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

项目摘要 我们实验室的研究提供了范可尼贫血（FA）和α地中海贫血迟缓的证据， 综合征X连锁（ATRX）途径相互关联，以促进基因组在DNA面前的稳定性 通过支持同源重组（HR）机制来复制应激。我们之前的观察 假设ATRX与FANCD 2合作促进HR介导的DNA复制叉拯救，但 还具有额外的、独立的活动，以支持DNA修复步骤的不同子集。 到目前为止，我们的研究结果主要是使用人类癌症来源的细胞系获得的。有趣的是，我们最近 研究结果表明，FANCD 2和ATRX依赖的DNA复制应激反应活动不同， 在人类癌细胞和非癌（“正常”）细胞之间的机制上。 为了验证我们的假设，我们建议研究FANCD 2和ATRX在正常人类细胞系中的功能， 即RPE 1和BJ细胞。为此，我们将产生对ATRX基因无效的正常人类细胞， FANCD 2或两者，以及携带ATRX功能丧失突变的细胞。重要的是，高效的ATRX基因 正常人细胞中的敲除或敲入需要使用温和的细胞分选器，其（a）允许单个活细胞（例如， 细胞分选，同时保持高ATRX突变细胞活力，和（B）允许特异性批量纯化S- 期细胞以增加随后的ATRX敲入靶向效率。我们建议使用索尼台式机 SH 800细胞分选仪配备了两个激光器和微流控分选芯片，以便： (1)对CRISPR/Cas9靶向的、ATRX-和ATRX/FANCD 2-无效RPE 1和 BJ细胞 (2)进行RPE 1和BJ细胞的S期特异性批量预分选，然后进行CRISPR/Cas9介导的细胞分选。 ATRX基因敲入。 利用SONY SH 800分选仪产生这些突变的RPE 1和BJ细胞将允许我们（i）阐明 ATRX-FANCD 2蛋白复合物的分子和结构组成;（ii）确定分子 ATRX/FANCD 2介导的复制叉恢复的机制;（iii）剖析FANCD 2依赖性和- ATRX在DNA修复过程中的独立作用。

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.