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Differential contribution of HP1 proteins to DNA end resection and homology-directed repair.

ABSTRACT: Heterochromatin protein 1 paralogs (HP1?, ? and ? in mammals) are not only central in heterochromatin organization, but have also been linked to transcriptional activation at euchromatic regions, maintenance of telomere stability and, most recently, to the DNA damage response (DDR). However, how HP1 proteins contribute to the DDR at a molecular level, and whether HP1 paralogs within the same organism, as well as their respective orthologs, have overlapping or unique roles in the DDR, remain to be elucidated. Herein, we have combined the analysis of the efficiency and kinetics of recruitment of key repair proteins to sites of DNA damage with specific DNA repair assays to demonstrate that human HP1 paralogs differentially modulate homology-directed repair (HDR) pathways, including homologous recombination (HR) and single-strand annealing (SSA). We find that while HP1? and ? stimulate HR and SSA, HP1? has an inhibitory role. In addition, we show that the stimulatory role of HP1? and ? in HDR is linked to the DNA-end resection step of DNA breaks, through the promotion of RPA loading and phosphorylation at damage sites. Altogether, our findings provide mechanistic insight into how human HP1 proteins participate in the recombination process, emerging as important chromatin regulators during HDR.

SUBMITTER: Soria G

PROVIDER: S-EPMC3587443 | biostudies-literature | 2013 Feb

REPOSITORIES: biostudies-literature

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Differential contribution of HP1 proteins to DNA end resection and homology-directed repair.

Soria Gaston G Almouzni Geneviève G

Cell cycle (Georgetown, Tex.) 20120201 3

Heterochromatin protein 1 paralogs (HP1α, β and γ in mammals) are not only central in heterochromatin organization, but have also been linked to transcriptional activation at euchromatic regions, maintenance of telomere stability and, most recently, to the DNA damage response (DDR). However, how HP1 proteins contribute to the DDR at a molecular level, and whether HP1 paralogs within the same organism, as well as their respective orthologs, have overlapping or unique roles in the DDR, remain to b ...[more]

PMID: 23287531

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Project description:BACKGROUND:The C9ORF72 hexanucleotide repeat expansion is the most common known genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), two fatal age-related neurodegenerative diseases. The C9ORF72 expansion encodes five dipeptide repeat proteins (DPRs) that are produced through a non-canonical translation mechanism. Among the DPRs, proline-arginine (PR), glycine-arginine (GR), and glycine-alanine (GA) are the most neurotoxic and increase the frequency of DNA double strand breaks (DSBs). While the accumulation of these genotoxic lesions is increasingly recognized as a feature of disease, the mechanism(s) of DPR-mediated DNA damage are ill-defined and the effect of DPRs on the efficiency of each DNA DSB repair pathways has not been previously evaluated. METHODS AND RESULTS:Using DNA DSB repair assays, we evaluated the efficiency of specific repair pathways, and found that PR, GR and GA decrease the efficiency of non-homologous end joining (NHEJ), single strand annealing (SSA), and microhomology-mediated end joining (MMEJ), but not homologous recombination (HR). We found that PR inhibits DNA DSB repair, in part, by binding to the nucleolar protein nucleophosmin (NPM1). Depletion of NPM1 inhibited NHEJ and SSA, suggesting that NPM1 loss-of-function in PR expressing cells leads to impediments of both non-homologous and homology-directed DNA DSB repair pathways. By deleting NPM1 sub-cellular localization signals, we found that PR binds NPM1 regardless of the cellular compartment to which NPM1 was directed. Deletion of the NPM1 acidic loop motif, known to engage other arginine-rich proteins, abrogated PR and NPM1 binding. Using confocal and super-resolution immunofluorescence microscopy, we found that levels of RAD52, a component of the SSA repair machinery, were significantly increased iPSC neurons relative to isogenic controls in which the C9ORF72 expansion had been deleted using CRISPR/Cas9 genome editing. Western analysis of post-mortem brain tissues confirmed that RAD52 immunoreactivity is significantly increased in C9ALS/FTD samples as compared to controls. CONCLUSIONS:Collectively, we characterized the inhibitory effects of DPRs on key DNA DSB repair pathways, identified NPM1 as a facilitator of DNA repair that is inhibited by PR, and revealed deficits in homology-directed DNA DSB repair pathways as a novel feature of C9ORF72-related disease.

Project description:Genome editing using transcription-activator like effector nucleases or RNA guided nucleases allows one to precisely engineer desired changes within a given target sequence. The genome editing reagents introduce double stranded breaks (DSBs) at the target site which can then undergo DNA repair by non-homologous end joining (NHEJ) or homology directed recombination (HDR) when a template DNA molecule is available. NHEJ repair results in indel mutations at the target site. As PCR amplified products from mutant target regions are likely to exhibit different melting profiles than PCR products amplified from wild type target region, we designed a high resolution melting analysis (HRMA) for rapid identification of efficient genome editing reagents. We also designed TaqMan assays using probes situated across the cut site to discriminate wild type from mutant sequences present after genome editing. The experiments revealed that the sensitivity of the assays to detect NHEJ-mediated DNA repair could be enhanced by selection of transfected cells to reduce the contribution of unmodified genomic DNA from untransfected cells to the DNA melting profile. The presence of donor template DNA lacking the target sequence at the time of genome editing further enhanced the sensitivity of the assays for detection of mutant DNA molecules by excluding the wild-type sequences modified by HDR. A second TaqMan probe that bound to an adjacent site, outside of the primary target cut site, was used to directly determine the contribution of HDR to DNA repair in the presence of the donor template sequence. The TaqMan qPCR assay, designed to measure the contribution of NHEJ and HDR in DNA repair, corroborated the results from HRMA. The data indicated that genome editing reagents can produce DSBs at high efficiency in HEK293T cells but a significant proportion of these are likely masked by reversion to wild type as a result of HDR. Supplying a donor plasmid to provide a template for HDR (that eliminates a PCR amplifiable target) revealed these cryptic DSBs and facilitated the determination of the true efficacy of genome editing reagents. The results indicated that in HEK293T cells, approximately 40% of the DSBs introduced by genome editing, were available for participation in HDR.

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Differential contribution of HP1 proteins to DNA end resection and homology-directed repair.

Publications

Differential contribution of HP1 proteins to DNA end resection and homology-directed repair.

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