Proteomic characterization of chromosomal common fragile site (CFS)-associated proteins uncovers ATRX as a regulator of CFS stability

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.

Transcription-mediated organization of the replication initiation program across large genes sets up common fragile sites genome-wide

“…Focussing on 5 large genes, a pioneer work has correlated the tissue-dependent expression of these genes to the instability of associated CFSs 10 . ChIP-Seq of FANCD2, a factor binding preferentially CFSs from S-phase to mitosis upon replication stress 11 , has recently confirmed genome-wide this co-localization of CFSs with large transcribed genes in human 12,13 and chicken 9 cells grown in vitro. Remarkably, the same correlation has been reached upon extensive mapping of copy number variations in large series of tumours 14 .…”

Transcription-replication conflicts as a source of common fragile site instability caused by BMI1-RNF2 deficiency

“…A few studies may have suggested how FANCD2 facilitates the R-loop processing; FANCD2 may recruit RNA processing factors (50), or facilitate FANCM translocase activity (20). FANCD2 may also facilitate the recruitment of chromatin remodeler ATRX to CFSs (42) to resolve R-loop-mediated replication stresses (52). FANCD2 is necessary for recovery of perturbed replication forks through recruiting CtIP nuclease (53), which can facilitate the R-loop removal (54).…”

Transcription-replication conflicts as a source of common fragile site instability caused by BMI1-RNF2 deficiency

“…Consistently, ChIP using anti DNA-RNA hybrid antibody (S9.6) showed similar results ( Fig 4B). A series of reports have suggested that FA proteins engaged in resolving R-loops (see Discussion), and recent reports found that FANCD2 becomes enriched at CFSs when cells were challenged with replication stressors [18,42]. We thus tested if FANCD2 proteins are differentially enriched at CFSs in WT versus RNF2 KO cells.…”

“…A few studies may have suggested how FANCD2 facilitates the R-loop processing; FANCD2 may recruit RNA processing factors [50], or facilitate FANCM translocase activity [20]. FANCD2 may also facilitate the recruitment of chromatin remodeler ATRX to CFSs [42] to resolve R-loop-mediated replication stresses [52]. FANCD2 is necessary for recovery of perturbed replication forks through recruiting CtIP nuclease [53], which can facilitate the R-loop removal [54].…”