RNA:DNA hybrids in the human genome have distinctive nucleotide characteristics, chromatin composition, and transcriptional relationships

Epigenetics & Chromatin

John M. Greally²  Cathal Seoighe¹  Aaron Golden⁶  Cristina Montagna⁶  Jeffrey Jeddeloh⁵  Zhengdong Zhang⁶  Hanae Sato⁶  Pilib Ó Broin⁶  N. Ari Wijetunga⁶  Christophe Lemetre³  Rodoniki Athanasiadou⁴  Julie Nadel⁶ 

[1] School of Mathematics, Statistics and Applied Mathematics, National University of Ireland Galway, Galway, Ireland;Department of Genetics, Center for Epigenomics and Division of Computational Genetics, Albert Einstein College of Medicine, 1301 Morris Park Avenue, Bronx 10461, NY, USA;Integrated Genomics Operation, Memorial Sloan-Kettering Cancer Center, New York 10065, NY, USA;Department of Biology, Center for Genomics and Systems Biology, New York University, 12 Waverly Place, New York 10003, NY, USA;Roche-NimbleGen, Madison 53711, WI, USA;Department of Genetics, Albert Einstein College of Medicine, Bronx 10461, NY, USA

关键词: Mass spectrometry;    Non-coding RNA;    Transcription;    Transcription factor;    DNA methylation;    Chromatin;    R-loop;    RNA:DNA hybrid;   

Others  :  1233994
DOI  :  10.1186/s13072-015-0040-6

 received in 2015-07-22, accepted in 2015-10-29,  发布年份 2015

【 摘 要 】

Background

RNA:DNA hybrids represent a non-canonical nucleic acid structure that has been associated with a range of human diseases and potential transcriptional regulatory functions. Mapping of RNA:DNA hybrids in human cells reveals them to have a number of characteristics that give insights into their functions.

Results

We find RNA:DNA hybrids to occupy millions of base pairs in the human genome. A directional sequencing approach shows the RNA component of the RNA:DNA hybrid to be purine-rich, indicating a thermodynamic contribution to their in vivo stability. The RNA:DNA hybrids are enriched at loci with decreased DNA methylation and increased DNase hypersensitivity, and within larger domains with characteristics of heterochromatin formation, indicating potential transcriptional regulatory properties. Mass spectrometry studies of chromatin at RNA:DNA hybrids shows the presence of the ILF2 and ILF3 transcription factors, supporting a model of certain transcription factors binding preferentially to the RNA:DNA conformation.

Conclusions

Overall, there is little to indicate a dependence for RNA:DNA hybrids forming co-transcriptionally, with results from the ribosomal DNA repeat unit instead supporting the intriguing model of RNA generating these structures in trans. The results of the study indicate heterogeneous functions of these genomic elements and new insights into their formation and stability in vivo.

【 授权许可】

   
2015 Nadel et al.

【 图 表 】

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【 参考文献 】

• [1]Wang J, Zhuang J, Iyer S, Lin X, Whitfield TW, Greven MC, Pierce BG, Dong X, Kundaje A, Cheng Y et al.. Sequence features and chromatin structure around the genomic regions bound by 119 human transcription factors. Genome Res. 2012; 22:1798-1812.
• [2]Neph S, Vierstra J, Stergachis AB, Reynolds AP, Haugen E, Vernot B, Thurman RE, John S, Sandstrom R, Johnson AK et al.. An expansive human regulatory lexicon encoded in transcription factor footprints. Nature. 2012; 489:83-90.
• [3]Natarajan A, Yardimci GG, Sheffield NC, Crawford GE, Ohler U. Predicting cell-type-specific gene expression from regions of open chromatin. Genome Res. 2012; 22:1711-1722.
• [4]Yip KY, Cheng C, Bhardwaj N, Brown JB, Leng J, Kundaje A, Rozowsky J, Birney E, Bickel P, Snyder M, Gerstein M. Classification of human genomic regions based on experimentally determined binding sites of more than 100 transcription-related factors. Genome Biol. 2012; 13:R48. BioMed Central Full Text
• [5]Hu S, Wan J, Su Y, Song Q, Zeng Y, Nguyen HN, Shin J, Cox E, Rho HS, Woodard C, et al. DNA methylation presents distinct binding sites for human transcription factors. eLife. 2013;2:e00726.
• [6]Medvedeva YA, Khamis AM, Kulakovskiy IV, Ba-Alawi W, Bhuyan MS, Kawaji H, Lassmann T, Harbers M, Forrest AR, Bajic VB. Effects of cytosine methylation on transcription factor binding sites. BMC Genom. 2014; 15:119. BioMed Central Full Text
• [7]Schlick T. Topics in nucleic acids structure: noncanonical helices and rna structure. In: Molecular Modeling and simulation: an interdisciplinary guide. Interdisciplinary Applied Mathematics, vol. 21. New York: Springer; 2010. pp. 205–36.
• [8]Zhou T, Shen N, Yang L, Abe N, Horton J, Mann RS, Bussemaker HJ, Gordan R, Rohs R. Quantitative modeling of transcription factor binding specificities using DNA shape. Proc Natl Acad Sci USA. 2015; 112:4654-4659.
• [9]Raiber EA, Kranaster R, Lam E, Nikan M, Balasubramanian S. A non-canonical DNA structure is a binding motif for the transcription factor SP1 in vitro. Nucleic Acids Res. 2012; 40:1499-1508.
• [10]Khrapunov S, Warren C, Cheng H, Berko ER, Greally JM, Brenowitz M. Unusual characteristics of the DNA binding domain of epigenetic regulatory protein MeCP2 determine its binding specificity. Biochemistry. 2014; 53:3379-3391.
• [11]Aguilera A, Garcia-Muse T. R loops: from transcription byproducts to threats to genome stability. Mol Cell. 2012; 46:115-124.
• [12]Roberts RW, Crothers DM. Stability and properties of double and triple helices: dramatic effects of RNA or DNA backbone composition. Science. 1992; 258:1463-1466.
• [13]Roy D, Yu K, Lieber MR. Mechanism of R-loop formation at immunoglobulin class switch sequences. Mol Cell Biol. 2008; 28:50-60.
• [14]Roy D, Lieber MR. G clustering is important for the initiation of transcription-induced R-loops in vitro, whereas high G density without clustering is sufficient thereafter. Mol Cell Biol. 2009; 29:3124-3133.
• [15]Murat P, Balasubramanian S. Existence and consequences of G-quadruplex structures in DNA. Curr Opin Genet Dev. 2014; 25:22-29.
• [16]Ginno PA, Lott PL, Christensen HC, Korf I, Chedin F. R-loop formation is a distinctive characteristic of unmethylated human CpG island promoters. Mol Cell. 2012; 45:814-825.
• [17]Dunn K, Griffith JD. The presence of RNA in a double helix inhibits its interaction with histone protein. Nucleic Acids Res. 1980; 8:555-566.
• [18]Roy D, Zhang Z, Lu Z, Hsieh CL, Lieber MR. Competition between the RNA transcript and the nontemplate DNA strand during R-loop formation in vitro: a nick can serve as a strong R-loop initiation site. Mol Cell Biol. 2010; 30:146-159.
• [19]Ginno PA, Lim YW, Lott PL, Korf I, Chedin F. GC skew at the 5′ and 3′ ends of human genes links R-loop formation to epigenetic regulation and transcription termination. Genome Res. 2013; 23:1590-1600.
• [20]Ratmeyer L, Vinayak R, Zhong YY, Zon G, Wilson WD. Sequence specific thermodynamic and structural properties for DNA.RNA duplexes. Biochemistry. 1994; 33:5298-5304.
• [21]Wanrooij PH, Uhler JP, Shi Y, Westerlund F, Falkenberg M, Gustafsson CM. A hybrid G-quadruplex structure formed between RNA and DNA explains the extraordinary stability of the mitochondrial R-loop. Nucleic Acids Res. 2012; 40:10334-10344.
• [22]Loomis EW, Sanz LA, Chedin F, Hagerman PJ. Transcription-associated R-loop formation across the human FMR1 CGG-repeat region. PLoS Genet. 2014; 10:e1004294.
• [23]Tracy RB, Lieber MR. Transcription-dependent R-loop formation at mammalian class switch sequences. EMBO J. 2000; 19:1055-1067.
• [24]Wahba L, Gore SK, Koshland D. The homologous recombination machinery modulates the formation of RNA-DNA hybrids and associated chromosome instability. eLife. 2013;2:e00505.
• [25]Wahba L, Amon JD, Koshland D, Vuica-Ross M. RNase H and multiple RNA biogenesis factors cooperate to prevent RNA:DNA hybrids from generating genome instability. Mol Cell. 2011; 44:978-988.
• [26]Mischo HE, Gomez-Gonzalez B, Grzechnik P, Rondon AG, Wei W, Steinmetz L, Aguilera A, Proudfoot NJ. Yeast Sen1 helicase protects the genome from transcription-associated instability. Mol Cell. 2011; 41:21-32.
• [27]Tuduri S, Crabbe L, Conti C, Tourriere H, Holtgreve-Grez H, Jauch A, Pantesco V, De Vos J, Thomas A, Theillet C et al.. Topoisomerase I suppresses genomic instability by preventing interference between replication and transcription. Nat Cell Biol. 2009; 11:1315-1324.
• [28]Chan YA, Aristizabal MJ, Lu PY, Luo Z, Hamza A, Kobor MS, Stirling PC, Hieter P. Genome-wide profiling of yeast DNA:RNA hybrid prone sites with DRIP-chip. PLoS Genet. 2014; 10:e1004288.
• [29]Reaban ME, Griffin JA. Induction of RNA-stabilized DNA conformers by transcription of an immunoglobulin switch region. Nature. 1990; 348:342-344.
• [30]Daniels GA, Lieber MR. RNA:DNA complex formation upon transcription of immunoglobulin switch regions: implications for the mechanism and regulation of class switch recombination. Nucleic Acids Res. 1995; 23:5006-5011.
• [31]Sun Q, Csorba T, Skourti-Stathaki K, Proudfoot NJ, Dean C. R-loop stabilization represses antisense transcription at the Arabidopsis FLC locus. Science. 2013; 340:619-621.
• [32]Pfeiffer V, Crittin J, Grolimund L, Lingner J. The THO complex component Thp2 counteracts telomeric R-loops and telomere shortening. EMBO J. 2013; 32:2861-2871.
• [33]Castellano-Pozo M, Santos-Pereira JM, Rondon AG, Barroso S, Andujar E, Perez-Alegre M, Garcia-Muse T, Aguilera A. R loops are linked to histone H3 S10 phosphorylation and chromatin condensation. Mol Cell. 2013; 52:583-590.
• [34]Nakama M, Kawakami K, Kajitani T, Urano T, Murakami Y. DNA-RNA hybrid formation mediates RNAi-directed heterochromatin formation. Genes Cells. 2012; 17:218-233.
• [35]Sollier J, Stork CT, Garcia-Rubio ML, Paulsen RD, Aguilera A, Cimprich KA. Transcription-coupled nucleotide excision repair factors promote R-loop-induced genome instability. Mol Cell. 2014; 56:777-785.
• [36]Hatchi E, Skourti-Stathaki K, Ventz S, Pinello L, Yen A, Kamieniarz-Gdula K, Dimitrov S, Pathania S, McKinney KM, Eaton ML et al.. BRCA1 recruitment to transcriptional pause sites is required for R-loop-driven DNA damage repair. Mol Cell. 2015; 57:636-647.
• [37]Bhatia V, Barroso SI, Garcia-Rubio ML, Tumini E, Herrera-Moyano E, Aguilera A. BRCA2 prevents R-loop accumulation and associates with TREX-2 mRNA export factor PCID2. Nature. 2014; 511:362-365.
• [38]Chen YZ, Bennett CL, Huynh HM, Blair IP, Puls I, Irobi J, Dierick I, Abel A, Kennerson ML, Rabin BA et al.. DNA/RNA helicase gene mutations in a form of juvenile amyotrophic lateral sclerosis (ALS4). Am J Hum Genet. 2004; 74:1128-1135.
• [39]Gunther C, Kind B, Reijns MA, Berndt N, Martinez-Bueno M, Wolf C, Tungler V, Chara O, Lee YA, Hubner N et al.. Defective removal of ribonucleotides from DNA promotes systemic autoimmunity. J Clin Invest. 2015; 125:413-424.
• [40]Lin Y, Dent SY, Wilson JH, Wells RD, Napierala M. R loops stimulate genetic instability of CTG.CAG repeats. Proc Natl Acad Sci USA. 2010; 107:692-697.
• [41]Colak D, Zaninovic N, Cohen MS, Rosenwaks Z, Yang WY, Gerhardt J, Disney MD, Jaffrey SR. Promoter-bound trinucleotide repeat mRNA drives epigenetic silencing in fragile X syndrome. Science. 2014; 343:1002-1005.
• [42]Groh M, Lufino MM, Wade-Martins R, Gromak N. R-loops associated with triplet repeat expansions promote gene silencing in Friedreich ataxia and fragile X syndrome. PLoS Genet. 2014; 10:e1004318.
• [43]Haeusler AR, Donnelly CJ, Periz G, Simko EA, Shaw PG, Kim MS, Maragakis NJ, Troncoso JC, Pandey A, Sattler R et al.. C9orf72 nucleotide repeat structures initiate molecular cascades of disease. Nature. 2014; 507:195-200.
• [44]Groh M, Gromak N. Out of balance: R-loops in human disease. PLoS Genet. 2014; 10:e1004630.
• [45]Skourti-Stathaki K, Proudfoot NJ. A double-edged sword: R loops as threats to genome integrity and powerful regulators of gene expression. Genes Dev. 2014; 28:1384-1396.
• [46]Boguslawski SJ, Smith DE, Michalak MA, Mickelson KE, Yehle CO, Patterson WL, Carrico RJ. Characterization of monoclonal antibody to DNA.RNA and its application to immunodetection of hybrids. J Immunol Methods. 1986; 89:123-130.
• [47]Chadwick LH. The NIH roadmap epigenomics program data resource. Epigenomics. 2012; 4:317-324.
• [48]Zhang ZZ, Pannunzio NR, Hsieh CL, Yu K, Lieber MR. Complexities due to single-stranded RNA during antibody detection of genomic rna:dna hybrids. BMC Res Notes. 2015; 8:127. BioMed Central Full Text
• [49]Parkhomchuk D, Borodina T, Amstislavskiy V, Banaru M, Hallen L, Krobitsch S, Lehrach H, Soldatov A. Transcriptome analysis by strand-specific sequencing of complementary DNA. Nucleic Acids Res. 2009; 37:e123.
• [50]Yu K, Chedin F, Hsieh CL, Wilson TE, Lieber MR. R-loops at immunoglobulin class switch regions in the chromosomes of stimulated B cells. Nat Immunol. 2003; 4:442-451.
• [51]Yeo AJ, Becherel OJ, Luff JE, Cullen JK, Wongsurawat T, Jenjaroenpoon P, Kuznetsov VA, McKinnon PJ, Lavin MF. R-loops in proliferating cells but not in the brain: implications for AOA2 and other autosomal recessive ataxias. PLoS One. 2014; 9:e90219.
• [52]Koo CX, Kobiyama K, Shen YJ, LeBert N, Ahmad S, Khatoo M, Aoshi T, Gasser S, Ishii KJ. RNA Polymerase III regulates cytosolic RNA:DNA hybrids and intracellular MicroRNA expression. J Biol Chem. 2015; 290:7463-7473.
• [53]Brown TA, Tkachuk AN, Clayton DA. Native R-loops persist throughout the mouse mitochondrial DNA genome. J Biol Chem. 2008; 283:36743-36751.
• [54]Gonzalez IL, Sylvester JE. Complete sequence of the 43-kb human ribosomal DNA repeat: analysis of the intergenic spacer. Genomics. 1995; 27:320-328.
• [55]Zentner GE, Saiakhova A, Manaenkov P, Adams MD, Scacheri PC. Integrative genomic analysis of human ribosomal DNA. Nucleic Acids Res. 2011; 39:4949-4960.
• [56]Skourti-Stathaki K, Proudfoot NJ, Gromak N. Human senataxin resolves RNA/DNA hybrids formed at transcriptional pause sites to promote Xrn2-dependent termination. Mol Cell. 2011; 42:794-805.
• [57]Belotserkovskii BP, Liu R, Tornaletti S, Krasilnikova MM, Mirkin SM, Hanawalt PC. Mechanisms and implications of transcription blockage by guanine-rich DNA sequences. Proc Natl Acad Sci USA. 2010; 107:12816-12821.
• [58]Nellaker C, Keane TM, Yalcin B, Wong K, Agam A, Belgard TG, Flint J, Adams DJ, Frankel WN, Ponting CP. The genomic landscape shaped by selection on transposable elements across 18 mouse strains. Genome Biol. 2012; 13:R45. BioMed Central Full Text
• [59]Medstrand P, van de Lagemaat LN, Mager DL. Retroelement distributions in the human genome: variations associated with age and proximity to genes. Genome Res. 2002; 12:1483-1495.
• [60]Core LJ, Waterfall JJ, Lis JT. Nascent RNA sequencing reveals widespread pausing and divergent initiation at human promoters. Science. 2008; 322:1845-1848.
• [61]Kim TK, Hemberg M, Gray JM, Costa AM, Bear DM, Wu J, Harmin DA, Laptewicz M, Barbara-Haley K, Kuersten S et al.. Widespread transcription at neuronal activity-regulated enhancers. Nature. 2010; 465:182-187.
• [62]De Santa F, Barozzi I, Mietton F, Ghisletti S, Polletti S, Tusi BK, Muller H, Ragoussis J, Wei CL, Natoli G. A large fraction of extragenic RNA pol II transcription sites overlap enhancers. PLoS Biol. 2010; 8:e1000384.
• [63]Core LJ, Martins AL, Danko CG, Waters CT, Siepel A, Lis JT. Analysis of nascent RNA identifies a unified architecture of initiation regions at mammalian promoters and enhancers. Nat Genet. 2014; 46:1311-1320.
• [64]Mao X, Miesfeldt S, Yang H, Leiden JM, Thompson CB. The FLI-1 and chimeric EWS-FLI-1 oncoproteins display similar DNA binding specificities. J Biol Chem. 1994; 269:18216-18222.
• [65]Fazzari MJ, Greally JM. Epigenomics: beyond CpG islands. Nat Rev Genet. 2004; 5:446-455.
• [66]Tibshirani R. Regression shrinkage and selection via the Lasso. J R Stat Soc. 1996; 58:267-288.
• [67]Efron B, Hastie T. Johnstone I. Least angle regression, Tibshirani R; 2004.
• [68]Lockhart R, Taylor J. Tibshirani RJ. A significance test for the lasso, Tibshirani R; 2014.
• [69]Chakraborty P, Grosse F. Human DHX9 helicase preferentially unwinds RNA-containing displacement loops (R-loops) and G-quadruplexes. DNA Repair (Amst). 2011; 10:654-665.
• [70]Toretsky JA, Erkizan V, Levenson A, Abaan OD, Parvin JD, Cripe TP, Rice AM, Lee SB, Uren A. Oncoprotein EWS-FLI1 activity is enhanced by RNA helicase A. Cancer Res. 2006; 66:5574-5581.
• [71]Stein U, Jurchott K, Walther W, Bergmann S, Schlag PM, Royer HD. Hyperthermia-induced nuclear translocation of transcription factor YB-1 leads to enhanced expression of multidrug resistance-related ABC transporters. J Biol Chem. 2001; 276:28562-28569.
• [72]Aoki Y, Zhao G, Qiu D, Shi L, Kao PN. CsA-sensitive purine-box transcriptional regulator in bronchial epithelial cells contains NF45, NF90, and Ku. Am J Physiol. 1998; 275:L1164-L1172.
• [73]Kao PN, Chen L, Brock G, Ng J, Kenny J, Smith AJ, Corthesy B. Cloning and expression of cyclosporin A- and FK506-sensitive nuclear factor of activated T-cells: NF45 and NF90. J Biol Chem. 1994; 269:20691-20699.
• [74]Langland JO, Kao PN, Jacobs BL. Nuclear factor-90 of activated T-cells: a double-stranded RNA-binding protein and substrate for the double-stranded RNA-dependent protein kinase, PKR. Biochemistry. 1999; 38:6361-6368.
• [75]Boeva V, Surdez D, Guillon N, Tirode F, Fejes AP, Delattre O, Barillot E. De novo motif identification improves the accuracy of predicting transcription factor binding sites in ChIP-Seq data analysis. Nucleic Acids Res. 2010; 38:e126.
• [76]Pimkin M, Kossenkov AV, Mishra T, Morrissey CS, Wu W, Keller CA, Blobel GA, Lee D, Beer MA, Hardison RC, Weiss MJ. Divergent functions of hematopoietic transcription factors in lineage priming and differentiation during erythro-megakaryopoiesis. Genome Res. 2014; 24:1932-1944.
• [77]Li Y, Luo H, Liu T, Zacksenhaus E, Ben-David Y. The ets transcription factor Fli-1 in development, cancer and disease. Oncogene. 2015; 34:2022-2031.
• [78]Erkizan HV, Schneider JA, Sajwan K, Graham GT, Griffin B, Chasovskikh S, Youbi SE, Kallarakal A, Chruszcz M, Padmanabhan R et al.. RNA helicase A activity is inhibited by oncogenic transcription factor EWS-FLI1. Nucleic Acids Res. 2015; 43:1069-1080.
• [79]Erkizan HV, Kong Y, Merchant M, Schlottmann S, Barber-Rotenberg JS, Yuan L, Abaan OD, Chou TH, Dakshanamurthy S, Brown ML et al.. A small molecule blocking oncogenic protein EWS-FLI1 interaction with RNA helicase A inhibits growth of Ewing’s sarcoma. Nat Med. 2009; 15:750-756.
• [80]Riggi N, Knoechel B, Gillespie SM, Rheinbay E, Boulay G, Suva ML, Rossetti NE, Boonseng WE, Oksuz O, Cook EB et al.. EWS-FLI1 utilizes divergent chromatin remodeling mechanisms to directly activate or repress enhancer elements in Ewing sarcoma. Cancer Cell. 2014; 26:668-681.
• [81]Szczelkun MD, Tikhomirova MS, Sinkunas T, Gasiunas G, Karvelis T, Pschera P, Siksnys V, Seidel R. Direct observation of R-loop formation by single RNA-guided Cas9 and cascade effector complexes. Proc Natl Acad Sci USA. 2014; 111:9798-9803.
• [82]Zheng R, Shen Z, Tripathi V, Xuan Z, Freier SM, Bennett CF, Prasanth SG, Prasanth KV. Polypurine-repeat-containing RNAs: a novel class of long non-coding RNA in mammalian cells. J Cell Sci. 2010; 123:3734-3744.
• [83]Bacolla A, Cooper DN, Vasquez KM. Non-B DNA structure and mutations causing human genetic disease. In: eLS. New York: John Wiley & Sons Ltd; 2001.
• [84]Montagna C, Andrechek ER, Padilla-Nash H, Muller WJ, Ried T. Centrosome abnormalities, recurring deletions of chromosome 4, and genomic amplification of HER2/neu define mouse mammary gland adenocarcinomas induced by mutant HER2/neu. Oncogene. 2002; 21:890-898.
• [85]Miller SA, Dykes DD, Polesky HF. A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res. 1988; 16:1215.
• [86]Zhang Y, Liu T, Meyer CA, Eeckhoute J, Johnson DS, Bernstein BE, Nusbaum C, Myers RM, Brown M, Li W, Liu XS. Model-based analysis of ChIP-Seq (MACS). Genome Biol. 2008; 9:R137. BioMed Central Full Text
• [87]Diaz A, Nellore A, Song JS. CHANCE: comprehensive software for quality control and validation of ChIP-seq data. Genome Biol. 2012; 13:R98. BioMed Central Full Text
• [88]Searle BC. Scaffold: a bioinformatic tool for validating MS/MS-based proteomic studies. Proteomics. 2010; 10:1265-1269.