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

• 批准号: 8458596
• 负责人: Mark G. Alexandrow
• 金额: $ 28.44万
• 依托单位: H. LEE MOFFITT CANCER CTR & RES INST
• 依托单位国家: 美国
• 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
• 来源: https://reporter.nih.gov/project-details/8458596
• 关键词: Address; Binding; Biochemical; Cells; Characteristics; Chromatin; Chromosomes; Complex; DNA; DNA Sequence Rearrangement; DNA biosynthesis; Eels; Enzymes; Event; G1 Phase; Geminin; Genes; Genomic Instability; Growth; Histone Deacetylase; Histones; Human; Lead; Maintenance; Malignant Neoplasms; Mediating; Modeling; Molecular; Physiological; Pre-Replication Complex; Process; Proteins; Replication Licensing; Role; Site; Source; Structure; Testing; base; cancer cell; chromatin remodeling; chromosome replication; in vivo; mutant; novel; overexpression; public health relevance; tumor; tumorigenesis

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）：该提案的长期目标是确定CDT1和Geminin在分子水平上的功能如何控制恢复前复合物的组装，以及其在此过程中角色背后的机制如何创造异常重复的倾向。 CDT1刺激PRERCS上的微型染色体维持（MCM）蛋白的负载，而双子素会阻止这种作用，但其背后的分子机制尚不清楚。初步结果表明，当靶向体内特定染色体位点时，CDT1可以诱导染色质大规模取消，然后在这些位点急剧富集MCMS和PCNA（因此）。双子素有效，有效地抑制了CDT1的这种影响，并且CDT1的重塑仅在已知MCMS加载时才发生在G1细胞中。已经鉴定出了两种物理结合CDT1的染色质重塑酶：一个称为HBO1的帽子，一个称为HDAC11的HDAC。这些酶调节CDT1在体内展开的染色质。 HBO1是通过CDT1展开染色质所必需的，而HDAC11反对展开。与CDT1一样，HBO1在体内也需要MCM加载。基于此，我们假设CDT1和GEMININ通过控制染色质的访问来调节PRERC的MCM加载，并且这种对染色质的访问是由HBO1和HDAC11介导的。因此，升高的CDT1或降低的双子素（有时在癌细胞中看到）会过度刺激染色质，并允许不适当的MCM重新加载和重复复制。提出的三个目标将阐明HBO1和HDAC11在CDT1/Geminin对DNA的控制（RE）复制中的作用：（i）HBO1和HDAC11在由CDT1控制的生理事件中的功能。 HBO1和HDAC11将过表达或减少，并将确定对CDT1诱导的重新复制和MCM加载的影响。同样，将分析HBO1和HDAC11对来自模型来源的DNA复制的直接控制。 （ii）将确定Geminin和几种双子素突变体调节HBO1和HDAC11与CDT1的结合的能力，它们的能力也将阻止CDT1诱导的染色质重塑的能力。 （iii）最后，将进一步分析CDT1诱导的染色质重塑中涉及的机制和组蛋白变化，将研究带有CDT1 IN VIVO的复合物中的新型蛋白质，并且通过DELETION结构函数研究将确定CDT1的CDT1功能结构域。总的来说，这些目标将阐明HBO1和HDAC11参与CDT1/GEMININ功能的程度，并在分子级解释一种新型机制，细胞调节复制许可/MCM负载。