Fanconi Anemia Pathway Suppression of R Loops Is a Mechanism of Maintenance of Genomic Stability

Background
The FA pathway involvement in homologous recombination and translesion synthesis repair requires binding to DNA by central proteins of the pathway, but interaction with RNA and modulation of genotoxic intermediates such as R loops has not been explored.  In this study we explore the incidence of R loops in FA mutant cells.

Methods
DRIP:  duplex RNA hybrid immunoprecipitation-genomic DNA was extracted, sonicated, and subjected to immunoprecipitation using S9.6 DNA:RNA hybrid antibody.   Resulting immunoprecipitates were run on agarose gel and stained using ethidium bromide.
Bisulfite modification/DNA Sequencing:  genomic DNA was extracted and a portion was denatured.  After bisulfite modification, coding sequence in GAPDH was amplified by PCR, and the resulting products sequenced.  Similarly prepared genomic DNA was prepared, exomic sequencing was performed.  Bioinformatic analysis was undertaken on each sample.
Immunofluorescence microscopy:   cells were cultured in chamber slides, treated or not with mitomycin C, fixed, and stained.
Cell culture:  mutant and complemented versions of each

Results
Using anti-RNA:DNA hybrid antibodies, we used a series of assays to test for the presence of R loops in FA mutant cells.  Agarose gel electrophoresis demonstrated increased pulldown of RNA:DNA hybrids in FA-D2 and FA-A mutant cells but not FA-J.  Similar results were seen in qPCR data designed to detect R loops in multiple sites in G-C rich areas of beta-actin and GAPDH.  Immunofluorescence revealed increased signal using the S9.6 antibody in a DNA damage inducible manner in mutant FA-D2 and FA-A cells but not FA-J mutant cells.  BRCA2 mutant cells also demonstrated increases in R loops.  Exome sequencing revealed that R loops were globally present in far greater amounts in FA-D2 mutant cells.
Conclusions
RNA:DNA hybrids as represented by R loops are present in greater amounts in FA-D2, FA-A, and BRCA2/FA-D1 mutant cells but not in FA-J mutant cells.  Suppression of R loops represents a non-canonical function for the FA pathway in maintenance of genomic stability.

Translational potential statement
The net cause of genomic instability in FA may be formation of DNA:RNA hybrids, which could be prevented by means of small molecules.  Understanding the biology can lead to enhanced treatment of bone marrow failure as well as of FA-associated tumors.