Developmental gene poising by Oct transcription factors

项目概要： Oct 4是多能性的主要转录调节因子。有趣的是，胚胎干细胞（ESC）和 诱导多能干细胞（iPSC）共表达Oct 4以及两种旁系同源物Oct 1和Oct 6。这些 蛋白质在体外结合相同的序列，在体内结合许多相同的基因。其功能在预- 植入胚胎和ESC，以及紧随分化之后，是未知的。虽然早期很关键 当ESC首先失去多能性并且Oct 4丢失时，发育命运决定被做出， 在分子水平上起作用仍然是一个黑盒子。例如，在发展上保持平衡的Oct 4目标可以是 在10月4日失去后的许多天，引发了如何维持平衡的问题。这项建议 专注于Oct 1/Oct 6/Oct 4在多能细胞和分化早期的协作。我们之前 表明Oct 1要么通过与NuRD结合抑制基因转录，要么在细胞中维持沉默基因。 通过与Jmjd 1a/KDM 3A相关联以减少局部组蛋白， H3 K9甲基化。最近，我们发现Oct 1在未分化的ESCs中表达，但在 分化是诱导发育特异性基因和抑制发育特异性基因所必需的。 替代发展谱系。因此，Oct 1缺陷型ESC表现正常，但表现出严重的 差异化的缺陷。Oct 1在ESC中不占据这些靶点，可能是由于来自 更丰富的Oct 4蛋白。相反，随着ESC分化，Oct 1占据了这些基因，而Oct 4丢失。 我们提出了一个“交接”模型，即沉默但稳定的基因被Oct 1占据，可能还有Oct 6， 保持正常的发育基因表达。我们将通过识别共同的和独特的来测试这个模型 Oct 1/Oct 4/Oct 6在分化ESCs中的全基因组靶向位点，决定辅因子的传递是否 伴随着Oct 1在分化细胞中Oct 4的替代，并研究Oct 1介导的对 谱系不适当基因，其在Oct 1缺陷型ESC分化时异位表达。 目的1：测试切换模型的有效性。 目的2：定义Oct 1和Oct 4在多能细胞中发育诱导靶点使用的辅因子， 分化后立即 目的3：确定Oct 1抑制谱系不适当基因的机制。

PROJECT SUMMARY: Oct4 is a master transcriptional regulator of pluripotency. Intriguingly, embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) co-express Oct4 together with two paralogs, Oct1 and Oct6. These proteins bind the same sequences in vitro and many of the same genes in vivo. Their functions in pre- implantation embryos and ESCs, and immediately following differentiation, are unknown. Though critical early developmental fate decisions are made as ESCs first lose pluripotency and Oct4 is lost, how this process works at a molecular level remains a black box. For example, developmentally poised Oct4 targets can be induced many days after loss of Oct4 itself, raising the question of how poising is maintained. This proposal focuses on Oct1/Oct6/Oct4 collaboration in pluripotent cells and early during differentiation. We previously showed that Oct1 either represses gene transcription, by associating with NuRD, or maintains silent genes in a configuration that allows for later expression, by associating with Jmjd1a/KDM3A to decrease local histone H3K9 methylation. More recently we showed that Oct1 is dispensable in undifferentiated ESCs, but following differentiation is required both to induce developmental-specific genes, and to suppress genes specific for alternative developmental lineages. As a consequence, Oct1 deficient ESCs appear normal but show severe defects upon differentiation. Oct1 does not occupy these targets in ESCs, presumably due to competition from the more abundant Oct4 protein. Instead, Oct1 occupies these genes as ESCs differentiate and Oct4 is lost. We propose a “handoff” model whereby silent but poised genes are occupied by Oct1, and possibly Oct6, to maintain proper developmental gene expression. We will test this model by identifying common and unique Oct1/Oct4/Oct6 target sites genome-wide in differentiating ESCs, determining whether handoff of cofactors accompanies Oct4 replacement by Oct1 in differentiating cells, and studying Oct1-mediated repression of lineage-inappropriate genes, which are ectopically expressed upon differentiation of Oct1 deficient ESCs. Aim 1: Test the validity of the handoff model. Aim 2: Define the cofactors used by Oct1 and Oct4 at developmentally inducible targets in pluripotent cells and immediately after differentiation. Aim 3: Determine the mechanism by which Oct1 represses lineage-inappropriate genes.