Abstract
Background: Fanconi anemia proteins, encoded by at least 22genes (FANCA-W), constitute the Interstrand Cross Link (ICL) repair pathway. While FANCD2 is a master regulator of ICL repair, it accumulates at common fragile sites (CFS) during mild replication stress stimulated by low-dose Aphidicolin (APH) treatment. A recent study indicated that FANCD2 is required for efficient genome replication across the CFS regions. FANCD2 is also implicated in the regulation of R-loops levels. R-loops, which consist of DNA: RNA hybrids and displaced single-stranded DNA, are physiologically relevant in the genome and associate with immunoglobulin class switching, replication of mitochondrial DNA as well as transcriptional promoters or terminators. However, in any case, untimely formation of R-loops is a major threat to genome instability. Furthermore, it has been reported that R-loops which are induced by common slicing factor mutations in cases with myelodysplastic syndrome are linked to compromised proliferation of hematopoietic progenitors. It is also interesting to note that a recent study shows an interaction of FANCD2 with splicing factor 3B1 (SF3B1) and proposes their role in organizing chromatin domains to ensure coordination of replication and co-transcriptional processes.
Methods: To examine the genome-wide distribution of FANCD2 protein, we set out to create a derivative of human osteosarcoma cell line, U2OS, which incorporated a 3×FLAG tag into the FANCD2 termination codon by genome editing. We performed chromatin-immunoprecipitation and sequencing (ChIP-Seq) analysis, and provide a genome-wide landscape of replication stress response involving FANCD2 in this cell line. Moreover, we purified the FANCD2 complex and analyzed by liquid chromatography-tandem mass spectrometry, and confirmed this interaction by co-immunoprecipitation (Co-IP) and proximal ligation assay (PLA) with FANCD2-3xFLAG. R-loops levels were assayed as the number of S9.6 (anti DNA:RNA hybrid antibody) stained foci per nucleus.
Results: FANCD2 accumulation mostly occurs in the central portion of large transcribed genes, including CFS, and its accumulation appeared to be dependent on R-loop formation induced by transcription-replication collisions during mild replication stress. Moreover, our mass spectrometry analysis identified that FANCD2 interacts with several RNA processing factors including heterogeneous nucleoprotein U (hnRNP U), or DEAD box protein 47 (DDX47). We confirmed the interaction of these factors with FANCD2 by Co-IP as well as PLA. It was previously reported that defects in RNA-processing factors result in R-loop accumulation associated genome instability. Indeed, we found that treatment with siRNA against hnRNP U or DDX47 resulted in the increased number of the S9.6 foci. Furthermore, FANCD2 and hnRNP U or DDX47 appeared to function in an epistatic manner in suppressing APH-induced transcription-replication collisions as detected by PLA between PCNA and RNA polymerase II.
Conclusion: We suggest that FANCD2 protects genome stability by recruiting RNA processing enzymes, including hnRNP U or DDX47, to resolve or prevent accumulation of R-loops induced by transcription-replication collisions during mild replication stress. Thus, our study may provide a novel insight to understand the mechanism of bone marrow failure and leukemogenesis in Fanconi anemia patients.
Disclosures
Takaori-Kondo: Bristol-Myers Squibb: Honoraria; Pfizer: Honoraria; Celgene: Honoraria, Research Funding; Novartis: Honoraria; Janssen Pharmaceuticals: Honoraria.
Mammalian NET-seq analysis defines nascent RNA profiles and associated RNA processing genome-wide