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Cdt1和Geminin如何在分子水平上控制复制前复合体的组装，以及它们在这一过程中作用背后的机制如何产生异常再复制的倾向。Cdt1刺激迷你染色体维持（MCM）蛋白在preRCs上的负载，而gemini阻断了这种作用，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点时，可以诱导大规模的染色质去浓缩，随后在这些位点上显著富集mcm和PCNA（以及prcs）。Geminin有效且特异性地抑制Cdt1的这种作用，并且Cdt1的重塑仅在已知mcm加载时发生在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：称为HBO1的HAT和称为HDAC11的HDAC。这些酶在体内通过Cdt1调节染色质展开。HBO1是Cdt1展开染色质所必需的，而HDAC11则反对染色质展开。与Cdt1一样，HBO1也是体内MCM加载所必需的。基于此，我们假设Cdt1和Geminin通过控制染色质通路来调节MCM在preRCs上的负载，而这种染色质通路是由HBO1和HDAC11介导的。因此，在癌细胞中，Cdt1升高或gemini减少会过度刺激染色质通路，并允许不适当的MCM重新加载和再复制。提出的三个目标将阐明HBO1和HDAC11在Cdt1/Geminin控制DNA（再）复制中的作用：(i)将测试HBO1和HDAC11在Cdt1控制的生理事件中的功能。HBO1和HDAC11会过表达或减少，并确定对cdt1诱导的再复制和MCM加载的影响。同样，HBO1和HDAC11对模型起源DNA复制的直接控制将被分析。（ii）将确定Geminin和几个Geminin突变体调节HBO1和HDAC11与Cdt1结合的能力，以及它们阻断Cdt1诱导的染色质重塑的能力。（iii）最后，将进一步分析Cdt1诱导染色质重塑的机制和组蛋白变化，研究体内与Cdt1复合物中的新蛋白，并通过缺失结构-功能研究确定Cdt1负责染色质重塑作用的功能域。总的来说，这些目标将阐明HBO1和HDAC11参与Cdt1/Geminin功能的程度，并在分子水平上解释细胞调节复制许可/MCM装载的新机制。

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. PUBLIC HEALTH RELEVANCE: Human cancers often display abnormal chromosomal numbers or rearrangements that lead to unwanted overexpression of genes that stimulate growth, or loss of genes that normally limit growth. This characteristic of tumors, called genomic instability, derives from several sources, one of which being abnormal re-replication of chromosomes or parts of chromosomes. The preliminary results and aims of this proposal directly address a poorly understood mechanism involved in causing abnormal re-replication, and will clarify the molecular and biochemical events involved in one type of genomic instability that can lead to tumor formation or progression.