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

项目总结 我们实验室的研究提供了范可尼贫血(FA)和阿尔法地中海贫血迟缓的证据 综合征X连锁(ATRX)途径相互连接以促进面对DNA的基因组稳定性 通过支持同源重组(HR)机制实现复制应激。我们之前的观察让我们 假设ATRX与FANCD2合作促进HR介导的DNA复制分叉救援，但 还拥有额外的、独立的活动来支持DNA修复步骤的不同子集。 到目前为止，我们的研究结果大多是使用人类癌症来源的细胞系获得的。耐人寻味的是，我们最近 研究结果表明，依赖FANCD2和ATRX的DNA复制应激反应活动不同 人类癌细胞和非癌症(“正常”)细胞之间的机械联系。 为了验证我们的假设，我们建议研究FANCD2和ATRX在正常人类细胞系中的功能， 即RPE1和BJ细胞。为此，我们将产生正常的人类细胞对ATRX基因为零， FANCD2，或两者兼而有之，以及携带ATRX功能丧失突变的细胞。重要的是，高效的ATRX基因 正常人类细胞的敲除或敲入需要使用温和的细胞分选器，该分选器(A)允许单个活细胞 在保持高ATRX突变细胞存活率的同时进行细胞分选，以及(B)允许对S进行特异性批量纯化- 阶段细胞，以提高后续的ATRX敲入靶向效率。我们建议使用索尼台式电脑 SH800细胞分选机配备了两个激光和微流控分选芯片，以： (1)对CRISPR/Cas9靶向、ATRX-和ATRX/FANCD2-空RPE1和 BJ细胞 (2)对RPE1和BJ细胞进行S时相特异性预分选，然后进行CRISPR/Cas9介导 ATRX基因敲入。 使用Sony SH800分选器产生这些突变的RPE1和BJ细胞将使我们能够(I)阐明 ATRX-FANCD2蛋白复合体的分子和结构组成；(II)分子测定 ATRX/FANCD2介导的复制分叉恢复机制；(Iii)剖析FANCD2依赖和- ATRX在DNA修复中的独立作用。

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.