One of the most remarkable gene reprogramming senarios in all of biology is the transformation of the transcriptionally silent fully grown oocyte into the totipotent embryonic stem cell. Epigenetic regulatory factors, including histone modifications and nucleosome remodeling complexes, play essential roles in this oocyte-to-embryo transition (OET). However, little is known about the specific factors involved, the program of histone modifications required for normal repropramming, or the modifications that occur at individual down-stream target genes. For over 25 years, my laboratory has used the budding yeast, Saccharomyces cerevisiae, as a model organism for dissecting the roles of histone modifications and variants in mRNA transcription, DNA replication and repair, and centromere function. Recently, our studies have expanded to focus on histone H4 acetylation by Myst2 in the preimplantation mouse embryo. Results from these preliminary experiments suggest that Myst2 is the enzyme responsible for H4 acetylation in reprogramming and zygotic gene activation. This is a major change in my research direction and success will require advanced expertise in mouse molecular genetics. To accomplish this goal, I have arranged to take a sabbatical in the laboratory of Dr. Barbara Knowles at the Jackson Laboratory. This Kirschstein-NRSA Senior Fellowship will enable a full year's residency, rather than six months, and ensure a complete training program in techniques and approaches that cannot be completed in a short tenure. During my sabbatical, I propose to address three specific aims. First, we will determine the pattern of histone modifications during OET, globally by high resolution 4Pi microscopy, and specifically at key reporter genes identified by the transcriptome analysis of the Knowles group. Second, we will engineer transgenic and knockout mouse strains that permit maternal depletion of Myst2 expression in the oocyte. We will use these strains to test directly for the requirement of Myst2 during OET. Third, we will examine the fate of H4 acetylation in Myst2- depleted embryos during OET, specifically at reporter genes and globally using tiled promoter microarrays. The results of these studies will have a significant impact on how we think about, and manage, a wide range of human health issues including assisted reproductive techniques, somatic nuclear transfer, and stem cell therapy. ? ? ?
