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 监管机构要保持正常的自我更新。对这一能力的精确控制对于抑制异常是必不可少的 扩散。因此，了解自我更新的规律对于洞察正常至关重要 和肿瘤性造血。PHF6，一种神秘的、白血病突变的染色质结合蛋白 抑制HSC的自我更新，为剖析潜在的监管网络提供了一个有吸引力的模型。 这项提议的目的是阐明正常的自我更新和变态之间的机械联系。 通过解剖PHF6如何调节关键的造血因子结合的增强剂来促进细胞增殖。 我们的体内研究表明，Phf6造血基因敲除特异性地增加了HSC的自我更新，而 使下游的造血系统基本不受干扰。中试实验表明，本构HOXA9 基因的表达与Phf6的缺失协同作用，导致快速、致命的祖细胞扩张。因此，我们确定了 深刻对比动态平衡和HOXA9驱动的Phf6丢失表型，提供了一个理想的系统 研究HSC的自我更新是如何参与异常增殖的。我们的初步数据显示PHF6结合了 并抑制由转录因子RUNX1、PU.1、IRF8共同占据的增强子。因此，我们确定了一种机械论 PHF6活性的基础(染色质与关键的造血因子共占)。 我们假设PHF6通过一种途径抑制HSC的自我更新和异常的髓系祖细胞扩增。 调节RUNX1、PU.1和IRF8结合的增强子的共同机制。 具体目标1：我们将确定PHF6在抑制HSC自我更新和髓系祖细胞中的作用 通过确定Phf6缺失是否加速HOXA9驱动的髓系祖细胞的扩张， R274Q突变是否取消了HSCs和髓系祖细胞的PHF6功能，以及Phf6是否丢失 激活RUNX1/PU.1/IRF8绑定的增强子。目标1中的实验将增进我们对 HSC生物学通过下游的核心调控回路将自我更新与异常扩张联系起来 PHF6。 具体目标2：我们将通过确定PHF6在体外的活性机制 RUNX1/PU.1/IRF8使PHF6与染色质结合，R274Q突变是否取消了PHF6染色质结合， 以及PHF6是否招募更多的复合体来抑制增强子的活性。目标2中的实验将 阐明从关键的转录因子招募PHF6到染色质，再到下游的一系列事件 PHF6对增强子功能的影响，从而影响基因表达。 这些研究，如果成功，将确定一种与疾病相关的机制，将HSC的自我更新与异常联系起来 通过PHF6与造血因子联合调节增强剂扩增祖细胞。这 这将是我们理解HSC自我更新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.