Abstract
Repair of ribosomal DNA (rDNA) is a very important nuclear process due to the most active transcription of ribosomal genes. Proper repair of rDNA is required for physiological biogenesis of ribosomes. Here, we analyzed the epigenetics of the DNA damage response in a nucleolar compartment, thus in the ribosomal genes studied in nonirradiated and UVA-irradiated mouse embryonic fibroblasts (MEFs). We found that the promoter of ribosomal genes is not abundant on H4K20me2, but it is densely occupied by H4K20me3. Ribosomal genes, regulated via UBF1/2 proteins, were characterized by an interaction between UBF1/2 and H4K20me2/me3. This interaction was strengthened by UVA irradiation that additionally causes a focal accumulation of H4K20me3 in the nucleolus. No interaction has been found between UBF1/2 and H3K9me3. Interestingly, UVA irradiation decreases the levels of H3K9me3 and H4K20me3 at 28S rDNA. Altogether, the UVA light affects the epigenetic status of ribosomal genes at 28S rDNA and strengthens an interaction between UBF1/2 proteins and H4K20me2/me3.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
Abbreviations
- ATM:
-
Ataxia telangiectasia mutated
- ATR:
-
ATM and Rad3 related
- ChIP:
-
Chromatin immunoprecipitation
- CPDs:
-
Cyclobutane pyrimidine dimers
- DDR:
-
DNA damage response
- DFC:
-
Dense fibrillar compartment
- DMEM:
-
Dulbecco’s modified Eagle’s medium
- DSBs:
-
Double-strand breaks
- FC:
-
Fibrillar centers
- FLIM:
-
Fluorescence-lifetime imaging microscopy
- FRET:
-
Förster resonance energy transfer
- GC:
-
Granular component
- GFP:
-
Green fluorescence protein
- GG-NER:
-
Global genome NER
- H3K9me3:
-
Tri-methylated lysine 9 of histone H3
- H4K20me3/me2:
-
Tri-methylated/di-methylated lysine 20 of histone H4
- HR:
-
Homologous recombination
- MEFs:
-
Mouse embryonic fibroblasts
- NBS1:
-
Nijmegen breakage syndrome
- NER:
-
Nucleotide excision repair
- NHEJ:
-
Nonhomologous end joining
- NORs:
-
Nucleolar organizer regions
- PBS:
-
Phosphate-buffered saline
- PCR:
-
Polymerase chain reaction
- PTMs:
-
Posttranslational modifications
- rDNA:
-
Ribosomal DNA
- RNA Pol I/II:
-
RNA polymerase I/II
- rRNA:
-
Ribosomal RNA
- SDS:
-
Sodium dodecyl sulfate
- TC-NER:
-
Transcription-coupled NER
- TCOF-1:
-
Treacle ribosome biogenesis factor 1
- UBF 1/2:
-
Upstream binding factor 1/2
- UVA, UVC:
-
Ultraviolet A, ultraviolet C
References
Andersen JS, Lam YW, Leung AKL, Ong SE, Lyon CE, Lamond AI, Mann M (2005) Nucleolar proteome dynamics. Nature 433:77–83. https://doi.org/10.1038/nature03207
Ayrapetov MK, Gursoy-Yuzugullu O, Xu C, Xu Y, Price BD (2014) 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 111:9169–9174. https://doi.org/10.1073/pnas.1403565111
Bacskai BJ, Skoch J, Hickey GA, Allen R, Hyman BT (2003) Fluorescence resonance energy transfer determinations using multiphoton fluorescence lifetime imaging microscopy to characterize amyloid-beta plaques. J Biomed Opt 8:368–375. https://doi.org/10.1117/1.1584442
Bian Q, Anderson EC, Brejc K, Meyer BJ (2017) Dynamic control of chromosome topology and gene expression by a chromatin modification. Cold Spring Harb Symp Quant Biol LXXXII:034439. https://doi.org/10.1101/sqb.2017.82.034439
Botuyan MV, Lee J, Ward IM, Kim JE, Thompson JR, Chen J, Mer G (2006) Structural basis for the methylation state-specific recognition of histone H4-K20 by 53BP1 and Crb2 in DNA repair. Cell 127:1361–1373. https://doi.org/10.1016/j.cell.2006.10.043
Boulon S, Westman BJ, Hutten S, Boisvert FM, Lamond AI (2010) The nucleolus under stress. Mol Cell 40:216–227. https://doi.org/10.1016/j.molcel.2010.09.024
Bromberg KD, Mitchell TRH, Upadhyay AK, Jakob CG, Jhala MA, Comess KM, Lasko LM, Li C, Tuzon CT, Dai Y, Li F, Eram MS, Nuber A, Soni NB, Manaves V, Algire MA, Sweis RF, Torrent M, Schotta G, Sun C, Michaelides MR, Shoemaker AR, Arrowsmith CH, Brown PJ, Santhakumar V, Martin A, Rice JC, Chiang GG, Vedadi M, Barsyte-Lovejoy D, Pappano WN (2017) The SUV4-20 inhibitor A-196 verifies a role for epigenetics in genomic integrity. Nat Chem Biol 13:317–324. https://doi.org/10.1038/nchembio.2282
Chitale S, Richly H (2018) DIC ER- and MMS ET-catalyzed H4K20me2 recruits the nucleotide excision repair factor XPA to DNA damage sites. J Cell Biol 217:527–540. https://doi.org/10.1083/jcb.201704028
de Laat W, Jaspers N, Hoeijmakers J (1999) Molecular mechanism of nucleotide excision repair. Genes Dev 13:768–785. https://doi.org/10.1101/gad.13.7.768
Elangovan M, Day RN, Periasamy A (2002) Nanosecond fluorescence resonance energy transfer-fluorescence lifetime imaging microscopy to localize the protein interactions in a single living cell. J Microsc 205:3–14
Farley KI, Surovtseva Y, Merkel J, Baserga SJ (2015) Determinants of mammalian nucleolar architecture. Chromosoma 124:323–331. https://doi.org/10.1007/s00412-015-0507-z
Franek M, Kovaříková A, Bártová E, Kozubek S (2016) Nucleolar reorganization upon site-specific double-strand break induction. J Histochem Cytochem 64:669–686. https://doi.org/10.1369/0022155416668505
Goodarzi AA, Noon AT, Deckbar D, Ziv Y, Shiloh Y, Löbrich M, Jeggo PA (2008) ATM signaling facilitates repair of DNA double-strand breaks associated with heterochromatin. Mol Cell 31:167–177. https://doi.org/10.1016/J.MOLCEL.2008.05.017
Grummt I (2007) Different epigenetic layers engage in complex crosstalk to define the epigenetic state of mammalian rRNA genes. Hum Mol Genet 16:R21–R27. https://doi.org/10.1093/hmg/ddm020
Harničarová Horáková A, Bártová E, Galiová G, Uhlířová R, Matula P, Kozubek S (2010) SUV39h-independent association of HP1β with fibrillarin-positive nucleolar regions. Chromosoma 119:227–241. https://doi.org/10.1007/s00412-009-0252-2
Haugland RP, Yguerabide J, Stryer L (1969) Dependence of the kinetics of singlet-singlet energy transfer on spectral overlap. Proc Natl Acad Sci U S A 63:23–30
Jørgensen S, Schotta G, Sørensen CS (2013) Histone H4 lysine 20 methylation: key player in epigenetic regulation of genomic integrity. Nucleic Acids Res 41:2797–2806. https://doi.org/10.1093/nar/gkt012
Kakarougkas A, Ismail A, Klement K, Goodarzi AA, Conrad S, Freire R, Shibata A, Lobrich M, Jeggo PA (2013) Opposing roles for 53BP1 during homologous recombination. Nucleic Acids Res 41:9719–9731. https://doi.org/10.1093/nar/gkt729
Krishnan RV, Saitoh H, Terada H, Centonze VE, Herman B (2003) Development of a multiphoton fluorescence lifetime imaging microscopy system using a streak camera. Rev Sci Instrum 74:2714–2721. https://doi.org/10.1063/1.1569410
Kruhlak M, Crouch EE, Orlov M, Montaño C, Gorski SA, Nussenzweig A, Misteli T, Phair RD, Casellas R (2007) The ATM repair pathway inhibits RNA polymerase I transcription in response to chromosome breaks. Nature 447:730–734. https://doi.org/10.1038/nature05842
Lakowicz JR (1999) Principles of fuorescence spectroscopy, 3rd edn. Springer US, New York
Lans H, Marteijn JA, Vermeulen W (2012) ATP-dependent chromatin remodeling in the DNA-damage response. Epigenetics Chromatin 5:4. https://doi.org/10.1186/1756-8935-5-4
Larsen DH, Hari F, Clapperton JA, Gwerder M, Gutsche K, Altmeyer M, Jungmichel S, Toledo LI, Fink D, Rask MB, Grøfte M, Lukas C, Nielsen ML, Smerdon SJ, Lukas J, Stucki M (2014) The NBS1-treacle complex controls ribosomal RNA transcription in response to DNA damage. Nat Cell Biol 16:792–803. https://doi.org/10.1038/ncb3007
Lindström MS, Jurada D, Bursac S, Orsolic I, Bartek J, Volarevic S (2018) Nucleolus as an emerging hub in maintenance of genome stability and cancer pathogenesis. Oncogene 37:2351–2366. https://doi.org/10.1038/s41388-017-0121-z
McStay B, Grummt I (2008) The epigenetics of rRNA genes: from molecular to chromosome biology. Annu Rev Cell Dev Biol 24:131–157. https://doi.org/10.1146/annurev.cellbio.24.110707.175259
Mikkelsen TS, Ku M, Jaffe DB, Issac B, Lieberman E, Giannoukos G, Alvarez P, Brockman W, Kim TK, Koche RP, Lee W, Mendenhall E, O’Donovan A, Presser A, Russ C, Xie X, Meissner A, Wernig M, Jaenisch R, Nusbaum C, Lander ES, Bernstein BE (2007) Genome-wide maps of chromatin state in pluripotent and lineage-committed cells. Nature 448:553–560. https://doi.org/10.1038/nature06008
Moore HM, Bai B, Boisvert F-M, Latonen L, Rantanen V, Simpson JC, Pepperkok R, Lamond AI, Laiho M (2011) Quantitative proteomics and dynamic imaging of the nucleolus reveal distinct responses to UV and ionizing radiation. Mol Cell Proteomics 10:M111.009241. https://doi.org/10.1074/mcp.M111.009241
Németh A, Grummt I (2018) Dynamic regulation of nucleolar architecture. Curr Opin Cell Biol 52:105–111. https://doi.org/10.1016/J.CEB.2018.02.013
Ogawa LM, Baserga SJ (2017) Crosstalk between the nucleolus and the DNA damage response. Mol BioSyst 13:443–455. https://doi.org/10.1039/c6mb00740f
Pederson T (2010) Cytologically “compact” nuclear domains. Nucleus 1:444–445. https://doi.org/10.4161/nucl.1.5.13056
Pesavento JJ, Yang H, Kelleher NL, Mizzen CA (2008) Certain and progressive methylation of histone H4 at lysine 20 during the cell cycle. Mol Cell Biol 28:468–486. https://doi.org/10.1128/MCB.01517-07
Raska I, Rychter Z, Smetana K (1983) Fibrillar centres and condensed nucleolar chromatin in resting and stimulated human lymphocytes. Z Mikrosk Anat Forsch 97:15–32
Reardon JT, Sancar A (2003) Recognition and repair of the cyclobutane thymine dimer, a major cause of skin cancers by the human excision nuclease. Genes Dev 17:2539–2551. https://doi.org/10.1101/gad.1131003
Santoro R, Grummt I (2005) Epigenetic mechanism of rRNA gene silencing: temporal order of NoRC-mediated histone modification, chromatin remodeling, and DNA methylation. Mol Cell Biol 25:2539–2546. https://doi.org/10.1128/MCB.25.7.2539-2546.2005
Schwarzacher HG, Wachtler F (1993) The nucleolus. Anat Embryol (Berl) 188:515–536
Sorokin DV, Stixová L, Sehnalová P, Legartová S, Suchánková J, Šimara P, Kozubek S, Matula P, Skalníková M, Raška I, Bártová E (2015) Localized movement and morphology of UBF1-positive nucleolar regions are changed by γ-irradiation in G2 phase of the cell cycle. Nucleus 6:301–313. https://doi.org/10.1080/19491034.2015.1075111
Stixová L, Sehnalová P, Legartová S, Suchánková J, Hrušková T, Kozubek S, Sorokin DV, Matula P, Raška I, Kovařík A, Fulneček J, Bártová E (2014) HP1β-dependent recruitment of UBF1 to irradiated chromatin occurs simultaneously with CPDs. Epigenetics Chromatin 7:39. https://doi.org/10.1186/1756-8935-7-39
Strašák L, Bártová E, Harničarová A et al (2009) H3K9 acetylation and radial chromatin positioning. J Cell Physiol 220:91–101. https://doi.org/10.1002/jcp.21734
Stryer L, Haugland RP (1967) Energy transfer: a spectroscopic ruler. Proc Natl Acad Sci U S A 58:719–726
Stryer L (1978) Fluorescence energy transfer as a spectroscopic ruler. Annu Rev Biochem 47:819–846. https://doi.org/10.1146/annurev.bi.47.070178.00413
Svobodová Kovaříková A, Legartová S, Krejčí J, Bártová E (2018) H3K9me3 and H4K20me3 represent the epigenetic landscape for 53BP1 binding to DNA lesions. Aging (Albany NY) 10:2585–2605. https://doi.org/10.18632/aging.101572
Thiry M, Lafontaine DLJ (2005) Birth of a nucleolus: the evolution of nucleolar compartments. Trends Cell Biol 15:194–199. https://doi.org/10.1016/j.tcb.2005.02.007
Tong Q, Cui G, Botuyan MV, Rothbart SB, Hayashi R, Musselman CA, Singh N, Appella E, Strahl BD, Mer G, Kutateladze TG (2015) Structural plasticity of methyllysine recognition by the tandem tudor domain of 53BP1. Structure 23:312–321. https://doi.org/10.1016/j.str.2014.11.013
Večeřa J, Bártová E, Krejčí J, Legartová S, Komůrková D, Rudá-Kučerová J, Štark T, Dražanová E, Kašpárek T, Šulcová A, Dekker FJ, Szymanski W, Seiser C, Weitzer G, Mechoulam R, Micale V, Kozubek S (2018) HDAC1 and HDAC3 underlie dynamic H3K9 acetylation during embryonic neurogenesis and in schizophrenia-like animals. J Cell Physiol 233:530–548. https://doi.org/10.1002/jcp.25914
Wakeman TP, Wang Q, Feng J, Wang X-F (2012) Bat3 facilitates H3K79 dimethylation by DOT1L and promotes DNA damage-induced 53BP1 foci at G1/G2 cell-cycle phases. EMBO J 31:2169–2181. https://doi.org/10.1038/emboj.2012.50
Warmerdam DO, van den Berg J, Medema RH (2016) Breaks in the 45S rDNA lead to recombination-mediated loss of repeats. Cell Rep 14:2519–2527. https://doi.org/10.1016/J.CELREP.2016.02.048
Wood K, Tellier M, Murphy S (2018) DOT1L and H3K79 methylation in transcription and genomic stability. Biomolecules 8. https://doi.org/10.3390/biom8010011
Zentner GE, Balow SA, Scacheri PC (2014) Genomic characterization of the mouse ribosomal DNA locus. G3(Bethesda) : Genes|Genomes|Genetics 4:243–254. https://doi.org/10.1534/g3.113.009290
Zentner GE, Saiakhova A, Manaenkov P, Adams MD, Scacheri PC (2011) Integrative genomic analysis of human ribosomal DNA. Nucleic Acids Res 39:4949–4960. https://doi.org/10.1093/nar/gkq1326
Zimmermann M, de Lange T (2014) 53BP1: pro choice in DNA repair. Trends Cell Biol 24:108–117. https://doi.org/10.1016/j.tcb.2013.09.003
Acknowledgements
We thank Soňa Legartová for the description of FLIM-FRET methodology.
Funding
This work was supported by the Czech Science Foundation (grant number: 18-07384S). Work was also supported by Strategie AV21, programme Qualitas, the Center for Epigenetics (68081707).
Author information
Authors and Affiliations
Contributions
LS was responsible for western blotting, immunofluorescence, immunoprecipitation, confocal microscopy, FLIM-FRET, and UVA irradiation. LS wrote a draft of the manuscript. DK performed the ChIP-PCR analyses and data quantifications. AK provided help with the western blotting and with the interpretation of FLIM-FRET results. EB coordinated experimental efforts, suggested experiments, finalized all images, and wrote the final version of this paper. All authors read and approved the final version of this manuscript.
Corresponding author
Ethics declarations
Conflict of interest
The authors declare that they have no conflict of interest.
Additional information
Responsible Editor: Jennifer Gerton and Lev Porokhovnik
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Rights and permissions
About this article
Cite this article
Stixová, L., Komůrková, D., Svobodová Kovaříková, A. et al. UVA irradiation strengthened an interaction between UBF1/2 proteins and H4K20 di-/tri-methylation. Chromosome Res 27, 41–55 (2019). https://doi.org/10.1007/s10577-018-9596-x
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10577-018-9596-x