FANCD2 Monoubiquitination in DNA Damage Responses

描述（由申请人提供）： 范可尼贫血（FA）是一种多基因疾病，可导致进行性骨髓衰竭，并具有患骨髓增生异常综合征和白血病的强烈倾向。FA蛋白通过一种很大程度上未知的机制参与细胞对DNA损伤的反应。FANCD 2的单泛素化需要至少8个FA蛋白的核复合物。FANCD 2的单泛素化是抵抗交联剂丝裂霉素C所必需的。ATR检查点激酶还调节FANCD 2单泛素化，并可能将FANCD 2与复制叉处的DNA损伤检测联系起来。本研究的目的是了解FANCD 2单泛素化的调节和功能，从而更好地了解FA蛋白在促进正常造血中的集体作用。某些FA蛋白在一般人群中的自发性癌症中发生突变，因此这些研究也与了解癌症如何发展有关。本研究的具体目的如下：1）阐明FANCD 2单泛素化的ATR依赖性调控机制。将确定MCM 7和Rad 17以及Rad 9沿着如何以及是否合作以将FANCD 2单泛素化偶联到复制叉处的DNA损伤的检测。将利用shRNA介导的这些蛋白在原代人CD 34+细胞中的抑制，以及突变体在永生化髓样前体细胞系中的表达。这些DNA损伤传感器是否防止染色体不稳定性和细胞凋亡，以应对MMC也将被确定。2)确定FANCD 2单泛素化和FANCD 2聚集点的组装缺陷是否会导致CD 34+造血干/祖细胞的染色体不稳定性和对MMC的敏感性。FANCD 2单泛素化将被shRNA介导的FANCA抑制所抑制。此外，FANCD 2募集到阻断的复制叉中所涉及的机制将在用EGFP-FANCD 2重建的细胞中使用实时显微镜来阐明。越来越清楚的是，一些范可尼贫血途径蛋白的改变与普通人群中观察到的一些自发性癌症有关。因此，本申请中描述的研究可能会对正常和异常干细胞功能以及癌症形成提供新的见解。

DESCRIPTION (provided by applicant): Fanconi Anemia (FA) is a multi-genic disorder that results in progressive bone marrow failure and a strong predisposition to myelodysplastic syndrome and leukemia. FA proteins are involved in the cellular response to DNA damage by a largely unknown mechanism. A nuclear complex of at least 8 FA proteins is required for the monoubiquitination of FANCD2. Monoubiquitination of FANCD2 is required for resistance to the crosslinker mitomycin C. The ATR checkpoint kinase also regulates FANCD2 monoubiquitination and potentially links FANCD2 to the detection of DNA damage at the replication fork. The goal of this study is to understand the regulation and function of FANCD2 monoubiquitination, and thereby to better understand the collective role of FA proteins in promoting normal hematopoiesis. Certain FA proteins are mutated in spontaneous cancers in the general population, so these studies are also relevant to understanding how cancer develops. The specific aims of the study are as follows: 1) Delineate mechanisms of the ATR-dependent regulation of FANCD2 monoubiquitination. How, and whether, MCM7 and Rad17, along with Rad9, cooperate to couple FANCD2 monoubiquitination to the detection of DNA damage at the replication fork will be determined. ShRNA-mediated suppression of these proteins in primary human CD34+ cells, and expression of mutants in an immortalized myeloid precursor line, will be utilized. Whether these DNA damage sensors prevent chromosomal instability and apoptosis in response to MMC will also be determined. 2) Determine whether defects in FANCD2 monoubiquitination and the assembly of FANCD2 foci results in chromosomal instability and sensitivity to MMC in CD34+ hematopoietic stem/progenitor cells. FANCD2 monoubiquitination will be inhibited by shRNA-mediated suppression of FANCA. Also, mechanisms involved in the recruitment of FANCD2 to blocked replication forks will be elucidated using real-time microscopy in cells reconstituted with EGFP- FANCD2. PROJECT NARRATIVE It is increasingly clear that alterations in some Fanconi anemia pathway proteins are involved in some spontanous cancers seen in the general population. Thus the studies described in this application may shed new light on both normal and abnormal stem cell function and on cancer formation.