Administrative Supplement to : Functional crosstalk between the Fanconi Anemia and ATRX/DAXX histone chaperone pathways

项目总结

PROJECT SUMMARY Studies from our laboratory provided evidence that the Fanconi Anemia (FA) and Alpha Thalassemia Retardation Syndrome X-linked (ATRX) pathways are interconnected to promote genome stability in the face of DNA replication stress by supporting homologous recombination (HR) mechanisms. Our previous observations led us to hypothesized that ATRX cooperates with FANCD2 to promote HR-mediated DNA replication fork rescue, but also possesses additional, independent activities to support a distinct subset of DNA repair steps. So far, our study results were mostly obtained using human cancer-derived cell lines. Intriguingly, our recent findings indicate that the FANCD2- and ATRX-dependent DNA replication stress responses activities differ mechanistically between human cancer cells and non-cancer (“normal”) cells. To test our hypothesis, we propose to investigate FANCD2 and ATRX functions in normal human cell lines, namely RPE1 and BJ cells. To this end, we will generate normal human cells genetically null for ATRX, FANCD2, or both, as well as cells carrying ATRX loss-of-function mutations. Importantly, efficient ATRX gene knock-out or knock-in in normal human cells requires the use of a gentle cell sorter that (a) allows single live cell sorting while maintaining high ATRX mutant cell viability and (b) allows for specific bulk purification of S- phase cells to increase subsequent ATRX knock-in targeting efficiency. We propose to use a Sony benchtop SH800 cell sorter equipped with two lasers and microfluidics sorting chips, in order to: (1) Perform single live cell sorting of CRISPR/Cas9 targeted, ATRX- and ATRX/FANCD2-null RPE1 and BJ cells (2) Perform S-phase-specific bulk pre-sorting of RPE1 and BJ cells, followed by CRISPR/Cas9 mediated ATRX gene knock-in. Generating these mutant RPE1 and BJ cells utilizing the SONY SH800 sorter will then allow us to (i) elucidate the molecular and structural makeup of ATRX-FANCD2 protein complexes; (ii) Determine molecular mechanisms of ATRX/FANCD2-mediated replication fork recovery; (iii) Dissect FANCD2-dependent and - independent roles of ATRX during DNA repair.