The Role of PHF6 in HSC self-renewal and myeloid expansion

摘要/项目摘要 造血干细胞（HSC）需要转录因子（TF）和表观遗传学的微调合作 监管机构保持正常的自我更新。精确控制这种能力对于抑制异常的 增殖因此，了解自我更新的调节对于了解正常的 和肿瘤性造血。PHF 6是一种神秘的白血病突变的染色质结合蛋白， 抑制HSC自我更新，为解剖潜在的调控网络提供了一个有吸引力的模型。 这个提议的目的是阐明正常自我更新和异常自我更新之间的机械联系。 通过解剖PHF 6如何调节关键造血TF结合的增强子来研究细胞增殖。 我们的体内研究表明，Phf 6造血敲除特异性地增加HSC自我更新， 使下游造血基本上不受干扰。初步实验表明，组成型HOXA 9 表达与Phf 6损失协同作用，引起快速、致死的祖细胞扩增。因此，我们确定了 深刻对比了Phf 6丢失的稳态和HOXA 9驱动的表型，提供了一个理想的系统， 研究HSC自我更新如何在异常增殖中被选择。我们的初步数据显示PHF 6结合 并抑制由TF RUNX 1、PU.1、IRF 8共同占据的增强子。因此，我们确定了一种机制， PHF 6活性的基础（与关键造血TF的染色质共占据）。 我们假设PHF 6通过抑制HSC的自我更新和异常的髓系祖细胞扩增， 调节RUNX 1、PU.1和IRF 8结合的增强子的共同机制。 具体目标1：我们将确定PHF 6在抑制HSC自我更新和髓系祖细胞中的作用。 通过确定Phf 6缺失是否加速HOXA 9驱动的骨髓祖细胞扩增， R274 Q突变是否废除了HSC和髓系祖细胞中的PHF 6功能，以及Phf 6缺失是否 激活RUNX 1/PU.1/IRF 8结合增强子。目标1中的实验将促进我们对 HSC生物学通过将自我更新与异常扩增通过核心调控回路下游的 PHF 6。 具体目标2：我们将确定PHF 6活性的体外机制， RUNX 1/PU.1/IRF 8将PHF 6募集至染色质，R274 Q突变是否消除PHF 6染色质结合， 以及PHF 6是否募集额外的复合物来抑制增强子活性。目标2中的实验将 阐明了从关键TF将PHF 6募集到染色质，到下游的事件序列。 PHF 6对增强子功能的影响，从而对基因表达的影响。 这些研究，如果成功，将明确一个疾病相关的机制，连接HSC自我更新异常， 通过PHF 6与造血TF联合调节增强子来扩增祖细胞。这 将是我们理解HSC自我更新的表观遗传调控的重要进展。

Abstract / Project Summary Hematopoietic stem cells (HSCs) require fine-tuned cooperation of transcription factors (TFs) and epigenetic regulators to maintain normal self-renewal. Precise control of this ability is essential to suppress aberrant proliferation. Understanding the regulation of self-renewal is therefore crucial for gaining insight into normal and neoplastic hematopoiesis. PHF6, an enigmatic, leukemia-mutated, chromatin-binding protein specifically represses HSC self-renewal, providing an attractive model for dissecting the underlying regulatory network. The goal of this proposal is to illuminate a mechanistic link between normal self-renewal and aberrant proliferation through dissection of how PHF6 modulates enhancers bound by key hematopoietic TFs. Our in vivo studies show that Phf6 hematopoietic knockout specifically increases HSC self-renewal while leaving downstream hematopoiesis largely unperturbed. Pilot experiments indicate that constitutive HOXA9 expression cooperates with Phf6 loss to cause rapid, lethal progenitor expansion. We have thus identified profoundly contrasting homeostatic and HOXA9-driven phenotypes of Phf6 loss, providing an ideal system to study how HSC self-renewal is co-opted in aberrant proliferation. Our preliminary data show that PHF6 binds and represses enhancers co-occupied by the TFs RUNX1, PU.1, IRF8. We have thus identified a mechanistic basis for PHF6 activity (chromatin co-occupancy with key hematopoietic TFs). We hypothesize that PHF6 represses HSC self-renewal and aberrant myeloid progenitor expansion through a common mechanism of modulating enhancers bound by RUNX1, PU.1, and IRF8. Specific Aim 1: We will determine the role of PHF6 in repressing HSC self-renewal and myeloid progenitor expansion in vivo by determining whether Phf6 loss accelerates HOXA9-driven myeloid progenitor expansion, whether R274Q mutation abrogates PHF6 functions in HSCs and myeloid progenitors, and whether Phf6 loss activates RUNX1/PU.1/IRF8-bound enhancers. The experiments in Aim 1 will advance our understanding of HSC biology by linking self-renewal to aberrant expansion through a core regulatory circuit downstream of PHF6. Specific Aim 2: We will determine the mechanism of PHF6 activity in vitro by determining whether RUNX1/PU.1/IRF8 recruit PHF6 to chromatin, whether R274Q mutation abrogates PHF6 chromatin binding, and whether PHF6 recruits additional complexes to repress enhancer activity. The experiments in Aim 2 will illuminate the sequence of events from recruitment of PHF6 to chromatin by key TFs, to the downstream effects of PHF6 on enhancer function and consequently on gene expression. These studies, if successful, will pinpoint a disease-relevant mechanism linking HSC self-renewal to aberrant progenitor expansion through modulation of enhancers by PHF6 in conjunction with hematopoietic TFs. This will be an important advance in our understanding of the epigenetic regulation of HSC self-renewal.