excess of low-affinity degenerate motifs. Here, using the melanoma lineage survival oncogene MITF as a model, we show that low-affinity binding sites act as a competitive reservoir in vivo from which transcription factors are released by mitogen-activated protein kinase (MAPK)-stimulated acetylation to promote increased occupancy of their regulatory elements. Consequently, a low-DNA-binding-affinity acetylation-mimetic MITF mutation supports melanocyte development and drives tumorigenesis, whereas a high-affinity non-acetylatable mutant does not. The results reveal a paradoxical acetylation-mediated molecular clutch that tunes transcription factor availability via genome-wide redistribution and couples BRAF to tumorigenesis. Our results further suggest that p300/CREB-binding protein-mediated transcription factor acetylation may represent a common mechanism to control transcription factor availability.
    Keywords:  DNA-binding affinity; E-box; MITF; acetylation; bHLH-LZ; melanocyte; melanoma; transcription factor
    DOI:  https://doi.org/10.1016/j.molcel.2020.05.025
  4. Nat Genet. 2020 Jun 08.
    Charlton J, Jung EJ, Mattei AL, Bailly N, Liao J, Martin EJ, Giesselmann P, Brändl B, Stamenova EK, Müller FJ, Kiskinis E, Gnirke A, Smith ZD, Meissner A.
      Mammalian cells stably maintain high levels of DNA methylation despite expressing both positive (DNMT3A/B) and negative (TET1-3) regulators. Here, we analyzed the independent and combined effects of these regulators on the DNA methylation landscape using a panel of knockout human embryonic stem cell (ESC) lines. The greatest impact on global methylation levels was observed in DNMT3-deficient cells, including reproducible focal demethylation at thousands of normally methylated loci. Demethylation depends on TET expression and occurs only when both DNMT3s are absent. Dynamic loci are enriched for hydroxymethylcytosine and overlap with subsets of putative somatic enhancers that are methylated in ESCs and can be activated upon differentiation. We observe similar dynamics in mouse ESCs that were less frequent in epiblast stem cells (EpiSCs) and scarce in somatic tissues, suggesting a conserved pluripotency-linked mechanism. Taken together, our data reveal tightly regulated competition between DNMT3s and TETs at thousands of somatic regulatory sequences within pluripotent cells.
    DOI:  https://doi.org/10.1038/s41588-020-0639-9
  5. Nucleic Acids Res. 2020 Jun 08. pii: gkaa482. [Epub ahead of print]
    Sun X, Ren Z, Cun Y, Zhao C, Huang X, Zhou J, Hu R, Su X, Ji L, Li P, Mak KLK, Gao F, Yang Y, Xu H, Ding J, Cao N, Li S, Zhang W, Lan P, Sun H, Wang J, Yuan P.
      Hippo-YAP signaling pathway functions in early lineage differentiation of pluripotent stem cells, but the detailed mechanisms remain elusive. We found that knockout (KO) of Mst1 and Mst2, two key components of the Hippo signaling in mouse embryonic stem cells (ESCs), resulted in a disruption of differentiation into mesendoderm lineage. To further uncover the underlying regulatory mechanisms, we performed a series of ChIP-seq experiments with antibodies against YAP, ESC master transcription factors and some characterized histone modification markers as well as RNA-seq assays using wild type and Mst KO samples at ES and day 4 embryoid body stage respectively. We demonstrate that YAP is preferentially co-localized with super-enhancer (SE) markers such as Nanog, Sox2, Oct4 and H3K27ac in ESCs. The hyper-activation of nuclear YAP in Mst KO ESCs facilitates the binding of Nanog, Sox2 and Oct4 as well as H3K27ac modification at the loci where YAP binds. Moreover, Mst depletion results in novel SE formation and enhanced liquid-liquid phase-separated Med1 condensates on lineage associated genes, leading to the upregulation of these genes and the distortion of ESC differentiation. Our study reveals a novel mechanism on how Hippo-YAP signaling pathway dictates ESC lineage differentiation.
    DOI:  https://doi.org/10.1093/nar/gkaa482
  6. Cancer Cell. 2020 May 13. pii: S1535-6108(20)30216-6. [Epub ahead of print]
    Su R, Dong L, Li Y, Gao M, Han L, Wunderlich M, Deng X, Li H, Huang Y, Gao L, Li C, Zhao Z, Robinson S, Tan B, Qing Y, Qin X, Prince E, Xie J, Qin H, Li W, Shen C, Sun J, Kulkarni P, Weng H, Huang H, Chen Z, Zhang B, Wu X, Olsen MJ, Müschen M, Marcucci G, Salgia R, Li L, Fathi AT, Li Z, Mulloy JC, Wei M, Horne D, Chen J.
      Fat mass and obesity-associated protein (FTO), an RNA N6-methyladenosine (m6A) demethylase, plays oncogenic roles in various cancers, presenting an opportunity for the development of effective targeted therapeutics. Here, we report two potent small-molecule FTO inhibitors that exhibit strong anti-tumor effects in multiple types of cancers. We show that genetic depletion and pharmacological inhibition of FTO dramatically attenuate leukemia stem/initiating cell self-renewal and reprogram immune response by suppressing expression of immune checkpoint genes, especially LILRB4. FTO inhibition sensitizes leukemia cells to T cell cytotoxicity and overcomes hypomethylating agent-induced immune evasion. Our study demonstrates that FTO plays critical roles in cancer stem cell self-renewal and immune evasion and highlights the broad potential of targeting FTO for cancer therapy.
    Keywords:  FTO; LILRB4; LSC/LIC self-renewal; N(6)-methyladenosine (m(6)A) modification; immune checkpoint genes; immune evasion; inhibitors; leukemia; solid tumors; therapeutics
    DOI:  https://doi.org/10.1016/j.ccell.2020.04.017
  7. Nat Commun. 2020 Jun 10. 11(1): 2919
    Bobkov GOM, Huang A, van den Berg SJW, Mitra S, Anselm E, Lazou V, Schunter S, Feederle R, Imhof A, Lusser A, Jansen LET, Heun P.
      Replication and transcription of genomic DNA requires partial disassembly of nucleosomes to allow progression of polymerases. This presents both an opportunity to remodel the underlying chromatin and a danger of losing epigenetic information. Centromeric transcription is required for stable incorporation of the centromere-specific histone dCENP-A in M/G1 phase, which depends on the eviction of previously deposited H3/H3.3-placeholder nucleosomes. Here we demonstrate that the histone chaperone and transcription elongation factor Spt6 spatially and temporarily coincides with centromeric transcription and prevents the loss of old CENP-A nucleosomes in both Drosophila and human cells. Spt6 binds directly to dCENP-A and dCENP-A mutants carrying phosphomimetic residues alleviate this association. Retention of phosphomimetic dCENP-A mutants is reduced relative to wildtype, while non-phosphorylatable dCENP-A retention is increased and accumulates at the centromere. We conclude that Spt6 acts as a conserved CENP-A maintenance factor that ensures long-term stability of epigenetic centromere identity during transcription-mediated chromatin remodeling.
    DOI:  https://doi.org/10.1038/s41467-020-16695-7
  8. Mol Cell. 2020 Jun 08. pii: S1097-2765(20)30317-8. [Epub ahead of print]
    Xiang Y, Tanaka Y, Patterson B, Hwang SM, Hysolli E, Cakir B, Kim KY, Wang W, Kang YJ, Clement EM, Zhong M, Lee SH, Cho YS, Patra P, Sullivan GJ, Weissman SM, Park IH.
      Rett syndrome (RTT), mainly caused by mutations in methyl-CpG binding protein 2 (MeCP2), is one of the most prevalent intellectual disorders without effective therapies. Here, we used 2D and 3D human brain cultures to investigate MeCP2 function. We found that MeCP2 mutations cause severe abnormalities in human interneurons (INs). Surprisingly, treatment with a BET inhibitor, JQ1, rescued the molecular and functional phenotypes of MeCP2 mutant INs. We uncovered that abnormal increases in chromatin binding of BRD4 and enhancer-promoter interactions underlie the abnormal transcription in MeCP2 mutant INs, which were recovered to normal levels by JQ1. We revealed cell-type-specific transcriptome impairment in MeCP2 mutant region-specific human brain organoids that were rescued by JQ1. Finally, JQ1 ameliorated RTT-like phenotypes in mice. These data demonstrate that BRD4 dysregulation is a critical driver for RTT etiology and suggest that targeting BRD4 could be a potential therapeutic opportunity for RTT.
    Keywords:  BRD4; JQ1; MeCP2; Rett syndrome; brain organoid; interneuron
    DOI:  https://doi.org/10.1016/j.molcel.2020.05.016
  9. Nat Genet. 2020 Jun 08.
    Stik G, Vidal E, Barrero M, Cuartero S, Vila-Casadesús M, Mendieta-Esteban J, Tian TV, Choi J, Berenguer C, Abad A, Borsari B, le Dily F, Cramer P, Marti-Renom MA, Stadhouders R, Graf T.
      Three-dimensional organization of the genome is important for transcriptional regulation1-7. In mammals, CTCF and the cohesin complex create submegabase structures with elevated internal chromatin contact frequencies, called topologically associating domains (TADs)8-12. Although TADs can contribute to transcriptional regulation, ablation of TAD organization by disrupting CTCF or the cohesin complex causes modest gene expression changes13-16. In contrast, CTCF is required for cell cycle regulation17, embryonic development and formation of various adult cell types18. To uncouple the role of CTCF in cell-state transitions and cell proliferation, we studied the effect of CTCF depletion during the conversion of human leukemic B cells into macrophages with minimal cell division. CTCF depletion disrupts TAD organization but not cell transdifferentiation. In contrast, CTCF depletion in induced macrophages impairs the full-blown upregulation of inflammatory genes after exposure to endotoxin. Our results demonstrate that CTCF-dependent genome topology is not strictly required for a functional cell-fate conversion but facilitates a rapid and efficient response to an external stimulus.
    DOI:  https://doi.org/10.1038/s41588-020-0643-0
  10. Proc Natl Acad Sci U S A. 2020 Jun 10. pii: 201912074. [Epub ahead of print]
    Boulard M, Rucli S, Edwards JR, Bestor TH.
      The mechanisms by which methylated mammalian promoters are transcriptionally silenced even in the presence of all of the factors required for their expression have long been a major unresolved issue in the field of epigenetics. Repression requires the assembly of a methylation-dependent silencing complex that contains the TRIM28 protein (also known as KAP1 and TIF1β), a scaffolding protein without intrinsic repressive or DNA-binding properties. The identity of the key effector within this complex that represses transcription is unknown. We developed a methylation-sensitized interaction screen which revealed that TRIM28 was complexed with O-linked β-N-acetylglucosamine transferase (OGT) only in cells that had normal genomic methylation patterns. OGT is the only glycosyltransferase that modifies cytoplasmic and nuclear protein by transfer of N-acetylglucosamine (O-GlcNAc) to serine and threonine hydroxyls. Whole-genome analysis showed that O-glycosylated proteins and TRIM28 were specifically bound to promoters of active retrotransposons and to imprinting control regions, the two major regulatory sequences controlled by DNA methylation. Furthermore, genome-wide loss of DNA methylation caused a loss of O-GlcNAc from multiple transcriptional repressor proteins associated with TRIM28. A newly developed Cas9-based editing method for targeted removal of O-GlcNAc was directed against retrotransposon promoters. Local chromatin de-GlcNAcylation specifically reactivated the expression of the targeted retrotransposon family without loss of DNA methylation. These data revealed that O-linked glycosylation of chromatin factors is essential for the transcriptional repression of methylated retrotransposons.
    Keywords:  DNA methylation; gene silencing; protein O-glycosylation
    DOI:  https://doi.org/10.1073/pnas.1912074117
  11. RNA Biol. 2020 Jun 07.
    Zhang Z, Yu W, Tang D, Zhou Y, Bi M, Wang H, Zheng Y, Chen M, Li L, Xu X, Zhang W, Tao H, Jin VX, Liu Z, Chen L.
      Breast cancer is one of the most prevalent cancers in women worldwide. Through the regulation of many coding and non-coding target genes, estrogen (E2 or 17β-estradiol) and its nuclear receptor ERα play important roles in breast cancer development and progression. Despite the astounding advances in our understanding of estrogen-regulated coding genes over the past decades, our knowledge on estrogen-regulated non-coding targets has just begun to expand. Here we leverage epigenomic approaches to systematically analyze estrogen-regulated long non-coding RNAs (lncRNAs).Similar to the coding targets of ERα, the transcription of estrogen-regulatedlncRNAs correlates with the activation status of ERα enhancers, measured by eRNA production, chromatin accessibility, and the occupancy of the enhancer regulatory components including P300, MED1, and ARID1B. Our 3D chromatin architecture analyses suggest that lncRNAs and their neighboring E2-resonsive coding genes, exemplified by LINC00160 and RUNX1, might be regulated as a 3D structural unit resulted from enhancer-promoter interactions. Finally, we evaluated the expression levels of LINC00160 and RUNX1 in various types of breast cancer and found that their expression positively correlated with the survival rate in ER+ breast cancer patients, implying that the estrogen-regulated LINC00160 and its neighboring RUNX1 might represent potential biomarkers for ER+ breast cancers.
    Keywords:  breast cancer; epigenomics-basedidentification; estrogen; long noncoding RNA
    DOI:  https://doi.org/10.1080/15476286.2020.1777769
  12. Cancer Res. 2020 Jun 10. pii: canres.0233.2020. [Epub ahead of print]
    Xu S, Fan L, Jeon HY, Zhang F, Cui X, Mickle MB, Peng G, Hussain A, Fazli L, Gleave ME, Dong X, Qi J.
      The androgen receptor (AR) pathway plays a central role in the development of castration-resistant prostate cancer (CRPC). The histone demethylase JMJD1A has been shown to regulate activities of AR and c-Myc transcription factors and promote prostate cancer progression. Here we report that JMJD1A protein stability is controlled by the ubiquitin ligase STUB1. High levels of JMJD1A were strongly correlated with low STUB1 levels in human CRPC specimens. STUB1 inhibited AR activity, AR-V7 levels, and prostate cancer cell growth partly through degradation of JMJD1A. Furthermore, the acetyltransferase p300 acetylated JMJD1A at lysine (K) 421, a modification that recruits the BET family member BRD4 to block JMJD1A degradation and promote JMJD1A recruitment to AR targets. Increased levels of both total and K421-acetylated JMJD1A were observed in prostate cancer cells as they developed resistance to the AR antagonist enzalutamide. Treatment of prostate cancer cells with either p300 or BET inhibitors destabilized JMJD1A and enzalutamide-resistant prostate cancer cells were more sensitive than parental cells to these inhibitors. Together, our findings identify a critical role for acetylation of JMJD1A in regulating JMJD1A stability and AR activity in CRPC. These newly identified mechanisms controlling JMJD1A protein stability provide potential druggable targets to encourage the development of additional therapies for advanced prostate cancer.
    DOI:  https://doi.org/10.1158/0008-5472.CAN-20-0233
  13. Front Oncol. 2020 ;10 770
    van Gisbergen MW, Offermans K, Voets AM, Lieuwes NG, Biemans R, Hoffmann RF, Dubois LJ, Lambin P.
      mtDNA variations often result in bioenergetic dysfunction inducing a metabolic switch toward glycolysis resulting in an unbalanced pH homeostasis. In hypoxic cells, expression of the tumor-associated carbonic anhydrase IX (CAIX) is enhanced to maintain cellular pH homeostasis. We hypothesized that cells with a dysfunctional oxidative phosphorylation machinery display elevated CAIX expression levels. Increased glycolysis was observed for cytoplasmic 143B mutant hybrid (m.3243A>G, >94.5%) cells (p < 0.05) and 143B mitochondrial DNA (mtDNA) depleted cells (p < 0.05). Upon hypoxia (0.2%, 16 h), genetic or pharmacological oxidative phosphorylation (OXPHOS) inhibition resulted in decreased CAIX (p < 0.05), vascular endothelial growth factor (VEGF) and hypoxia-inducible factor 1-alpha (HIF-1α) expression levels. Reactive oxygen species (ROS) and prolyl-hydroxylase 2 (PHD2) levels could not explain these observations. In vivo, tumor take (>500 mm3) took longer for mutant hybrid xenografts, but growth rates were comparable with control tumors upon establishment. Previously, it has been shown that HIF-1α is responsible for tumor establishment. In agreement, we found that HIF-1α expression levels and the pimonidazole-positive hypoxic fraction were reduced for the mutant hybrid xenografts. Our results demonstrate that OXPHOS dysfunction leads to a decreased HIF-1α stabilization and subsequently to a reduced expression of its downstream targets and hypoxic fraction in vivo. In contrast, hypoxia-inducible factor 2-alpha (HIF-2α) expression levels in these xenografts were enhanced. Inhibition of mitochondrial function is therefore an interesting approach to increase therapeutic efficacy in hypoxic tumors.
    Keywords:  CAIX; HIF-1α; Metformin; OXPHOS; mitochondria; mtDNA
    DOI:  https://doi.org/10.3389/fonc.2020.00770
  14. Dev Cell. 2020 Jun 08. pii: S1534-5807(20)30396-8. [Epub ahead of print]53(5): 561-576.e9
    Liang YC, Wu P, Lin GW, Chen CK, Yeh CY, Tsai S, Yan J, Jiang TX, Lai YC, Huang D, Cai M, Choi R, Widelitz RB, Lu W, Chuong CM.
      Regional specification is critical for skin development, regeneration, and evolution. The contribution of epigenetics in this process remains unknown. Here, using avian epidermis, we find two major strategies regulate β-keratin gene clusters. (1) Over the body, macro-regional specificities (scales, feathers, claws, etc.) established by typical enhancers control five subclusters located within the epidermal differentiation complex on chromosome 25; (2) within a feather, micro-regional specificities are orchestrated by temporospatial chromatin looping of the feather β-keratin gene cluster on chromosome 27. Analyses suggest a three-factor model for regional specification: competence factors (e.g., AP1) make chromatin accessible, regional specifiers (e.g., Zic1) target specific genome regions, and chromatin regulators (e.g., CTCF and SATBs) establish looping configurations. Gene perturbations disrupt morphogenesis and histo-differentiation. This chicken skin paradigm advances our understanding of how regulation of big gene clusters can set up a two-dimensional body surface map.
    Keywords:  CTCF; KLF4; SATB2; beta-keratin; chromatin looping; development; enhancers; epigenetics; evolution; feather; higher-order; scale
    DOI:  https://doi.org/10.1016/j.devcel.2020.05.007
  15. Proc Natl Acad Sci U S A. 2020 Jun 08. pii: 201920725. [Epub ahead of print]
    Healton SE, Pinto HD, Mishra LN, Hamilton GA, Wheat JC, Swist-Rosowska K, Shukeir N, Dou Y, Steidl U, Jenuwein T, Gamble MJ, Skoultchi AI.
      Nearly 50% of mouse and human genomes are composed of repetitive sequences. Transcription of these sequences is tightly controlled during development to prevent genomic instability, inappropriate gene activation and other maladaptive processes. Here, we demonstrate an integral role for H1 linker histones in silencing repetitive elements in mouse embryonic stem cells. Strong H1 depletion causes a profound de-repression of several classes of repetitive sequences, including major satellite, LINE-1, and ERV. Activation of repetitive sequence transcription is accompanied by decreased H3K9 trimethylation of repetitive sequence chromatin. H1 linker histones interact directly with Suv39h1, Suv39h2, and SETDB1, the histone methyltransferases responsible for H3K9 trimethylation of chromatin within these regions, and stimulate their activity toward chromatin in vitro. However, we also implicate chromatin compaction mediated by H1 as an additional, dominant repressive mechanism for silencing of repetitive major satellite sequences. Our findings elucidate two distinct, H1-mediated pathways for silencing heterochromatin.
    Keywords:  chromatin; epigenetics; linker histones; repetitive elements
    DOI:  https://doi.org/10.1073/pnas.1920725117
  16. Cell Rep. 2020 Jun 09. pii: S2211-1247(20)30731-2. [Epub ahead of print]31(10): 107751
    DiFiore JV, Ptacek TS, Wang Y, Li B, Simon JM, Strahl BD.
      Set2 co-transcriptionally methylates lysine 36 of histone H3 (H3K36), producing mono-, di-, and trimethylation (H3K36me1/2/3). These modifications recruit or repel chromatin effector proteins important for transcriptional fidelity, mRNA splicing, and DNA repair. However, it was not known whether the different methylation states of H3K36 have distinct biological functions. Here, we use engineered forms of Set2 that produce different lysine methylation states to identify unique and shared functions for H3K36 modifications. Although H3K36me1/2 and H3K36me3 are functionally redundant in many SET2 deletion phenotypes, we found that H3K36me3 has a unique function related to Bur1 kinase activity and FACT (facilitates chromatin transcription) complex function. Further, during nutrient stress, either H3K36me1/2 or H3K36me3 represses high levels of histone acetylation and cryptic transcription that arises from within genes. Our findings uncover the potential for the regulation of diverse chromatin functions by different H3K36 methylation states.
    Keywords:  H3K36 methylation; RNA Polymerase II; Set2; chromatin; cryptic transcription; epigenetics; histone; nutrient stress; transcriptional regulation
    DOI:  https://doi.org/10.1016/j.celrep.2020.107751
  17. Haematologica. 2020 Jun 11. pii: haematol.2019.244541. [Epub ahead of print]
    Juban G, Sakakini N, Chagraoui H, Cruz Hernandez D, Cheng Q, Soady K, Stoilova B, Garnett C, Waithe D, Otto G, Doondeea J, Usukhbayar B, Karkoulia E, Alexiou M, Strouboulis J, Morrissey E, Roberts I, Porcher C, Vyas P.
      The megakaryocyte/erythroid Transient Myeloproliferative Disorder (TMD) in newborns with Down Syndrome (DS) occurs when N-terminal truncating mutations of the hemopoietic transcription factor GATA1, that produce GATA1short protein (GATA1s), are acquired early in development. Prior work has shown that murine GATA1s, by itself, causes a transient yolk sac myeloproliferative disorder. However, it is unclear where in the hemopoietic cellular hierarchy GATA1s exerts its effects to produce this myeloproliferative state. Here, through a detailed examination of hemopoiesis from murine GATA1s ES cells and GATA1s embryos we define defects in erythroid and megakaryocytic differentiation that occur relatively late in hemopoiesis. GATA1s causes an arrest late in erythroid differentiation in vivo, and even more profoundly in ES-cell derived cultures, with a marked reduction of Ter-119 cells and reduced erythroid gene expression. In megakaryopoiesis, GATA1s causes a differentiation delay at a specific stage, with accumulation of immature, kit-expressing CD41hi megakaryocytic cells. In this specific megakaryocytic compartment, there are increased numbers of GATA1s cells in S-phase of cell cycle and reduced number of apoptotic cells compared to GATA1 cells in the same cell compartment. There is also a delay in maturation of these immature GATA1s megakaryocytic lineage cells compared to GATA1 cells at the same stage of differentiation. Finally, even when GATA1s megakaryocytic cells mature, they mature aberrantly with altered megakaryocyte-specific gene expression and activity of the mature megakaryocyte enzyme, acetylcholinesterase. These studies pinpoint the hemopoietic compartment where GATA1s megakaryocyte myeloproliferation occurs, defining where molecular studies should now be focussed to understand the oncogenic action of GATA1s.
    Keywords:  GATA1s; Hematopoiesis; Megakaryopoiesis; Red Cells; Transient Myeloproliferative Disorder
    DOI:  https://doi.org/10.3324/haematol.2019.244541
  18. Epigenetics Chromatin. 2020 Jun 06. 13(1): 26
    Teif VB, Gould TJ, Clarkson CT, Boyd L, Antwi EB, Ishaque N, Olins AL, Olins DE.
      BACKGROUND: Histone H1 is the most mobile histone in the cell nucleus. Defining the positions of H1 on chromatin in situ, therefore, represents a challenge. Immunoprecipitation of formaldehyde-fixed and sonicated chromatin, followed by DNA sequencing (xChIP-seq), is traditionally the method for mapping histones onto DNA elements. But since sonication fragmentation precedes ChIP, there is a consequent loss of information about chromatin higher-order structure. Here, we present a new method, xxChIP-seq, employing antibody binding to fixed intact in situ chromatin, followed by extensive washing, a second fixation, sonication and immunoprecipitation. The second fixation is intended to prevent the loss of specifically bound antibody during washing and subsequent sonication and to prevent antibody shifting to epitopes revealed by the sonication process. In many respects, xxChIP-seq is comparable to immunostaining microscopy, which also involves interaction of the primary antibody with fixed and permeabilized intact cells. The only epitopes displayed after immunostaining are the "exposed" epitopes, not "hidden" by the fixation of chromatin higher-order structure. Comparison of immunoprecipitated fragments between xChIP-seq versus xxChIP-seq should indicate which epitopes become inaccessible with fixation and identify their associated DNA elements.RESULTS: We determined the genomic distribution of histone variants H1.2 and H1.5 in human myeloid leukemia cells HL-60/S4 and compared their epitope exposure by both xChIP-seq and xxChIP-seq, as well as high-resolution microscopy, illustrating the influences of preserved chromatin higher-order structure in situ. We found that xChIP and xxChIP H1 signals are in general negatively correlated, with differences being more pronounced near active regulatory regions. Among the intriguing observations, we find that transcription-related regions and histone PTMs (i.e., enhancers, promoters, CpG islands, H3K4me1, H3K4me3, H3K9ac, H3K27ac and H3K36me3) exhibit significant deficiencies (depletions) in H1.2 and H1.5 xxChIP-seq reads, compared to xChIP-seq. These observations suggest the existence of in situ transcription-related chromatin higher-order structures stabilized by formaldehyde.
    CONCLUSION: Comparison of H1 xxChIP-seq to H1 xChIP-seq allows the development of hypotheses on the chromosomal localization of (stabilized) higher-order structure, indicated by the generation of "hidden" H1 epitopes following formaldehyde crosslinking. Changes in H1 epitope exposure surrounding averaged chromosomal binding sites or epigenetic modifications can also indicate whether these sites have chromatin higher-order structure. For example, comparison between averaged active or inactive promoter regions suggests that both regions can acquire stabilized higher-order structure with hidden H1 epitopes. However, the H1 xChIP-seq comparison cannot define their differences. Application of the xxChIP-seq versus H1 xChIP-seq method is particularly relevant to chromatin-associated proteins, such as linker histones, that play dynamic roles in establishing chromatin higher-order structure.
    DOI:  https://doi.org/10.1186/s13072-020-00345-9
  19. Cell Rep. 2020 Jun 09. pii: S2211-1247(20)30719-1. [Epub ahead of print]31(10): 107739
    Jackson-Weaver O, Ungvijanpunya N, Yuan Y, Qian J, Gou Y, Wu J, Shen H, Chen Y, Li M, Richard S, Chai Y, Sucov HM, Xu J.
      Epicardial cells are cardiac progenitors that give rise to the majority of cardiac fibroblasts, coronary smooth muscle cells, and pericytes during development. An integral phase of epicardial fate transition is epithelial-to-mesenchymal transition (EMT) that confers motility. We uncover an essential role for the protein arginine methyltransferase 1 (PRMT1) in epicardial invasion and differentiation. Using scRNA-seq, we show that epicardial-specific deletion of Prmt1 reduced matrix and ribosomal gene expression in epicardial-derived cell lineages. PRMT1 regulates splicing of Mdm4, which is a key controller of p53 stability. Loss of PRMT1 leads to accumulation of p53 that enhances Slug degradation and blocks EMT. During heart development, the PRMT1-p53 pathway is required for epicardial invasion and formation of epicardial-derived lineages: cardiac fibroblasts, coronary smooth muscle cells, and pericytes. Consequently, this pathway modulates ventricular morphogenesis and coronary vessel formation. Altogether, our study reveals molecular mechanisms involving the PRMT1-p53 pathway and establish its roles in heart development.
    Keywords:  MDM4; PRMT1; RNA splicing; coronary development; epicardium; epithelial-mesenchymal transition; heart development; p53; protein arginine methylation
    DOI:  https://doi.org/10.1016/j.celrep.2020.107739
  20. Clin Sci (Lond). 2020 Jun 08. pii: CS20200244. [Epub ahead of print]
    Zhou Y, Xu Q, Lv T, Chen Y, Shu Y, Wu Z, Lu C, Shi Y, Bu H.
      The chromatin remodeling complex SWI/SNF regulates the accessibility of target genes to transcription factors and plays a critical role in the tumorigenesis of hepatocellular carcinoma (HCC). The SWI/SNF complex is assembled from approximately fifteen subunits, and most of these subunits have distinct roles and are often aberrantly expressed in HCC. A comprehensive exploration of the expression and clinical significance of these subunits would be of great value. In the present study, we obtained the gene expression profile of each SWI/SNF subunit and the corresponding clinical information from The Cancer Genome Atlas (TCGA). We found that fourteen out of the fifteen SWI/SNF subunits were significantly increased in HCC tissues compared to paired normal liver tissues, and eleven subunits were significantly associated with overall survival (OS). We identified a four-gene prognostic signature including ACTL6A, ARID1A, SMARCC1 and SMARCD1 that could effectively predict OS in HCC patients. Among the genes, SMARCD1 has the most prognostic value. We further conducted in vitro and in vivo experiments and revealed that SMARCD1 promotes liver cancer growth by activating the mTOR signaling pathway. In conclusion, our study has revealed that the expression of SWI/SNF complex subunits, especially SMARCD1, is highly associated with HCC development and acts as a promising prognostic predictor.
    Keywords:  Cell prolferation; SMARCD1; SWI/SNF complex; TCGA; hepatocellular carcinoma
    DOI:  https://doi.org/10.1042/CS20200244
  21. Oncogene. 2020 Jun 07.
    Portney BA, Arad M, Gupta A, Brown RA, Khatri R, Lin PN, Hebert AM, Angster KH, Silipino LE, Meltzer WA, Taylor RJ, Zalzman M.
      Cancer stem cells (CSCs) are cells within tumors that maintain the ability to self-renew, drive tumor growth, and contribute to therapeutic resistance and cancer recurrence. In this study, we investigate the role of Zinc finger and SCAN domain containing 4 (ZSCAN4) in human head and neck squamous cell carcinoma (HNSCC). The murine Zscan4 is involved in telomere maintenance and genomic stability of mouse embryonic stem cells. Our data indicate that the human ZSCAN4 is enriched for, marks and is co-expressed with CSC markers in HNSCC. We show that transient ZSCAN4 induction for just 2 days increases CSC frequency both in vitro and in vivo and leads to upregulation of pluripotency and CSC factors. Importantly, we define for the first time the role of ZSCAN4 in altering the epigenetic profile and regulating the chromatin state. Our data show that ZSCAN4 leads to a functional histone 3 hyperacetylation at the promoters of OCT3/4 and NANOG, leading to an upregulation of CSC factors. Consistently, ZSCAN4 depletion leads to downregulation of CSC markers, decreased ability to form tumorspheres and severely affects tumor growth. Our study suggests that ZSCAN4 plays an important role in the maintenance of the CSC phenotype, indicating it is a potential therapeutic target in HNSCC.
    DOI:  https://doi.org/10.1038/s41388-020-1333-1
  22. Nat Commun. 2020 Jun 09. 11(1): 2907
    Liu M, Lu Y, Yang B, Chen Y, Radda JSD, Hu M, Katz SG, Wang S.
      The three-dimensional architecture of the genome affects genomic functions. Multiple genome architectures at different length scales, including chromatin loops, domains, compartments, and lamina- and nucleolus-associated regions, have been discovered. However, how these structures are arranged in the same cell and how they are mutually correlated in different cell types in mammalian tissue are largely unknown. Here, we develop Multiplexed Imaging of Nucleome Architectures that measures multiscale chromatin folding, copy numbers of numerous RNA species, and associations of numerous genomic regions with nuclear lamina, nucleoli and surface of chromosomes in the same, single cells. We apply this method in mouse fetal liver, and identify de novo cell-type-specific chromatin architectures associated with gene expression, as well as cell-type-independent principles of chromatin organization. Polymer simulation shows that both intra-chromosomal self-associating interactions and extra-chromosomal interactions are necessary to establish the observed organization. Our results illustrate a multi-faceted picture and physical principles of chromatin organization.
    DOI:  https://doi.org/10.1038/s41467-020-16732-5
  23. Genome Biol. 2020 Jun 08. 21(1): 135
    Zhao Q, Dacre M, Nguyen T, Pjanic M, Liu B, Iyer D, Cheng P, Wirka R, Kim JB, Fraser HB, Quertermous T.
      BACKGROUND: To investigate the epigenetic and transcriptional mechanisms of coronary artery disease (CAD) risk, as well as the functional regulation of chromatin structure and function, we create a catalog of genetic variants associated with three stages of transcriptional cis-regulation in primary human coronary artery vascular smooth muscle cells (HCASMCs).RESULTS: We use a pooling approach with HCASMC lines to map regulatory variants that mediate binding of the CAD-associated transcription factor TCF21 with ChIPseq studies (bQTLs), variants that regulate chromatin accessibility with ATACseq studies (caQTLs), and chromosomal looping with Hi-C methods (clQTLs). We examine the overlap of these QTLs and their relationship to smooth muscle-specific genes and transcription factors. Further, we use multiple analyses to show that these QTLs are highly associated with CAD GWAS loci and correlate to lead SNPs where they show allelic effects. By utilizing genome editing, we verify that identified functional variants can regulate both chromatin accessibility and chromosomal looping, providing new insights into functional mechanisms regulating chromatin state and chromosomal structure. Finally, we directly link the disease-associated TGFB1-SMAD3 pathway to the CAD-associated FN1 gene through a response QTL that modulates both chromatin accessibility and chromosomal looping.
    CONCLUSIONS: Together, these studies represent the most thorough mapping of multiple QTL types in a highly disease-relevant primary cultured cell type and provide novel insights into their functional overlap and mechanisms that underlie these genomic features and their relationship to disease risk.
    Keywords:  Chromatin accessibility; Chromosomal looping; Coronary artery disease; Quantitative trait locus; Smooth muscle cells; TCF21
    DOI:  https://doi.org/10.1186/s13059-020-02049-5
  24. Elife. 2020 Jun 09. pii: e56611. [Epub ahead of print]9
    Srinivasan M, Fumasoni M, Petela NJ, Murray A, Nasmyth KA.
      Sister chromatid cohesion essential for mitotic chromosome segregation is thought to involve the co-entrapment of sister DNAs within cohesin rings. Although cohesin can load onto chromosomes throughout the cell cycle, it only builds cohesion during S phase. A key question is whether cohesion is generated by conversion of cohesin complexes associated with un-replicated DNAs ahead of replication forks into cohesive structures behind them, or from nucleoplasmic cohesin that is loaded de novo onto nascent DNAs associated with forks, a process that would be dependent on cohesin's Scc2 subunit. We show here that in S. cerevisiae, both mechanisms exist and that each requires a different set of replisome-associated proteins. Cohesion produced by cohesin conversion requires Tof1/Csm3, Ctf4 and Chl1 but not Scc2 while that created by Scc2-dependent de novo loading at replication forks requires the Ctf18-RFC complex. The association of specific replisome proteins with different types of cohesion establishment opens the way to a mechanistic understanding of an aspect of DNA replication unique to eukaryotic cells.
    Keywords:  S. cerevisiae; SMC; biochemistry; chemical biology; chromosomes; cohesin; gene expression; replication; replisome; shister chromatid cohesion
    DOI:  https://doi.org/10.7554/eLife.56611
  25. Mol Cancer. 2020 Jun 08. 19(1): 104
    Zhou Z, Lv J, Yu H, Han J, Yang X, Feng D, Wu Q, Yuan B, Lu Q, Yang H.
      Since the breakthrough discoveries of DNA and histone modifications, the field of RNA modifications has gained increasing interest in the scientific community. The discovery of N6-methyladenosine (m6A), a predominantly internal epigenetic modification in eukaryotes mRNA, heralded the creation of the field of epi-transcriptomics. This post-transcriptional RNA modification is dynamic and reversible, and is regulated by methylases, demethylases and proteins that preferentially recognize m6A modifications. Altered m6A levels affect RNA processing, degradation and translation, thereby disrupting gene expression and key cellular processes, ultimately resulting in tumor initiation and progression. Furthermore, inhibitors and regulators of m6A-related factors have been explored as therapeutic approaches for treating cancer. In the present review, the mechanisms of m6A RNA modification, the clinicopathological relevance of m6A alterations, the type and frequency of alterations and the multiple functions it regulates in different types of cancer are discussed.
    Keywords:  Cancer; N6-methyladenosine; RNA methylation
    DOI:  https://doi.org/10.1186/s12943-020-01216-3
  26. PLoS Genet. 2020 Jun 10. 16(6): e1008756
    Murphy PJ, Guo J, Jenkins TG, James ER, Hoidal JR, Huecksteadt T, Broberg DS, Hotaling JM, Alonso DF, Carrell DT, Cairns BR, Aston KI.
      Paternal cigarette smoke (CS) exposure is associated with increased risk of behavioral disorders and cancer in offspring, but the mechanism has not been identified. Here we use mouse models to investigate mechanisms and impacts of paternal CS exposure. We demonstrate that CS exposure induces sperm DNAme changes that are partially corrected within 28 days of removal from CS exposure. Additionally, paternal smoking is associated with changes in prefrontal cortex DNAme and gene expression patterns in offspring. Remarkably, the epigenetic and transcriptional effects of CS exposure that we observed in wild type mice are partially recapitulated in Nrf2-/- mice and their offspring, independent of smoking status. Nrf2 is a central regulator of antioxidant gene transcription, and mice lacking Nrf2 consequently display elevated oxidative stress, suggesting that oxidative stress may underlie CS-induced heritable epigenetic changes. Importantly, paternal sperm DNAme changes do not overlap with DNAme changes measured in offspring prefrontal cortex, indicating that the observed DNAme changes in sperm are not directly inherited. Additionally, the changes in sperm DNAme associated with CS exposure were not observed in sperm of unexposed offspring, suggesting the effects are likely not maintained across multiple generations.
    DOI:  https://doi.org/10.1371/journal.pgen.1008756
  27. Cell Rep. 2020 Jun 09. pii: S2211-1247(20)30722-1. [Epub ahead of print]31(10): 107742
    Christin JR, Wang C, Chung CY, Liu Y, Dravis C, Tang W, Oktay MH, Wahl GM, Guo W.
      Lineage plasticity is important for the development of basal-like breast cancer (BLBC), an aggressive cancer subtype. While BLBC is likely to originate from luminal progenitor cells, it acquires substantial basal cell features and contains a heterogenous collection of cells exhibiting basal, luminal, and hybrid phenotypes. Why luminal progenitors are prone to BLBC transformation and what drives luminal-to-basal reprogramming remain unclear. Here, we show that the transcription factor SOX9 acts as a determinant for estrogen-receptor-negative (ER-) luminal stem/progenitor cells (LSPCs). SOX9 controls LSPC activity in part by activating both canonical and non-canonical nuclear factor κB (NF-κB) signaling. Inactivation of TP53 and RB via expression of SV40 TAg in a BLBC mouse tumor model leads to upregulation of SOX9, which drives luminal-to-basal reprogramming in vivo. Furthermore, SOX9 deletion inhibits the progression of ductal carcinoma in situ (DCIS)-like lesions to invasive carcinoma. These data show that ER- LSPC determinant SOX9 acts as a lineage plasticity driver for BLBC progression.
    Keywords:  DCIS progression; SOX9; basal-like breast cancer; bipotent cells; lineage plasticity; luminal stem progenitor cells
    DOI:  https://doi.org/10.1016/j.celrep.2020.107742
  28. Elife. 2020 Jun 09. pii: e55526. [Epub ahead of print]9
    Kuwahara A, Lewis AE, Coombes C, Leung FS, Percharde M, Bush JO.
      The genome-scale transcriptional programs that specify the mammalian trachea and esophagus are unknown. Though NKX2-1 and SOX2 are hypothesized to be co-repressive master regulators of tracheoesophageal fates, this is untested at a whole transcriptomic scale and their downstream networks remain unidentified. By combining single-cell RNA-sequencing with bulk RNA-sequencing of Nkx2-1 mutants and NKX2-1 ChIP-sequencing in mouse embryos, we delineate the NKX2-1 transcriptional program in tracheoesophageal specification, and discover that the majority of the tracheal and esophageal transcriptome is NKX2-1 independent. To decouple the NKX2-1 transcriptional program from regulation by SOX2, we interrogate the expression of newly-identified tracheal and esophageal markers in Sox2/Nkx2-1 compound mutants. Finally, we discover that NKX2-1 binds directly to Shh and Wnt7b and regulates their expression to control mesenchymal specification to cartilage and smooth muscle, coupling epithelial identity with mesenchymal specification. These findings create a new framework for understanding early tracheoesophageal fate specification at the genome-wide level.
    Keywords:  Nkx2-1; Sox2; developmental biology; esophagus; genetics; genomics; lung; mouse; trachea; tracheoesophageal fistula
    DOI:  https://doi.org/10.7554/eLife.55526
  29. PLoS Comput Biol. 2020 Jun 09. 16(6): e1007770
    Lamparter D, Bhatnagar R, Hebestreit K, Belgard TG, Zhang A, Hanson-Smith V.
      A longstanding goal of regulatory genetics is to understand how variants in genome sequences lead to changes in gene expression. Here we present a method named Bayesian Annotation Guided eQTL Analysis (BAGEA), a variational Bayes framework to model cis-eQTLs using directed and undirected genomic annotations. We used BAGEA to integrate directed genomic annotations with eQTL summary statistics from tissues of various origins. This analysis revealed epigenetic marks that are relevant for gene expression in different tissues and cell types. We estimated the predictive power of the models that were fitted based on directed genomic annotations. This analysis showed that, depending on the underlying eQTL data used, the directed genomic annotations could predict up to 1.5% of the variance observed in the expression of genes with top nominal eQTL association p-values < 10-7. For genes with estimated effect sizes in the top 25% quantile, up to 5% of the expression variance could be predicted. Based on our results, we recommend the use of BAGEA for the analysis of cis-eQTL data to reveal annotations relevant to expression biology.
    DOI:  https://doi.org/10.1371/journal.pcbi.1007770
  30. iScience. 2020 May 20. pii: S2589-0042(20)30370-9. [Epub ahead of print]23(6): 101185
    Mandric I, Hill BL, Freund MK, Thompson M, Halperin E.
      Single-cell RNA-sequencing (scRNA-seq) is a set of technologies used to profile gene expression at the level of individual cells. Although the throughput of scRNA-seq experiments is steadily growing in terms of the number of cells, large datasets are not yet commonly generated owing to prohibitively high costs. Integrating multiple datasets into one can improve power in scRNA-seq experiments, and efficient integration is very important for downstream analyses such as identifying cell-type-specific eQTLs. State-of-the-art scRNA-seq integration methods are based on the mutual nearest neighbor paradigm and fail to both correct for batch effects and maintain the local structure of the datasets. In this paper, we propose a novel scRNA-seq dataset integration method called BATMAN (BATch integration via minimum-weight MAtchiNg). Across multiple simulations and real datasets, we show that our method significantly outperforms state-of-the-art tools with respect to existing metrics for batch effects by up to 80% while retaining cell-to-cell relationships.
    Keywords:  Algorithms; Bioinformatics; Transcriptomics
    DOI:  https://doi.org/10.1016/j.isci.2020.101185
  31. BMC Bioinformatics. 2020 Jun 11. 21(1): 240
    Qi Y, Guo Y, Jiao H, Shang X.
      BACKGROUND: Single-cell RNA sequencing (scRNA-seq) provides an effective tool to investigate the transcriptomic characteristics at the single-cell resolution. Due to the low amounts of transcripts in single cells and the technical biases in experiments, the raw scRNA-seq data usually includes large noise and makes the downstream analyses complicated. Although many methods have been proposed to impute the noisy scRNA-seq data in recent years, few of them take into account the prior associations across genes in imputation and integrate multiple types of imputation data to identify cell types.RESULTS: We present a new framework, NetImpute, towards the identification of cell types from scRNA-seq data by integrating multiple types of biological networks. We employ a statistic method to detect the noise data items in scRNA-seq data and develop a new imputation model to estimate the real values of data noise by integrating the PPI network and gene pathways. Meanwhile, based on the data imputed by multiple types of biological networks, we propose an integrated approach to identify cell types from scRNA-seq data. Comprehensive experiments demonstrate that the proposed network-based imputation model can estimate the real values of noise data items accurately and integrating the imputation data based on multiple types of biological networks can improve the identification of cell types from scRNA-seq data.
    CONCLUSIONS: Incorporating the prior gene associations in biological networks can potentially help to improve the imputation of noisy scRNA-seq data and integrating multiple types of network-based imputation data can enhance the identification of cell types. The proposed NetImpute provides an open framework for incorporating multiple types of biological network data to identify cell types from scRNA-seq data.
    Keywords:  Biological networks; Cell types; Data integration; Dropout events; scRNA-seq
    DOI:  https://doi.org/10.1186/s12859-020-03547-w
  32. Genome Biol. 2020 Jun 09. 21(1): 127
    Sakthikumar S, Roy A, Haseeb L, Pettersson ME, Sundström E, Marinescu VD, Lindblad-Toh K, Forsberg-Nilsson K.
      BACKGROUND: Glioblastoma (GBM) has one of the worst 5-year survival rates of all cancers. While genomic studies of the disease have been performed, alterations in the non-coding regulatory regions of GBM have largely remained unexplored. We apply whole-genome sequencing (WGS) to identify non-coding mutations, with regulatory potential in GBM, under the hypothesis that regions of evolutionary constraint are likely to be functional, and somatic mutations are likely more damaging than in unconstrained regions.RESULTS: We validate our GBM cohort, finding similar copy number aberrations and mutated genes based on coding mutations as previous studies. Performing analysis on non-coding constraint mutations and their position relative to nearby genes, we find a significant enrichment of non-coding constraint mutations in the neighborhood of 78 genes that have previously been implicated in GBM. Among them, SEMA3C and DYNC1I1 show the highest frequencies of alterations, with multiple mutations overlapping transcription factor binding sites. We find that a non-coding constraint mutation in the SEMA3C promoter reduces the DNA binding capacity of the region. We also identify 1776 other genes enriched for non-coding constraint mutations with likely regulatory potential, providing additional candidate GBM genes. The mutations in the top four genes, DLX5, DLX6, FOXA1, and ISL1, are distributed over promoters, UTRs, and multiple transcription factor binding sites.
    CONCLUSIONS: These results suggest that non-coding constraint mutations could play an essential role in GBM, underscoring the need to connect non-coding genomic variation to biological function and disease pathology.
    Keywords:  Cancer; Gene regulation; Glioblastoma; Non-coding constraint
    DOI:  https://doi.org/10.1186/s13059-020-02035-x
  33. Development. 2020 Jun 11. pii: dev190231. [Epub ahead of print]147(11):
    Zaytseva O, Mitchell NC, Guo L, Marshall OJ, Parsons LM, Hannan RD, Levens DL, Quinn LM.
      Here, we report novel tumour suppressor activity for the Drosophila Argonaute family RNA-binding protein AGO1, a component of the miRNA-dependent RNA-induced silencing complex (RISC). The mechanism for growth inhibition does not, however, involve canonical roles as part of the RISC; rather, AGO1 controls cell and tissue growth by functioning as a direct transcriptional repressor of the master regulator of growth, Myc. AGO1 depletion in wing imaginal discs drives a significant increase in ribosome biogenesis, nucleolar expansion and cell growth in a manner dependent on Myc abundance. Moreover, increased Myc promoter activity and elevated Myc mRNA in AGO1-depleted animals requires RNA polymerase II transcription. Further support for transcriptional AGO1 functions is provided by physical interaction with the RNA polymerase II transcriptional machinery (chromatin remodelling factors and Mediator Complex), punctate nuclear localisation in euchromatic regions and overlap with Polycomb Group transcriptional silencing loci. Moreover, significant AGO1 enrichment is observed on the Myc promoter and AGO1 interacts with the Myc transcriptional activator Psi. Together, our data show that Drosophila AGO1 functions outside of the RISC to repress Myc transcription and inhibit developmental cell and tissue growth.This article has an associated 'The people behind the papers' interview.
    Keywords:  Argonaute; Drosophila; Myc; Proliferation; Psi; Transcription
    DOI:  https://doi.org/10.1242/dev.190231
  34. Nat Commun. 2020 Jun 12. 11(1): 2807
    Leonards K, Almosailleakh M, Tauchmann S, Bagger FO, Thirant C, Juge S, Bock T, Méreau H, Bezerra MF, Tzankov A, Ivanek R, Losson R, Peters AHFM, Mercher T, Schwaller J.
      The nuclear receptor binding SET domain protein 1 (NSD1) is recurrently mutated in human cancers including acute leukemia. We show that NSD1 knockdown alters erythroid clonogenic growth of human CD34+ hematopoietic cells. Ablation of Nsd1 in the hematopoietic system of mice induces a transplantable erythroleukemia. In vitro differentiation of Nsd1-/- erythroblasts is majorly impaired despite abundant expression of GATA1, the transcriptional master regulator of erythropoiesis, and associated with an impaired activation of GATA1-induced targets. Retroviral expression of wildtype NSD1, but not a catalytically-inactive NSD1N1918Q SET-domain mutant induces terminal maturation of Nsd1-/- erythroblasts. Despite similar GATA1 protein levels, exogenous NSD1 but not NSDN1918Q significantly increases the occupancy of GATA1 at target genes and their expression. Notably, exogenous NSD1 reduces the association of GATA1 with the co-repressor SKI, and knockdown of SKI induces differentiation of Nsd1-/- erythroblasts. Collectively, we identify the NSD1 methyltransferase as a regulator of GATA1-controlled erythroid differentiation and leukemogenesis.
    DOI:  https://doi.org/10.1038/s41467-020-16179-8
  35. Proc Natl Acad Sci U S A. 2020 Jun 12. pii: 202000467. [Epub ahead of print]
    Gurdon JB, Javed K, Vodnala M, Garrett N.
      Some lineage-determining transcription factors are overwhelmingly important in directing embryonic cells to a particular differentiation pathway, such as Ascl1 for nerve. They also have an exceptionally strong ability to force cells to change from an unrelated pathway to one preferred by their action. Transcription factors are believed to have a very short residence time of only a few seconds on their specific DNA or chromatin-binding sites. We have developed a procedure in which DNA containing one copy of the binding site for the neural-inducing factor Ascl1 is injected directly into a Xenopus oocyte nucleus which has been preloaded with a limiting amount of the Ascl1 transcription factor protein. This is followed by a further injection of DNA as a competitor, either in a plasmid or in chromosomal DNA, containing the same binding site but with a different reporter. Importantly, expression of the reporter provides a measure of the function of the transcription factor in addition to its residence time. The same long residence time and resistance to competition are seen with the estrogen receptor and its DNA response elements. We find that in this nondividing oocyte, the nerve-inducing factor Ascl1 can remain bound to a specific chromatin site for hours or days and thereby help to stabilize gene expression. This stability of transcription factor binding to chromatin is a necessary part of its action because removal of this factor causes discontinuation of its effect on gene expression. Stable transcription factor binding may be a characteristic of nondividing cells.
    Keywords:  Ascl1 gene transcription; Xenopus; dwell time; oocytes; transcription factor
    DOI:  https://doi.org/10.1073/pnas.2000467117
  36. Dev Biol. 2020 Jun 03. pii: S0012-1606(20)30152-4. [Epub ahead of print]
    Basta JM, Singh A, Robbins L, Stout L, Pherson M, Rauchman M.
      Chromatin-remodeling complexes play critical roles in establishing gene expression patterns in response to developmental signals. How these epigenetic regulators determine the fate of progenitor cells during development of specific organs is not well understood. We found that genetic deletion of Brg1 (Smarca4), the core enzymatic protein in SWI/SNF, in nephron progenitor cells leads to severe renal hypoplasia. Nephron progenitor cells were depleted in Six2-Cre, Brg1flx/flx mice due to reduced cell proliferation. This defect in self-renewal, together with impaired differentiation resulted in a profound nephron deficit in Brg1 mutant kidneys. Sall1, a transcription factor that is required for expansion and maintenance of nephron progenitors, associates with SWI/SNF. Brg1 and Sall1 bind promoters of many progenitor cell genes and regulate expression of key targets that promote their proliferation.
    DOI:  https://doi.org/10.1016/j.ydbio.2020.05.008
  37. Nat Struct Mol Biol. 2020 Jun;27(6): 521-528
    Sartorelli V, Lauberth SM.
      Noncoding RNAs (ncRNAs) direct a remarkable number of diverse functions in development and disease through their regulation of transcription, RNA processing and translation. Leading the charge in the RNA revolution is a class of ncRNAs that are synthesized at active enhancers, called enhancer RNAs (eRNAs). Here, we review recent insights into the biogenesis of eRNAs and the mechanisms underlying their multifaceted functions and consider how these findings could inform future investigations into enhancer transcription and eRNA function.
    DOI:  https://doi.org/10.1038/s41594-020-0446-0
  38. Nat Biotechnol. 2020 Jun;38(6): 747-755
    Mereu E, Lafzi A, Moutinho C, Ziegenhain C, McCarthy DJ, Álvarez-Varela A, Batlle E, Sagar , Grün D, Lau JK, Boutet SC, Sanada C, Ooi A, Jones RC, Kaihara K, Brampton C, Talaga Y, Sasagawa Y, Tanaka K, Hayashi T, Braeuning C, Fischer C, Sauer S, Trefzer T, Conrad C, Adiconis X, Nguyen LT, Regev A, Levin JZ, Parekh S, Janjic A, Wange LE, Bagnoli JW, Enard W, Gut M, Sandberg R, Nikaido I, Gut I, Stegle O, Heyn H.
      Single-cell RNA sequencing (scRNA-seq) is the leading technique for characterizing the transcriptomes of individual cells in a sample. The latest protocols are scalable to thousands of cells and are being used to compile cell atlases of tissues, organs and organisms. However, the protocols differ substantially with respect to their RNA capture efficiency, bias, scale and costs, and their relative advantages for different applications are unclear. In the present study, we generated benchmark datasets to systematically evaluate protocols in terms of their power to comprehensively describe cell types and states. We performed a multicenter study comparing 13 commonly used scRNA-seq and single-nucleus RNA-seq protocols applied to a heterogeneous reference sample resource. Comparative analysis revealed marked differences in protocol performance. The protocols differed in library complexity and their ability to detect cell-type markers, impacting their predictive value and suitability for integration into reference cell atlases. These results provide guidance both for individual researchers and for consortium projects such as the Human Cell Atlas.
    DOI:  https://doi.org/10.1038/s41587-020-0469-4
  39. Elife. 2020 Jun 11. pii: e53159. [Epub ahead of print]9
    Gupta A, Stocker H.
      The transcription factor FoxO has been shown to block proliferation and progression in mTORC1-driven tumorigenesis but the picture of the relevant FoxO target genes remains incomplete. Here, we employed RNA-seq profiling on single clones isolated using laser capture microdissection from Drosophila larval eye imaginal discs to identify FoxO targets that restrict the proliferation of Tsc1-deficient cells under nutrient restriction (NR). Transcriptomics analysis revealed downregulation of endoplasmic reticulum-associated protein degradation pathway components upon foxo knockdown. Induction of ER stress pharmacologically or by suppression of other ER stress response pathway components led to an enhanced overgrowth of Tsc1 knockdown tissue. Increase of ER stress in Tsc1 loss-of-function cells upon foxo knockdown was also confirmed by elevated expression levels of known ER stress markers. These results highlight the role of FoxO in limiting ER stress to regulate Tsc1 mutant overgrowth.
    Keywords:  D. melanogaster; ER stress; FoxO; Tsc1; cancer biology; genetics; genomics; laser capture microdissection
    DOI:  https://doi.org/10.7554/eLife.53159