DNA damage response (DDR) in ribosomal genes and mechanisms of DNA repair in embryonic stem cells (ESCs) are less explored nuclear events. DDR in ESCs should be unique due to their high proliferation rate, expression of pluripotency factors, and specific chromatin signature. Given short population doubling time and fast progress through G1 phase, ESCs require a sustained production of rRNA, which leads to the formation of large and prominent nucleoli. Although transcription of rRNA in the nucleolus is relatively well understood, little is known about DDR in this nuclear compartment. Here, we directed formation of double-strand breaks in rRNA genes with I- PpoI endonuclease, and we studied nucleolar morphology, DDR, and chromatin modifications. We observed a pronounced formation of I- PpoI-induced nucleolar caps, positive on BRCA1, NBS1, MDC1, γH2AX, and UBF1 proteins. We showed interaction of nucleolar protein TCOF1 with HDAC1 and TCOF1 with CARM1 after DNA injury. Moreover, H3R17me2a modification mediated by CARM1 was found in I- PpoI-induced nucleolar caps. Finally, we report that heterochromatin protein 1 is not involved in DNA repair of nucleolar caps.

Institute of Biophysics Academy of Sciences of the Czech Republic Brno Czech Republic

Zobrazit více v PubMed

Iyama T, Wilson DM., 3rd DNA repair mechanisms in dividing and non-dividing cells. DNA repair (Amst). 2013;12:620–36. PubMed PMC

Misteli T, Soutoglou E. The emerging role of nuclear architecture in DNA repair and genome maintenance. Nat Rev Mol Cell Biol. 2009;10:243–54. PubMed PMC

Kinner A, Wu W, Staudt Ch, Iliakis G. Gamma-H2Ax in recognition and signaling of DNA double-strand breaks in the context of chromatin. Nucleic Acids Res. 2008;36:5678–94. PubMed PMC

Goldstein M, Derheimer FA, Tait-Mulder J, Kastan MB. Nucleolin mediates nucleosome disruption critical for DNA double-strand break repair. Proc Natl Acad Sci U S A. 2013;110:16874–9. PubMed PMC

Gursoy-Yuzugullu O, House N, Price BD. Patching broken DNA: nucleosome dynamics and the repair of DNA breaks. J Mol Biol. 2016;428(9 Pt B):1846–60. PubMed PMC

Rogakou EP, Pilch DR, Orr AH, Ivanova VS, Bonner WM. DNA double-stranded breaks induce histone H2AX phosphorylation on serine 139. J Biol Chem. 1998;273:5858–68. PubMed

Kuo LJ, Yang L. Gamma-H2AX—a novel biomarker for DNA double-strand breaks. In Vivo. 2008;22: 305–10. PubMed

Mailand N, Bekker-Jensen S, Faustrup H, Melander F, Bartek J, Lukas C, Lukas J. RNF8 ubiquitylates histones at DNA double-strand breaks and promotes assembly of repair proteins. Cell. 2007;131:887–900. PubMed

Huen MS, Grant R, Manke I, Minn K, Yu X, Yaffe MB, Chen J. RNF8 transduces the DNA-damage signal via histone ubiquitylation and checkpoint protein assembly. Cell. 2007;131:901–14. PubMed PMC

Doil C, Mailand N, Bekker-Jensen S, Menard P, Larsen DH, Pepperkok R, Ellenberg J, Panier S, Durocher D, Bartek J, Lukas J, Lukas C. RNF168 binds and amplifies ubiquitin conjugates on damaged chromosomes to allow accumulation of repair proteins. Cell. 2009;136:435–46. PubMed

Ayrapetov MK, Gursoy-Yuzugullu O, Xu C, Xu Y, Price BD. DNA double-strand breaks promote methylation of histone H3 on lysine 9 and transient formation of repressive chromatin. Proc Natl Acad Sci U S A. 2014;111:9169–74. PubMed PMC

Mosammaparast N, Kim H, Laurent B, Zhao Y, Lim HJ, Majid MC, Dango S, Luo Y, Hempel K, Sowa ME, Gygi SP, Steen H, Harper JW, Yanker B, Shi Y. The histone demethylase LSD1/KDM1A promotes the DNA damage response. J Cell Biol. 2013;203:457–70. PubMed PMC

Li F, Mao G, Tong D, Huang J, Gu L, Yang W, Li GM. The histone mark H3K36me3 regulates human DNA mismatch repair through its interaction with MutSα. Cell. 2013;153:590–600. PubMed PMC

Brown DD, Gurdon JB. Absence of ribosomal RNA synthesis in the anucleolate mutant of Xenopus laevis. Proc Natl Acad Sci U S A. 1964;51:139–46. PubMed PMC

Tollervey D, Lehtonen H, Carmo-Fonseca M, Hurt EC. The small nucleolar RNP protein NOP1 (fibrillarin) is required for pre-rRNA processing in yeast. EMBO J. 1991;10:573–83. PubMed PMC

Boisvert FM, van Koningsbruggen S, Navascues J, Lamond AI. The multifunctional nucleolus. Nat Rev Mol Cell Biol. 2007;8:574–85. PubMed

Boulon S, Westman BJ, Hutten S, Boisvert FM, Lamond AI. The nucleolus under stress. Cell. 2010;40:216–27. PubMed PMC

Sirri V, Urcuqui-Inchima S, Roussel P, Hernandez-Verdun D. Nucleolus: the fascinating nuclear body. Histochem Cell Biol. 2008;129:13–31. PubMed PMC

McClintock B. The relation of a particular chromosomal element to the development of the nucleoli in Zea mays. Z Zellforsch Mikrosk Anat. 1934;21:294–328.

Bártová E, Horakova AH, Uhlirova R, Raska I, Galiova G, Orlova D, Kozubek S. Structure and epigenetics of nucleoli in comparison with non-nucleolar compartments. J Histochem Cytochem. 2010;58:391–403. PubMed PMC

McStay B, Grummt I. The epigenetics of rRNA genes: from molecular to chromosome biology. Annu Rev Cell Dev Biol. 2008;24:131–57. PubMed

Zorn C, Cremer T, Cremer C, Zimmer J. Laser UV microirradiation of interphase nuclei and post-treatment with caffeine. A new approach to establish the arrangement of interphase chromosomes. Hum Genet. 1976;35:83–89. PubMed

Cremer C, Cremer T. Induction of chromosome shattering by ultraviolet light and caffeine: the influence of different distributions of photolesions. Mutat Res. 1986;163:33–40. PubMed

Fenina M, Simon-Chazottes D, Vandormael-Pournin S, Soueid J, Langa F, Cohen-Tannoudji M, Bernard BA, Panthier JJ. I-SceI-mediated double-strand break does not increase the frequency of homologous recombination at the Dct locus in mouse embryonic stem cells. PLoS ONE. 2012;7:e39895. doi:10.1371/journal.pone.0039895. PubMed DOI PMC

Murga M, Jaco I, Fan Y, Soria R, Martinez-Pastor B, Cuadrado M, Yang SM, Blasco MA, Skoultchi AI, Fernandez-Capetillo O. Global chromatin compaction limits the strength of the DNA damage response. J Cell Biol. 2007;178:1101–8. PubMed PMC

Joshi R, Ho KK, Tenney K, Chen JH, Golden BL, Gimble FS. Evolution of I-SceI homing endonucleases with increased DNA recognition site specificity. J Mol Biol. 2011;405:185–200. PubMed PMC

Smith BL, Bauer GB, Povirk LF. DNA damage induced by bleomycin, neocarzinostatin, and melphalan in a precisely positioned nucleosome. Asymmetry in protection at the periphery of nucleosome-bound DNA. J Biol Chem. 1994;269:30587–94. PubMed

Chen J, Ghorai MK, Kenney G, Stubbe J. Mechanistic studies on bleomycin-mediated DNA damage: multiple binding modes can result in double-stranded DNA cleavage. Nucleic Acids Res. 2008;36:3781–90. PubMed PMC

Nakayama Y, Igarashi A, Kikuchi I, Obata Y, Fukumoto Y, Yamaguchi N. Bleomycin-induced over-replication involves sustained inhibition of mitotic entry through the ATM/ATR pathway. Exp Cell Res. 2009;315:2515–28. PubMed

Muscarella DE, Ellison EL, Ruoff BM, Vogt VM. Characterization of I-Ppo, an intron-encoded endonuclease that mediates homing of a group I intron in the ribosomal DNA of Physarum polycephalum. Mol Cell Biol. 1990;10:3386–96. PubMed PMC

Berkovich E, Monnat RJ, Kastan MB. Roles of ATM and NBS1 in chromatin structure modulation and DNA double-strand break repair. Nat Cell Biol. 2007;9:683–90. PubMed

Britton S, Coates J, Jackson SP. A new method for high-resolution imaging of Ku foci to decipher mechanisms of DNA double-strand break repair. J Cell Biol. 2013;202:579–95. PubMed PMC

Larsen DH, Hari F, Clapperton JA, Gwerder M, Gutsche K, Altmeyer M, Jungmichel S, Toledo LI, Fink D, Rask MB, Grofte M, Lukas C, Nielsen ML, Smerdon SJ, Lukas J, Stucki M. The NBS1-Treacle complex controls ribosomal RNA transcription in response to DNA damage. Nat Cell Biol. 2014;16:792–803. PubMed PMC

Ciccia A, Huang JW, Izhar L, Sowa ME, Harper JW, Elledge SJ. Treacher Collins syndrome TCOF1 protein cooperates with NBS1 in the DNA damage response. Proc Natl Acad Sci U S A. 2014;111:18631–6. PubMed PMC

van Sluis M, McStay B. A localized nucleolar DNA damage response facilitates recruitment of the homology-directed repair machinery independent of cell cycle stage. Genes Dev. 2015;29:1151–63. PubMed PMC

Manders EMM, Verbeek FJ, Aten JA. Measurement of co-localization of objects in dual-colour confocal images. J Microsc. 1993;169:375–82. PubMed

Costes SV, Daelemans D, Cho EH, Dobbin Z, Pavlakis G, Lockett S. Automatic and quantitative measurement of protein-protein colocalization in live cells. Biophys J. 2004;86:3993–4003. PubMed PMC

Rohlf FJ, Sokal RR. Statistical tables, 3rd ed. New York: W.H. Freeman and Company; 1995.

Dunn KW, Kamocka MM, McDonald JH. A practical guide to evaluating colocalization in biological microscopy. Am J Physiol Cell Physiol. 2011;300:C723–42. PubMed PMC

Reynolds RC, Montgomery PO, Hughes B. Nucleolar “caps” produced by actinomycin D. Cancer Res. 1964;24:1269–77. PubMed

Sorokin DV, Stixova L, Sehnalova P, Legartova S, Suchankova J, Simara P, Kozubek S, Matula P, Skalnikova M, Raska I, Bártová E. Localized movement and morphology of UBF1-positive nucleolar regions are changed by γ-irradiation in G2 phase of the cell cycle. Nucleus. 2015;6:301–13. PubMed PMC

Mischo HE, Hemmerich P, Grosse F, Zhang S. Actinomycin D induces histone γ-H2AX foci and complex formation of γ-H2AX with Ku70 and nuclear DNA helicase II. J Biol Chem. 2005;280:9586–94. PubMed

Neumuller RA, Gross T, Samsonova AA, Vinayagam A, Buckner M, Founk K, Hu Y, Sharifpoor S, Rosebrock AP, Andrews B, Winston F, Perrimon N. Conserved regulators of nucleolar size revealed by global phenotypic analyses. Sci Signal. 2013;6(289):ra70. doi:10.1126/scisignal.2004145. PubMed DOI PMC

Derenzini M, Montanaro L, Trere D. What the nucleolus says to a tumour pathologist. Histopathology. 2009;54:753–62. PubMed

Brangwynne CP, Mitchison TJ, Hyman AA. Active liquid-like behavior of nucleoli determines their size and shape in Xenopus laevis oocytes. Proc Natl Acad Sci U S A. 2011;108:4334–9. PubMed PMC

Berry J, Weber SC, Vaidya N, Haataja M, Brangwynne CP. RNA transcription modulates phase transition-driven nuclear body assembly. Proc Natl Acad Sci U S A. 2015;112;E5237–45. PubMed PMC

Mitrea DM, Kriwacki RW. Phase separation in biology; functional organization of a higher order. Cell Commun Signal. 2016;14. doi:10.1186/s12964-015-0125-7. PubMed DOI PMC

Schenkwein D, Turkki V, Ahlroth MK, Timonen O, Airenne KJ, Yla-Herttuala S. rRNA-directed integration by an HIV-1 integrase-I-PpoI fusion protein. Nucleic Acids Res. 2013;41:e61. doi:10.1093/nar/gks1438. PubMed DOI PMC

Martin RM, Ter-Avetisyan G, Herce HD, Ludwig AK, Lattig-Tunnemann G, Cardoso MC. Principles of protein targeting to the nucleolus. Nucleus. 2015;6:314–25. PubMed PMC

Zheng H, Chen L, Pledger WJ, Fang J, Chen J. P53 promotes repair of heterochromatin DNA by regulating Jmjd2b and Suv39h1 expression. Oncogene. 2014;33:734–44. PubMed PMC

Sun Y, Jiang X, Xu Y, Ayrapetov M, Moreau LA, Whetstine JR, Price BD. Histone H3 methylation links DNA damage detection to activation of the Tip60 tumor suppressor. Nat Cell Biol. 2009;11:1376–82. PubMed PMC

Yuan X, Feng W, Imhof A, Grummt I, Zhou Y. Activation of RNA polymerase I transcription by cockayne syndrome group B protein and histone methyltransferase G9a. Mol Cell. 2007;27:585–95. PubMed

Shen M, Zhou T, Xie W, Ling T, Zhu Q, Zong L, Lyu G, Gao Q, Zhang F, Tao W. The chromatin remodeling factor CSB recruits histone acetyltransferase PCAF to rRNA gene promoters in active state for transcription initiation. PLoS ONE. 2013;8:e62668. doi:10.1371/journal.pone.0062668. PubMed DOI PMC

Zhou Y, Santoro R, Grummt I. The chromatin remodeling complex NoRC targets HDAC1 to the ribosomal gene promoter and represses RNA polymerase I transcription. EMBO J. 2002;21:4632–40. PubMed PMC

Santoro R, Grummt I. Epigenetic mechanism of rRNA gene silencing: temporal order of NoRC-mediated histone modification, chromatin remodeling, and DNA methylation. Mol Cell Biol. 2005;25:2539–46. PubMed PMC

Miller KM, Tjeertes JV, Coates J, Legube G, Polo SE, Britton S, Jackson SP. Human HDAC1 and HDAC2 function in the DNA-damage response to promote DNA nonhomologous end-joining. Nat Struct Mol Biol. 2010;17:1144–51. PubMed PMC

Fukuda T, Wu W, Okada M, Maeda I, Kojima Y, Hayami R, Miyoshi Y, Tsugawa K, Ohta T. Class I histone deacetylase inhibitors inhibit the retention of BRCA1 and 53BP1 at the site of DNA damage. Cancer Sci. 2015;106:1050–6. PubMed PMC

Wu J, Xu W. Histone H3R17me2a mark recruits human RNA polymerase-associated factor 1 complex to activate transcription. Proc Natl Acad Sci U S A. 2012;109:5675–80. PubMed PMC

Franek M, Legartova S, Suchankova J, Milite C, Castellano S, Sbardella G, Kozubek S, Bártová E. CARM1 modulators affect epigenome of stem cells and change morphology of nucleoli. Physiol Res. 2015;64:769–82. PubMed

Legartova S, Sbardella G, Kozubek S, Bártová E. Ellagic acid-changed epigenome of ribosomal genes and condensed RPA194-positive regions of nucleoli in tumour cells. Folia Biol (Praha). 2015;61:49–59. PubMed

Kobayashi J, Fujimoto H, Sato J, Hayashi I, Burma S, Matsuura S, Chen DJ, Komatsu K. Nucleolin participates in DNA double-strand break-induced damage response through MDC1-dependent pathway. PLoS ONE. 2012;7:e49245. doi:10.1371/journal.pone.0049245. PubMed DOI PMC

Kwon SH, Workman JL. The changing faces of HP1: from heterochromatin formation and gene silencing to euchromatic gene expression: HP1 acts as a positive regulator of transcription. Bioessays. 2011;33:280–9. PubMed

Ayoub N, Jeyasekharan AD, Bernal JA, Venkitaraman AR. HP1-beta mobilization promotes chromatin changes that initiate the DNA damage response. Nature. 2008;453:682–6. PubMed

Luijsterburg MS, Dinant C, Lans H, Stap J, Wiernasz E, Lagerwerf S, Warmerdam DO, Lindh M, Brink MC, Dobrucki JW, Aten JA, Fousteri MI, Jansen G, Dantuma NP, Vermeulen W, Mullenders LH, Houtsmuller AB, Verschure PJ, van Driel R. Heterochromatin protein 1 is recruited to various types of DNA damage. J Cell Biol. 2009;185:577–86. PubMed PMC

Stixova L, Sehnalova P, Legarotva S, Suchankova J, Hruskova T, Kozubek S, Sorokin DV, Matula P, Raska I, Kovarik A, Fulnecek J, Bártová E. HP1β-dependent recruitment of UBF1 to irradiated chromatin occurs simultaneously with CPDs. Epigenetics Chromatin. 2014;7(1):39. doi:10.1186/1756-8935-7-39. PubMed DOI PMC

Condemine W, Takahashi Y, Le Bras M, de The H. A nucleolar targeting signal in PML-I addresses PML to nucleolar caps in stressed or senescent cells. J Cell Sci. 2007;120:3219–27. PubMed

Shav-Tal Y, Blechman J, Darzacq X, Montagna C, Dye BT, Patton JG, Singer RH, Zipori D. Dynamic sorting of nuclear components into distinct nucleolar caps during transcriptional inhibition. Mol Biol Cell. 2005;16:2395–413. PubMed PMC

Dellaire G, Ching RW, Ahmed K, Jalali F, Tse KCK, Bristow RG, Bazett-Jones DP. Promyelocytic leukemia nuclear bodies behave as DNA damage sensors whose response to DNA double-strand breaks is regulated by NBS1 and the kinases ATM, Chk2, and ATR. J Cell Biol. 2006;175:55–66. PubMed PMC

Carbone R, Pearson M, Minucci S, Pelicci PG. PML NBs associate with the hMre11 complex and p53 at sites of irradiation induced DNA damage. Oncogene. 2002;21:1633–40. PubMed

Guan D, Kao H. The function, regulation and therapeutic implications of the tumor suppressor protein, PML. Cell Biosci. 2015;5. doi:10.1186/s13578-015-0051-9. PubMed DOI PMC

UVA irradiation strengthened an interaction between UBF1/2 proteins and H4K20 di-/tri-methylation

H3K9me3 and H4K20me3 represent the epigenetic landscape for 53BP1 binding to DNA lesions