The histone variant macroH2A1.1 is recruited to DSBs through a mechanism involving PARP1 - PubMed (original) (raw)

The histone variant macroH2A1.1 is recruited to DSBs through a mechanism involving PARP1

Chang Xu et al. FEBS Lett. 2012.

Abstract

The repair of DNA double-strand breaks (DSBs) requires remodeling of the local chromatin architecture to allow the repair machinery to access sites of damage. Here, we report that the histone variant macroH2A1.1 is recruited to DSBs. Cells lacking macroH2A1 have defective recruitment of 53BP1, defective activation of chk2 kinase and increased radiosensitivity. Importantly, macroH2A1.1 is not incorporated into nucleosomes at DSBs, but instead associates with the chromatin through a mechanism which requires PARP1 activity. These results reveal an unusual mechanism involving a direct association of macroH2A1.1 with PARylated chromatin which is critical for retaining 53BP1 at sites of damage.

Copyright © 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

PubMed Disclaimer

Figures

Figure 1

Figure 1. Depletion of macroH2A1 increases radiosensitivity

(A) 293T cells were infected with a non-specific shRNA (shCon) or shRNA targeting mH2A1.1 or mH2A1.2 (shmH2A1). mH2A1 was measured by western blot. (B) mH2A1.1 and mH2A1.2 mRNA levels were measured by RT-qPCR in shCon and shmH2A1 cells. (C) and (D) 293T cells expressing a non-specific shRNA (shCon: ●) or shmH2A1 (○) were irradiated (C) or incubated with MMS for 1 hr (D). Clonogenic cell survival was measured 10 days later. Results ± SE (n = 3). (E) HeLa cells with an integrated NHEJ-GFP reporter were stably infected with control shRNA or shRNA targeting mH2A1. I-SceI was transiently expressed and GFP+ cells counted by FACS 48hr later. Results ± SD (n = 3).

Figure 2

Figure 2. mH2A1 is required for recruitment of 53BP1 to DSBs

(A) Cells expressing control shRNA or shRNA targeting mH2A1 were irradiated (2Gy), fixed and processed for immunofluorescent staining using γH2AX antibody. The number of cells with >5 foci were counted. Results ± SD (n > 150 cells). (B) Cells expressing control shRNA or shRNA targeting mH2A1 were irradiated (10Gy) and analyzed by western blot with antibodies to phosphoserine 1981 of ATM (pATM), ATM, mH2A1 and tubulin (loading control). (C) Cells expressing control shRNA or shRNA targeting mH2A1 were irradiated (2Gy), fixed and processed for immunofluorescent staining using 53BP1 antibody. The number of cells with >5 foci were counted. Results ± SD (n > 150 cells). (D) Cells expressing control shRNA or shRNA targeting mH2A1 were irradiated (10Gy) followed by western blot analysis with antibodies specific for chk2 and phospho-chk2.

Figure 3

Figure 3. mH2A1.1 is recruited to DSBs

(A) Expression of myc-HA-mH2A1.1 and myc-HA-mH2A1.2 in 293T cells. (B) and (C): 293T cells expressing myc-HA-mH2A1.1 were transiently transfected with vector or p84-ZFN. 18h later cells were processed for ChIP analysis using either (B) γH2AX antibody or (C) HA antibody to detect the HA-mH2A1.1. Samples were processed for ChIP using the indicated primer pairs. ChIP data points were calculated as IP DNA/input DNA and were normalized to the uncut sample, which was assigned a value of 1. Results ± SD (n = 2). (D) 293T cells expressing myc-HA-mH2A1.1 or myc-HA-mH2A1.2 were transiently transfected with vector or p84-ZFN. 18h later cells were processed for ChIP analysis using HA antibody as described above.

Figure 4

Figure 4. mH2A1.1 is not incorporated into nucleosomes at DSBs

(A) 293T cells stably expressing myc-HA-mH2A1.1 were transfected with p84-ZFN as indicated. 18hr later, cells were either mock cross-linked or cross-linked with formaldehyde (X-link). Cells extracts were processed for ChIP using antibodies (Ab) to γH2AX, Ku70 or HA-mH2A1.1. Results ± SD (n = 3). (B) 293T cells expressing myc-HA-mH2A1.1 were transiently transfected with p84-ZFN in the presence or absence of PARPi (20µM AZD2281). Cells were processed for ChIP and myc-HA-mH2A1.1 accumulation at the DSB (−0.5kB) or at a distant site (-50kb) analyzed by RT-qPCR.

Similar articles

Cited by

References

    1. Lukas J, Lukas C, Bartek J. More than just a focus: The chromatin response to DNA damage and its role in genome integrity maintenance. Nature cell biology. 2011;13:1161–1169. - PubMed
    1. Xu Y, Price BD. Chromatin dynamics and the repair of DNA double strand breaks. Cell Cycle. 2011;10:261–267. - PMC - PubMed
    1. Xu Y, Sun Y, Jiang X, Ayrapetov MK, Moskwa P, Yang S, Weinstock DM, Price BD. The p400 ATPase regulates nucleosome stability and chromatin ubiquitination during DNA repair. J Cell Biol. 2010;191:31–43. - PMC - PubMed
    1. Murr R, Loizou JI, Yang YG, Cuenin C, Li H, Wang ZQ, Herceg Z. Histone acetylation by Trrap-Tip60 modulates loading of repair proteins and repair of DNA double-strand breaks. Nat Cell Biol. 2006;8:91–99. - PubMed
    1. Messick TE, Greenberg RA. The ubiquitin landscape at DNA double-strand breaks. The Journal of cell biology. 2009;187:319–326. - PMC - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources