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，导致进行性骨髓衰竭和骨髓增生异常综合征和白血病的强烈易感性。FA蛋白参与细胞对DNA损伤的反应，其机制尚不清楚。FANCD2的单泛素化需要至少8个FA蛋白的核复合体。FANCD2的单泛素化是对交联剂丝裂霉素c的抗性所必需的。ATR检查点激酶也调节FANCD2的单泛素化，并可能将FANCD2与复制叉上DNA损伤的检测联系起来。本研究的目的是了解FANCD2单泛素化的调控和功能，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蛋白在普通人群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atr依赖性调控FANCD2单泛素化的机制。MCM7和Rad17以及Rad9如何以及是否合作将FANCD2单泛素化偶联到复制叉处DNA损伤的检测将被确定。shrna介导的这些蛋白在原代人CD34+细胞中的抑制，以及在永生化骨髓前体细胞中的突变表达，将被利用。这些DNA损伤传感器是否能防止MMC反应中的染色体不稳定和细胞凋亡也将被确定。2)确定FANCD2单泛素化和FANCD2灶组装缺陷是否导致CD34+造血干细胞/祖细胞染色体不稳定和对MMC的敏感性。shrna介导的FANCA抑制可抑制FANCD2单泛素化。此外，在EGFP- FANCD2重组的细胞中，使用实时显微镜将阐明FANCD2募集到阻断复制叉的机制。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的一些自发性癌症有关。因此，在本应用中描述的研究可能为正常和异常干细胞功能以及癌症形成提供新的线索。

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.