Regulation of Growth Factors and Embryogenesis

描述（由申请人提供）：本研究的目的是扩大我们对胚胎发生过程中起关键作用的基因调控网络的理解。最近的研究强烈认为，转录因子Sox 2和Oct-3/4各自表现出协调哺乳动物胚胎发生和胚胎干细胞（ES）自我更新的主调节因子的重要标志。Sox 2和Oct-3/4已经显示协同工作以协调在胚胎发生期间和由ES细胞表达的至少6个基因（FGF-4、UTF 1、Fbx 15、Nanog、Sox 2和Oct-3/4）的转录。这些基因被称为Sox 2：Oct-3/4靶基因。本文提出的工作基于3个新的发现和假设。首先，我们最近发现了19个可能是Sox 2：Oct-3/4基因调控网络的一部分的额外基因。考虑到6个已知的Sox 2：Oct-3/4靶基因中的4个，可能5个是胚胎发生所必需的，我们的中心假设是大多数Sox 2：Oct-3/4靶基因在胚胎发生过程中起关键作用，并且许多是ES细胞自我更新所需的。第二，6个已知的Sox 2：Oct-3/4靶基因的表达显示非常严重地依赖于每个基因的增强子中的Sox 2和Oct-3/4的紧密间隔的DNA结合位点。这些和其他研究使我们假设Sox 2：Oct-3/4基因调控网络由一组共享的共激活因子协调控制，其中至少有一些本身在分化过程中受到调控，因此是细胞命运决定的关键参与者。第三，尽管已知Sox 2与Oct-3/4组合刺激干细胞中Sox 2：Oct-3/4靶基因的表达，但我们确定，当Sox 2过表达时，6个Sox 2：Oct-3/4靶基因中的至少5个（包括Sox 2基因本身）被抑制。这些发现导致我们假设Sox 2：Oct-3/4靶基因受到负反馈环和正反馈环的仔细调节，以确保这些基因以胚胎发生所需的水平表达。提出了三个具体目标来检验这些假设。1)研究一组新发现的推定Sox 2：Oct-3/4靶基因对ES细胞自我更新和哺乳动物发育的影响。2)检查特异性共激活因子在Sox 2：Oct-3/4靶基因表达中的作用。3)确定Sox 2过表达抑制Sox 2：Oct-3/4靶基因表达的分子机制。总之，这些研究将提供一个关键的基因调控网络，协调胚胎发生和控制ES细胞的自我更新的机制的理解。因此，这些研究将提供新的见解，并对发育生物学，再生医学和癌症生物学产生重大影响。

DESCRIPTION (provided by applicant): The goal of this study is to broaden our understanding of a gene regulatory network that plays key roles during embryogenesis. Recent studies argue strongly that the transcription factors Sox2 and Oct-3/4 each exhibit important hallmarks of master regulators that orchestrate mammalian embryogenesis and the self-renewal of embryonic stem (ES) cells. Sox2 and Oct-3/4 have been shown to work together cooperatively to coordinate the transcription of at least 6 genes (FGF-4, UTF1, Fbx15, Nanog, Sox2 and Oct-3/4) expressed during embryogenesis and by ES cells. These genes are referred to as Sox2:Oct-3/4 target genes. The work proposed here is based on 3 novel findings and hypotheses. First, we recently identified 19 additional genes that are likely to be part of the Sox2:Oct-3/4 gene regulatory network. Given that 4, and possibly 5, of the 6 known Sox2:Oct-3/4 target genes are essential for embryogenesis it is our central hypothesis that the majority of Sox2:Oct-3/4 target genes play critical roles during embryogenesis, and that many are required for the self-renewal of ES cells. Second, the expression of the 6 known Sox2:Oct-3/4 target genes was shown to be very heavily dependent on closely spaced DNA binding sites for Sox2 and Oct-3/4 in the enhancer of each gene. These and other studies led us to hypothesize that the Sox2:Oct-3/4 gene regulatory network is controlled coordinately by a shared set of co-activators, at least some of which are themselves regulated during differentiation and, thus, are key players in cell fate determination. Third, although Sox2, in combination with Oct-3/4, is known to stimulate the expression of Sox2:Oct-3/4 target genes in stem cells, we determined that at least 5 of the 6 Sox2:Oct-3/4 target genes, including the Sox2 gene itself, are inhibited when Sox2 is overexpressed. These findings led to our hypothesis that Sox2:Oct-3/4 target genes are regulated carefully by a negative, as well as a positive, feedback loop to ensure that these genes are expressed at levels required for embryogenesis. Three Specific Aims are proposed to test these hypotheses. 1) Examine a newly identified set of putative Sox2:Oct-3/4 target genes for their effects on the self-renewal of ES cells and mammalian development. 2) Examine the roles of specific co-activators in the expression of Sox2:Oct-3/4 target genes. 3) Determine the molecular mechanisms by which Sox2 overexpression inhibits the expression of Sox2:Oct-3/4 target genes. Together, these studies will provide a mechanistic understanding of a critical gene regulatory network that orchestrates embryogenesis and controls the self-renewal of ES cells. As such, these studies will provide novel insights and have significant impact on developmental biology, regenerative medicine and cancer biology.