bims-crepig Biomed News
on Chromatin regulation and epigenetics in cell fate and cancer
Issue of 2020‒04‒05
seventy-six papers selected by
Connor Rogerson
University of Cambridge, MRC Cancer Unit

  1. Mol Cell. 2020 Mar 31. pii: S1097-2765(20)30158-1. [Epub ahead of print]
    Michowski W, Chick JM, Chu C, Kolodziejczyk A, Wang Y, Suski JM, Abraham B, Anders L, Day D, Dunkl LM, Li Cheong Man M, Zhang T, Laphanuwat P, Bacon NA, Liu L, Fassl A, Sharma S, Otto T, Jecrois E, Han R, Sweeney KE, Marro S, Wernig M, Geng Y, Moses A, Li C, Gygi SP, Young RA, Sicinski P.
      The cyclin-dependent kinase 1 (Cdk1) drives cell division. To uncover additional functions of Cdk1, we generated knockin mice expressing an analog-sensitive version of Cdk1 in place of wild-type Cdk1. In our study, we focused on embryonic stem cells (ESCs), because this cell type displays particularly high Cdk1 activity. We found that in ESCs, a large fraction of Cdk1 substrates is localized on chromatin. Cdk1 phosphorylates many proteins involved in epigenetic regulation, including writers and erasers of all major histone marks. Consistent with these findings, inhibition of Cdk1 altered histone-modification status of ESCs. High levels of Cdk1 in ESCs phosphorylate and partially inactivate Dot1l, the H3K79 methyltransferase responsible for placing activating marks on gene bodies. Decrease of Cdk1 activity during ESC differentiation de-represses Dot1l, thereby allowing coordinated expression of differentiation genes. These analyses indicate that Cdk1 functions to maintain the epigenetic identity of ESCs.
    Keywords:  analog sensitive kinase; cell cycle; cyclin; cyclin dependent kinase; differentiation; embryonic stem cells; epigenetics; phosphoproteomics
  2. Nat Cell Biol. 2020 Mar 30.
    Lee QY, Mall M, Chanda S, Zhou B, Sharma KS, Schaukowitch K, Adrian-Segarra JM, Grieder SD, Kareta MS, Wapinski OL, Ang CE, Li R, Südhof TC, Chang HY, Wernig M.
      The on-target pioneer factors Ascl1 and Myod1 are sequence-related but induce two developmentally unrelated lineages-that is, neuronal and muscle identities, respectively. It is unclear how these two basic helix-loop-helix (bHLH) factors mediate such fundamentally different outcomes. The chromatin binding of Ascl1 and Myod1 was surprisingly similar in fibroblasts, yet their transcriptional outputs were drastically different. We found that quantitative binding differences explained differential chromatin remodelling and gene activation. Although strong Ascl1 binding was exclusively associated with bHLH motifs, strong Myod1-binding sites were co-enriched with non-bHLH motifs, possibly explaining why Ascl1 is less context dependent. Finally, we observed that promiscuous binding of Myod1 to neuronal targets results in neuronal reprogramming when the muscle program is inhibited by Myt1l. Our findings suggest that chromatin access of on-target pioneer factors is primarily driven by the protein-DNA interaction, unlike ordinary context-dependent transcription factors, and that promiscuous transcription factor binding requires specific silencing mechanisms to ensure lineage fidelity.
  3. Curr Top Dev Biol. 2020 ;pii: S0070-2153(19)30089-4. [Epub ahead of print]138 1-71
    Klein DC, Hainer SJ.
      In eukaryotes, DNA is highly compacted within the nucleus into a structure known as chromatin. Modulation of chromatin structure allows for precise regulation of gene expression, and thereby controls cell fate decisions. Specific chromatin organization is established and preserved by numerous factors to generate desired cellular outcomes. In embryonic stem (ES) cells, chromatin is precisely regulated to preserve their two defining characteristics: self-renewal and pluripotent state. This action is accomplished by a litany of nucleosome remodelers, histone variants, epigenetic marks, and other chromatin regulatory factors. These highly dynamic regulatory factors come together to precisely define a chromatin state that is conducive to ES cell maintenance and development, where dysregulation threatens the survival and fitness of the developing organism.
    Keywords:  Bivalent promoters; Chromatin remodelers; Gene expression; Genome organization; Histone chaperones; Pluripotency; Transcription; Transcription factors
  4. J Neurochem. 2020 Mar 31.
    Ripamonti S, Shomroni O, Rhee JS, Chowdhury K, Jahn O, Hellmann KP, Bonn S, Brose N, Tirard M.
      SUMOylation is a dynamic post-translational protein modification that primarily takes place in cell nuclei, where it plays a key role in multiple DNA-related processes. In neurons, the SUMOylation-dependent control of a subset of neuronal transcription factors is known to regulate various aspects of nerve cell differentiation, development, and function. In an unbiased screen for endogenous SUMOylation targets in the developing mouse brain, based on a His6 -HA-SUMO1 KI mouse line, we previously identified the transcription factor Zbtb20 as a new SUMO1-conjugate. We show here that the three key SUMO paralogues SUMO1, SUMO2, and SUMO3 can all be conjugated to Zbtb20 in vitro in HEK293FT cells, and we confirm the SUMOylation of Zbtb20 in vivo in mouse brain. Using primary hippocampal neurons from wild-type and Zbtb20 KO mice as a model system, we then demonstrate that the expression of Zbtb20 is required for proper nerve cell development and neurite growth and branching. Furthermore, we show that the SUMOylation of Zbtb20 is essential for its function in this context, and provide evidence indicating that SUMOylation affects the Zbtb20-dependent transcriptional profile of neurons. Our data highlight the role of SUMOylation in the regulation of neuronal transcription factors that determine nerve cell development, and they demonstrate that key functions of the transcription factor Zbtb20 in neuronal development and neurite growth are under obligatory SUMOylation control.
    Keywords:  SUMOylation; Zbtb20; neurite branching; neurite growth; primary hippocampal neurons; transcription factors
  5. Nat Cell Biol. 2020 Mar 30.
    Pijuan-Sala B, Wilson NK, Xia J, Hou X, Hannah RL, Kinston S, Calero-Nieto FJ, Poirion O, Preissl S, Liu F, Göttgens B.
      During mouse embryonic development, pluripotent cells rapidly divide and diversify, yet the regulatory programs that define the cell repertoire for each organ remain ill-defined. To delineate comprehensive chromatin landscapes during early organogenesis, we mapped chromatin accessibility in 19,453 single nuclei from mouse embryos at 8.25 days post-fertilization. Identification of cell-type-specific regions of open chromatin pinpointed two TAL1-bound endothelial enhancers, which we validated using transgenic mouse assays. Integrated gene expression and transcription factor motif enrichment analyses highlighted cell-type-specific transcriptional regulators. Subsequent in vivo experiments in zebrafish revealed a role for the ETS factor FEV in endothelial identity downstream of ETV2 (Etsrp in zebrafish). Concerted in vivo validation experiments in mouse and zebrafish thus illustrate how single-cell open chromatin maps, representative of a mammalian embryo, provide access to the regulatory blueprint for mammalian organogenesis.
  6. Mol Cell. 2020 Apr 02. pii: S1097-2765(20)30186-6. [Epub ahead of print]78(1): 112-126.e12
    Rosencrance CD, Ammouri HN, Yu Q, Ge T, Rendleman EJ, Marshall SA, Eagen KP.
      Delineating how chromosomes fold at length scales beyond one megabase remains obscure relative to smaller-scale folding into TADs, loops, and nucleosomes. We find that rather than simply unfolding chromatin, histone hyperacetylation results in interactions between distant genomic loci separated by tens to hundreds of megabases, even in the absence of transcription. These hyperacetylated "megadomains" are formed by the BRD4-NUT fusion oncoprotein, interact both within and between chromosomes, and form a specific nuclear subcompartment that has elevated gene activity with respect to other subcompartments. Pharmacological degradation of BRD4-NUT results in collapse of megadomains and attenuation of the interactions between them. In contrast, these interactions persist and contacts between newly acetylated regions are formed after inhibiting RNA polymerase II initiation. Our structure-function approach thus reveals that broad chromatin domains of identical biochemical composition, independent of transcription, form nuclear subcompartments, and also indicates the potential of altering chromosome structure for treating human disease.
    Keywords:  BRD4-NUT; Hi-C; NUT carcinoma; PROTAC; Proteolysis Targeted Chimera; chromatin acetylation; chromosome architecture; nuclear organization; nuclear subcompartments
  7. Nucleic Acids Res. 2020 Mar 30. pii: gkaa179. [Epub ahead of print]
    Takaku M, Grimm SA, De Kumar B, Bennett BD, Wade PA.
      Estrogen receptors (ER) are activated by the steroid hormone 17β-estradiol. Estrogen receptor alpha (ER-α) forms a regulatory network in mammary epithelial cells and in breast cancer with the transcription factors FOXA1 and GATA3. GATA3 is one of the most frequently mutated genes in breast cancer and is capable of specifying chromatin localization of FOXA1 and ER-α. How GATA3 mutations found in breast cancer impact genomic localization of ER-α and the transcriptional network downstream of ER-α and FOXA1 remains unclear. Here, we investigate the function of a recurrent patient-derived GATA3 mutation (R330fs) on this regulatory network. Genomic analysis indicates that the R330fs mutant can disrupt localization of ER-α and FOXA1. Loci co-bound by all three factors are enriched for genes integral to mammary gland development as well as epithelial cell biology. This gene set is differentially regulated in GATA3 mutant cells in culture and in tumors bearing similar mutations in vivo. The altered distribution of ER-α and FOXA1 in GATA3-mutant cells is associated with altered chromatin architecture, which leads to differential gene expression. These results suggest an active role for GATA3 zinc finger 2 mutants in ER-α positive breast tumors.
  8. JCI Insight. 2020 Mar 31. pii: 135826. [Epub ahead of print]
    Hwang I, Pan H, Yao J, Elemento O, Zheng H, Paik J.
      Capicua (CIC), a member of the high mobility group (HMG)-box superfamily of transcriptional repressors, is frequently mutated in human oligodendrogliomas. But its function in brain development and tumorigenesis remains poorly understood. Here, we report that brain-specific deletion of Cic compromises developmental transition of neuroblast to immature neurons in mouse hippocampus and compromises normal neuronal differentiation. Combined gene expression and ChIP-seq analyses identified VGF as an important CIC-repressed transcriptional surrogate involved in neuronal lineage regulation. Aberrant VGF expression promotes neural progenitor cell proliferation by suppressing their differentiation. Mechanistically, we demonstrated that CIC represses VGF expression by tethering SIN3-HDAC to form a transcriptional corepressor complex. Mass spectrometry analysis of CIC-interacting proteins further identified BRG1 containing mSWI/SNF complex whose function is necessary for transcriptional repression by CIC. Together, this study uncovers a novel regulatory pathway of CIC-dependent neuronal differentiation and may implicate these molecular mechanisms in CIC-dependent brain tumorigenesis.
    Keywords:  Brain cancer; Neuronal stem cells; Oncology; Stem cells; Transcription
  9. Am J Hum Genet. 2020 Mar 31. pii: S0002-9297(20)30084-7. [Epub ahead of print]
    Choufani S, Gibson WT, Turinsky AL, Chung BHY, Wang T, Garg K, Vitriolo A, Cohen ASA, Cyrus S, Goodman S, Chater-Diehl E, Brzezinski J, Brudno M, Ming LH, White SM, Lynch SA, Clericuzio C, Temple IK, Flinter F, McConnell V, Cushing T, Bird LM, Splitt M, Kerr B, Scherer SW, Machado J, Imagawa E, Okamoto N, Matsumoto N, Testa G, Iascone M, Tenconi R, Caluseriu O, Mendoza-Londono R, Chitayat D, Cytrynbaum C, Tatton-Brown K, Weksberg R.
      Weaver syndrome (WS), an overgrowth/intellectual disability syndrome (OGID), is caused by pathogenic variants in the histone methyltransferase EZH2, which encodes a core component of the Polycomb repressive complex-2 (PRC2). Using genome-wide DNA methylation (DNAm) data for 187 individuals with OGID and 969 control subjects, we show that pathogenic variants in EZH2 generate a highly specific and sensitive DNAm signature reflecting the phenotype of WS. This signature can be used to distinguish loss-of-function from gain-of-function missense variants and to detect somatic mosaicism. We also show that the signature can accurately classify sequence variants in EED and SUZ12, which encode two other core components of PRC2, and predict the presence of pathogenic variants in undiagnosed individuals with OGID. The discovery of a functionally relevant signature with utility for diagnostic classification of sequence variants in EZH2, EED, and SUZ12 supports the emerging paradigm shift for implementation of DNAm signatures into diagnostics and translational research.
    Keywords:  DNA methylation signature; EED; SUZ12; intellectual disability; overgrowth syndromes
  10. Blood. 2020 Mar 31. pii: blood.2019002326. [Epub ahead of print]
    Mujahed H, Miliara S, Neddermeyer AH, Bengtzen S, Nilsson C, Deneberg S, Cordeddu L, Ekwall K, Lennartsson A, Lehmann S.
      CTCF is a key regulator of gene expression through organization of the chromatin structure. Still, it is unclear how CTCF binding is perturbed in leukemia or in cancer in general. We studied CTCF binding by ChIP-Seq in cells from patients with acute myeloid leukemia (AML) and in normal bone marrow (NBM) in the context of gene expression, DNA methylation and azacytidine exposure. CTCF binding was increased in AML compared to NBM. Aberrant CTCF binding was enriched for motifs for key myeloid transcription factors such as CEBPA, PU.1 and RUNX1. AML with TET2 mutations was characterized by a particularly strong gain of CTCF binding, highly enriched for gain in promoter regions while AML in general was enriched for changes at enhancers. There was a strong anti-correlation between CTCF binding and DNA methylation. Gain of CTCF occupancy was associated with increased gene expression, however, the genomic location (promoter vs. distal regions) and enrichment of motifs (for repressing vs. activating co-factors) were decisive for the gene expression pattern. Knock-down of CTCF in K562 cells caused loss of CTCF binding and transcriptional repression of genes with changed CTCF binding in AML, as well as loss of RUNX1 binding at RUNX1/CTCF binding sites. In addition, CTCF knock-down caused increased differentiation. Azacytidine exposure caused major changes in CTCF occupancy in AML patient cells, partly by restoring a CTCF binding pattern similar to NBM. We conclude that AML displays an aberrant increase in CTCF occupancy that targets key genes for AML development and impacts on gene expression.
  11. Cell Death Dis. 2020 Apr 01. 11(4): 214
    Fan L, Xu S, Zhang F, Cui X, Fazli L, Gleave M, Clark DJ, Yang A, Hussain A, Rassool F, Qi J.
      The DNA damage response (DDR) pathway is a promising target for anticancer therapies. The androgen receptor and myeloblastosis transcription factors have been reported to regulate expression of an overlapping set of DDR genes in prostate cancer cells. Here, we found that histone demethylase JMJD1A regulates expression of a different set of DDR genes largely through c-Myc. Inhibition of JMJD1A delayed the resolution of γ-H2AX foci, reduced the formation of foci containing ubiquitin, 53BP1, BRCA1 or Rad51, and inhibited the reporter activity of double-strand break (DSB) repair. Mechanistically, JMJD1A regulated expression of DDR genes by increasing not only the level but also the chromatin recruitment of c-Myc through H3K9 demethylation. Further, we found that ubiquitin ligase HUWE1 induced the K27-/K29-linked noncanonical ubiquitination of JMJD1A at lysine-918. Ablation of the JMJD1A noncanonical ubiquitination lowered DDR gene expression, impaired DSB repair, and sensitized response of prostate cells to irradiation, topoisomerase inhibitors or PARP inhibitors. Thus, development of agents that target JMJD1A or its noncanonical ubiquitination may sensitize the response of prostate cancer to radiotherapy and possibly also genotoxic therapy.
  12. Dev Biol. 2020 Mar 31. pii: S0012-1606(20)30101-9. [Epub ahead of print]
    Kyono Y, Raj S, Sifuentes CJ, Buisine N, Sachs L, Denver RJ.
      Methylation of cytosine residues in DNA influences chromatin structure and gene transcription, and its regulation is crucial for brain development. There is mounting evidence that DNA methylation can be modulated by hormone signaling. We analyzed genome-wide changes in DNA methylation and their relationship to gene regulation in the brain of Xenopus tadpoles during metamorphosis, a thyroid hormone-dependent developmental process. We studied the region of the tadpole brain containing neurosecretory neurons that control pituitary hormone secretion, a region that is highly responsive to thyroid hormone action. Using Methylated DNA Capture sequencing (MethylCap-seq) we discovered a diverse landscape of DNA methylation across the tadpole neural cell genome, and pairwise stage comparisons identified several thousand differentially methylated regions (DMRs). During the pre-to pro-metamorphic period, the number of DMRs was lowest (1,163), with demethylation predominating. From pre-metamorphosis to metamorphic climax DMRs nearly doubled (2,204), with methylation predominating. The largest changes in DNA methylation were seen from metamorphic climax to the completion of metamorphosis (2960 DMRs), with 80% of the DMRs representing demethylation. Using RNA sequencing, we found negative correlations between differentially expressed genes and DMRs localized to gene bodies and regions upstream of transcription start sites. DNA demethylation at metamorphosis revealed by MethylCap-seq was corroborated by increased immunoreactivity for the DNA demethylation intermediates 5-hydroxymethylcytosine and 5-carboxymethylcytosine, and the cytosine dioxygenase ten eleven translocation 3 that catalyzes DNA demethylation. Our findings show that the genome of tadpole neural cells undergoes significant changes in DNA methylation during metamorphosis, and these changes likely influence chromatin architecture, and gene regulation programs occurring during this developmental period.
    Keywords:  Brain development; DNA methylation; Metamorphosis; TET enzymes; Transcription; Xenopus
  13. Curr Genet. 2020 Apr 01.
    Lin A, Du Y, Xiao W.
      The nucleosome is a small unit of chromatin, which is dynamic in eukaryotes. Chromatin conformation and post-translational modifications affect nucleosome dynamics under certain conditions, playing an important role in the epigenetic regulation of transcription, replication and reprogramming. The Snf2 remodeling family is one of the crucial remodeling complexes that tightly regulate chromatin structure and affect nucleosome dynamics. This family alters nucleosome positioning, exchanges histone variants, and assembles and disassembles nucleosomes at certain locations. Moreover, the Snf2 family, in conjunction with other co-factors, regulates gene expression in Saccharomyces cerevisiae. Here we first review recent findings on the Snf2 family remodeling complexes and then use some examples to illustrate the cooperation between different members of Snf2 family, and the cooperation between Snf2 family and other co-factors in gene regulation especially during transcription initiation.
    Keywords:  Histone nucleosome; Mediator; Remodeling complex; SAGA; Transcription; Yeast
  14. Leukemia. 2020 Apr 02.
    Godfrey L, Crump NT, O'Byrne S, Lau IJ, Rice S, Harman JR, Jackson T, Elliott N, Buck G, Connor C, Thorne R, Knapp DJHF, Heidenreich O, Vyas P, Menendez P, Inglott S, Ancliff P, Geng H, Roberts I, Roy A, Milne TA.
      MLL gene rearrangements (MLLr) are a common cause of aggressive, incurable acute lymphoblastic leukemias (ALL) in infants and children, most of which originate in utero. The most common MLLr produces an MLL-AF4 fusion protein. MLL-AF4 promotes leukemogenesis by activating key target genes, mainly through recruitment of DOT1L and increased histone H3 lysine-79 methylation (H3K79me2/3). One key MLL-AF4 target gene is PROM1, which encodes CD133 (Prominin-1). CD133 is a pentaspan transmembrane glycoprotein that represents a potential pan-cancer target as it is found on multiple cancer stem cells. Here we demonstrate that aberrant PROM1/CD133 expression is essential for leukemic cell growth, mediated by direct binding of MLL-AF4. Activation is controlled by an intragenic H3K79me2/3 enhancer element (KEE) leading to increased enhancer-promoter interactions between PROM1 and the nearby gene TAPT1. This dual locus regulation is reflected in a strong correlation of expression in leukemia. We find that in PROM1/CD133 non-expressing cells, the PROM1 locus is repressed by polycomb repressive complex 2 (PRC2) binding, associated with reduced expression of TAPT1, partially due to loss of interactions with the PROM1 locus. Together, these results provide the first detailed analysis of PROM1/CD133 regulation that explains CD133 expression in MLLr ALL.
  15. Cancer Cell. 2020 Mar 13. pii: S1535-6108(20)30101-X. [Epub ahead of print]
    Zhang Z, Zhou C, Li X, Barnes SD, Deng S, Hoover E, Chen CC, Lee YS, Zhang Y, Wang C, Metang LA, Wu C, Tirado CR, Johnson NA, Wongvipat J, Navrazhina K, Cao Z, Choi D, Huang CH, Linton E, Chen X, Liang Y, Mason CE, de Stanchina E, Abida W, Lujambio A, Li S, Lowe SW, Mendell JT, Malladi VS, Sawyers CL, Mu P.
      Metastatic prostate cancer is characterized by recurrent genomic copy number alterations that are presumed to contribute to resistance to hormone therapy. We identified CHD1 loss as a cause of antiandrogen resistance in an in vivo small hairpin RNA (shRNA) screen of 730 genes deleted in prostate cancer. ATAC-seq and RNA-seq analyses showed that CHD1 loss resulted in global changes in open and closed chromatin with associated transcriptomic changes. Integrative analysis of this data, together with CRISPR-based functional screening, identified four transcription factors (NR3C1, POU3F2, NR2F1, and TBX2) that contribute to antiandrogen resistance, with associated activation of non-luminal lineage programs. Thus, CHD1 loss results in chromatin dysregulation, thereby establishing a state of transcriptional plasticity that enables the emergence of antiandrogen resistance through heterogeneous mechanisms.
    Keywords:  CHD1; NR2F1; NR3C1 (GR); POU3F2 (BRN2); TBX2; antiandrogen resistantce; castration-resistant prostate cancer; chromatin remodeling; lineage plasticity; tumor heterogeneity
  16. Nucleic Acids Res. 2020 Apr 02. pii: gkaa214. [Epub ahead of print]
    Peng L, Qian M, Liu Z, Tang X, Sun J, Jiang Y, Sun S, Cao X, Pang Q, Liu B.
      SIRT6 deacetylase activity improves stress resistance via gene silencing and genome maintenance. Here, we reveal a deacetylase-independent function of SIRT6, which promotes anti-apoptotic gene expression via the transcription factor GATA4. SIRT6 recruits TIP60 acetyltransferase to acetylate GATA4 at K328/330, thus enhancing its chromatin binding capacity. In turn, GATA4 inhibits the deacetylase activity of SIRT6, thus ensuring the local chromatin accessibility via TIP60-promoted H3K9 acetylation. Significantly, the treatment of doxorubicin (DOX), an anti-cancer chemotherapeutic, impairs the SIRT6-TIP60-GATA4 trimeric complex, blocking GATA4 acetylation and causing cardiomyocyte apoptosis. While GATA4 hyperacetylation-mimic retains the protective effect against DOX, the hypoacetylation-mimic loses such ability. Thus, the data reveal a novel SIRT6-TIP60-GATA4 axis, which promotes the anti-apoptotic pathway to prevent DOX toxicity. Targeting the trimeric complex constitutes a new strategy to improve the safety of DOX chemotherapy in clinical application.
  17. Curr Opin Nephrol Hypertens. 2020 May;29(3): 280-285
    Wilson PC, Ledru N, Humphreys BD.
      PURPOSE OF REVIEW: Epigenetic modifications are reversible changes to a cell's DNA or histones that alter gene expression but not DNA sequence. The present review will explore epigenomic profiling and bioinformatics techniques for the study of kidney development and disease.RECENT FINDINGS: Reversible DNA and histone modifications influence chromatin accessibility and can be measured by a variety of recent techniques including DNase-seq, ATAC-seq, and single cell ATAC-seq. These approaches have been used to demonstrate that DNA methylation is critical for nephron progenitor maturation, for example. New bioinformatics techniques allow the prediction of chromatin loops that connect regulatory elements to target genes. Recent studies have demonstrated that DNA elements regulate transcription in the kidney via long-range physical interactions and create a new framework for understanding how genome wide association studies risk loci contribute to kidney disease. Increasingly, epigenomic approaches are being combined with transcriptomic analyses to generate multimodal datasets.
    SUMMARY: Epigenomics has expanded our knowledge of gene architecture and regulation. Novel tools and techniques have led to the emergence of 'multiomics' in which epigenomic profiling, transcriptomics, and additional methods complement each other to improve our understanding of kidney disease and development.
  18. Nat Commun. 2020 Apr 03. 11(1): 1673
    Reyes-Palomares A, Gu M, Grubert F, Berest I, Sa S, Kasowski M, Arnold C, Shuai M, Srivas R, Miao S, Li D, Snyder MP, Rabinovitch M, Zaugg JB.
      Environmental and epigenetic factors often play an important role in polygenic disorders. However, how such factors affect disease-specific tissues at the molecular level remains to be understood. Here, we address this in pulmonary arterial hypertension (PAH). We obtain pulmonary arterial endothelial cells (PAECs) from lungs of patients and controls (n = 19), and perform chromatin, transcriptomic and interaction profiling. Overall, we observe extensive remodeling at active enhancers in PAH PAECs and identify hundreds of differentially active TFs, yet find very little transcriptomic changes in steady-state. We devise a disease-specific enhancer-gene regulatory network and predict that primed enhancers in PAH PAECs are activated by the differentially active TFs, resulting in an aberrant response to endothelial signals, which could lead to disturbed angiogenesis and endothelial-to-mesenchymal-transition. We validate these predictions for a selection of target genes in PAECs stimulated with TGF-β, VEGF or serotonin. Our study highlights the role of chromatin state and enhancers in disease-relevant cell types of PAH.
  19. Nature. 2020 Apr;580(7801): 147-150
    Yin Y, Lu JY, Zhang X, Shao W, Xu Y, Li P, Hong Y, Cui L, Shan G, Tian B, Zhang QC, Shen X.
      Long noncoding RNAs (lncRNAs) and promoter- or enhancer-associated unstable transcripts locate preferentially to chromatin, where some regulate chromatin structure, transcription and RNA processing1-13. Although several RNA sequences responsible for nuclear localization have been identified-such as repeats in the lncRNA Xist and Alu-like elements in long RNAs14-16-how lncRNAs as a class are enriched at chromatin remains unknown. Here we describe a random, mutagenesis-coupled, high-throughput method that we name 'RNA elements for subcellular localization by sequencing' (mutREL-seq). Using this method, we discovered an RNA motif that recognizes the U1 small nuclear ribonucleoprotein (snRNP) and is essential for the localization of reporter RNAs to chromatin. Across the genome, chromatin-bound lncRNAs are enriched with 5' splice sites and depleted of 3' splice sites, and exhibit high levels of U1 snRNA binding compared with cytoplasm-localized messenger RNAs. Acute depletion of U1 snRNA or of the U1 snRNP protein component SNRNP70 markedly reduces the chromatin association of hundreds of lncRNAs and unstable transcripts, without altering the overall transcription rate in cells. In addition, rapid degradation of SNRNP70 reduces the localization of both nascent and polyadenylated lncRNA transcripts to chromatin, and disrupts the nuclear and genome-wide localization of the lncRNA Malat1. Moreover, U1 snRNP interacts with transcriptionally engaged RNA polymerase II. These results show that U1 snRNP acts widely to tether and mobilize lncRNAs to chromatin in a transcription-dependent manner. Our findings have uncovered a previously unknown role of U1 snRNP beyond the processing of precursor mRNA, and provide molecular insight into how lncRNAs are recruited to regulatory sites to carry out chromatin-associated functions.
  20. Genome Biol. 2020 Apr 01. 21(1): 77
    Horii T, Morita S, Hino S, Kimura M, Hino Y, Kogo H, Nakao M, Hatada I.
      BACKGROUND: Epigenetic modifications, including DNA methylation, play an important role in gene silencing and genome stability. Consequently, epigenetic dysregulation can cause several diseases, such as cancer, obesity, diabetes, autism, and imprinting disorders.RESULTS: We validate three methods for the generation of epigenome-edited mice using the dCas9-SunTag and single-chain variable fragment-TET1 catalytic domain. We generate model mice for Silver-Russell syndrome (SRS), an imprinting disorder, by target-specific DNA demethylation in the H19 differentially methylated region. Like SRS patients, these mice show H19 upregulation and Igf2 downregulation, leading to severe intrauterine and postnatal growth retardation.
    CONCLUSION: This is the first report of an imprinting disease model animal generated by targeted demethylation of specific loci of the epigenome in fertilized eggs. Epigenome-edited animals are also useful for exploring the causative epimutations in epigenetic diseases.
    Keywords:  CRISPR/Cas9; Demethylation; Epigenome editing; Silver-Russell syndrome; dCas9
  21. Cell Stem Cell. 2020 Mar 30. pii: S1934-5909(20)30095-3. [Epub ahead of print]
    Sommerkamp P, Altamura S, Renders S, Narr A, Ladel L, Zeisberger P, Eiben PL, Fawaz M, Rieger MA, Cabezas-Wallscheid N, Trumpp A.
      Alternative polyadenylation (APA) is emerging as an important regulatory mechanism of RNA and protein isoform expression by controlling 3' untranslated region (3'-UTR) composition. The relevance of APA in stem cell hierarchies remains elusive. Here, we first demonstrate the requirement of the APA regulator Pabpn1 for hematopoietic stem cell (HSC) function. We then determine the genome-wide APA landscape (APAome) of HSCs and progenitors by performing low-input 3' sequencing paired with bioinformatic pipelines. This reveals transcriptome-wide dynamic APA patterns and an overall shortening of 3'-UTRs during differentiation and upon homeostatic or stress-induced transition from quiescence to proliferation. Specifically, we show that APA regulates activation-induced Glutaminase (Gls) isoform switching by Nudt21. This adaptation of the glutamine metabolism by increasing the GAC:KGA isoform ratio fuels versatile metabolic pathways necessary for HSC self-renewal and proper stress response. Our study establishes APA as a critical regulatory layer orchestrating HSC self-renewal, behavior, and commitment.
    Keywords:  3′-seq; APAome; GAC; KGA; Nudt21; Pabpn1; alternative polyadenylation; glutaminolysis; hematopoietic stem cell; inflammation
  22. Dev Cell. 2020 Apr 01. pii: S1534-5807(20)30189-1. [Epub ahead of print]
    Bhattacharya D, Azambuja AP, Simoes-Costa M.
      The Warburg effect is one of the metabolic hallmarks of cancer cells, characterized by enhanced glycolysis even under aerobic conditions. This physiological adaptation is associated with metastasis , but we still have a superficial understanding of how it affects cellular processes during embryonic development. Here we report that the neural crest, a migratory stem cell population in vertebrate embryos, undergoes an extensive metabolic remodeling to engage in aerobic glycolysis prior to delamination. This increase in glycolytic flux promotes Yap/Tead signaling, which activates the expression of a set of transcription factors to drive epithelial-to-mesenchymal transition. Our results demonstrate how shifts in carbon metabolism can trigger the gene regulatory circuits that control complex cell behaviors. These findings support the hypothesis that the Warburg effect is a precisely regulated developmental mechanism that is anomalously reactivated during tumorigenesis and metastasis.
    Keywords:  Warburg effect; Yap/Tead signaling; cell metabolism; cell migration; epithelial to mesenchymal transition; glycolysis; neural crest
  23. Anal Chem. 2020 Apr 03.
    Chen P, Zhuo Y, Tian S, Zhang T, Zhai G, Fan E, Ma Z, Zhang Y, Zhang K.
      Histone post-translational modifications (HPTMs) serve as signal platforms for recruitment of binding proteins (readers) to regulate gene expression. Accumulated evidence suggests that the intensive distribution of HPTMs may result in crosstalk, which increases or inhibits the recruitment of reader proteins, further altering the functional outcome of HPTMs. Therefore, the comprehensive identification of multiple interactions between combinatorial HPTMs and reading domains is essential to understand the chromatin-templated processes. However it is still a big challenge to profile these complicated interactions due to various limitations including rather weak, transient and multiple interactions between HPTMs and readers, the high dynamic property of HPTMs as well as the low abundance of reader proteins. Here we developed an integrated approach to profile the complicated interactions between combinatorial HPTMs and dual domains. Based on a combinatorial HPTM peptide library (tri-methylation of histone H3 lysine 4 and its neighboring PTMs) and five affinity tag proteins containing tandem-domain probes, histone interactions can be profiled by pull-down assay combined with mass spectrometry analysis. The interactions were further verified by Isothermal Titration Calorimetry and Proximity Ligation Assay, as well as molecular docking. By use of combinatorial HPTMs, we demonstrated that this integrated approach can be successfully utilized for the characterization of multiple interactions between reading domains and combinatorial HPTMs including novel HPTMs with low stoichiometry. Thus a novel chemical proteomics tool for profiling of multiple PTM-mediated protein-protein inter-actions was successfully developed and can be adapted for broad biomedical applications.
  24. Nat Cell Biol. 2020 Mar 30.
    Sankar A, Lerdrup M, Manaf A, Johansen JV, Gonzalez JM, Borup R, Blanshard R, Klungland A, Hansen K, Andersen CY, Dahl JA, Helin K, Hoffmann ER.
      The importance of germline-inherited post-translational histone modifications on priming early mammalian development is just emerging1-4. Histone H3 lysine 9 (H3K9) trimethylation is associated with heterochromatin and gene repression during cell-fate change5, whereas histone H3 lysine 4 (H3K4) trimethylation marks active gene promoters6. Mature oocytes are transcriptionally quiescent and possess remarkably broad domains of H3K4me3 (bdH3K4me3)1,2. It is unknown which factors contribute to the maintenance of the bdH3K4me3 landscape. Lysine-specific demethylase 4A (KDM4A) demethylates H3K9me3 at promoters marked by H3K4me3 in actively transcribing somatic cells7. Here, we report that KDM4A-mediated H3K9me3 demethylation at bdH3K4me3 in oocytes is crucial for normal pre-implantation development and zygotic genome activation after fertilization. The loss of KDM4A in oocytes causes aberrant H3K9me3 spreading over bdH3K4me3, resulting in insufficient transcriptional activation of genes, endogenous retroviral elements and chimeric transcripts initiated from long terminal repeats during zygotic genome activation. The catalytic activity of KDM4A is essential for normal epigenetic reprogramming and pre-implantation development. Hence, KDM4A plays a crucial role in preserving the maternal epigenome integrity required for proper zygotic genome activation and transfer of developmental control to the embryo.
  25. J Biol Chem. 2020 Apr 02. pii: jbc.RA120.013338. [Epub ahead of print]
    Basu U, Mishra N, Farooqui M, Shen J, Johnson LC, Patel SS.
      The structurally homologous Mtf1 and TFB2M proteins serve as transcription initiation factors of the mitochondrial RNA polymerases in Saccharomyces cerevisiae and humans, respectively. These transcription factors directly interact with the non-template strand of the transcription bubble to drive promoter melting. Given the key roles of Mtf1 and TFB2M in promoter-specific transcription initiation, it can be expected that the DNA-binding activity of the mitochondrial transcription factors is regulated to prevent DNA binding at inappropriate times. However, little information is available on how mitochondrial DNA transcription is regulated. While studying C-terminal (C-tail) deletion mutants of Mtf1 and TFB2M, here we stumbled upon a finding suggesting that the flexible C-tail region of these factors autoregulates their DNA-binding activity. Quantitative DNA-binding studies with fluorescence anisotropy-based titrations revealed that Mtf1 with an intact C-tail has no affinity for DNA, but that deletion of the C-tail greatly increases Mtf1's DNA-binding affinity. Similar observations were made with TFB2M, although autoinhibition by the C-tail of TFB2M was not as complete as in Mtf1. Analysis of available TFB2M structures disclosed that the C-tail engages in intramolecular interactions with the DNA-binding groove in the free factor, which we propose inhibit its DNA-binding activity. Further experiments showed that RNA polymerase relieves this autoinhibition by interacting with the C-tail and engaging it in complex formation. In conclusion, our biochemical and structural analyses reveal autoinhibitory and activation mechanisms of mitochondrial transcription factors that regulate their DNA-binding activities and aid in the specific assembly of the transcription initiation complexes.
    Keywords:  Mtf1; RNA polymerase; TFB2M; autoinhibition; fluorescence anisotropy; mitochondria; mitochondrial RNA polymerase; mitochondrial transcription factors; protein-DNA interaction; transcriptional coactivator
  26. Cancer Cell. 2020 Mar 23. pii: S1535-6108(20)30106-9. [Epub ahead of print]
    Alam H, Tang M, Maitituoheti M, Dhar SS, Kumar M, Han CY, Ambati CR, Amin SB, Gu B, Chen TY, Lin YH, Chen J, Muller FL, Putluri N, Flores ER, DeMayo FJ, Baseler L, Rai K, Lee MG.
      Epigenetic modifiers frequently harbor loss-of-function mutations in lung cancer, but their tumor-suppressive roles are poorly characterized. Histone methyltransferase KMT2D (a COMPASS-like enzyme, also called MLL4) is among the most highly inactivated epigenetic modifiers in lung cancer. Here, we show that lung-specific loss of Kmt2d promotes lung tumorigenesis in mice and upregulates pro-tumorigenic programs, including glycolysis. Pharmacological inhibition of glycolysis preferentially impedes tumorigenicity of human lung cancer cells bearing KMT2D-inactivating mutations. Mechanistically, Kmt2d loss widely impairs epigenomic signals for super-enhancers/enhancers, including the super-enhancer for the circadian rhythm repressor Per2. Loss of Kmt2d decreases expression of PER2, which regulates multiple glycolytic genes. These findings indicate that KMT2D is a lung tumor suppressor and that KMT2D deficiency confers a therapeutic vulnerability to glycolytic inhibitors.
    Keywords:  KMT2D; epigenetic modifier; glycolysis; histone methylation; histone methyltransferase; inhibitor; lung cancer; metabolism; super-enhancer; tumor suppressor
  27. Nucleic Acids Res. 2020 Apr 04. pii: gkaa210. [Epub ahead of print]
    Zhang T, Pilko A, Wollman R.
      Therapeutic targeting of epigenetic modulators offers a novel approach to the treatment of multiple diseases. The cellular consequences of chemical compounds that target epigenetic regulators (epi-drugs) are complex. Epi-drugs affect global cellular phenotypes and cause local changes to gene expression due to alteration of a gene chromatin environment. Despite increasing use in the clinic, the mechanisms responsible for cellular changes are unclear. Specifically, to what degree the effects are a result of cell-wide changes or disease related locus specific effects is unknown. Here we developed a platform to systematically and simultaneously investigate the sensitivity of epi-drugs at hundreds of genomic locations by combining DNA barcoding, unique split-pool encoding, and single cell expression measurements. Internal controls are used to isolate locus specific effects separately from any global consequences these drugs have. Using this platform we discovered wide-spread loci specific sensitivities to epi-drugs for three distinct epi-drugs that target histone deacetylase, DNA methylation and bromodomain proteins. By leveraging ENCODE data on chromatin modification, we identified features of chromatin environments that are most likely to be affected by epi-drugs. The measurements of loci specific epi-drugs sensitivities will pave the way to the development of targeted therapy for personalized medicine.
  28. Epigenomics. 2020 Mar 30.
    Li X, Chen Y, Fu C, Li H, Yang K, Bi J, Huo R.
      Aim: This study was conducted to reveal epigenetic landscape in infantile hemangiomas (IHs) and identify transcription factors (TFs) and their downstream genes active in IHs. Materials & methods: We performed Assay for Transposase Accessible Chromatin (ATAC-seq) with RNA-seq in three pairs of IHs and their adjacent normal tissues. Functions of candidate TFs were investigated in human umbilical vein endothelial cells (HUVECs). Results: Chromatin of IH tissues is less compact. Some candidate genes and TFs were identified. In HUVECs, SPDEF inhibited cell viability and tube formation, and promoted apoptosis; SOX4 exerted the opposite effect. SPDEF may act through EPHA5, ZBTB46 and SASH1; SOX4 may act through MMP12 and HIVEP3. Conclusion: Epigenetics plays a role in IHs. SPDEF and SOX4 may act in IHs.
    Keywords:  ATAC-seq; RNA-seq; SOX4; SPDEF; infantile hemangioma; open chromatin
  29. Methods. 2020 Mar 30. pii: S1046-2023(20)30059-1. [Epub ahead of print]
    Nakato R, Sakata T.
      Chromatin immunoprecipitation followed by sequencing (ChIP-seq) is a central method in epigenomic research. Genome-wide analysis of histone modifications, such as enhancer analysis and genome-wide chromatin state annotation, enables systematic analysis of how the epigenomic landscape contributes to cell identity, development, lineage specification, and disease. In this review, we first present a typical ChIP-seq analysis workflow, from quality assessment to chromatin-state annotation. We focus on practical, rather than theoretical, approaches for biological studies. Next, we outline various advanced ChIP-seq applications and introduce several state-of-the-art methods, including prediction of gene expression level and chromatin loops from epigenome data and data imputation. Finally, we discuss recently developed single-cell ChIP-seq analysis methodologies that elucidate the cellular diversity within complex tissues and cancers.
    Keywords:  ChIP-seq; Chromatin state; Histone modifications; Machine learning; Quality assessment; Single-cell analysis
  30. Genome Biol. 2020 Apr 02. 21(1): 84
    Wang G, Meng Q, Xia B, Zhang S, Lv J, Zhao D, Li Y, Wang X, Zhang L, Cooke JP, Cao Q, Chen K.
      We present TADsplimer, the first computational tool to systematically detect topologically associating domain (TAD) splits and mergers across the genome between Hi-C samples. TADsplimer recaptures splits and mergers of TADs with high accuracy in simulation analyses and defines hundreds of TAD splits and mergers between pairs of different cell types, such as endothelial cells and fibroblasts. Our work reveals a key role for TAD remodeling in epigenetic regulation of transcription and delivers the first tool for the community to perform dynamic analysis of TAD splits and mergers in numerous biological and disease models.
    Keywords:  Bioinformatics; Chromatin conformation; Computational biology; Epigenomics; Hi-C; Histone modification; Topologically associating domains
  31. Nat Cell Biol. 2020 Mar 30.
    Stewart-Morgan KR, Petryk N, Groth A.
      Propagation of the chromatin landscape across cell divisions is central to epigenetic cell memory. Mechanistic analysis of the interplay between DNA replication, the cell cycle, and the epigenome has provided insights into replication-coupled chromatin assembly and post-replicative chromatin maintenance. These breakthroughs are critical for defining how proliferation impacts the epigenome during cell identity changes in development and disease. Here we review these findings in the broader context of epigenetic inheritance across mitotic cell division.
  32. Mol Cell. 2020 Mar 26. pii: S1097-2765(20)30155-6. [Epub ahead of print]
    Sati S, Bonev B, Szabo Q, Jost D, Bensadoun P, Serra F, Loubiere V, Papadopoulos GL, Rivera-Mulia JC, Fritsch L, Bouret P, Castillo D, Gelpi JL, Orozco M, Vaillant C, Pellestor F, Bantignies F, Marti-Renom MA, Gilbert DM, Lemaitre JM, Cavalli G.
      To understand the role of the extensive senescence-associated 3D genome reorganization, we generated genome-wide chromatin interaction maps, epigenome, replication-timing, whole-genome bisulfite sequencing, and gene expression profiles from cells entering replicative senescence (RS) or upon oncogene-induced senescence (OIS). We identify senescence-associated heterochromatin domains (SAHDs). Differential intra- versus inter-SAHD interactions lead to the formation of senescence-associated heterochromatin foci (SAHFs) in OIS but not in RS. This OIS-specific configuration brings active genes located in genomic regions adjacent to SAHDs in close spatial proximity and favors their expression. We also identify DNMT1 as a factor that induces SAHFs by promoting HMGA2 expression. Upon DNMT1 depletion, OIS cells transition to a 3D genome conformation akin to that of cells in replicative senescence. These data show how multi-omics and imaging can identify critical features of RS and OIS and discover determinants of acute senescence and SAHF formation.
    Keywords:  3D genome architecture; DNMT1; Hi-C; chromatin compartments; gene regulation; oncogene-induced senescence; replicative senescence; senescence
  33. Genome Biol. 2020 Mar 30. 21(1): 81
    Schreiber J, Durham T, Bilmes J, Noble WS.
      The human epigenome has been experimentally characterized by thousands of measurements for every basepair in the human genome. We propose a deep neural network tensor factorization method, Avocado, that compresses this epigenomic data into a dense, information-rich representation. We use this learned representation to impute epigenomic data more accurately than previous methods, and we show that machine learning models that exploit this representation outperform those trained directly on epigenomic data on a variety of genomics tasks. These tasks include predicting gene expression, promoter-enhancer interactions, replication timing, and an element of 3D chromatin architecture.
  34. Int J Mol Sci. 2020 Mar 29. pii: E2356. [Epub ahead of print]21(7):
    von Knethen A, Heinicke U, Weigert A, Zacharowski K, Brüne B.
      Regulatory T cells (Tregs) are important mediators of immunological self-tolerance and homeostasis. Being cluster of differentiation 4+Forkhead box protein3+ (CD4+FOXP3+), these cells are a subset of CD4+ T lymphocytes and can originate from the thymus (tTregs) or from the periphery (pTregs). The malfunction of CD4+ Tregs is associated with autoimmune responses such as rheumatoid arthritis (RA), multiple sclerosis (MS), type 1 diabetes (T1D), inflammatory bowel diseases (IBD), psoriasis, systemic lupus erythematosus (SLE), and transplant rejection. Recent evidence supports an opposed role in sepsis. Therefore, maintaining functional Tregs is considered as a therapy regimen to prevent autoimmunity and allograft rejection, whereas blocking Treg differentiation might be favorable in sepsis patients. It has been shown that Tregs can be generated from conventional naïve T cells, called iTregs, due to their induced differentiation. Moreover, Tregs can be effectively expanded in vitro based on blood-derived tTregs. Taking into consideration that the suppressive role of Tregs has been mainly attributed to the expression and function of the transcription factor Foxp3, modulating its expression and binding to the promoter regions of target genes by altering the chromatin histone acetylation state may turn out beneficial. Hence, we discuss the role of histone deacetylation inhibitors as epigenetic modulators of Tregs in this review in detail.
    Keywords:  Foxp3 expression; Treg; autoimmunity; epigenetics; histone deacetylase inhibitor; sepsis; tolerance induction; transplantation
  35. Mol Cell. 2020 Mar 17. pii: S1097-2765(20)30156-8. [Epub ahead of print]
    Fant CB, Levandowski CB, Gupta K, Maas ZL, Moir J, Rubin JD, Sawyer A, Esbin MN, Rimel JK, Luyties O, Marr MT, Berger I, Dowell RD, Taatjes DJ.
      RNA polymerase II (RNAPII) transcription is governed by the pre-initiation complex (PIC), which contains TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH, RNAPII, and Mediator. After initiation, RNAPII enzymes pause after transcribing less than 100 bases; precisely how RNAPII pausing is enforced and regulated remains unclear. To address specific mechanistic questions, we reconstituted human RNAPII promoter-proximal pausing in vitro, entirely with purified factors (no extracts). As expected, NELF and DSIF increased pausing, and P-TEFb promoted pause release. Unexpectedly, the PIC alone was sufficient to reconstitute pausing, suggesting RNAPII pausing is an inherent PIC function. In agreement, pausing was lost upon replacement of the TFIID complex with TATA-binding protein (TBP), and PRO-seq experiments revealed widespread disruption of RNAPII pausing upon acute depletion (t = 60 min) of TFIID subunits in human or Drosophila cells. These results establish a TFIID requirement for RNAPII pausing and suggest pause regulatory factors may function directly or indirectly through TFIID.
    Keywords:  DSIF; NELF; P-TEFb; PRO-seq; RNA polymerase II; TAF1; TBP; TFIID; TRIM-Away; pausing
  36. EMBO J. 2020 Apr 01. e104759
    Bärthel S, Schneider G, Saur D.
      Epithelial differentiation of normal and tumor cells is orchestrated by lineage-determining transcriptional regulatory networks that enforce cell identity. Recent research by Kalisz et al (2020) in the EMBO Journal elucidates the molecular mechanisms by which a transcriptional differentiation program governed by HNF1A and KDM6A maintains acinar differentiation and the epithelial identity of pancreatic ductal adenocarcinoma (PDAC). Loss of function of either transcriptional regulator induces tumor progression to a poorly differentiated and highly aggressive PDAC subtype with a squamous transcriptome and poor prognosis.
  37. Theranostics. 2020 ;10(9): 4134-4149
    Bi Z, Zhang Q, Fu Y, Wadgaonkar P, Zhang W, Almutairy B, Xu L, Rice M, Qiu Y, Thakur C, Chen F.
      In this report, we demonstrated that inorganic arsenic (iAs) induces generation of the cancer stem-like cells (CSCs) through Nrf2-dependent HIF1α activation, and the subsequent metabolic reprogramming from mitochondrial oxidative phosphorylation to glycolysis in epithelial cells. Methods: Genome-wide ChIP-seq analysis was performed to investigate the global binding of Nrf2 and/or HIF1α on the genome in the cells treated with iAs. Both untargeted metabolomics and UDP-13C-glucose flux were applied to determine metabolic reprogramming in the iAs-induced CSCs. The role of Nrf2 on iAs-induced HIF1α and other stemness gene expression was validated by lentiviral transfection of Nrf2 inhibitor Keap1 and CRISPR-Cas9-mediated Nrf2 gene knockout, respectively. Results: The CSCs induced by iAs exhibit a diminished mitochondrial oxidative phosphorylation and an enhanced glycolysis that is actively shunted to the hexosamine biosynthetic pathway (HBP) and serine/glycine pathway. ChIP-seq data revealed that treatment of the cells with iAs amplified Nrf2 enrichment peaks in intergenic region, promoter and gene body. In contrast, a shift of the HIF1α peaks from distal intergenic region to gene promoter and the first exon was noted. Both Nrf2 and HIF1α are responsible for the iAs-induced expression of the glycolytic genes and the genes important for the stemness of the CSCs. Intriguingly, we also discovered a mutual transcriptional regulation between Nrf2 and HIF1α. Inhibition of Nrf2 by lentiviral infection of Keap1, or knockout of Nrf2 by CRISPR-Cas9 gene editing, not only blocked iAs-induced HIF1α activation, but reduced the expression of the key stemness genes for the formation of CSCs also. Conclusion: We demonstrated that Nrf2 activation is an initiating signal for iAs-induced HIF1α activation, and Nrf2 and HIF1α played a concerted role on inducing metabolic reprogramming and the CSCs.
    Keywords:  HIF1α; Nrf2; arsenic; cancer stem cells; metabolic reprogramming
  38. Mol Metab. 2020 Feb 12. pii: S2212-8778(20)30027-2. [Epub ahead of print]
    Bheda P.
      BACKGROUND: Organisms can be primed by metabolic exposures to continue expressing response genes even once the metabolite is no longer available, and can affect the speed and magnitude of responsive gene expression during subsequent exposures. This "metabolic transcriptional memory" can have a profound impact on the survivability of organisms in fluctuating environments.SCOPE OF REVIEW: Here I present several examples of metabolic transcriptional memory in the microbial world and discuss what is known so far regarding the underlying mechanisms, which mainly focus on chromatin modifications, protein inheritance, and broad changes in metabolic network. From these lessons learned in microbes, some insights into the yet understudied human metabolic memory can be gained. I thus discuss the implications of metabolic memory in disease progression in humans - i.e., the memory of high blood sugar exposure and the resulting effects on diabetic complications.
    MAJOR CONCLUSIONS: Carbon source shifts from glucose to other less preferred sugars such as lactose, galactose, and maltose for energy metabolism as well as starvation of a signal transduction precursor sugar inositol are well-studied examples of metabolic transcriptional memory in Escherichia coli and Saccharomyces cerevisiae. Although the specific factors guiding metabolic transcriptional memory are not necessarily conserved from microbes to humans, the same basic mechanisms are in play, as is observed in hyperglycemic memory. Exploration of new metabolic transcriptional memory systems as well as further detailed mechanistic analyses of known memory contexts in microbes is therefore central to understanding metabolic memory in humans, and may be of relevance for the successful treatment of the ever-growing epidemic of diabetes.
    Keywords:  Bistability; Chromatin modification; Diabetes; Galactose; Glucose; Hyperglycemia; Hysteresis; Inositol; Lactose; Maltose; Metabolic memory; Metabolic network; Protein inheritance; Reinduction memory; Transcriptional memory
  39. Stem Cell Reports. 2020 Mar 29. pii: S2213-6711(20)30099-0. [Epub ahead of print]
    Boles NC, Fernandes M, Swigut T, Srinivasan R, Schiff L, Rada-Iglesias A, Wang Q, Saini JS, Kiehl T, Stern JH, Wysocka J, Blenkinsop TA, Temple S.
      Epithelial to mesenchymal transition (EMT) is a biological process involved in tissue morphogenesis and disease that causes dramatic changes in cell morphology, migration, proliferation, and gene expression. The retinal pigment epithelium (RPE), which supports the neural retina, can undergo EMT, producing fibrous epiretinal membranes (ERMs) associated with vision-impairing clinical conditions, such as macular pucker and proliferative vitreoretinopathy (PVR). We found that co-treatment with TGF-β and TNF-α (TNT) accelerates EMT in adult human RPE stem cell-derived RPE cell cultures. We captured the global epigenomic and transcriptional changes elicited by TNT treatment of RPE and identified putative active enhancers associated with actively transcribed genes, including a set of upregulated transcription factors that are candidate regulators. We found that the vitamin B derivative nicotinamide downregulates these key transcriptional changes, and inhibits and partially reverses RPE EMT, revealing potential therapeutic routes to benefit patients with ERM, macular pucker and PVR.
    Keywords:  contractility; epigenetics; epithelial to mesenchymal transition; mesenchymal-to-epithelial transition; nicotinamide; proliferative vitreoretinopathy; retinal pigment epithelium; whole transcriptome
  40. Mol Metab. 2020 Feb 06. pii: S2212-8778(20)30030-2. [Epub ahead of print]35 100958
    Dumayne C, Tarussio D, Sanchez-Archidona AR, Picard A, Basco D, Berney XP, Ibberson M, Thorens B.
      OBJECTIVES: In the pathogenesis of type 2 diabetes, development of insulin resistance triggers an increase in pancreatic β-cell insulin secretion capacity and β-cell number. Failure of this compensatory mechanism is caused by a dedifferentiation of β-cells, which leads to insufficient insulin secretion and diabetic hyperglycemia. The β-cell factors that normally protect against dedifferentiation remain poorly defined. Here, through a systems biology approach, we identify the transcription factor Klf6 as a regulator of β-cell adaptation to metabolic stress.METHODS: We used a β-cell specific Klf6 knockout mouse model to investigate whether Klf6 may be a potential regulator of β-cell adaptation to a metabolic stress.
    RESULTS: We show that inactivation of Klf6 in β-cells blunts their proliferation induced by the insulin resistance of pregnancy, high-fat high-sucrose feeding, and insulin receptor antagonism. Transcriptomic analysis showed that Klf6 controls the expression of β-cell proliferation genes and, in the presence of insulin resistance, it prevents the down-expression of genes controlling mature β-cell identity and the induction of disallowed genes that impair insulin secretion. Its expression also limits the transdifferentiation of β-cells into α-cells.
    CONCLUSION: Our study identifies a new transcription factor that protects β-cells against dedifferentiation, and which may be targeted to prevent diabetes development.
    Keywords:  Dedifferentiation; Insulin resistance; Transdifferentiation; Type 2 diabetes; β-Cell proliferation
  41. J Biol Chem. 2020 Apr 03. pii: jbc.RA120.013196. [Epub ahead of print]
    Meriesh HA, Lerner AM, Chandrasekharan MB, Strahl BD.
      Histone H2B monoubiquitylation (H2Bub1) has central functions in multiple DNA-templated processes, including gene transcription, DNA repair, and replication. H2Bub1 also is required for the trans-histone regulation of H3K4 and H3K79 methylation. Although previous studies have elucidated the basic mechanisms that establish and remove H2Bub1, we have only an incomplete understanding of how H2Bub1 is regulated. We report here that the histone H4 basic patch regulates H2Bub1. Yeast cells with arginine-to-alanine mutations in the H4 basic patch (H42RA) exhibited significant loss of global H2Bub1. H42RA mutant yeast strains also displayed chemotoxin sensitivities similar to, but less severe than, strains containing a complete loss of H2Bub1. We found that the H4 basic patch regulates H2Bub1 levels independently of interactions with chromatin remodelers and separately from its regulation of H3K79 methylation. To measure H2B ubiquitylation and deubiquitination kinetics in vivo, we used a rapid and reversible optogenetic tool, the light-inducible nuclear exporter (LINX), to control the subcellular location of the H2Bub1 E3-ligase, Bre1. The ability of Bre1 to ubiquitylate H2B was unaffected in the H42RA mutant. In contrast, H2Bub1 deubiquitination by SAGA-associated Ubp8, but not by Ubp10, increased in the H42RA mutant. Consistent with a function for the H4 basic patch in regulating SAGA deubiquitinase activity, we also detected increased SAGA-mediated histone acetylation in H4 basic patch mutants. Our findings uncover that the H4 basic patch has a regulatory function in SAGA-mediated histone modifications.
    Keywords:  chromatin; chromatin regulation; gene transcription; histone; histone modification; yeast transcription
  42. Cancers (Basel). 2020 Mar 21. pii: E748. [Epub ahead of print]12(3):
    Li J, Zhang B, Liu M, Fu X, Ci X, A J, Fu C, Dong G, Wu R, Zhang Z, Fu L, Dong JT.
      Androgen/androgen receptor (AR) signaling drives both the normal prostate development and prostatic carcinogenesis, and patients with advanced prostate cancer often develop resistance to androgen deprivation therapy. The transcription factor Krüppel-like factor 5 (KLF5) also regulates both normal and cancerous development of the prostate. In this study, we tested whether and how KLF5 plays a role in the function of AR signaling in prostate cancer cells. We found that KLF5 is upregulated by androgen depending on AR in LNCaP and C4-2B cells. Silencing KLF5, in turn, reduced AR transcriptional activity and inhibited androgen-induced cell proliferation and tumor growth in vitro and in vivo. Mechanistically, KLF5 occupied the promoter of AR, and silencing KLF5 repressed AR transcription. In addition, KLF5 and AR physically interacted with each other to regulate the expression of multiple genes (e.g., MYC, CCND1 and PSA) to promote cell proliferation. These findings indicate that, while transcriptionally upregulated by AR signaling, KLF5 also regulates the expression and transcriptional activity of AR in androgen-sensitive prostate cancer cells. The KLF5-AR interaction could provide a therapeutic opportunity for the treatment of prostate cancer.
    Keywords:  KLF5; androgen receptor; cell proliferation; prostate cancer; tumorigenesis
  43. Nat Immunol. 2020 Mar 30.
    Abdelsamed HA, Zebley CC, Nguyen H, Rutishauser RL, Fan Y, Ghoneim HE, Crawford JC, Alfei F, Alli S, Ribeiro SP, Castellaw AH, McGargill MA, Jin H, Boi SK, Speake C, Serti E, Turka LA, Busch ME, Stone M, Deeks SG, Sekaly RP, Zehn D, James EA, Nepom GT, Youngblood B.
      The pool of beta cell-specific CD8+ T cells in type 1 diabetes (T1D) sustains an autoreactive potential despite having access to a constant source of antigen. To investigate the long-lived nature of these cells, we established a DNA methylation-based T cell 'multipotency index' and found that beta cell-specific CD8+ T cells retained a stem-like epigenetic multipotency score. Single-cell assay for transposase-accessible chromatin using sequencing confirmed the coexistence of naive and effector-associated epigenetic programs in individual beta cell-specific CD8+ T cells. Assessment of beta cell-specific CD8+ T cell anatomical distribution and the establishment of stem-associated epigenetic programs revealed that self-reactive CD8+ T cells isolated from murine lymphoid tissue retained developmentally plastic phenotypic and epigenetic profiles relative to the same cells isolated from the pancreas. Collectively, these data provide new insight into the longevity of beta cell-specific CD8+ T cell responses and document the use of this methylation-based multipotency index for investigating human and mouse CD8+ T cell differentiation.
  44. Cell Rep. 2020 Mar 31. pii: S2211-1247(20)30329-6. [Epub ahead of print]30(13): 4551-4566.e7
    Morin A, Goncalves J, Moog S, Castro-Vega LJ, Job S, Buffet A, Fontenille MJ, Woszczyk J, Gimenez-Roqueplo AP, Letouzé E, Favier J.
      Loss-of-function mutations in the SDHB subunit of succinate dehydrogenase predispose patients to aggressive tumors characterized by pseudohypoxic and hypermethylator phenotypes. The mechanisms leading to DNA hypermethylation and its contribution to SDH-deficient cancers remain undemonstrated. We examine the genome-wide distribution of 5-methylcytosine and 5-hydroxymethylcytosine and their correlation with RNA expression in SDHB-deficient tumors and murine Sdhb-/- cells. We report that DNA hypermethylation results from TET inhibition. Although it preferentially affects PRC2 targets and known developmental genes, PRC2 activity does not contribute to the DNA hypermethylator phenotype. We also prove, in vitro and in vivo, that TET silencing, although recapitulating the methylation profile of Sdhb-/- cells, is not sufficient to drive their EMT-like phenotype, which requires additional HIF2α activation. Altogether, our findings reveal synergistic roles of TET repression and pseudohypoxia in the acquisition of metastatic traits, providing a rationale for targeting HIF2α and DNA methylation in SDH-associated malignancies.
    Keywords:  SDH; SDHB; epithelial-to-mesenchymal transition; hydroxymethylation; hypoxia; methylation; paraganglioma; pheochromocytoma; succinate dehydrogenase
  45. Cancer Cell. 2020 Mar 19. pii: S1535-6108(20)30105-7. [Epub ahead of print]
    Guerra SL, Maertens O, Kuzmickas R, De Raedt T, Adeyemi RO, Guild CJ, Guillemette S, Redig AJ, Chambers ES, Xu M, Tiv H, Santagata S, Jänne PA, Elledge SJ, Cichowski K.
      While KRAS mutations are common in non-small cell lung cancer (NSCLC), effective treatments are lacking. Here, we report that half of KRAS-mutant NSCLCs aberrantly express the homeobox protein HOXC10, largely due to unappreciated defects in PRC2, which confers sensitivity to combined BET/MEK inhibitors in xenograft and PDX models. Efficacy of the combination is dependent on suppression of HOXC10 by BET inhibitors. We further show that HOXC10 regulates the expression of pre-replication complex (pre-RC) proteins in sensitive tumors. Accordingly, BET/MEK inhibitors suppress pre-RC proteins in cycling cells, triggering stalled replication, DNA damage, and death. These studies reveal a promising therapeutic strategy for KRAS-mutant NSCLCs, identify a predictive biomarker of response, and define a subset of NSCLCs with a targetable epigenetic vulnerability.
    Keywords:  BET; EED; HOXC10; KRAS; MEK; NSCLC; PRC2; SUZ12; combination therapy; lung cancer
  46. Nucleic Acids Res. 2020 Mar 30. pii: gkaa203. [Epub ahead of print]
    Zhang LF, Tan-Tai WJ, Li XH, Liu MF, Shi HJ, Martin-DeLeon PA, O WS, Chen H.
      Previously, we have shown that human sperm Prohibitin (PHB) expression is significantly negatively correlated with mitochondrial ROS levels but positively correlated with mitochondrial membrane potential and motility. However, the possible role of PHB in mammalian spermatogenesis has not been investigated. Here we document the presence of PHB in spermatocytes and its functional roles in meiosis by generating the first male germ cell-specific Phb-cKO mouse. Loss of PHB in spermatocytes resulted in complete male infertility, associated with not only meiotic pachytene arrest with accompanying apoptosis, but also apoptosis resulting from mitochondrial morphology and function impairment. Our mechanistic studies show that PHB in spermatocytes regulates the expression of STAG3, a key component of the meiotic cohesin complex, via a non-canonical JAK/STAT pathway, and consequently promotes meiotic DSB repair and homologous recombination. Furthermore, the PHB/JAK2 axis was found as a novel mechanism in the maintenance of stabilization of meiotic STAG3 cohesin complex and the modulation of heterochromatin formation in spermatocytes during meiosis. The observed JAK2-mediated epigenetic changes in histone modifications, reflected in a reduction of histone 3 tyrosine 41 phosphorylation (H3Y41ph) and a retention of H3K9me3 at the Stag3 locus, could be responsible for Stag3 dysregulation in spermatocytes with the loss of PHB.
  47. BMC Med Genomics. 2020 Apr 03. 13(Suppl 5): 49
    Dong C, Liu J, Chen SX, Dong T, Jiang G, Wang Y, Wu H, Reiter JL, Liu Y.
      BACKGROUND: While several multigene signatures are available for predicting breast cancer prognosis, particularly in early stage disease, effective molecular indicators are needed, especially for triple-negative carcinomas, to improve treatments and predict diagnostic outcomes. The objective of this study was to identify transcriptional regulatory networks to better understand mechanisms giving rise to breast cancer development and to incorporate this information into a model for predicting clinical outcomes.METHODS: Gene expression profiles from 1097 breast cancer patients were retrieved from The Cancer Genome Atlas (TCGA). Breast cancer-specific transcription regulatory information was identified by considering the binding site information from ENCODE and the top co-expressed targets in TCGA using a nonlinear approach. We then used this information to predict breast cancer patient survival outcome.
    RESULT: We built a multiple regulator-based prediction model for breast cancer. This model was validated in more than 5000 breast cancer patients from the Gene Expression Omnibus (GEO) databases. We demonstrated our regulator model was significantly associated with clinical stage and that cell cycle and DNA replication related pathways were significantly enriched in high regulator risk patients.
    CONCLUSION: Our findings demonstrate that transcriptional regulator activities can predict patient survival. This finding provides additional biological insights into the mechanisms of breast cancer progression.
    Keywords:  Breast cancer; Prognostic model; Transcription regulators
  48. Epigenetics. 2020 Mar 31.
    Blanco-Luquin I, Acha B, Urdánoz-Casado A, Sánchez-Ruiz de Gordoa J, Vicuña-Urriza J, Roldán M, Labarga A, Zelaya MV, Cabello C, Méndez-López I, Mendioroz M.
      The discovery of new biomarkers would be very valuable to improve the detection of early Alzheimer's disease (AD). DNA methylation marks may serve as epigenetic biomarkers of early AD. Here we identified epigenetic marks that are present in the human hippocampus from the earliest stages of AD.A previous methylome dataset of the human AD hippocampus was used to select a set of 8 differentially methylated positions (DMPs) since early AD stages. Next, bisulfite pyrosequencing was performed in an expanded homogeneous cohort of 18 pure controls and 35 hippocampal samples with neuropathological changes of pure AD. Correlation between DNA methylation levels in DMPs and phospho-tau protein burden assessed by immunohistochemistry in the hippocampus was also determined.We found 4 DMPs showing higher levels of DNA methylation at early AD stages compared to controls, involving ELOVL2, GIT1/TP53I13 and the histone gene locus at chromosome 6. DNA methylation levels assessed by bisulfite pyrosequencing correlated with phospho-tau protein burden for ELOVL2 and HIST1H3E/HIST1H3F genes.In this discovery study a set of 4 epigenetic marks of early AD stages have been identified in the human hippocampus. These epigenetic changes point to specific pathways that might be involved in AD pathogenesis, such as the long-chain fatty acid biogenesis or histone functions. It would be worth studying in depth the specific pathways related to these epigenetic marks. These early alterations in DNA methylation in the AD hippocampus could be regarded as candidate biomarkers to be explored in future translational studies.
    Keywords:  DNA methylation; ELOVL2; Early Alzheimer’s disease; GIT1; Hippocampus; biomarkers; epigenetics; fatty acid; histone
  49. Nature. 2020 Apr;580(7801): 142-146
    Collombet S, Ranisavljevic N, Nagano T, Varnai C, Shisode T, Leung W, Piolot T, Galupa R, Borensztein M, Servant N, Fraser P, Ancelin K, Heard E.
      Paternal and maternal epigenomes undergo marked changes after fertilization1. Recent epigenomic studies have revealed the unusual chromatin landscapes that are present in oocytes, sperm and early preimplantation embryos, including atypical patterns of histone modifications2-4 and differences in chromosome organization and accessibility, both in gametes5-8 and after fertilization5,8-10. However, these studies have led to very different conclusions: the global absence of local topological-associated domains (TADs) in gametes and their appearance in the embryo8,9 versus the pre-existence of TADs and loops in the zygote5,11. The questions of whether parental structures can be inherited in the newly formed embryo and how these structures might relate to allele-specific gene regulation remain open. Here we map genomic interactions for each parental genome (including the X chromosome), using an optimized single-cell high-throughput chromosome conformation capture (HiC) protocol12,13, during preimplantation in the mouse. We integrate chromosome organization with allelic expression states and chromatin marks, and reveal that higher-order chromatin structure after fertilization coincides with an allele-specific enrichment of methylation of histone H3 at lysine 27. These early parental-specific domains correlate with gene repression and participate in parentally biased gene expression-including in recently described, transiently imprinted loci14. We also find TADs that arise in a non-parental-specific manner during a second wave of genome assembly. These de novo domains are associated with active chromatin. Finally, we obtain insights into the relationship between TADs and gene expression by investigating structural changes to the paternal X chromosome before and during X chromosome inactivation in preimplantation female embryos15. We find that TADs are lost as genes become silenced on the paternal X chromosome but linger in regions that escape X chromosome inactivation. These findings demonstrate the complex dynamics of three-dimensional genome organization and gene expression during early development.
  50. Urol Oncol. 2020 Mar 25. pii: S1078-1439(19)30490-9. [Epub ahead of print]
    Syed JS, Brito J, Pooli A, Boutros PC, Shuch B.
      Improvements in chemistry, molecular biology, genetics, and bioinformatics have allowed broad use of transcriptomic profiling. Understanding the population of ribonucleic acid (RNA) transcripts can provide important clinical information relevant to kidney cancer care. This includes a better understanding of kidney cancer subtype and distinct clusters within these categories. RNA-sequencing (RNA-seq) is typically done on a region within the tumor, which represents thousands to millions of heterogeneous cells and various components of the microenvironment. Computational tools can deconvolute these populations to provide insight into the microenvironment. Specific signatures of hypoxia, proliferation, angiogenesis and immune infiltration can predict response and survival. Prognostic signatures can risk stratify tumors to aid in identification of patients who might derive benefit from adjuvant therapy. As the cost of sequencing continues to decline and improved bioinformatic tools are developed, the barriers to clinical use of transcriptomic data continue to crumble. Here we review the current literature around the use of transcriptomics in kidney cancer diagnosis and management.
    Keywords:  Microarray; Prognosis; Renal cell carcinoma; Transcriptome
  51. Nat Commun. 2020 Apr 03. 11(1): 1685
    Zhu S, Wang JZ, Chen , He YT, Meng N, Chen M, Lu RX, Chen XH, Zhang XL, Yan GR.
      N6-methyladenosine (m6A) is the most prevalent modification in eukaryotic RNAs. The biological importance of m6A relies on m6A readers, which control mRNA fate and function. However, it remains unexplored whether additional regulatory subunits of m6A readers are involved in the m6A recognition on RNAs. Here we discover that the long noncoding RNA (lncRNA) LINC00266-1 encodes a 71-amino acid peptide. The peptide mainly interacts with the RNA-binding proteins, including the m6A reader IGF2BP1, and is thus named "RNA-binding regulatory peptide" (RBRP). RBRP binds to IGF2BP1 and strengthens m6A recognition by IGF2BP1 on RNAs, such as c-Myc mRNA, to increase the mRNA stability and expression of c-Myc, thereby promoting tumorigenesis. Cancer patients with RBRPhigh have a poor prognosis. Thus, the oncopeptide RBRP encoded by LINC00266-1 is a regulatory subunit of m6A readers and strengthens m6A recognition on the target RNAs by the m6A reader to exert its oncogenic functions.
  52. PLoS Genet. 2020 Apr 03. 16(4): e1008663
    Xu D, Gokcumen O, Khurana E.
      Previous studies have surveyed the potential impact of loss-of-function (LoF) variants and identified LoF-tolerant protein-coding genes. However, the tolerance of human genomes to losing enhancers has not yet been evaluated. Here we present the catalog of LoF-tolerant enhancers using structural variants from whole-genome sequences. Using a conservative approach, we estimate that individual human genomes possess at least 28 LoF-tolerant enhancers on average. We assessed the properties of LoF-tolerant enhancers in a unified regulatory network constructed by integrating tissue-specific enhancers and gene-gene interactions. We find that LoF-tolerant enhancers tend to be more tissue-specific and regulate fewer and more dispensable genes relative to other enhancers. They are enriched in immune-related cells while enhancers with low LoF-tolerance are enriched in kidney and brain/neuronal stem cells. We developed a supervised learning approach to predict the LoF-tolerance of all enhancers, which achieved an area under the receiver operating characteristics curve (AUROC) of 98%. We predict 3,519 more enhancers would be likely tolerant to LoF and 129 enhancers that would have low LoF-tolerance. Our predictions are supported by a known set of disease enhancers and novel deletions from PacBio sequencing. The LoF-tolerance scores provided here will serve as an important reference for disease studies.
  53. Sci Adv. 2020 03;6(13): eaaz3152
    Kyrchanova O, Maksimenko O, Ibragimov A, Sokolov V, Postika N, Lukyanova M, Schedl P, Georgiev P.
      In mammals, a C2H2 zinc finger (C2H2) protein, CTCF, acts as the master regulator of chromosomal architecture and of the expression of Hox gene clusters. Like mammalian CTCF, the Drosophila homolog, dCTCF, localizes to boundaries in the bithorax complex (BX-C). Here, we have determined the minimal requirements for the assembly of a functional boundary by dCTCF and two other C2H2 zinc finger proteins, Pita and Su(Hw). Although binding sites for these proteins are essential for the insulator activity of BX-C boundaries, these binding sites alone are insufficient to create a functional boundary. dCTCF cannot effectively bind to a single recognition sequence in chromatin or generate a functional insulator without the help of additional proteins. In addition, for boundary elements in BX-C at least four binding sites for dCTCF or the presence of additional DNA binding factors is required to generate a functional insulator.
  54. Stem Cell Reports. 2020 Mar 24. pii: S2213-6711(20)30096-5. [Epub ahead of print]
    Hartman AA, Scalf SM, Zhang J, Hu X, Chen X, Eastman AE, Yang C, Guo S.
      Yes-associated protein (YAP) is known to promote the stemness of multiple stem cell types, including pluripotent stem cells, while also antagonizing pluripotency during early embryogenesis. How YAP accomplishes these distinct functions remains unclear. Here, we report that, depending on the specific cells in which it is expressed, YAP could exhibit opposing effects on pluripotency induction from mouse somatic cells. Specifically, YAP inhibits pluripotency induction cell-autonomously but promotes it non-cell-autonomously. For its non-cell-autonomous role, YAP alters the expression of many secreted and matricellular proteins, including CYR61. YAP's non-cell-autonomous promoting effect could be recapitulated by recombinant CYR61 and abrogated by CYR61 depletion. Thus, we define a YAP-driven effect on enhancing pluripotency induction largely mediated by CYR61. Our work highlights the importance of considering the distinct contributions from heterologous cell types in deciphering cell fate control mechanisms and calls for careful re-examination of the co-existing bystander cells in complex cultures and tissues.
    Keywords:  CYR61; YAP; Yamanaka reprogramming; iPSCs; non-cell-autonomous; pluripotency; stem Cell; yes-associated protein
  55. Nat Commun. 2020 Mar 27. 11(1): 1446
    Cresswell GD, Nichol D, Spiteri I, Tari H, Zapata L, Heide T, Maley CC, Magnani L, Schiavon G, Ashworth A, Barry P, Sottoriva A.
      Circulating tumour DNA (ctDNA) allows tracking of the evolution of human cancers at high resolution, overcoming many limitations of tissue biopsies. However, exploiting ctDNA to determine how a patient's cancer is evolving in order to aid clinical decisions remains difficult. This is because ctDNA is a mix of fragmented alleles, and the contribution of different cancer deposits to ctDNA is largely unknown. Profiling ctDNA almost invariably requires prior knowledge of what genomic alterations to track. Here, we leverage on a rapid autopsy programme to demonstrate that unbiased genomic characterisation of several metastatic sites and concomitant ctDNA profiling at whole-genome resolution reveals the extent to which ctDNA is representative of widespread disease. We also present a methylation profiling method that allows tracking evolutionary changes in ctDNA at single-molecule resolution without prior knowledge. These results have critical implications for the use of liquid biopsies to monitor cancer evolution in humans and guide treatment.
  56. Mol Cancer. 2020 Apr 03. 19(1): 72
    Shen C, Xuan B, Yan T, Ma Y, Xu P, Tian X, Zhang X, Cao Y, Ma D, Zhu X, Zhang Y, Fang JY, Chen H, Hong J.
      BACKGROUND: Epigenetic alterations are involved in various aspects of colorectal carcinogenesis. N6-methyladenosine (m6A) modifications of RNAs are emerging as a new layer of epigenetic regulation. As the most abundant chemical modification of eukaryotic mRNA, m6A is essential for the regulation of mRNA stability, splicing, and translation. Alterations of m6A regulatory genes play important roles in the pathogenesis of a variety of human diseases. However, whether this mRNA modification participates in the glucose metabolism of colorectal cancer (CRC) remains uncharacterized.METHODS: Transcriptome-sequencing and liquid chromatography-tandem mass spectrometry (LC-MS) were performed to evaluate the correlation between m6A modifications and glucose metabolism in CRC. Mass spectrometric metabolomics analysis, in vitro and in vivo experiments were conducted to investigate the effects of METTL3 on CRC glycolysis and tumorigenesis. RNA MeRIP-sequencing, immunoprecipitation and RNA stability assay were used to explore the molecular mechanism of METTL3 in CRC.
    RESULTS: A strong correlation between METTL3 and 18F-FDG uptake was observed in CRC patients from Xuzhou Central Hospital. METTL3 induced-CRC tumorigenesis depends on cell glycolysis in multiple CRC models. Mechanistically, METTL3 directly interacted with the 5'/3'UTR regions of HK2, and the 3'UTR region of SLC2A1 (GLUT1), then further stabilized these two genes and activated the glycolysis pathway. M6A-mediated HK2 and SLC2A1 (GLUT1) stabilization relied on the m6A reader IGF2BP2 or IGF2BP2/3, respectively.
    CONCLUSIONS: METTL3 is a functional and clinical oncogene in CRC. METTL3 stabilizes HK2 and SLC2A1 (GLUT1) expression in CRC through an m6A-IGF2BP2/3- dependent mechanism. Targeting METTL3 and its pathway offer alternative rational therapeutic targets in CRC patients with high glucose metabolism.
    Keywords:  Colorectal cancer; GLUT1; Glycolysis; HK2; METTL3; m6A modification
  57. Proc Natl Acad Sci U S A. 2020 Mar 27. pii: 201922159. [Epub ahead of print]
    Wasmuth EV, Hoover EA, Antar A, Klinge S, Chen Y, Sawyers CL.
      The androgen receptor (AR) is a type I nuclear hormone receptor and the primary drug target in prostate cancer due to its role as a lineage survival factor in prostate luminal epithelium. In prostate cancer, the AR cistrome is reprogrammed relative to normal prostate epithelium and particularly in cancers driven by oncogenic ETS fusion genes. The molecular basis for this change has remained elusive. Using purified proteins, we report a minimal cell-free system that demonstrates interdomain cooperativity between the ligand (LBD) and DNA binding domains (DBD) of AR, and its autoinhibition by the N terminus of AR. Furthermore, we identify ERG as a cofactor that activates AR's ability to bind DNA in both high and lower affinity contexts through direct interaction within a newly identified AR-interacting motif (AIM) in the ETS domain, independent of ERG's own DNA binding ability. Finally, we present evidence that this interaction is conserved among ETS factors whose expression is altered in prostate cancer. Our work highlights, at a biochemical level, how tumor-initiating ETS translocations result in reprogramming of the AR cistrome.
    Keywords:  antiandrogen; cistrome; prostate cancer
  58. Genes Dev. 2020 Apr 01. 34(7-8): 465-488
    Schier AC, Taatjes DJ.
      RNA polymerase II (Pol II) transcribes all protein-coding genes and many noncoding RNAs in eukaryotic genomes. Although Pol II is a complex, 12-subunit enzyme, it lacks the ability to initiate transcription and cannot consistently transcribe through long DNA sequences. To execute these essential functions, an array of proteins and protein complexes interact with Pol II to regulate its activity. In this review, we detail the structure and mechanism of over a dozen factors that govern Pol II initiation (e.g., TFIID, TFIIH, and Mediator), pausing, and elongation (e.g., DSIF, NELF, PAF, and P-TEFb). The structural basis for Pol II transcription regulation has advanced rapidly in the past decade, largely due to technological innovations in cryoelectron microscopy. Here, we summarize a wealth of structural and functional data that have enabled a deeper understanding of Pol II transcription mechanisms; we also highlight mechanistic questions that remain unanswered or controversial.
    Keywords:  DSIF; Mediator; NELF; P-TEFb; TBP; TFIID; TFIIH; cryo-EM; pausing; preinitiation complex
  59. Stem Cell Reports. 2020 Mar 17. pii: S2213-6711(20)30095-3. [Epub ahead of print]
    Bernitz JM, Rapp K, Daniel MG, Shcherbinin D, Yuan Y, Gomes A, Waghray A, Brosh R, Lachmann A, Ma'ayan A, Papatsenko D, Moore KA.
      Hematopoietic stem cells (HSCs) exist in a dormant state and progressively lose regenerative potency as they undergo successive divisions. Why this functional decline occurs and how this information is encoded is unclear. To better understand how this information is stored, we performed RNA sequencing on HSC populations differing only in their divisional history. Comparative analysis revealed that genes upregulated with divisions are enriched for lineage genes and regulated by cell-cycle-associated transcription factors, suggesting that proliferation itself drives lineage priming. Downregulated genes are, however, associated with an HSC signature and targeted by the Polycomb Repressive Complex 2 (PRC2). The PRC2 catalytic subunits Ezh1 and Ezh2 promote and suppress the HSC state, respectively, and successive divisions cause a switch from Ezh1 to Ezh2 dominance. We propose that cell divisions drive lineage priming and Ezh2 accumulation, which represses HSC signature genes to consolidate information on divisional history into memory.
    Keywords:  cell heterogeneity; divisional history; epigenetics; hematopoietic stem cells; leukemia; memory; polycomb repressive complex
  60. Nat Rev Genet. 2020 Mar 31.
    Wagner DE, Klein AM.
      A fundamental goal of developmental and stem cell biology is to map the developmental history (ontogeny) of differentiated cell types. Recent advances in high-throughput single-cell sequencing technologies have enabled the construction of comprehensive transcriptional atlases of adult tissues and of developing embryos from measurements of up to millions of individual cells. Parallel advances in sequencing-based lineage-tracing methods now facilitate the mapping of clonal relationships onto these landscapes and enable detailed comparisons between molecular and mitotic histories. Here we review recent progress and challenges, as well as the opportunities that emerge when these two complementary representations of cellular history are synthesized into integrated models of cell differentiation.
  61. Nat Commun. 2020 Apr 03. 11(1): 1648
    Wang Y, Gao M, Zhu F, Li X, Yang Y, Yan Q, Jia L, Xie L, Chen Z.
      Brown adipose tissue (BAT) undergoes rapid postnatal development and then protects against cold and obesity into adulthood. However, the molecular mechanism that determines postnatal development and maturation of BAT is largely unknown. Here we show that METTL3 (a key RNA methyltransferase) expression increases significantly in interscapular brown adipose tissue (iBAT) after birth and plays an essential role in the postnatal development and maturation of iBAT. BAT-specific deletion of Mettl3 severely impairs maturation of BAT in vivo by decreasing m6A modification and expression of Prdm16, Pparg, and Ucp1 transcripts, which leads to a marked reduction in BAT-mediated adaptive thermogenesis and promotes high-fat diet (HFD)-induced obesity and systemic insulin resistance. These data demonstrate that METTL3 is an essential regulator that controls iBAT postnatal development and energy homeostasis.
  62. Cancer Res. 2020 Mar 31. pii: canres.1764.2019. [Epub ahead of print]
    Chen Y, Wang B, Huang M, Wang T, Hu C, Liu Q, Han D, Chen C, Zhang J, Li Z, Liu C, Lei W, Chang Y, Wu M, Xiang D, Chen Y, Wang R, Huang W, Lei Z, Chu X.
      Despite the fact that osteosarcoma is one of the most common primary bone malignancies with poor prognosis, the mechanism behind the pathogenesis of osteosarcoma is only partially known. Here we characterized differentially expressed genes (DEG) by extensive analysis of several publicly available gene expression profile datasets and identified MAFB as a key transcriptional regulator in osteosarcoma progression. MAFB was highly expressed in tumor tissues and required for proliferation and tumorigenicity of osteosarcoma cells. MAFB expression was elevated in osteosarcoma stem cells to maintain their self-renewal potential in vitro and in vivo through upregulation of stem cell regulator Sox9 at the transcriptional level. Sox9 in turn activated MAFB expression via direct recognition of its sequence binding enrichment (SBE) motif on the MAFB locus, thereby forming a positive feedback regulatory loop. Sox9-mediated feedback activation of MAFB was pivotal to tumorsphere-forming and tumor-initiating capacities of osteosarcoma stem cells. Moreover, expression of MAFB and Sox9 was highly correlated in osteosarcoma and associated with disease progression. Combined detection of both MAFB and Sox9 represented a promising prognostic biomarker that stratified a subset of osteosarcoma patients with shortest overall survival. Taken together, these findings reveal a MAFB-Sox9 reciprocal regulatory axis driving cancer stemness and malignancy in osteosarcoma and identify novel molecular targets that might be therapeutically applicable in clinical settings.
  63. Nature. 2020 Apr;580(7801): 93-99
    Li J, Xu C, Lee HJ, Ren S, Zi X, Zhang Z, Wang H, Yu Y, Yang C, Gao X, Hou J, Wang L, Yang B, Yang Q, Ye H, Zhou T, Lu X, Wang Y, Qu M, Yang Q, Zhang W, Shah NM, Pehrsson EC, Wang S, Wang Z, Jiang J, Zhu Y, Chen R, Chen H, Zhu F, Lian B, Li X, Zhang Y, Wang C, Wang Y, Xiao G, Jiang J, Yang Y, Liang C, Hou J, Han C, Chen M, Jiang N, Zhang D, Wu S, Yang J, Wang T, Chen Y, Cai J, Yang W, Xu J, Wang S, Gao X, Wang T, Sun Y.
      Prostate cancer is the second most common cancer in men worldwide1. Over the past decade, large-scale integrative genomics efforts have enhanced our understanding of this disease by characterizing its genetic and epigenetic landscape in thousands of patients2,3. However, most tumours profiled in these studies were obtained from patients from Western populations. Here we produced and analysed whole-genome, whole-transcriptome and DNA methylation data for 208 pairs of tumour tissue samples and matched healthy control tissue from Chinese patients with primary prostate cancer. Systematic comparison with published data from 2,554 prostate tumours revealed that the genomic alteration signatures in Chinese patients were markedly distinct from those of Western cohorts: specifically, 41% of tumours contained mutations in FOXA1 and 18% each had deletions in ZNF292 and CHD1. Alterations of the genome and epigenome were correlated and were predictive of disease phenotype and progression. Coding and noncoding mutations, as well as epimutations, converged on pathways that are important for prostate cancer, providing insights into this devastating disease. These discoveries underscore the importance of including population context in constructing comprehensive genomic maps for disease.
  64. Elife. 2020 Mar 30. pii: e51317. [Epub ahead of print]9
    Zhang SC, Wang MY, Feng JR, Chang Y, Ji SR, Wu Y.
      Acute phase reactants (APRs) are secretory proteins exhibiting large expression changes in response to proinflammatory cytokines. Here we show that the expression pattern of a major APR, i.e. human C-reactive protein (CRP), is casually determined by DNMT3A and TET2-tuned promoter methylation status. CRP features a CpG-poor promoter with its CpG motifs located in binding sites of STAT3, C/EBP-β and NF-κB. These motifs are highly methylated at the resting state, but undergo STAT3- and NF-κB-dependent demethylation upon cytokine stimulation, leading to markedly enhanced recruitment of C/EBP-β that boosts CRP expression. Withdrawal of cytokines, by contrast, results in a rapid recovery of promoter methylation and termination of CRP induction. Further analysis suggests that reversible methylation also regulates the expression of highly inducible genes carrying CpG-poor promoters with APRs as representatives. Therefore, these CpG-poor promoters may evolve CpG-containing TF binding sites to harness dynamic methylation for prompt and reversible responses.
    Keywords:  immunology; inflammation; mouse
  65. Genomics. 2020 Mar 28. pii: S0888-7543(19)30944-9. [Epub ahead of print]
    Seal DB, Das V, Goswami S, De RK.
      Gene expression analysis plays a significant role for providing molecular insights in cancer. Various genetic and epigenetic factors (being dealt under multi-omics) affect gene expression giving rise to cancer phenotypes. A recent growth in understanding of multi-omics seems to provide a resource for integration in interdisciplinary biology since they altogether can draw the comprehensive picture of an organism's developmental and disease biology in cancers. Such large scale multi-omics data can be obtained from public consortium like The Cancer Genome Atlas (TCGA) and several other platforms. Integrating these multi-omics data from varied platforms is still challenging due to high noise and sensitivity of the platforms used. Currently, a robust integrative predictive model to estimate gene expression from these genetic and epigenetic data is lacking. In this study, we have developed a deep learning-based predictive model using Deep Denoising Auto-encoder (DDAE) and Multi-layer Perceptron (MLP) that can quantitatively capture how genetic and epigenetic alterations correlate with directionality of gene expression for liver hepatocellular carcinoma (LIHC). The DDAE used in the study has been trained to extract significant features from the input omics data to estimate the gene expression. These features have then been used for back-propagation learning by the multilayer perceptron for the task of regression and classification. We have benchmarked the proposed model against state-of-the-art regression models. Finally, the deep learning-based integration model has been evaluated for its disease classification capability, where an accuracy of 95.1% has been obtained.
    Keywords:  Copy number variation; DNA methylation; Denoising auto-encoder; Gene expression; Multi-omics integration; Multilayer perceptron; Regression
  66. BMC Genomics. 2020 Apr 02. 21(Suppl 3): 163
    Su SY, Lu IH, Cheng WC, Chung WC, Chen PY, Ho JM, Chen SH, Lin CY.
      BACKGROUND: DNA methylation is a crucial epigenomic mechanism in various biological processes. Using whole-genome bisulfite sequencing (WGBS) technology, methylated cytosine sites can be revealed at the single nucleotide level. However, the WGBS data analysis process is usually complicated and challenging.RESULTS: To alleviate the associated difficulties, we integrated the WGBS data processing steps and downstream analysis into a two-phase approach. First, we set up the required tools in Galaxy and developed workflows to calculate the methylation level from raw WGBS data and generate a methylation status summary, the mtable. This computation environment is wrapped into the Docker container image DocMethyl, which allows users to rapidly deploy an executable environment without tedious software installation and library dependency problems. Next, the mtable files were uploaded to the web server EpiMOLAS_web to link with the gene annotation databases that enable rapid data retrieval and analyses.
    CONCLUSION: To our knowledge, the EpiMOLAS framework, consisting of DocMethyl and EpiMOLAS_web, is the first approach to include containerization technology and a web-based system for WGBS data analysis from raw data processing to downstream analysis. EpiMOLAS will help users cope with their WGBS data and also conduct reproducible analyses of publicly available data, thereby gaining insights into the mechanisms underlying complex biological phenomenon. The Galaxy Docker image DocMethyl is available at EpiMOLAS_web is publicly accessible at
    Keywords:  DNA methylation data analysis; Docker; Galaxy platform; WGBS pipeline
  67. J Exp Clin Cancer Res. 2020 Mar 30. 39(1): 55
    Yu C, Cheng Z, Cui S, Mao X, Li B, Fu Y, Wang H, Jin H, Ye Q, Zhao X, Jiang L, Qin W.
      BACKGROUND: Biological role and clinical significance of circular RNAs (circRNAs) remain largely unknown. Herein, we aimed to investigate biological function, molecular mechanism, and clinical significance of a circular RNA FOXM1 (circFOXM1) in non-small cell lung cancer (NSCLC).METHODS: Expression of circFOXM1 was measured in 48 paired samples of NSCLC by qRT-PCR. Functional roles of circFOXM1 on tumor cells were explored by in vitro and in vivo assays. Transcriptome sequencing was employed to screen the molecules involved in circFOXM1 regulatory network. RNA immunoprecipitation, luciferase analysis, RNA pull-down, and rescue assay were used to investigate potential mechanisms of circFOXM1.
    RESULTS: We found that circFOXM1 was significantly upregulated in NSCLC tissues, and its upregulation was positively correlated with advanced clinical stage and poor prognosis of NSCLC patients. Gain or loss-of-function assay showed that circFOXM1 promoted cell proliferation and cell cycle progression. In vivo assays showed that silencing circFOXM1 inhibited xenograft tumor growth. Mechanically, transcriptome sequencing data indicated that silencing circFOXM1 led to the downregulation of cell cycle-related mRNAs. RNA pull-down and dual-luciferase reporter assay suggested that circFOXM1 could bind to miR-614, and FAM83D was an essential gene involved in the circFOXM1/miR-614 regulatory network.
    CONCLUSIONS: circFOXM1promotes NSCLC progression by interacting with miR-614 and thus inactivating the function of miR-614, which will further release the suppression of FAM83D. circFOXM1/miR-614/FAM83D regulatory network may serve as a potential therapeutic target for NSCLC patients.
    Keywords:  FAM83D; Non-small cell lung cancer; circFOXM1; miR-614
  68. Oncogene. 2020 Mar 31.
    He J, McLaughlin RP, van der Beek L, Canisius S, Wessels L, Smid M, Martens JWM, Foekens JA, Zhang Y, van de Water B.
      The genetically heterogeneous triple-negative breast cancer (TNBC) continues to be an intractable disease, due to lack of effective targeted therapies. Gene amplification is a major event in tumorigenesis. Genes with amplification-dependent expression are being explored as therapeutic targets for cancer treatment. In this study, we have applied Analytical Multi-scale Identification of Recurring Events analysis and transcript quantification in the TNBC genome across 222 TNBC tumors and identified 138 candidate genes with positive correlation in copy number gain (CNG) and gene expression. siRNA-based loss-of-function screen of the candidate genes has validated EGFR, MYC, ASAP1, IRF2BP2, and CCT5 genes as drivers promoting proliferation in different TNBC cells. MYC, ASAP1, IRF2BP2, and CCT5 display frequent CNG and concurrent expression over 2173 breast cancer tumors (cBioPortal dataset). More frequently are MYC and ASAP1 amplified in TNBC tumors (>30%, n = 320). In particular, high expression of ASAP1, the ADP-ribosylation factor GTPase-activating protein, is significantly related to poor metastatic relapse-free survival of TNBC patients (n = 257, bc-GenExMiner). Furthermore, we have revealed that silencing of ASAP1 modulates numerous cytokine and apoptosis signaling components, such as IL1B, TRAF1, AIFM2, and MAP3K11 that are clinically relevant to survival outcomes of TNBC patients. ASAP1 has been reported to promote invasion and metastasis in various cancer cells. Our findings that ASAP1 is an amplification-dependent TNBC driver gene promoting TNBC cell proliferation, functioning upstream apoptosis components, and correlating to clinical outcomes of TNBC patients, support ASAP1 as a potential actionable target for TNBC treatment.
  69. BMC Med Genomics. 2020 Apr 03. 13(Suppl 5): 44
    Mostavi M, Chiu YC, Huang Y, Chen Y.
      BACKGROUND: Precise prediction of cancer types is vital for cancer diagnosis and therapy. Through a predictive model, important cancer marker genes can be inferred. Several studies have attempted to build machine learning models for this task however none has taken into consideration the effects of tissue of origin that can potentially bias the identification of cancer markers.RESULTS: In this paper, we introduced several Convolutional Neural Network (CNN) models that take unstructured gene expression inputs to classify tumor and non-tumor samples into their designated cancer types or as normal. Based on different designs of gene embeddings and convolution schemes, we implemented three CNN models: 1D-CNN, 2D-Vanilla-CNN, and 2D-Hybrid-CNN. The models were trained and tested on gene expression profiles from combined 10,340 samples of 33 cancer types and 713 matched normal tissues of The Cancer Genome Atlas (TCGA). Our models achieved excellent prediction accuracies (93.9-95.0%) among 34 classes (33 cancers and normal). Furthermore, we interpreted one of the models, 1D-CNN model, with a guided saliency technique and identified a total of 2090 cancer markers (108 per class on average). The concordance of differential expression of these markers between the cancer type they represent and others is confirmed. In breast cancer, for instance, our model identified well-known markers, such as GATA3 and ESR1. Finally, we extended the 1D-CNN model for the prediction of breast cancer subtypes and achieved an average accuracy of 88.42% among 5 subtypes. The codes can be found at
    CONCLUSIONS: Here we present novel CNN designs for accurate and simultaneous cancer/normal and cancer types prediction based on gene expression profiles, and unique model interpretation scheme to elucidate biologically relevance of cancer marker genes after eliminating the effects of tissue-of-origin. The proposed model has light hyperparameters to be trained and thus can be easily adapted to facilitate cancer diagnosis in the future.
    Keywords:  Breast cancer subtype prediction; Cancer gene markers; Cancer type prediction; Convolutional neural networks; Deep learning; The Cancer Genome Atlas
  70. Hum Pathol. 2020 Mar 25. pii: S0046-8177(20)30058-7. [Epub ahead of print]
    Villatoro TM, Ma C, Pai RK.
      The switch/sucrose-nonfermenting (SWI/SNF) nucleosome complex consists of several proteins that are involved in cellular proliferation and tumor suppression. The aim of this study was to correlate immunohistochemical expression of four SWI/SNF complex subunits, SMARCA2, SMARCB1, SMARCA4, and ARID1A, with clinicopathologic and molecular features and patient survival in 338 patients with colorectal adenocarcinoma using a tissue microarray approach. Twenty-three (7%) colorectal adenocarcinomas demonstrated deficient SWI/SNF expression: 7 had SMARCA2 deficiency, 12 had ARID1A deficiency, and 4 had both SMARCA2 and ARID1A deficiency. No cases were SMARCB1 or SMARCA4-deficient. Twelve (52%) SWI/SNF complex-deficient tumors demonstrated MMR deficiency (p=0.02), 6 (26%) showed medullary differentiation (p=0.001), and 9 were negative for CDX2 expression (p<0.001). Among the MMR deficient SWI/SNF complex-deficient tumors, 8 were sporadic MLH1 deficient and 4 were seen in Lynch syndrome patients. Compared to ARID1A-deficient alone tumors, SMARCA2-deficient tumors were less likely to exhibit MMR deficiency (27% vs. 75%, p=0.04), medullary differentiation (0% vs. 50%, p=0.01), and mucinous differentiation (0% vs. 42%, p=0.04). Conventional gland-forming histology was more often identified in SMARCA2-deficient tumors (11/11, 100%) compared to tumors with ARID1A deficiency alone (4/12, 33%) (p=0.001). There was no difference in KRAS mutation, BRAF mutation, stage, disease-specific survival, or disease-free survival for patients stratified by SWI/SNF expression (all with p>0.05). In conclusion, SMARCA2-deficient and ARID1A-deficient colorectal carcinomas had distinctly different clinicopathologic features with ARID1A-deficient tumors exhibiting medullary and mucinous differentiation and MMR deficiency, and SMARCA2-deficient tumors demonstrating conventional gland-forming histologic growth with less frequent MMR deficiency.
    Keywords:  ARID1A; BRAF; DNA mismatch repair; KRAS; SMARCA2; SMARCA4; SMARCB1; colorectal carcinoma; immunohistochemistry; microsatellite instability; survival
  71. Comput Struct Biotechnol J. 2020 ;18 558-570
    Xu H, Zhang S, Yi X, Plewczynski D, Li MJ.
      Mechanisms underlying gene regulation are key to understand how multicellular organisms with various cell types develop from the same genetic blueprint. Dynamic interactions between enhancers and genes are revealed to play central roles in controlling gene transcription, but the determinants to link functional enhancer-promoter pairs remain elusive. A major challenge is the lack of reliable approach to detect and verify functional enhancer-promoter interactions (EPIs). In this review, we summarized the current methods for detecting EPIs and described how developing techniques facilitate the identification of EPI through assessing the merits and drawbacks of these methods. We also reviewed recent state-of-art EPI prediction methods in terms of their rationale, data usage and characterization. Furthermore, we briefly discussed the evolved strategies for validating functional EPIs.
    Keywords:  Chromatin Conformation Capture; Chromatin loop; Computational method; Enhancer-promoter interaction; Machine learning; cis-Regulatory element
  72. J Mol Endocrinol. 2020 Mar 01. pii: JME-19-0246.R3. [Epub ahead of print]
    Hanel A, Malmberg HR, Carlberg C.
      The transcription factor vitamin D receptor (VDR) is the exclusive nuclear target of the biologically active form of vitamin D (1,25(OH)2D3). In THP-1 human monocytes we obtained a highly accurate VDR cistrome after 2 and 24 h ligand stimulation comprising more than 11,600 genomic loci, 78% of which were detected exclusively after 24 h. In contrast, a group of 510 persistent VDR sites occurred at all conditions and some 2,100 VDR loci were only transiently occupied. Machine learning and statistical analysis as well as a comparison with the re-analyzed B cell VDR cistrome indicated a subgroup of 339 highly conserved persistent VDR sites that were suited best for describing vitamin D-triggered gene regulatory scenarios. The 1,25(OH)2D3-dependent transcriptome of THP-1 cells comprised 587 genes, 311 of which were primary targets with main functions in the immune system. More than 97% of the latter genes were located within 1,25(OH)2D3-modulated topologically associated domains (TADs). The number of persistent and transient VDR sites was found to be the main discriminator for sorting these TADs into five classes carrying vitamin D target genes involved in distinct biological processes. In conclusion, specific regulation of biological processes by vitamin D depends on differences in time-dependent VDR binding.
  73. Dev Dyn. 2020 Apr 03.
    Takahashi M, Ikeda K, Ohmuraya M, Nakagawa Y, Sakuma T, Yamamoto T, Kawakami K.
      BACKGROUND: The structure of the mouse incisor is characterized by its asymmetric accumulation of enamel matrix proteins on the labial side. The asymmetric structure originates from the patterning of the epithelial incisor placode through the interaction with dental mesenchymal cells. However, the molecular basis for the asymmetric patterning of the incisor germ is largely unknown.RESULTS: A homeobox transcription factor SIX1 was shown to be produced in the mandibular mesenchyme, and its localization patterns changed dynamically during lower incisor development. Six1-/- mice exhibited smaller lower incisor primordia than wild-type mice. Furthermore, Six1-/- mice showed enamel matrix production on both the lingual and labial sides and disturbed odontoblast maturation. In the earlier stages of development, the formation of signaling centers, the initiation knot and the enamel knot, which are essential for the morphogenesis of tooth germs, were impaired in Six1-/- embryos. Notably, Wnt signaling activity, which shows an anterior-posterior gradient, and the expression patterns of genes involved in incisor formation were altered in the mesenchyme in Six1-/- embryos.
    CONCLUSION: Our results indicate that Six1 is required for signaling center formation in lower incisor germs and the labial-lingual asymmetry of the lower incisors by regulating the anterior-posterior patterning of the mandibular mesenchyme. This article is protected by copyright. All rights reserved.
    Keywords:  A-P patterning; Incisor placode; Wnt signaling activity; ameloblasts; dental mesenchyme; odontoblasts
  74. PLoS Genet. 2020 Mar 30. 16(3): e1008667
    Dai JY, Wang X, Wang B, Sun W, Jordahl KM, Kolb S, Nyame YA, Wright JL, Ostrander EA, Feng Z, Stanford JL.
      Genome-wide association studies have identified more than 100 SNPs that increase the risk of prostate cancer (PrCa). We identify and compare expression quantitative trait loci (eQTLs) and CpG methylation quantitative trait loci (meQTLs) among 147 established PrCa risk SNPs in primary prostate tumors (n = 355 from a Seattle-based study and n = 495 from The Cancer Genome Atlas, TCGA) and tumor-adjacent, histologically benign samples (n = 471 from a Mayo Clinic study). The role of DNA methylation in eQTL regulation of gene expression was investigated by data triangulation using several causal inference approaches, including a proposed adaptation of the Causal Inference Test (CIT) for causal direction. Comparing eQTLs between tumors and benign samples, we show that 98 of the 147 risk SNPs were identified as eQTLs in the tumor-adjacent benign samples, and almost all 33 eQTL identified in tumor sets were also eQTLs in the benign samples. Three lines of results support the causal role of DNA methylation. First, nearly 100 of the 147 risk SNPs were identified as meQTLs in one tumor set, and almost all eQTLs in tumors were meQTLs. Second, the loss of eQTLs in tumors relative to benign samples was associated with altered DNA methylation. Third, among risk SNPs identified as both eQTLs and meQTLs, mediation analyses suggest that over two-thirds have evidence of a causal role for DNA methylation, mostly mediating genetic influence on gene expression. In summary, we provide a comprehensive catalog of eQTLs, meQTLs and putative cancer genes for known PrCa risk SNPs. We observe that a substantial portion of germline eQTL regulatory mechanisms are maintained in the tumor development, despite that somatic alterations in tumor genome. Finally, our mediation analyses illuminate the likely intermediary role of CpG methylation in eQTL regulation of gene expression.