Biology of the Myb-MuvB Oncoprotein-Tumor Suppressor Protein Complex

描述(申请人提供)：视网膜母细胞瘤(RB)肿瘤抑制通路在大多数(如果不是全部)人类癌症中发生突变。该途径调节S期进程所需的基因以及细胞分化所必需的基因，并与异染色质的建立和维持有关。脊椎动物Rb途径的生化研究一直受到从天然状态下的细胞核中提取Rb复合体的困难的阻碍。脊椎动物Rb途径的遗传学研究因冗余、世代时间长以及遗传筛选的高昂成本而变得复杂。幸运的是，RB途径在后生动物进化过程中是保守的，这使得对更简单的有机体进行信息分析成为可能。这项建议的重点是最近发现的和相当意想不到的Rb肿瘤抑制途径和Myb癌基因家族之间的联系。对果蝇细胞的核提取物进行分级，发现了一个以前未知的大型多蛋白复合体，称为Myb-MuvB。Myb DNA结合蛋白是由脊椎动物Myb癌基因家族中唯一的果蝇同源物编码的，这在线虫线虫中是不存在的。复合体中的其他蛋白质由果蝇编码的synMuvB基因的同源物编码，这些基因是在线虫中发现的，是由于功能丧失突变导致的多外阴表型，与RTK-RAS-MAP激酶通路中功能获得突变所引起的表型相同。Rb和Ras通路都包含大多数人类癌症发生过程中突变的关键靶点。此外，最近对人类乳腺癌分子标志物的筛查显示，B-MYB/MYBL2(果蝇Myb的人类同源基因)表达增加是预后不良的有力预测因素。因此，更好地了解Myb-MuvB复合体可能会改进人类癌症的诊断和治疗。我们建议在果蝇中使用遗传学、细胞生物学和生化分析来确定Myb-MuvB复合体组件的急性功能丧失或获得的后果。我们的初步数据显示，在没有Myb的情况下，染色体凝聚的正常进程会失败。此外，我们还发现，着丝粒特异性组蛋白H3变异体CID/CENP-A的沉积在没有Myb的情况下发生了变化。我们的假设是，Myb-MuvB复合体的其他成分也是染色体生物学中这些关键步骤所必需的。因此，我们的具体目标是：(1)确定Myb-MuvB的哪些成分调节染色体凝聚；(2)描述Myb-MuvB在异染色质的建立、维持和扩散中的作用；(3)测试Myb-MuvB的成分是否调节组蛋白H3变体的交换。

DESCRIPTION (provided by applicant): The retinoblastoma (Rb) tumor suppressor pathway is mutated in most, if not all, human cancers. This pathway regulates genes required for S phase progression as well as genes essential for cellular differentiation, and has also been implicated in the establishment and maintenance of heterochromatin. Biochemical studies of the Rb pathway in vertebrates have been hampered by difficulties in extracting Rb complexes from cell nuclei in their native state. Genetic studies of the Rb pathway in vertebrates have been complicated by redundancy, long generation times, and the high cost of genetic screens. Fortunately, the Rb pathway has been conserved during metazoan evolution, permitting the informative analysis of simpler organisms. This proposal focuses on a recently discovered and quite unexpected connection between the Rb tumor suppressor pathway and the Myb oncogene family. Fractionation of nuclear extracts from Drosophila cells revealed a large, previously unknown multiprotein complex called Myb-MuvB. The Myb DNA-binding protein is encoded by the sole Drosophila homologue of the vertebrate Myb oncogene family which is absent in the nematode C elegans. The other proteins in the complex are encoded by Drosophila homologues of the synMuvB genes which were discovered in C elegans due to loss-of-funotion mutants that cause a multivulval phenotype identical to that caused by gain-of- function mutants in the RTK-RAS-MAP kinase pathway. Both the Rb and RAS pathways contain critical targets for mutation in the development of most human cancers. In addition, recent screens for molecular markers in human breast cancer have revealed that increased expression of B-MYB/MYBL2 (the human orthologue of Drosophila Myb) is a strong predictor of poor prognosis. Therefore, better understanding of the Myb-MuvB complex will likely lead to improvements in the diagnosis and treatment of human cancer. We propose to use genetic, cell biological, and biochemical analyses in Drosophila to determine the consequences of acute loss or gain of function of components of the Myb-MuvB complex. Our preliminary data show that a failure of the normal progression of chromosome condensation occurs in the absence of Myb. In addition, we found that the deposition of the centromere-specific histone H3 variant CID/CENP-A is altered in the absence of Myb. Our hypothesis is that other components of the Myb-MuvB complex are also required for these critical steps in chromosome biology. Therefore, our specific aims are: (1) To determine which components of Myb-MuvB regulate chromosome condensation; (2) To delineate the role of Myb-MuvB in the establishment, maintenance, and spread of heterochromatin; (3) To test whether components of Myb- MuvB regulate the exchange of histone H3 variants.