bims-crepig Biomed News
on Chromatin regulation and epigenetics in cell fate and cancer
Issue of 2020‒05‒10
thirty-two papers selected by
Connor Rogerson
University of Cambridge, MRC Cancer Unit

  1. J Biol Chem. 2020 May 06. pii: jbc.RA120.013268. [Epub ahead of print]
    Safgren SL, Olson RLO, Vrabel AM, Almada LL, Marks DL, Hernandez-Alvarado N, Gaspar-Maia A, Fernandez-Zapico ME.
      The transcription factor GLI family zinc finger 1 (GLI1) plays a key role in the development and progression of multiple malignancies. To date, regulation of transcriptional activity at target gene promoters is the only molecular event known to underlie the oncogenic function of GLI1. Here, we provide evidence that GLI1 controls chromatin accessibility at distal regulatory regions by modulating the recruitment of SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily A, member 2 (SMARCA2) to these elements. We demonstrate that SMARCA2 endogenously interacts with GLI1 and enhances its transcriptional activity. Mapping experiments indicated that the C-terminal transcriptional activation domain of GLI1 and SMARCA2's central domains, including its ATPase motif, are required for this interaction. Interestingly, similar to SMARCA2, GLI1 overexpression increased chromatin accessibility, as indicated by results of the micrococcal nuclease (MNase) assay. Further, results of assays for transposase-accessible chromatin with sequencing (ATAC-Seq) after GLI1 knockdown supported these findings, revealing that GLI1 regulates chromatin accessibility at several regions distal to gene promoters. Integrated RNA-Seq and ATAC-Seq data analyses identified a subset of differentially expressed genes located in cis to these regulated chromatin sites. Finally, using the GLI1-regulated gene Hedgehog-interacting protein (HHIP) as a model, we demonstrate that GLI1 and SMARCA2 co-occupy a distal chromatin peak and that SMARCA2 recruitment to this HHIP putative enhancer requires intact GLI1. These findings provide insights into how GLI1 controls gene expression in cancer cells and may inform approaches targeting this oncogenic transcription factor to manage malignancies.
    Keywords:  GLI1; SMARC2; cancer; chromatin; chromatin remodeling; gene transcription; transcription enhancer
  2. Cell Rep. 2020 May 05. pii: S2211-1247(20)30550-7. [Epub ahead of print]31(5): 107601
    Toledo EM, Yang S, Gyllborg D, van Wijk KE, Sinha I, Varas-Godoy M, Grigsby CL, Lönnerberg P, Islam S, Steffensen KR, Linnarsson S, Arenas E.
      Liver X receptors (LXRs) and their ligands are potent regulators of midbrain dopaminergic (mDA) neurogenesis and differentiation. However, the molecular mechanisms by which LXRs control these functions remain to be elucidated. Here, we perform a combined transcriptome and chromatin immunoprecipitation sequencing (ChIP-seq) analysis of midbrain cells after LXR activation, followed by bioinformatic analysis to elucidate the transcriptional networks controlling mDA neurogenesis. Our results identify the basic helix-loop-helix transcription factor sterol regulatory element binding protein 1 (SREBP1) as part of a cluster of proneural transcription factors in radial glia and as a regulator of transcription factors controlling mDA neurogenesis, such as Foxa2. Moreover, loss- and gain-of-function experiments in vitro and in vivo demonstrate that Srebf1 is both required and sufficient for mDA neurogenesis. Our data, thus, identify Srebf1 as a central player in mDA neurogenesis.
    Keywords:  FOXA2; LXR; Parkinson disease; bHLH; chromatin immunoprecipitation; development; nuclear receptor; oxysterol; radial glia; single-cell RNA sequencing; transcriptional network
  3. Cell Rep. 2020 May 05. pii: S2211-1247(20)30561-1. [Epub ahead of print]31(5): 107612
    Gill JK, Maffioletti A, García-Molinero V, Stutz F, Soudet J.
      Eukaryotic genomes are almost entirely transcribed by RNA polymerase II. Consequently, the transcription of long noncoding RNAs often overlaps with coding gene promoters, triggering potential gene repression through a poorly characterized mechanism of transcription interference. Here, we propose a comprehensive model of chromatin-based transcription interference in Saccharomyces cerevisiae (S. cerevisiae). By using a noncoding transcription-inducible strain, we analyze the relationship between antisense elongation and coding sense repression, nucleosome occupancy, and transcription-associated histone modifications using near-base pair resolution techniques. We show that antisense noncoding transcription leads to the deacetylation of a subpopulation of -1/+1 nucleosomes associated with increased H3K36me3. Reduced acetylation results in the decreased binding of the RSC chromatin remodeler at -1/+1 nucleosomes and subsequent sliding into the nucleosome-depleted region hindering pre-initiation complex association. Finally, we extend our model by showing that natural antisense noncoding transcription significantly represses ∼20% of S. cerevisiae genes through this chromatin-based transcription interference mechanism.
    Keywords:  RSC; antisense; chromatin remodeler; histone modifications; noncoding transcription; nucleosome-depleted regions; nucleosomes; yeast
  4. Elife. 2020 May 05. pii: e47980. [Epub ahead of print]9
    Tsujimura T, Takase O, Yoshikawa M, Sano E, Hayashi M, Hoshi K, Takato T, Toyoda A, Okano H, Hishikawa K.
      While regulation of gene-enhancer interaction is intensively studied, its application remains limited. Here, we reconstituted arrays of CTCF-binding sites and devised a synthetic topological insulator with tetO for chromatin-engineering (STITCH). By coupling STITCH with tetR linked to the KRAB domain to induce heterochromatin and disable the insulation, we developed a drug-inducible system to control gene activation by enhancers. In human induced pluripotent stem cells, STITCH inserted between MYC and the enhancer down-regulated MYC. Progressive mutagenesis of STITCH led to a preferential escalation of the gene-enhancer interaction, corroborating the strong insulation ability of STITCH. STITCH also altered epigenetic states around MYC. Time-course analysis by drug induction uncovered deposition and removal of H3K27me3 repressive marks follows and reflects, but does not precede and determine, the expression change. Finally, STITCH inserted near NEUROG2 impaired the gene activation in differentiating neural progenitor cells. Thus, STITCH should be broadly useful for functional genetic studies.
    Keywords:  CTCF; MYC; chromatin conformation; chromosomes; enhancer; epigenetic manipulation; gene expression; genetics; genomics; human; mouse
  5. Nat Commun. 2020 May 08. 11(1): 2271
    Tam J, van Werven FJ.
      Intrinsic signals and external cues from the environment drive cell fate decisions. In budding yeast, the decision to enter meiosis is controlled by nutrient and mating-type signals that regulate expression of the master transcription factor for meiotic entry, IME1. How nutrient signals control IME1 expression remains poorly understood. Here, we show that IME1 transcription is regulated by multiple sequence-specific transcription factors (TFs) that mediate association of Tup1-Cyc8 co-repressor to its promoter. We find that at least eight TFs bind the IME1 promoter when nutrients are ample. Remarkably, association of these TFs is highly regulated by different nutrient cues. Mutant cells lacking three TFs (Sok2/Phd1/Yap6) displayed reduced Tup1-Cyc8 association, increased IME1 expression, and earlier onset of meiosis. Our data demonstrate that the promoter of a master regulator is primed for rapid activation while repression by multiple TFs mediating Tup1-Cyc8 recruitment dictates the fate decision to enter meiosis.
  6. Biochim Biophys Acta Gene Regul Mech. 2020 Apr 28. pii: S1874-9399(19)30464-X. [Epub ahead of print] 194567
    Hughes AL, Kelley JR, Klose RJ.
      The precise regulation of gene transcription is required to establish and maintain cell type-specific gene expression programs during multicellular development. In addition to transcription factors, chromatin, and its chemical modification, play a central role in regulating gene expression. In vertebrates, DNA is pervasively methylated at CG dinucleotides, a modification that is repressive to transcription. However, approximately 70% of vertebrate gene promoters are associated with DNA elements called CpG islands (CGIs) that are refractory to DNA methylation. CGIs integrate the activity of a range of chromatin-regulating factors that can post-translationally modify histones and modulate gene expression. This is exemplified by the trimethylation of histone H3 at lysine 4 (H3K4me3), which is enriched at CGI-associated gene promoters and correlates with transcriptional activity. Through studying H3K4me3 at CGIs it has become clear that CGIs shape the distribution of H3K4me3 and, in turn, H3K4me3 influences the chromatin landscape at CGIs. Here we will discuss our understanding of the emerging relationship between CGIs, H3K4me3, and gene expression.
    Keywords:  Chromatin; CpG islands; DNA methylation; H3K4me3; Transcription
  7. J Clin Invest. 2020 May 04. pii: 126726. [Epub ahead of print]
    Xing M, Ooi WF, Tan J, Qamra A, Lee PH, Li Z, Xu C, Padmanabhan N, Lim JQ, Guo YA, Yao X, Amit M, Ng LM, Sheng T, Wang J, Huang KK, Anene-Nzelu CG, Ho SWT, Ray M, Ma L, Fazzi G, Lim KJ, Wijaya GC, Zhang S, Nandi T, Yan T, Chang MM, Das K, Isa ZFA, Wu J, Poon PSY, Lam YN, Lin JS, Tay ST, Lee MH, Tan ALK, Ong X, White K, Rozen SG, Beer M, Foo RSY, Grabsch HI, Skanderup AJ, Li S, Teh BT, Tan P.
      Transcriptional reactivation of telomerase catalytic subunit (TERT) is a frequent hallmark of cancer, occurring in 90% of human malignancies. However, specific mechanisms driving TERT reactivation remain obscure for many tumor types and in particular gastric cancer (GC), a leading cause of global cancer mortality. Here, through comprehensive genomic and epigenomic analysis of primary GCs and GC cell lines, we identified the transcription factor early B cell factor 1 (EBF1) as a TERT transcriptional repressor and inactivation of EBF1 function as a major cause of TERT upregulation. Abolishment of EBF1 function occurs through 3 distinct (epi)genomic mechanisms. First, EBF1 is epigenetically silenced via DNA methyltransferase, polycomb-repressive complex 2 (PRC2), and histone deacetylase activity in GCs. Second, recurrent, somatic, and heterozygous EBF1 DNA-binding domain mutations result in the production of dominant-negative EBF1 isoforms. Third, more rarely, genomic deletions and rearrangements proximal to the TERT promoter remobilize or abolish EBF1-binding sites, derepressing TERT and leading to high TERT expression. EBF1 is also functionally required for various malignant phenotypes in vitro and in vivo, highlighting its importance for GC development. These results indicate that multimodal genomic and epigenomic alterations underpin TERT reactivation in GC, converging on transcriptional repressors such as EBF1.
    Keywords:  Gastric cancer; Gastroenterology; Molecular genetics; Oncology
  8. Nat Commun. 2020 May 07. 11(1): 2243
    Gaub A, Sheikh BN, Basilicata MF, Vincent M, Nizon M, Colson C, Bird MJ, Bradner JE, Thevenon J, Boutros M, Akhtar A.
      Cells rely on a diverse repertoire of genes for maintaining homeostasis, but the transcriptional networks underlying their expression remain poorly understood. The MOF acetyltransferase-containing Non-Specific Lethal (NSL) complex is a broad transcription regulator. It is essential in Drosophila, and haploinsufficiency of the human KANSL1 subunit results in the Koolen-de Vries syndrome. Here, we perform a genome-wide RNAi screen and identify the BET protein BRD4 as an evolutionary conserved co-factor of the NSL complex. Using Drosophila and mouse embryonic stem cells, we characterise a recruitment hierarchy, where NSL-deposited histone acetylation enables BRD4 recruitment for transcription of constitutively active genes. Transcriptome analyses in Koolen-de Vries patient-derived fibroblasts reveals perturbations with a cellular homeostasis signature that are evoked by the NSL complex/BRD4 axis. We propose that BRD4 represents a conserved bridge between the NSL complex and transcription activation, and provide a new perspective in the understanding of their functions in healthy and diseased states.
  9. Cell Death Discov. 2020 ;6 30
    He F, Wu H, Zhou L, Lin Q, Cheng Y, Sun YE.
      DNA methylation and demethylation at CpG di-nucleotide sites plays important roles in cell fate specification of neural stem cells (NSCs). We have previously reported that DNA methyltransferases, Dnmt1and Dnmt3a, serve to suppress precocious astrocyte differentiation from NSCs via methylation of astroglial lineage genes. However, whether active DNA demethylase also participates in astrogliogenesis remains undetermined. In this study, we discovered that a Ten-eleven translocation (Tet) protein, Tet2, which was critically involved in active DNA demethylation through oxidation of 5-Methylcytosine (5mC), drove astrocyte differentiation from NSCs by demethylation of astroglial lineage genes including Gfap. Moreover, we found that an NSC-specific bHLH transcription factor Olig2 was an upstream inhibitor for Tet2 expression through direct association with the Tet2 promoter, and indirectly inhibited astrocyte differentiation. Our research not only revealed a brand-new function of Tet2 to promote NSC differentiation into astrocytes, but also a novel mechanism for Olig2 to inhibit astrocyte formation.
    Keywords:  Cellular neuroscience; Neural stem cells
  10. Nat Commun. 2020 May 06. 11(1): 2219
    Ren W, Medeiros N, Warneford-Thomson R, Wulfridge P, Yan Q, Bian J, Sidoli S, Garcia BA, Skordalakes E, Joyce E, Bonasio R, Sarma K.
      Heterochromatin in the eukaryotic genome is rigorously controlled by the concerted action of protein factors and RNAs. Here, we investigate the RNA binding function of ATRX, a chromatin remodeler with roles in silencing of repetitive regions of the genome and in recruitment of the polycomb repressive complex 2 (PRC2). We identify ATRX RNA binding regions (RBRs) and discover that the major ATRX RBR lies within the N-terminal region of the protein, distinct from its PHD and helicase domains. Deletion of this ATRX RBR (ATRXΔRBR) compromises ATRX interactions with RNAs in vitro and in vivo and alters its chromatin binding properties. Genome-wide studies reveal that loss of RNA interactions results in a redistribution of ATRX on chromatin. Finally, our studies identify a role for ATRX-RNA interactions in regulating PRC2 localization to a subset of polycomb target genes.
  11. Oncogene. 2020 May 04.
    Zhang X, Zegar T, Lucas A, Morrison-Smith C, Knox T, French CA, Knapp S, Müller S, Siveke JT.
      Nuclear protein of the testis (NUT) midline carcinoma (NMC), is a rare and highly aggressive form of undifferentiated squamous cell carcinoma. NMC is molecularly characterized by chromosomal rearrangement of the NUT gene to another gene, most commonly the bromodomain and extraterminal domain (BET) gene BRD4, forming the BRD4-NUT fusion oncogene. Therefore, inhibiting BRD4-NUT oncogenic function directly by BET inhibitors represents an attractive therapeutic approach but toxicity may limit the use of pan-BET inhibitors treating this cancer. We thus performed a drug screening approach using a library consisting of epigenetic compounds and 'Donated Chemical Probes' collated by the Structural Genomics Consortium (SGC) and identified the p300/CBP HAT inhibitor A-485, in addition to the well-known BET inhibitor JQ1, to be the most active candidate for NMC treatment. In contrast to JQ1, A-485 was selectively potent in NMC compared to other cell lines tested. Mechanistically, A-485 inhibited p300-mediated histone acetylation, leading to disruption of BRD4-NUT binding to hyperacetylated megadomains. Consistently, BRD4-NUT megadomain-associated genes MYC, CCAT1 and TP63 were downregulated by A-485. A-485 strongly induced squamous differentiation, cell cycle arrest and apoptosis. Combined inhibition of p300/CBP and BET showed synergistic effects. In summary, we identified the p300/CBP HAT domain as a putative therapeutic target in highly therapy-resistant NMC.
  12. Dev Cell. 2020 Apr 26. pii: S1534-5807(20)30270-7. [Epub ahead of print]
    Hu R, Walker E, Huang C, Xu Y, Weng C, Erickson GE, Coldren A, Yang X, Brissova M, Kaverina I, Appakalai BN, Wright CVE, Li Y, Stein R, Gu G.
      Although cellular stress response is important for maintaining function and survival, overactivation of late-stage stress effectors cause dysfunction and death. We show that the myelin transcription factors (TFs) Myt1 (Nzf2), Myt2 (Myt1l, Nztf1, and Png-1), and Myt3 (St18 and Nzf3) prevent such overactivation in islet β cells. Thus, we found that co-inactivating the Myt TFs in mouse pancreatic progenitors compromised postnatal β cell function, proliferation, and survival, preceded by upregulation of late-stage stress-response genes activating transcription factors (e.g., Atf4) and heat-shock proteins (Hsps). Myt1 binds putative enhancers of Atf4 and Hsps, whose overexpression largely recapitulated the Myt-mutant phenotypes. Moreover, Myt(MYT)-TF levels were upregulated in mouse and human β cells during metabolic stress-induced compensation but downregulated in dysfunctional type 2 diabetic (T2D) human β cells. Lastly, MYT knockdown caused stress-gene overactivation and death in human EndoC-βH1 cells. These findings suggest that Myt TFs are essential restrictors of stress-response overactivity.
    Keywords:  ATF4; Myelin transcription factors; beta cell compensation; beta cell death; beta cell failure; diabetes; heat-shock proteins; post-transcriptional control; proliferation; stress response
  13. Nat Commun. 2020 May 08. 11(1): 2316
    Treviño LS, Dong J, Kaushal A, Katz TA, Jangid RK, Robertson MJ, Grimm SL, Ambati CSR, Putluri V, Cox AR, Kim KH, May TD, Gallo MR, Moore DD, Hartig SM, Foulds CE, Putluri N, Coarfa C, Walker CL.
      Our early-life environment has a profound influence on developing organs that impacts metabolic function and determines disease susceptibility across the life-course. Using a rat model for exposure to an endocrine disrupting chemical (EDC), we show that early-life chemical exposure causes metabolic dysfunction in adulthood and reprograms histone marks in the developing liver to accelerate acquisition of an adult epigenomic signature. This epigenomic reprogramming persists long after the initial exposure, but many reprogrammed genes remain transcriptionally silent with their impact on metabolism not revealed until a later life exposure to a Western-style diet. Diet-dependent metabolic disruption was largely driven by reprogramming of the Early Growth Response 1 (EGR1) transcriptome and production of metabolites in pathways linked to cholesterol, lipid and one-carbon metabolism. These findings demonstrate the importance of epigenome:environment interactions, which early in life accelerate epigenomic aging, and later in adulthood unlock metabolically restricted epigenetic reprogramming to drive metabolic dysfunction.
  14. Exp Cell Res. 2020 May 03. pii: S0014-4827(20)30266-4. [Epub ahead of print] 112039
    Aspuria PJ, Cheon DJ, Gozo MC, Beach JA, Recouvreux MS, Walts AE, Karlan BY, Orsulic S.
      Expression of the homeodomain transcription factor HOXB13 has been demonstrated in several malignancies but its role in tumorigenesis remains elusive. We observed high levels of HOXB13 in poorly differentiated pediatric tumors including a highly aggressive childhood neoplasm - malignant rhabdoid tumor. In a xenograft model of rhabdoid tumor, knockout of HOXB13 diminished tumor growth while partial knockdown of HOXB13 promoted differentiation of tumor cells into bone. These results suggest that HOXB13 enhances rhabdoid malignancy by interfering with mesenchymal stem cell differentiation. Consistent with this hypothesis, overexpression of HOXB13 in mesenchymal progenitor cells inhibited adipogenic, myogenic, and osteogenic differentiation. Mechanistically, we demonstrated that HOXB13 binds to super-enhancer regions regulating genes involved in differentiation and tumorigenesis.
    Keywords:  Adipogenesis; Differentiation; HOXB13; Homeobox; Mesenchymal stem cells; Myogenesis; Osteogenesis; Rhabdoid tumor; Sarcoma; Super-enhancer
  15. Mol Cancer Res. 2020 May 04. pii: molcanres.0024.2020. [Epub ahead of print]
    Harachi M, Masui K, Honda H, Muragaki Y, Kawamata T, Cavenee WK, Mischel PS, Shibata N.
      Epigenetic regulation known for DNA methylation and histone modification is critical for securing proper gene expression and chromosomal function, and its aberration induces various pathological conditions including cancer. Trimethylation of histone H3 on lysine 27 (H3K27me3) is known to suppress various genes related to cancer cell survival and the level of H3K27me3 may have an influence on tumor progression and malignancy. However, it remains unclear how histone methylation is regulated in response to genetic mutation and microenvironmental cues to facilitate the cancer cell survival. Here, we report a novel mechanism of the specific regulation of H3K27me3 by cooperatively two mechanistic target of rapamycin (mTOR) complexes, mTORC1 and mTORC2 in human glioblastoma (GBM). Integrated analyses revealed that mTORC1 upregulates the protein expression of enhancer of zeste homolog 2 (EZH2), a main component of polycomb repressive complex 2 (PRC2) which is known as H3K27-specific methyltransferase. The other mTOR complex, mTORC2, regulates production of S-adenosylmethionine (SAM), an essential substrate for histone methylation. This cooperative regulation causes H3K27 hypermethylation which subsequently promotes tumor cell survival both in vitro and in vivo xenografted mouse tumor model. These results indicate that activated mTORC1 and mTORC2 complexes cooperatively contribute to tumor progression through specific epigenetic regulation, nominating them as an exploitable therapeutic target against cancer. Implications: A dynamic regulation of histone methylation by mTOR complexes promotes tumor growth in human GBM, but at the same time could be exploitable as a novel therapeutic target against this deadly tumor.
  16. Nat Commun. 2020 May 08. 11(1): 2306
    Fischer K, Fenzl A, Liu D, Dyar KA, Kleinert M, Brielmeier M, Clemmensen C, Fedl A, Finan B, Gessner A, Jastroch M, Huang J, Keipert S, Klingenspor M, Brüning JC, Kneilling M, Maier FC, Othman AE, Pichler BJ, Pramme-Steinwachs I, Sachs S, Scheideler A, Thaiss WM, Uhlenhaut H, Ussar S, Woods SC, Zorn J, Stemmer K, Collins S, Diaz-Meco M, Moscat J, Tschöp MH, Müller TD.
      During β-adrenergic stimulation of brown adipose tissue (BAT), p38 phosphorylates the activating transcription factor 2 (ATF2) which then translocates to the nucleus to activate the expression of Ucp1 and Pgc-1α. The mechanisms underlying ATF2 target activation are unknown. Here we demonstrate that p62 (Sqstm1) binds to ATF2 to orchestrate activation of the Ucp1 enhancer and Pgc-1α promoter. P62Δ69-251 mice show reduced expression of Ucp1 and Pgc-1α with impaired ATF2 genomic binding. Modulation of Ucp1 and Pgc-1α expression through p62 regulation of ATF2 signaling is demonstrated in vitro and in vivo in p62Δ69-251 mice, global p62-/- and Ucp1-Cre p62flx/flx mice. BAT dysfunction resulting from p62 deficiency is manifest after birth and obesity subsequently develops despite normal food intake, intestinal nutrient absorption and locomotor activity. In summary, our data identify p62 as a master regulator of BAT function in that it controls the Ucp1 pathway through regulation of ATF2 genomic binding.
  17. Nucleic Acids Res. 2020 May 08. pii: gkaa357. [Epub ahead of print]
    Williams JD, Houserova D, Johnson BR, Dyniewski B, Berroyer A, French H, Barchie AA, Bilbrey DD, Demeis JD, Ghee KR, Hughes AG, Kreitz NW, McInnis CH, Pudner SC, Reeves MN, Stahly AN, Turcu A, Watters BC, Daly GT, Langley RJ, Gillespie MN, Prakash A, Larson ED, Kasukurthi MV, Huang J, Jinks-Robertson S, Borchert GM.
      Mammalian antibody switch regions (∼1500 bp) are composed of a series of closely neighboring G4-capable sequences. Whereas numerous structural and genome-wide analyses of roles for minimal G4s in transcriptional regulation have been reported, Long G4-capable regions (LG4s)-like those at antibody switch regions-remain virtually unexplored. Using a novel computational approach we have identified 301 LG4s in the human genome and find LG4s prone to mutation and significantly associated with chromosomal rearrangements in malignancy. Strikingly, 217 LG4s overlap annotated enhancers, and we find the promoters regulated by these enhancers markedly enriched in G4-capable sequences suggesting G4s facilitate promoter-enhancer interactions. Finally, and much to our surprise, we also find single-stranded loops of minimal G4s within individual LG4 loci are frequently highly complementary to one another with 178 LG4 loci averaging >35 internal loop:loop complements of >8 bp. As such, we hypothesized (then experimentally confirmed) that G4 loops within individual LG4 loci directly basepair with one another (similar to characterized stem-loop kissing interactions) forming a hitherto undescribed, higher-order, G4-based secondary structure we term a 'G4 Kiss or G4K'. In conclusion, LG4s adopt novel, higher-order, composite G4 structures directly contributing to the inherent instability, regulatory capacity, and maintenance of these conspicuous genomic regions.
  18. Proc Natl Acad Sci U S A. 2020 May 08. pii: 201921484. [Epub ahead of print]
    Somerville TDD, Xu Y, Wu XS, Maia-Silva D, Hur SK, de Almeida LMN, Preall JB, Koo PK, Vakoc CR.
      Lineage plasticity is a prominent feature of pancreatic ductal adenocarcinoma (PDA) cells, which can occur via deregulation of lineage-specifying transcription factors. Here, we show that the zinc finger protein ZBED2 is aberrantly expressed in PDA and alters tumor cell identity in this disease. Unexpectedly, our epigenomic experiments reveal that ZBED2 is a sequence-specific transcriptional repressor of IFN-stimulated genes, which occurs through antagonism of IFN regulatory factor 1 (IRF1)-mediated transcriptional activation at cooccupied promoter elements. Consequently, ZBED2 attenuates the transcriptional output and growth arrest phenotypes downstream of IFN signaling in multiple PDA cell line models. We also found that ZBED2 is preferentially expressed in the squamous molecular subtype of human PDA, in association with inferior patient survival outcomes. Consistent with this observation, we show that ZBED2 can repress the pancreatic progenitor transcriptional program, enhance motility, and promote invasion in PDA cells. Collectively, our findings suggest that high ZBED2 expression is acquired during PDA progression to suppress the IFN response pathway and to promote lineage plasticity in this disease.
    Keywords:  IRF1; ZBED2; interferon; lineage plasticity; pancreatic ductal adenocarcinoma
  19. Nucleic Acids Res. 2020 May 04. pii: gkaa317. [Epub ahead of print]
    Kwon M, Park K, Hyun K, Lee JH, Zhou L, Cho YW, Ge K, Skalnik DG, Muir TW, Kim J.
      In mammalian cells, distinct H3K4 methylation states are created by deposition of methyl groups by multiple complexes of histone lysine methyltransferase 2 (KMT2) family proteins. For comprehensive analyses that directly compare the catalytic properties of all six human KMT2 complexes, we employed a biochemically defined system reconstituted with recombinant KMT2 core complexes (KMT2CoreCs) containing minimal components required for nucleosomal H3K4 methylation activity. We found that each KMT2CoreC generates distinct states and different levels of H3K4 methylation, and except for MLL3 all are stimulated by H2Bub. Notably, SET1BCoreC exhibited the strongest H3K4 methylation activity and, to our surprise, did not require H2B ubiquitylation (H2Bub); in contrast, H2Bub was required for the H3K4me2/3 activity of the paralog SET1ACoreC. We also found that WDR5, RbBP5, ASH2L and DPY30 are required for efficient H3K4 methyltransferase activities of all KMT2CoreCs except MLL3, which could produce H3K4me1 in the absence of WDR5. Importantly, deletion of the PHD2 domain of CFP1 led to complete loss of the H3K4me2/3 activities of SET1A/BCoreCs in the presence of H2Bub, indicating a critical role for this domain in the H2Bub-stimulated H3K4 methylation. Collectively, our results suggest that each KMT2 complex methylates H3K4 through distinct mechanisms in which individual subunits differentially participate.
  20. Cancer Res. 2020 May 04. pii: canres.0855.2019. [Epub ahead of print]
    Almairac F, Turchi L, Sakakini N, Debruyne DN, Elkeurti S, Gjernes E, Polo B, Bianchini L, Fontaine D, Paquis P, Chneiweiss H, Junier MP, Verrando P, Burel-Vandenbos F, Virolle T.
      There is great interest in understanding how the cancer stem cell population may be maintained in solid tumors. Here we show that tumor cells exhibiting stem-like properties and expression of pluripotency markers NANOG and OCT4 can arise from original differentiated tumor cells freshly isolated from human glioblastomas and which have never known any serum culture conditions. Induction of EGR1 by EGFR/ERK signaling promoted cell conversion from a less aggressive, more differentiated cellular state to a self-renewing and strongly tumorigenic state, expressing NANOG and OCT4. Expression of these pluripotency markers occurred before the cells re-entered the cell cycle, demonstrating their capacity to change and dedifferentiate without any cell divisions. In differentiated GBM cells, ERK-mediated repression of miR-199a-3p induced EGR1 protein expression and triggered dedifferentiation. Overall, this signaling pathway constitutes an ERK-mediated "toggle switch" that promotes pluripotency marker expression and stem-like features in GBM cells.
  21. Biochem Soc Trans. 2020 May 05. pii: BST20191106. [Epub ahead of print]
    Batie M, Rocha S.
      Oxygen sensing is an essential feature of metazoan biology and reductions in oxygen availability (hypoxia) have both physiological and pathophysiological implications. Co-ordinated mechanisms have evolved for sensing and responding to hypoxia, which involve diverse biological outputs, with the main aim of restoring oxygen homeostasis. This includes a dynamic gene transcriptional response, the central drivers of which are the hypoxia-inducible factor (HIF) family of transcription factors. HIFs are regulated in an oxygen-dependent manner and while their role in hypoxia is well established, it is apparent that other key players are required for gene expression control in hypoxia. In this review, we highlight the current understanding of the known and potential molecular mechanisms underpinning gene transcriptional responses to hypoxia in mammals, with a focus on oxygen-dependent effects on chromatin structure.
    Keywords:  chromatin; hypoxia; hypoxia-inducible factors; transcription
  22. Nat Commun. 2020 May 08. 11(1): 2294
    Anteneh H, Fang J, Song J.
      DNA methyltransferase DNMT3A is essential for establishment of mammalian DNA methylation during development. The R882H DNMT3A is a hotspot mutation in acute myeloid leukemia (AML) causing aberrant DNA methylation. However, how this mutation affects the structure and function of DNMT3A remains unclear. Here we report structural characterization of wild-type and R882H-mutated DNMT3A in complex with DNA substrates with different sequence contexts. A loop from the target recognition domain (TRD loop) recognizes the CpG dinucleotides in a +1 flanking site-dependent manner. The R882H mutation reduces the DNA binding at the homodimeric interface, as well as the molecular link between the homodimeric interface and TRD loop, leading to enhanced dynamics of TRD loop. Consistently, in vitro methylation analyses indicate that the R882H mutation compromises the enzymatic activity, CpG specificity and flanking sequence preference of DNMT3A. Together, this study uncovers multiple defects of DNMT3A caused by the R882H mutation in AML.
  23. Nucleic Acids Res. 2020 May 07. pii: gkaa306. [Epub ahead of print]
    Gerstner N, Kehl T, Lenhof K, Müller A, Mayer C, Eckhart L, Grammes NL, Diener C, Hart M, Hahn O, Walter J, Wyss-Coray T, Meese E, Keller A, Lenhof HP.
      We present GeneTrail 3, a major extension of our web service GeneTrail that offers rich functionality for the identification, analysis, and visualization of deregulated biological processes. Our web service provides a comprehensive collection of biological processes and signaling pathways for 12 model organisms that can be analyzed with a powerful framework for enrichment and network analysis of transcriptomic, miRNomic, proteomic, and genomic data sets. Moreover, GeneTrail offers novel workflows for the analysis of epigenetic marks, time series experiments, and single cell data. We demonstrate the capabilities of our web service in two case-studies, which highlight that GeneTrail is well equipped for uncovering complex molecular mechanisms. GeneTrail is freely accessible at:
  24. Sci Rep. 2020 May 04. 10(1): 7429
    Zhang JY, Xia Y, Hao YH, Tan Z.
      Bioinformatic analysis reveals an enrichment of putative DNA:RNA hybrid G-quadruplex-forming sequences (PHQS) on both sides of the transcription start sites (TSSs) in the genome of warm-blooded animals, suggesting a positive selection of PHQSs in evolution and functional role of DNA:RNA hybrid G-quadruplexes (HQs) in transcription. The formation of HQs downstream of TSS in transcribed DNA has been documented under in vitro conditions; however, it is still not known if such HQs can form at the upstream side of TSSs. In this study, we report that such HQs can form in transcription in DNA with two to three guanine tracts if RNA carrying the required number of G-tracts is supplied. We also show that the formation of such HQs is dependent on the negative supercoiling generated by RNA polymerases. These results suggest that HQs may also form at the upstream side of TSSs in vivo and play a role in transcription since the two requirements are satisfied in cells.
  25. Cell Death Dis. 2020 May 07. 11(5): 323
    Chen Z, Li S, Shen L, Wei X, Zhu H, Wang X, Yang M, Zheng X.
      In various malignant tumors, NF-kappa B interacting long noncoding RNA (NKILA) displays antitumor activity by inhibiting the NF-kappa B pathway. However, the role of NKILA in gliomas remains unclear. Surprisingly, this study showed that NKILA is significantly upregulated in gliomas, and the increased levels of NKILA were correlated with a decrease in patient survival time. NKILA increased the expression level of hypoxia-inducible factor-1α, and the activity of the hypoxia pathway in gliomas. Furthermore, we demonstrated that NKILA enhances the Warburg effect and angiogenesis in gliomas both in vitro and in vivo. Therefore, NKILA is a potential therapeutic target in gliomas. In addition, we showed that a 20(S)-Rg3 monomer suppresses NKILA accumulation and reverses its stimulation of the Warburg effect and angiogenesis in gliomas, both in vitro and in vivo. Therefore, this study not only identified NKILA as a potential therapeutic target in gliomas, but also demonstrated a practical approach to treatment.
  26. Comput Struct Biotechnol J. 2020 ;18 993-999
    Damiani C, Rovida L, Maspero D, Sala I, Rosato L, Di Filippo M, Pescini D, Graudenzi A, Antoniotti M, Mauri G.
      We present MaREA4Galaxy, a user-friendly tool that allows a user to characterize and to graphically compare groups of samples with different transcriptional regulation of metabolism, as estimated from cross-sectional RNA-seq data. The tool is available as plug-in for the widely-used Galaxy platform for comparative genomics and bioinformatics analyses. MaREA4Galaxy combines three modules. The Expression2RAS module, which, for each reaction of a specified set, computes a Reaction Activity Score (RAS) as a function of the expression level of genes encoding for the associated enzyme. The MaREA (Metabolic Reaction Enrichment Analysis) module that allows to highlight significant differences in reaction activities between specified groups of samples. The Clustering module which employs the RAS computed before as a metric for unsupervised clustering of samples into distinct metabolic subgroups; the Clustering tool provides different clustering techniques and implements standard methods to evaluate the goodness of the results.
    Keywords:  Galaxy; Metabolism; RNA-seq; Sample stratification; TCGA
  27. Cancer Res. 2020 May 06. pii: canres.1523.2019. [Epub ahead of print]
    Huang W, Navarro-Serer B, Jeong YJ, Chianchiano P, Xia L, Luchini C, Veronese N, Dowiak C, Ng T, Trujillo MA, Huang B, Pflüger MJ, Macgregor-Das AM, Lionheart G, Jones D, Fujikura K, Nguyen-Ngoc KV, Neumann NM, Groot VP, Hasanain A, van Oosten AF, Fischer SE, Gallinger S, Singhi AD, Zureikat AH, Brand RE, Gaida MM, Heinrich S, Burkhart RA, He J, Wolfgang CL, Goggins MG, Thompson ED, Roberts NJ, Ewald AJ, Wood LD.
      Pancreatic ductal adenocarcinoma (PDAC) is an aggressive malignancy characterized by extensive local invasion and systemic spread. In this study, we employed a three-dimensional organoid model of human pancreatic cancer to characterize the molecular alterations critical for invasion. Time lapse microscopy was used to observe invasion in organoids from 25 surgically resected human PDAC samples in collagen I. Subsequent lentiviral modification and small molecule inhibitors were used to investigate the molecular programs underlying invasion in PDAC organoids. When cultured in collagen I, PDAC organoids exhibited two distinct, morphologically defined invasive phenotypes, mesenchymal and collective. Each individual PDAC gave rise to organoids with a predominant phenotype, and PDAC that generated organoids with predominantly mesenchymal invasion showed a worse prognosis. Collective invasion predominated in organoids from cancers with somatic mutations in the driver gene SMAD4 (or its signaling partner TGFBR2). Re-expression of SMAD4 abrogated the collective invasion phenotype in SMAD4-mutant PDAC organoids, indicating that SMAD4 loss is required for collective invasion in PDAC organoids. Surprisingly, invasion in passaged SMAD4-mutant PDAC organoids required exogenous TGFβ, suggesting that invasion in SMAD4-mutant organoids is mediated through non-canonical TGFβ signaling. The Rho-like GTPases RAC1 and CDC42 acted as potential mediators of TGFβ-stimulated invasion in SMAD4-mutant PDAC organoids, as inhibition of these GTPases suppressed collective invasion in our model. These data suggest that PDAC utilizes different invasion programs depending on SMAD4 status, with collective invasion uniquely present in PDAC with SMAD4 loss.
  28. Biomolecules. 2020 May 05. pii: E719. [Epub ahead of print]10(5):
    Ishak M, Baharudin R, Rose IM, Sagap I, Mazlan L, Azman ZAM, Abu N, Jamal R, Lee LH, Mutalib NSA.
      The methylome of open chromatins was investigated in colorectal cancer (CRC) to explore cancer-specific methylation and potential biomarkers. Epigenome-wide methylome of open chromatins was studied in colorectal cancer tissues using the Infinium DNA MethylationEPIC assay. Differentially methylated regions were identified using the ChAMP Bioconductor. Our stringent analysis led to the discovery of 2187 significant differentially methylated open chromatins in CRCs. More hypomethylated probes were observed and the trend was similar across all chromosomes. The majority of hyper- and hypomethylated probes in open chromatin were in chromosome 1. Our unsupervised hierarchical clustering analysis showed that 40 significant differentially methylated open chromatins were able to segregate CRC from normal colonic tissues. Receiver operating characteristic analyses from the top 40 probes revealed several significant, highly discriminative, specific and sensitive probes such as OPLAH cg26256223, EYA4 cg01328892, and CCNA1 cg11513637, among others. OPLAH cg26256223 hypermethylation is associated with reduced gene expression in the CRC. This study reports many open chromatin loci with novel differential methylation statuses, some of which with the potential as candidate markers for diagnostic purposes.
    Keywords:  DNA methylation; Infinium MethylationEPIC; colorectal cancer; epigenetics aberrations; open chromatin
  29. Sci Rep. 2020 May 07. 10(1): 7733
    Liu S, Shah Z, Sav A, Russo C, Berkovsky S, Qian Y, Coiera E, Di Ieva A.
      Mutations in isocitrate dehydrogenase genes IDH1 and IDH2 are frequently found in diffuse and anaplastic astrocytic and oligodendroglial tumours as well as in secondary glioblastomas. As IDH is a very important prognostic, diagnostic and therapeutic biomarker for glioma, it is of paramount importance to determine its mutational status. The haematoxylin and eosin (H&E) staining is a valuable tool in precision oncology as it guides histopathology-based diagnosis and proceeding patient's treatment. However, H&E staining alone does not determine the IDH mutational status of a tumour. Deep learning methods applied to MRI data have been demonstrated to be a useful tool in IDH status prediction, however the effectiveness of deep learning on H&E slides in the clinical setting has not been investigated so far. Furthermore, the performance of deep learning methods in medical imaging has been practically limited by small sample sizes currently available. Here we propose a data augmentation method based on the Generative Adversarial Networks (GAN) deep learning methodology, to improve the prediction performance of IDH mutational status using H&E slides. The H&E slides were acquired from 266 grade II-IV glioma patients from a mixture of public and private databases, including 130 IDH-wildtype and 136 IDH-mutant patients. A baseline deep learning model without data augmentation achieved an accuracy of 0.794 (AUC = 0.920). With GAN-based data augmentation, the accuracy of the IDH mutational status prediction was improved to 0.853 (AUC = 0.927) when the 3,000 GAN generated training samples were added to the original training set (24,000 samples). By integrating also patients' age into the model, the accuracy improved further to 0.882 (AUC = 0.931). Our findings show that deep learning methodology, enhanced by GAN data augmentation, can support physicians in gliomas' IDH status prediction.
  30. Cell Death Dis. 2020 May 07. 11(5): 331
    Lei XX, Liu Y, Wang JX, Cai Q, Yan M, He HP, Liu Q, Long ZJ, Guan Z.
      Undifferentiation is a key feature of nasopharyngeal carcinoma (NPC), which presents as a unique opportunity for intervention by differentiation therapy. In this study, we found that SOX1 inhibited proliferation, promoted differentiation, and induced senescence of NPC cells, which depended on its transcriptional function. RNA-Seq-profiling analysis showed that multiple undifferentiated markers of keratin family, including KRT5, KRT13, and KRT19, were reduced in SOX1 overexpressed NPC cells. Interestingly, gene ontology (GO) analysis revealed genes in SOX1 overexpressed cells were enriched in extracellular functions. The data of LC/MS untargeted metabolomics showed that the content of retinoids in SOX1 overexpressed cells and culture medium was both higher than that in the control group. Subsequently, we screened mRNA level of genes in retinoic acid (RA) signaling or metabolic pathway and found that the expression of UDP-glucuronosyltransferases was significantly decreased. Furtherly, UGT2B7 could rescue the differentiation induced by SOX1 overexpression. Inhibition of UGTs by demethylzeylasteral (T-96) could mimic SOX1 to promote the differentiation of NPC cells. Thus, we described a mechanism by which SOX1 regulated the differentiation of NPC cells by activating retinoid metabolic pathway, providing a potential target for differentiation therapy of NPC.
  31. Genome Biol. 2020 May 07. 21(1): 97
    Begik O, Lucas MC, Liu H, Ramirez JM, Mattick JS, Novoa EM.
      BACKGROUND: RNA modifications play central roles in cellular fate and differentiation. However, the machinery responsible for placing, removing, and recognizing more than 170 RNA modifications remains largely uncharacterized and poorly annotated, and we currently lack integrative studies that identify which RNA modification-related proteins (RMPs) may be dysregulated in each cancer type.RESULTS: Here, we perform a comprehensive annotation and evolutionary analysis of human RMPs, as well as an integrative analysis of their expression patterns across 32 tissues, 10 species, and 13,358 paired tumor-normal human samples. Our analysis reveals an unanticipated heterogeneity of RMP expression patterns across mammalian tissues, with a vast proportion of duplicated enzymes displaying testis-specific expression, suggesting a key role for RNA modifications in sperm formation and possibly intergenerational inheritance. We uncover many RMPs that are dysregulated in various types of cancer, and whose expression levels are predictive of cancer progression. Surprisingly, we find that several commonly studied RNA modification enzymes such as METTL3 or FTO are not significantly upregulated in most cancer types, whereas several less-characterized RMPs, such as LAGE3 and HENMT1, are dysregulated in many cancers.
    CONCLUSIONS: Our analyses reveal an unanticipated heterogeneity in the expression patterns of RMPs across mammalian tissues and uncover a large proportion of dysregulated RMPs in multiple cancer types. We provide novel targets for future cancer research studies targeting the human epitranscriptome, as well as foundations to understand cell type-specific behaviors that are orchestrated by RNA modifications.
    Keywords:  Dysregulation in cancer; Epitranscriptomics; RNA modifications; Tissue specificity