Affiliation:
1. Department of Cellular and Molecular Medicine, Center for Chromosome Stability and Center for Healthy Aging, University of Copenhagen, Copenhagen 2200, Denmark
2. Proteomics Program, Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, 2200 Copenhagen, Denmark
Abstract
Abstract
Common fragile sites (CFSs) are conserved genomic regions prone to break under conditions of replication stress (RS). Thus, CFSs are hotspots for rearrangements in cancer and contribute to its chromosomal instability. Here, we have performed a global analysis of proteins that recruit to CFSs upon mild RS to identify novel players in CFS stability. To this end, we performed Chromatin Immunoprecipitation (ChIP) of FANCD2, a protein that localizes specifically to CFSs in G2/M, coupled to mass spectrometry to acquire a CFS interactome. Our strategy was validated by the enrichment of many known regulators of CFS maintenance, including Fanconi Anemia, DNA repair and replication proteins. Among the proteins identified with unknown functions at CFSs was the chromatin remodeler ATRX. Here we demonstrate that ATRX forms foci at a fraction of CFSs upon RS, and that ATRX depletion increases the occurrence of chromosomal breaks, a phenotype further exacerbated under mild RS conditions. Accordingly, ATRX depletion increases the number of 53BP1 bodies and micronuclei, overall indicating that ATRX is required for CFS stability. Overall, our study provides the first proteomic characterization of CFSs as a valuable resource for the identification of novel regulators of CFS stability.
Funder
Danish National Research Foundation
Danish Cancer Society
European Research Council
Novo Nordisk Foundation
Lundbeck Foundation
Publisher
Oxford University Press (OUP)
Cited by
23 articles.
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