Role of Transcription Pausing in Hematopoietic Stem Cell Development

 描述(申请人提供)：脊椎动物胚胎中的造血干细胞(HSCs)的规格受到RNA聚合酶II(Pol II)介导的转录的严格调控，转录过程经历了多个步骤，包括启动/激活、延长和终止。了解HSC发育的转录机制有助于深入了解人类血液疾病的病理生理学。近年来的广泛研究表明，转录参与的POLII经常在启动子下游20-40bp处停滞。POL II的这种启动子-近端的暂停是由暂停因子DSIF和NELF介导的，并且需要正的延伸因子P-TEFb来释放。中断的暂停到延长的转换通常会导致细胞特有的缺陷，这表明POLII暂停在细胞命运决定中发挥了作用。与此一致的是，我之前的工作表明，Pol II的伸长中断 特异性阻断斑马鱼胚胎中红系祖细胞的分化。在这项提议中，我将继续探索暂停到延长的转变如何有助于决定HSCs的细胞命运，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证明了胚胎HSC的发育需要良好控制地释放暂停的Pol II。失去暂停因子NELF或DSIF会导致HSCs显著减少，这可以通过抑制P-TEFb活性来减少Pol II的伸长来恢复。使用生化方法，我们已经确定了可能促进POL的蛋白质 第二，停顿与伸长与停顿因素的交互作用。此外，我们进行了化学筛选以挽救暂停缺陷斑马鱼胚胎中的HSC缺陷，并鉴定了多种参与不同信号通路的化合物。基于这些初步数据，我们假设受到严格控制的POL II延伸与转录调控因子协同作用 以及调节脊椎动物胚胎中HSC出现的发育信号。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，使用集成的方法来验证这一假设。目的1将结合遗传学、基因组学和生物化学方法来确定Pol II停顿因子调节HSC形成的机制，并确定Pol II停顿调节的主要HSC靶点。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将重点介绍类固醇激素受体信号通路与Pol II暂停/延长机制在HSC发育过程中的功能相互作用。这些目标的完成将为理解POL II停顿的发育作用提供重要的见解，并使我们能够识别HSC规范的新调节因子。鉴于转录伸长失控参与了多种人类疾病，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所起作用的理解，并可能识别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.