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Chromatin Remodeling by Cdt1: Role in DNA Replication and Tumorigenesis

Cdt1 的染色质重塑：在 DNA 复制和肿瘤发生中的作用

基本信息

批准号：
8658391
负责人：
Mark G. Alexandrow
金额：
$ 29.34万
依托单位：
H. LEE MOFFITT CANCER CTR & RES INST
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-07-01 至 2015-04-30
项目状态：
已结题

项目摘要

DESCRIPTION (provided by applicant): The long term objective of this proposal is to determine how Cdt1 and Geminin function at the molecular level to control assembly of pre-Replication Complexes, and how the mechanisms behind their roles in this process create the propensity for abnormal re-replication. Cdt1 stimulates the loading of Mini- Chromosome Maintenance (MCM) proteins at preRCs, and Geminin blocks this effect, but the molecular mechanisms behind this are unknown. Preliminary results show that Cdt1 can induce large-scale decondensation of chromatin when targeted to specific chromosomal sites in vivo, which is followed by dramatic enrichment of MCMs and PCNA (and thus preRCs) at these sites. Geminin efficiently and specifically suppresses this effect of Cdt1, and the remodeling by Cdt1 only occurs in G1 cells when MCMs are known to load. Two chromatin remodeling enzymes that physically bind Cdt1 in vivo have been identified: a HAT called HBO1, and an HDAC called HDAC11. These enzymes modulate chromatin unfolding by Cdt1 in vivo. HBO1 is required for the unfolding of chromatin by Cdt1, while HDAC11 opposes the unfolding. HBO1, like Cdt1, is also required for MCM loading in vivo. Based on this, we hypothesize that Cdt1 and Geminin modulate MCM loading at preRCs by controlling chromatin access, and this access to chromatin is mediated by HBO1 and HDAC11. Thus, elevated Cdt1 or reduced Geminin, as is sometimes seen in cancer cells, would over-stimulate chromatin access and allow inappropriate MCM re-loading and re-replication. Three proposed aims will clarify the roles of HBO1 and HDAC11 in Cdt1/Geminin control of DNA (re)replication: (i) The function of HBO1 and HDAC11 in physiological events controlled by Cdt1 will be tested. HBO1 and HDAC11 will be overexpressed or reduced, and the effects on Cdt1-induced re-replication and MCM loading will be determined. Similarly, direct control by HBO1 and HDAC11 over DNA replication from a model origin will be analyzed. (ii) The ability of Geminin, and several Geminin mutants, to modulate the binding of HBO1 and HDAC11 to Cdt1 will be determined, as will their ability to block Cdt1-induced chromatin remodeling. (iii) Finally, the mechanisms and histone changes involved in Cdt1-induced chromatin remodeling will be further analyzed, novel proteins in complexes with Cdt1 in vivo will be investigated, and functional domains of Cdt1 responsible for the chromatin remodeling effect will be determined by deletion structure-function studies. Collectively, these aims will clarify the extent to which HBO1 and HDAC11 are involved in Cdt1/Geminin functions, and explain at the molecular level a novel mechanism whereby cells regulate replication licensing/MCM loading.

描述(由申请人提供)：本提案的长期目标是确定CDT1和Gminin如何在分子水平上发挥作用以控制复制前复合体的组装，以及它们在这一过程中所起作用背后的机制如何创造异常重复复制的倾向。CDT1刺激微小染色体维持(MCM)蛋白在Pre-RCs上的加载，双子座阻断了这一作用，但其背后的分子机制尚不清楚。初步结果表明，当CDT1靶向体内特定的染色体位置时，可以诱导染色质的大规模去凝集，随后在这些位置上MCMS和增殖细胞核抗原(从而前RC)显著浓缩。双黄素有效且特异地抑制了CDT1的这种作用，而CDT1的重塑只有在已知加载MCM的情况下才发生在G1细胞中。已经确定了两种在体内物理结合CDT1的染色质重塑酶：一种名为HBO1的HAT，以及一种名为HDAC11的HDAC。这些酶在体内通过CDT1调节染色质的展开。CDT1对染色质的去折叠需要HBO1，而HDAC11反对这种去折叠。HBO1和CDT1一样，也是体内MCM负载所必需的。在此基础上，我们假设CDT1和Gminin通过控制染色质访问来调节前RCs的MCM负载，这种访问染色质是由HBO1和HDAC11介导的。因此，在癌细胞中有时可以看到，CDT1升高或Gminin减少，会过度刺激染色质的获取，并允许不适当的MCM重新加载和重新复制。提出的三个目标将阐明HBO1和HDAC11在CDT1/Gminin控制DNA(Re)复制中的作用：(I)测试HBO1和HDAC11在由CDT1控制的生理事件中的功能。HBO1和HDAC11将过度表达或降低，并将确定对CDT1诱导的重新复制和MCM负载的影响。同样，将分析HBO1和HDAC11对来自模型起源的DNA复制的直接控制。(Ii)双子座和几个双子座突变体调节HBO1和HDAC11与CDT1结合的能力将被确定，它们阻止CDT1诱导的染色质重塑的能力也将被确定。(3)进一步分析了CDT1诱导染色质重塑的机制和组蛋白变化，研究了体内与CDT1形成的复合体中新的蛋白质，并通过缺失结构-功能研究确定了与染色质重塑相关的CDT1功能结构域。总之，这些目标将阐明HBO1和HDAC11在多大程度上参与CDT1/Ginin功能，并在分子水平上解释细胞调节复制许可/MCM负载的新机制。