• DISCOVERY OF A ROLE OF FMRP IN R-LOOP REGULATION AND GENOME MAINTENANCE THROUGH BREAK-SEQ ANALYSIS OF THE FRAGILE X GENOME

      Chakraborty, Arijita (2020)
      Fragile X Syndrome (FXS) is a neurodevelopmental disorder caused by mutations in the FMRP translation regulator 1 (FMR1) gene and deficiency of its product, FMRP. FMRP is known as a translation repressor whose nuclear function is poorly understood. We investigated the global impact on genome stability due to FMRP loss. We applied Break-seq to a human cell line-based model for FXS and mapped genome-wide spontaneous and replication stress-induced DNA double strand breaks (DSBs) for the first time. We report that the genomes of FXS patient-derived cells are inherently unstable and accumulate more than twice as many DSBs as those fromnormal cells. The DSBs in FXS cells are enriched in neuron projection and synapse organization pathways. We further demonstrate that replication stress-induced DSBs in FXS cells correlate with R-loop forming sequences. FMRP, and not an RNA-binding mutant FMRP-I304N, abates R-loop-induced DSBs during programmed replication-transcription conflict.Moreover, exogenously expressed FMRP in FXS patient-derived cells reduces the replication stress-induced DSB formation. We conclude that the FXS cells are more susceptible to DNA replication stress. Furthermore, we identified chromatin binding sites of FMRP for the first time in human lymphoblastoid cells.Through mapping FMRP-bound chromatin loci in normal cells and correlating with FX-specific chromosome breaks, we identified novel FXS-susceptible genes. We show that FX cells have reduced expression of the uridine diphosphoglucuronosyl transferase 1 family enzymes, suggesting defective xenobioticmetabolism. In addition, using transcriptome analysis, we show that DNA repair genes are downregulated in FX cells under replication stress. Finally, we report a direct binding interaction between FMRP and R-loop and that the C-terminal domain is important for this interaction. Therefore, we proposethat FMRP is a novel genome maintenance protein required for preventing R-loop formation during replication stress. Our study provides newinsights into the etiological basis for FXS.