Chfr and RNF8 synergistically regulate ATM activation

Division of Molecular Medicine and Genetics, Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, Michigan, USA.
Nature Structural & Molecular Biology (Impact Factor: 13.31). 06/2011; 18(7):761-8. DOI: 10.1038/nsmb.2078
Source: PubMed


Protein ubiquitination is a crucial component of the DNA damage response. To study the mechanism of the DNA damage-induced ubiquitination pathway, we analyzed the impact of the loss of two E3 ubiquitin ligases, RNF8 and Chfr. Notably, DNA damage-induced activation of ATM kinase is suppressed in cells deficient in both RNF8 and Chfr (double-knockout, or DKO), and DKO mice develop thymic lymphomas that are nearly diploid but harbor clonal chromosome translocations. Moreover, DKO mice and cells are hypersensitive to ionizing radiation. We present evidence that RNF8 and Chfr synergistically regulate histone ubiquitination to control histone H4 Lys16 acetylation through MRG15-dependent acetyltransferase complexes. Through these complexes, RNF8 and Chfr affect chromatin relaxation and modulate ATM activation and DNA damage response pathways. Collectively, our findings demonstrate that two chromatin-remodeling factors, RNF8 and Chfr, function together to activate ATM and maintain genomic stability in vivo.

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    • "This is achieved by tyrosine phosphorylation of Tip60 in response to DNA damage which promotes its binding to H3K9me3 and subsequent acetylation and activation of ATM [61]. It is also evident that ATM is activated by gross changes in chromatin structure [53] and by RNF8 and CHFR-mediated chromatin relaxation by histone ubiquitination [62]. More recently it has been demonstrated that ATM is activated by R-loops (RNA/DNA hybrids) at transcription blocking lesions to impede spliceosome organization and augment alternative splicing triggered by DNA damage [63]. "
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    ABSTRACT: The recognition, signalling and repair of DNA double strand breaks (DSB) involves the participation of a multitude of proteins and post-translational events that ensure maintenance of genome integrity. Amongst the proteins involved are several which when mutated give rise to genetic disorders characterised by chromosomal abnormalities, cancer predisposition, neurodegeneration and other pathologies. ATM (mutated in ataxia-telangiectasia (A-T) and members of the Mre11/Rad50/Nbs1 (MRN complex) play key roles in this process. The MRN complex rapidly recognises and locates to DNA DSB where it acts to recruit and assist in ATM activation. ATM, in the company of several other DNA damage response proteins, in turn phosphorylates all three members of the MRN complex to initiate downstream signalling. While ATM has hundreds of substrates, members of the MRN complex play a pivotal role in mediating the downstream signalling events that give rise to cell cycle control, DNA repair and ultimately cell survival or apoptosis. Here we focus on the interplay between ATM and the MRN complex in initiating signaling of breaks and more specifically on the adaptor role of the MRN complex in mediating ATM signalling to downstream substrates to control different cellular processes.
    Preview · Article · Oct 2015
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    • "Interestingly, the localizations of all these three protein kinases were still preserved in spermatocytes lacking H2AX, MDC1, or RNF8 (Figure 7a–b). Since the activations of ATM and DNAPK at DNA damage sites require proper H2AX-MDC1-RNF8 signaling in somatic cells 37,38, this observation suggests that the regulation of these kinases at the unsynapsed axes is different from that in somatic DDR. Future studies will reveal their precise roles at the unsynapsed axes. "
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    ABSTRACT: During meiotic prophase in males, the sex chromosomes partially synapse to form the XY body, a unique structure that recruits proteins involved in the DNA damage response, which is believed to be important for silencing of the sex chromosomes. It remains elusive how the DNA damage response in the XY body is regulated. Here we show that H2AX-MDC1-RNF8 signaling, which is well characterized in somatic cells, is dispensable for the recruitment of proteins to the unsynapsed axes in the XY body. On the other hand, the DNA damage response that spreads over the sex chromosomes is largely similar to that in somatic cells. This analysis shows that there are important differences between the regulation of the DNA damage response at the XY body and at DNA damage sites in somatic cells.
    Preview · Article · Jun 2013 · Nature Communications
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    • "Following this idea, our attention was directed toward a chromatin remodeling complex consistently trapped in pADPr IP extracts, namely the FACT complex SUPT16H/SSRP1 that acts to reorganize nucleosomes. As a control, we used CHFR, a chromatin remodeling protein that regulates histone modifications and the ATM-dependent DNA damage response pathway after DSBs (72). CHFR possesses a PBZ domain known for its non-covalent interaction with pADPr. "
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    ABSTRACT: Upon DNA damage induction, DNA-dependent poly(ADP-ribose) polymerases (PARPs) synthesize an anionic poly(ADP-ribose) (pADPr) scaffold to which several proteins bind with the subsequent formation of pADPr-associated multiprotein complexes. We have used a combination of affinity-purification methods and proteomics approaches to isolate these complexes and assess protein dynamics with respect to pADPr metabolism. As a first approach, we developed a substrate trapping strategy by which we demonstrate that a catalytically inactive Poly(ADP-ribose) glycohydrolase (PARG) mutant can act as a physiologically selective bait for the isolation of specific pADPr-binding proteins through its macrodomain-like domain. In addition to antibody-mediated affinity-purification methods, we used a pADPr macrodomain affinity resin to recover pADPr-binding proteins and their complexes. Second, we designed a time course experiment to explore the changes in the composition of pADPr-containing multiprotein complexes in response to alkylating DNA damage-mediated PARP activation. Spectral count clustering based on GeLC-MS/MS analysis was complemented with further analyses using high precision quantitative proteomics through isobaric tag for relative and absolute quantitation (iTRAQ)- and Stable isotope labeling by amino acids in cell culture (SILAC)-based proteomics. Here, we present a valuable resource in the interpretation of systems biology of the DNA damage response network in the context of poly(ADP-ribosyl)ation and provide a basis for subsequent investigations of pADPr-binding protein candidates.
    Full-text · Article · Jun 2012 · Nucleic Acids Research
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