NSF Award
9305858
9305858 McCarrey This is an application for renewal of an ongoing project to study the regulation of X-chromosome inactivation (XCI) during spermatogenesis and oogenesis in mammals using the laboratory mouse as a model experimental system. The goal of these studies remains to understand the role of XCI during mammalian gametogenesis, and its relationship to meiosis and to the development and differentiation of functional gametes. Results from the previous grant period suggest that XCI in germ cells (gcXCI) of both sexes may differ from that in somatic cells (scXCI), especially in the stringency of repression of transcription from affected X-linked structural genes. This may, in turn, be related to the fact that gcXCI is a developmentally transient phenomenon, whereas scXCI leads to terminal repression of affected genes. Additional results indicate that a gene that may regulate XCI, the Xist gene, is expressed both in germ cells and somatic cells, suggesting that the primary mechanism of XCI may be similar in both cell types. In this application, we have proposed experiments focused at the molecular level to learn more about both the mechanism of transcriptional inactivation of X-linked structural genes and the mechanism by which gcXCI is initiated and terminated during germ cell development. We will examine two exemplary X-linked genes encoding common metabolic enzymes, phosphoglycerate kinase (Pgk-1) and hypoxanthine phosphoribosyl transferase (Hprt). We will measure parameters at the molecular level, including cessation of active transcription in spermatogenic cells and re-initiation of active transcription in oogenic cells, and potentially associated changes in nuclease sensitivity, protein-DNA interactions, and methylation at CpG dinucleotides in the promoter regions of these genes in premeiotic and meiotic germ cells. This will allow us to determine whether gcXCI affects genes in a similar manner in the germ cells of each sex, and to what extent thi s resembles the effects of scXCI in XX somatic cells. To learn more about the potential mechanism of gcXCI, we will study the Xist gene at the molecular level, including its regulation, its expression, and the developmental relationship between Xist expression and the occurrence of gcXCI. ***
