Mechanisms of Transcriptional Regulation in Stem Cells

描述(由申请人提供)：干细胞必须处理两种关键但相互对立的力量：持续自我更新的需要和对基因表达大规模变化的分化信号做出反应的能力。许多研究表明，染色质结构对细胞增殖和分化的调控有很大影响。这项提案的总体目标是确定负责在干细胞中建立和维持活性和非活性染色质模式的分子机制，从而确定基因表达模式。这一提议的一个极具创新性的方面是将染色质免疫沉淀与几种不同类型的小鼠基因组微阵列的使用相结合。第一组阵列将包含斑点CpG岛和其他在老鼠和人类基因组之间保守的调节区。第二种类型的阵列将由包含整个小鼠染色体的寡核苷酸组成。我们将通过使用RNA聚合酶II和改良组蛋白的抗体(目标1)，通过使用阵列来鉴定活性启动子和非活性启动子来开始我们的研究。我们将检验这一假设，即不同形式的基因调控在胚胎干细胞和类胚体中占主导地位。这些实验将提供未分化胚胎干细胞和分化胚胎干细胞中活跃染色质和非活跃染色质的数量和位置的全球快照，还将提供哪些基因以分化特有的方式开启和关闭的信息。在目标2A中，我们将确定Oct4的靶基因，Oct4是一种已知对维持ES细胞的多能性至关重要的转录因子。在确定Oct4靶基因后，我们将描述Oct4调控这些基因的机制，特别是验证Oct4在ES细胞中作为转录抑制因子的假设。在目标1中获得的关于活性染色质与非活性染色质的特征的知识将对这些研究至关重要。在目标2B中，我们将描述对Oct4基因进行干细胞特异性调控的分子机制。

DESCRIPTION (provided by applicant): Stem cells must deal with two critical, yet opposing, forces; the need for continual self-renewal and the ability to respond to differentiation signals with large scale changes in gene expression. Many studies have established that regulation of proliferation and differentiation is heavily influenced by chromatin structure. The overall goals of this proposal are to determine the molecular mechanisms responsible for establishing and maintaining patterns of active and inactive chromatin, and thus patterns of gene expression, in stem cells. A highly innovative aspect of this proposal is the combination of chromatin immunoprecipitation with the use of several different types of mouse genomic microarrays. The first set of arrays will contain spotted CpG islands and other regulatory regions that are conserved between the mouse and human genomes. The second type of array will consist of oligonucleotides that encompass an entire mouse chromosome. We will begin our studies by employing the arrays to identify active vs. inactive promoters, using antibodies to RNA polymerase II and modified histones (Aim 1). We will test the hypothesis that different forms of gene regulation predominate in embryonic stem cells vs. in embryoid bodies. These experiments will provide a global snapshot of the amount and location of active vs. inactive chromatin in undifferentiated vs. differentiating embryonic stem cells and will also provide information as to which genes turn on and off in a differentiation-specific manner. In Aim 2A, we will identify targets genes of Oct4, a transcription factor that is known to be critical for maintaining pluripotency of ES cells. After identification of Oct4 target genes, we will characterize the mechanisms by which Oct4 regulates these genes, specifically testing the hypothesis that Oct4 functions as a transcriptional repressor in ES cells. The knowledge gained in Aim 1 concerning the characteristics of active vs. inactive chromatin will be critical for these studies. In Aim 2B, we will characterize the molecular mechanisms responsible for the stem cell-specific regulation of the Oct4 gene.