FANCD2 Monoubiquitination in DNA Damage Responses

描述(申请人提供)：Fanconi贫血(FA)是一种多基因疾病，会导致进行性骨髓衰竭，并极易患上骨髓增生异常综合征和白血病。FA蛋白通过一种基本未知的机制参与细胞对DNA损伤的反应。FANCD2的单素化需要至少8个FA蛋白的核复合体。FANCD2的单素化是产生对交联剂丝裂霉素C的抗性所必需的。ATR检查点激酶还调节FANCD2的单素化，并潜在地将FANCD2与复制分叉处的DNA损伤检测联系起来。本研究的目的是了解FANCD2单素化的调节和功能，从而更好地了解FA蛋白在促进正常造血中的集体作用。在普通人群中，某些FA蛋白在自发性癌症中发生突变，因此这些研究对于了解癌症的发展也是相关的。本研究的具体目的如下：1)阐明ATR依赖的FANCD2单泛素化调控机制。MCM7和Rad17以及Rad9如何以及是否合作将FANCD2单核苷酸连接到复制分叉处的DNA损伤检测将被确定。将利用shRNA介导的在原代人类CD34细胞中抑制这些蛋白，以及在永生化的髓系前体细胞中表达突变。还将确定这些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。2)确定FANCD2单素化缺陷和FANCD2焦点组装缺陷是否导致CD34造血干/祖细胞染色体不稳定和对MMC敏感。FANCD2单素化将被shRNA介导的FANCA抑制所抑制。此外，在与EGFP-FANCD2重组的细胞中，将使用实时显微镜来阐明FANCD2招募到被阻断的复制分叉的机制。项目简介越来越清楚的是，一些Fanconi贫血途径蛋白的改变与普通人群中看到的一些自发性癌症有关。因此，本申请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.