Role of Transcription Pausing in Hematopoietic Stem Cell Development

 描述（由申请人提供）：脊椎动物胚胎中造血干细胞（HSC）的特化受到RNA聚合酶II（Pol II）介导的转录的严格调控，该转录通过多个步骤进行，包括起始/激活、延伸和终止。理解HSC发育的转录机制为人类血液疾病的病理生理学提供了重要的见解。近年来的大量研究表明，转录参与的Pol II通常在启动子下游20- 40 bp处停滞。Pol II的启动子近端暂停由暂停因子DSIF和NELF介导，并且需要正延伸因子P-TEFb释放。中断暂停延长开关往往会导致细胞特异性的缺陷，这表明在细胞命运决定的Pol II暂停的作用。与此一致，我以前的工作表明，破坏Pol II的延伸， 特异性阻断斑马鱼胚胎中红系祖细胞的分化。在这个提议中，我扩展了这项工作，以探索暂停到延长过渡如何有助于HSC的细胞命运决定，以及哪些信号通路调节这一过程。我们已经使用斑马鱼遗传学来证明胚胎HSC发育需要良好控制的暂停Pol II的释放。暂停因子NELF或DSIF的丧失导致HSC的显著减少，这可以通过抑制P-TEFb活性减少Pol II延伸来恢复。使用生物化学方法，我们已经确定了可能促进Pol 与停顿因子相互作用的停顿和延长。此外，我们已经进行了化学筛选，以挽救暂停缺陷斑马鱼胚胎中的HSC缺陷，并确定了参与不同信号通路的多种化合物。基于这些初步的数据，我们假设，一个严格控制的Pol II延伸与转录调节因子合作， 和发育信号来调节脊椎动物胚胎中HSC的出现。我们将使用一套互补的模型系统，包括斑马鱼和人类血细胞培养系统的综合方法来测试这一假设。目的1将使用遗传学，基因组学和生物化学方法的组合，以确定的机制，其中Pol II暂停因子调节HSC的形成，并确定主要的HSC的目标Pol II暂停调节。在目标2中，我们将确定与DSIF合作的相互作用伙伴，以控制Pol II暂停造血基因。目的3将集中在HSC发育过程中类固醇激素受体信号通路和Pol II暂停/延长机制之间的功能相互作用。这些目标的完成将提供重要的见解，了解发展中的作用，Pol II暂停，也使我们能够确定新的监管机构的HSC规格。鉴于在各种人类疾病中转录延长失调的参与，这些研究将促进我们对其在这些疾病的发病机制和进展中的作用的理解，并且还可以确定可用于开发新的靶向治疗策略的候选基因或途径。

 DESCRIPTION (provided by applicant): The specification of hematopoietic stem cells (HSCs) in vertebrate embryos is tightly regulated by RNA polymerase II (Pol II)-mediated transcription, which proceeds through multiple steps including initiation/activation, elongation and termination. Understanding the transcriptional mechanism of HSC development provides significant insight into the pathophysiology of human blood diseases. Extensive studies in recent years have demonstrated that transcriptionally engaged Pol II often stalls at 20-40bp downstream of the promoter. This promoter-proximal pausing of Pol II is mediated by pausing factors DSIF and NELF, and requires the positive elongation factor P-TEFb for release. Disrupted pausing-to-elongation switch often causes cell-specific defects, suggesting a role of Pol II pausing in cell fate determination. In line with this, my previous work showed that disruption of Pol II elongation specifically blocked the differentiation of erythroid progenitors in zebrafish embryos. In this proposal, I extend the work to explore how the pausing-to-elongation transition contributes to cell fate determination of HSCs, and which signaling pathways regulate this process. We have used zebrafish genetics to demonstrate that embryonic HSC development requires a well-controlled release of paused Pol II. Loss of pausing factors NELF or DSIF causes significant reduction of HSCs, which can be restored by reducing Pol II elongation via inhibition of P-TEFb activity. Using biochemical approaches, we have identified proteins that potentially facilitate Pol II pausing and elongation by interacting with pausing factors. Furthermore, we have performed a chemical screen to rescue the HSC defect in pausing-deficient zebrafish embryos and identified multiple compounds involved in distinct signaling pathways. Based on these preliminary data, we hypothesize that a tightly controlled Pol II elongation cooperates with transcription regulators and developmental signals to regulate HSC emergence in vertebrate embryos. We will test this hypothesis using an integrated approach with a complementary set of model systems including both zebrafish and human blood cell culture systems. Aim 1 will use a combination of genetic, genomic and biochemical approaches to determine the mechanisms by which Pol II pausing factors regulate HSC formation and identify primary HSC targets regulated by Pol II pausing. In Aim 2, we will identify interacting partners cooperating with DSIF to control Pol II pausing on hematopoietic genes. Aim 3 will focus on the functional interplay between steroid hormone receptor signaling pathways and the Pol II pausing/elongation machinery during HSC development. Completion of these aims will provide important insights for understanding the developmental role of Pol II pausing and also allow us to identify novel regulators of HSC specification. Given the involvement of disregulated transcription elongation in a variety of human disorders, these studies will advance our understanding of its role in the pathogenesis and progression of these maladies and may also identify candidate genes or pathways that can be used for developing novel targeted treatment strategies.