Epigenetic Regulation of Gene Expression During Early Mouse Embryogenesis

描述(申请人提供)：所有生物学中最显著的基因重新编程的前辈之一是将转录沉默的完全成熟的卵母细胞转变为全能的胚胎干细胞。表观遗传调节因子，包括组蛋白修饰和核小体重塑复合体，在卵母细胞到胚胎的转变(OET)中起着至关重要的作用。然而，对于涉及的具体因素，正常重排所需的组蛋白修饰程序，或者发生在单个下游靶基因的修饰，我们知之甚少。25年来，我的实验室一直使用萌芽酵母酿酒酵母作为模式生物，剖析组蛋白修饰和变异在mRNA转录、DNA复制和修复以及着丝粒功能中的作用。最近，我们的研究已经扩展到组蛋白H4在小鼠着床前胚胎中被Myst2乙酰化。这些初步实验的结果表明，Myst2是负责重编程和合子基因激活中H4乙酰化的酶。这是我研究方向的一个重大变化，成功将需要老鼠分子遗传学方面的高级专业知识。为了实现这个目标，我安排去杰克逊实验室的芭芭拉·诺尔斯博士的实验室休假。这项Kirschstein-NRSA高级奖学金将实现一整年的驻院实习，而不是六个月，并确保在短期内无法完成的技术和方法方面的完整培训计划。在我的休假期间，我提议解决三个具体目标。首先，我们将通过高分辨率4PI显微镜确定组蛋白在OET期间的修饰模式，特别是在Knowles小组的转录组分析中确定的关键报告基因。其次，我们将设计转基因和基因敲除小鼠品系，允许母体去除卵母细胞中Myst2的表达。我们将在OET期间使用这些菌株直接检测Myst2的要求。第三，我们将使用平铺启动子微阵列来研究Myst2缺失胚胎中H4乙酰化的命运，特别是在报告基因和全球范围内。这些研究的结果将对我们如何思考和管理广泛的人类健康问题产生重大影响，包括辅助生殖技术、体细胞核移植和干细胞治疗。

DESCRIPTION (provided by applicant): 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.