The centromere inheritance paradox is that although centromeres control genetic inheritance by directing chromosome segregation, centromeres themselves are not encoded by a particular DNA sequence. Instead, centromeres are defined epigenetically by the presence of nucleosomes containing the histone H3 variant CENP-A. That a protein is the mark poses a problem for maintaining centromere identity during oocyte development because oocytes arrest in prophase I for years or decades (in humans) before resuming the cell cycle to complete meiosis. In cycling somatic cells, CENP-A partitions between sister chromatids during DNA replication and reloads in early G1 phase, which maintains CENP-A levels between cell cycles. These mechanisms do not explain how centromere identity is maintained in the oocyte because there is no known mechanism to assemble new CENP-A nucleosomes according to the established paradigm for loading in G1. Oocytes are unique in that they arrest in prophase I, and then centromeres must be maintained until the cell cycle resumes in response to hormonal stimuli. In principle, two factors could contribute to maintain centromere identity: (1) the intrinsic stability of CENP-A nucleosomes and (2) a possible prophase I loading mechanism to replace CENP-A that is lost with age. Our preliminary data show that both make significant contributions.
Aim 1 will define CENP-A stability over the reproductive lifespan of the animal, determine the mechanism of loading during prophase I, and determine the functional significance of this loading mechanism. Our previous biophysical and structural studies showed that CENP-A nucleosomes are ~10-fold more rigid than their canonical H3 counterparts.
Aim 2 will test the hypothesis that structural rigidity of CENP-A underlies its long-term stability and centromere inheritance in the oocyte. We will identify point mutations that compromise structural rigidity and stability but localize normally and support centromere function in cycling somatic cells. We will also generate knock-in animals of selected mutants to test the functional consequences of reduced stability for maintaining centromere identity and function in oocytes. Overall, our experiments will provide the first insight into mechanisms underlying centromere inheritance in the mammalian female germline, and connect atomic level structural insights to long-term maintenance of centromere identity and ultimately reproductive fitness. Our approach is highly interdisciplinary, combining structural biology and biophysics with in vivo reproductive biology, to gain insight into how oocytes maintain structures assembled during entry into meiotic prophase I that must then function years or decades later during oocyte maturation. Such understanding has clear health implications given the decline in fertility associated with increasing maternal age and women delaying the time of childbirth.
The proposed studies will provide new information addressing fundamental questions of how essential long- lived protein structures are maintained during oocyte development with increasing maternal age. The results of these studies will likely impact on the treatment of human infertility and assisted reproduction technologies.
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