Centromeric heterochromatin formation is essential to regulate fundamental aspects of nuclear architecture, gene silencing through DNA methylation, and chromosome segregation. The appropriate regulation of these processes is critical for the successful completion of oogenesis. Although the mechanisms involved in the establishment of heterochromatin domains during oogenesis are currently unknown, chromatin-remodeling proteins are likely to be important determinants. The focus of this proposal will be on ATRX, a member of the SWI/SNF2 family of chromatin remodeling proteins. This proposal is based on the hypotheses that: 1) chromatin remodeling proteins such as ATRX are important components of centromeric heterochromatin domains in mammalian oocytes; 2) that ATRX is essential for proper chromosome segregation during meiosis; and 3) that binding of ATRX to centromeric domains is essential for DNA methylation at satellite centromeric sequences. The specific objectives include: (1) The functional characterization of the ATRX protein in mouse oocytes; (2) To determine the mechanisms by which ATRX is bound to centromeric heterochromatin during oogenesis; (3) To determine whether ATRX is involved in establishing the patterns of DNA methylation at centromeric satellite sequences during the transition from meiosis to the first mitosis. A transgenic RNA interference (RNAi) approach will be used to generate an oocyte-specific conditional knockdown of the ATRX transcript. This mouse model will be used to establish whether functional ablation of ATRX disrupts appropriate chromosome-microtubule interactions during meiosis and the transition from gamete to embryo. Moreover, whether ATRX is involved in methylation of minor or major satellite DNA sequences will be determined. These studies will provide new information regarding the relationship between DNA methylation, heterochromatin formation and chromosome segregation during female meiosis, as well as the role of meiotic centromere function in the transmission of a euploid chromosome complement.
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