Chromatin Remodeling by Cdt1: Role in DNA Replication and Tumorigenesis

描述（由申请人提供）：本提案的长期目标是确定Cdt 1和Geminin如何在分子水平上发挥功能以控制复制前复合物的组装，以及它们在此过程中的作用背后的机制如何产生异常再复制的倾向。Cdt 1刺激前RC上的微染色体维持（MCM）蛋白的加载，而Geminin阻断这种作用，但其背后的分子机制尚不清楚。初步结果表明，Cdt 1可以诱导大规模的染色质解凝聚时，靶向特定的染色体位点在体内，这是其次是显着富集的MCMs和PCNA（因此preRC）在这些网站。Geminin有效地和特异性地抑制Cdt 1的这种作用，并且Cdt 1的重塑仅发生在已知MCMs负载的G1细胞中。已经鉴定了两种在体内物理结合Cdt 1的染色质重塑酶：称为HBO 1的HAT和称为HDAC 11的HDAC。这些酶通过Cdt 1在体内调节染色质展开。HBO 1是Cdt 1展开染色质所必需的，而HDAC 11则反对展开。HBO 1和Cdt 1一样，也是体内MCM负载所必需的。基于此，我们假设Cdt 1和Geminin通过控制染色质进入来调节前RC处的MCM加载，并且这种进入染色质的方式是由HBO 1和HDAC 11介导的。因此，Cdt 1升高或Geminin降低，如有时在癌细胞中所见，会过度刺激染色质进入并允许不适当的MCM重新加载和重新复制。提出的三个目标将阐明HBO 1和HDAC 11在Cdt 1/Geminin控制DNA（再）复制中的作用：（i）将测试HBO 1和HDAC 11在由Cdt 1控制的生理事件中的功能。HBO 1和HDAC 11将过表达或减少，并将确定对Cdt 1诱导的再复制和MCM负载的影响。类似地，将分析HBO 1和HDAC 11对来自模型起点的DNA复制的直接控制。(ii)将测定Geminin和几种Geminin突变体调节HBO 1和HDAC 11与Cdt 1结合的能力，以及它们阻断Cdt 1诱导的染色质重塑的能力。(iii)最后，将进一步分析Cdt 1诱导的染色质重塑的机制和组蛋白的变化，研究Cdt 1在体内复合物中的新蛋白，并通过缺失结构-功能研究确定Cdt 1的功能结构域。总的来说，这些目标将阐明HBO 1和HDAC 11参与Cdt 1/Geminin功能的程度，并在分子水平上解释细胞调节复制许可/MCM加载的新机制。

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.