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

PHF6 在 HSC 自我更新和骨髓扩张中的作用

基本信息

批准号：
10095975
负责人：
Vikram R. Paralkar
金额：
$ 53.64万
依托单位：
UNIVERSITY OF PENNSYLVANIA
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-01-06 至 2024-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10095975
关键词：
Binding Binding Proteins Biotinylation Blood Cells Bone Marrow Bone Marrow Diseases Cell Line ChIP-seq Chromatin Complex Data Disease Dissection Enhancers Epigenetic Process Equilibrium Event Flow Cytometry Gene Expression Genes Genetic Genetic Transcription Goals HOXA9 gene Health Hematopoiesis Hematopoietic Hematopoietic stem cells Histology In Vitro Informal Social Control Knock-out Life Link Modeling Mus Mutate Mutation Myelogenous Names Nature Phenotype Point Mutation Polymerase Process Proteins RNA RUNX1 gene Recurrence Regulation Role System Testing clinically relevant epigenetic regulation experimental study hematopoietic stem cell self-renewal histone modification in vitro activity in vivo insight leukemia mutant neoplastic novel prevent progenitor recruit self-renewal stem cell biology time use tool transcription factor transcriptome sequencing

项目摘要

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.

摘要/项目摘要造血干细胞 (HSC) 需要转录因子 (TF) 和表观遗传的微调合作调节器维持正常的自我更新。精确控制这种能力对于抑制异常至关重要增殖。因此，了解自我更新的调节对于深入了解正常现象至关重要。和肿瘤性造血。 PHF6，一种神秘的白血病突变染色质结合蛋白抑制 HSC 自我更新，为剖析底层监管网络提供了一个有吸引力的模型。该提案的目的是阐明正常自我更新与异常自我更新之间的机制联系通过剖析 PHF6 如何调节关键造血 TF 结合的增强子来研究增殖。我们的体内研究表明 Phf6 造血基因敲除特异性地增加 HSC 的自我更新，同时下游造血基本不受干扰。初步实验表明，本构型 HOXA9 表达与 Phf6 损失协同作用，导致快速、致命的祖细胞扩增。我们由此确定了深刻对比 Phf6 损失的稳态和 HOXA9 驱动的表型，提供了一个理想的系统研究 HSC 自我更新如何参与异常增殖。我们的初步数据表明 PHF6 结合并抑制 TF RUNX1、PU.1、IRF8 共同占据的增强子。因此我们确定了一个机制 PHF6 活性的基础（染色质与关键造血转录因子的共存）。我们假设 PHF6 通过抑制 HSC 自我更新和异常骨髓祖细胞扩张调节由 RUNX1、PU.1 和 IRF8 结合的增强子的常见机制。具体目标 1：我们将确定 PHF6 在抑制 HSC 自我更新和骨髓祖细胞中的作用通过确定 Phf6 丢失是否加速 HOXA9 驱动的骨髓祖细胞扩张来进行体内扩张， R274Q 突变是否会消除 HSC 和骨髓祖细胞中的 PHF6 功能，以及 Phf6 是否丧失激活 RUNX1/PU.1/IRF8 结合增强子。目标 1 中的实验将加深我们对 HSC 生物学通过下游的核心调节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 的体外活性机制 RUNX1/PU.1/IRF8 将 PHF6 募集至染色质，R274Q 突变是否废除 PHF6 染色质结合，以及 PHF6 是否招募额外的复合物来抑制增强子活性。目标 2 中的实验将阐明从关键 TF 将 PHF6 招募到染色质到下游的事件顺序 PHF6 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，将查明 HSC 自我更新与异常之间的疾病相关机制通过 PHF6 与造血转录因子一起调节增强子来实现祖细胞扩增。这这将是我们理解 HSC 自我更新的表观遗传调控的重要进展。