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.
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.
