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

• 批准号: 10540364
• 负责人: Vikram R. Paralkar
• 金额: $ 54.13万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 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/10540364
• 关键词: Acceleration; 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; Proliferating; Proteins; RNA; RUNX1 gene; Recurrence; Regulation; Repression; 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

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）需要对转录因子（TFS）和表观遗传的微调合作 监管机构维持正常的自我更新。这种能力的精确控制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获得正常的洞察至关重要 和肿瘤造血。 PHF6，一种神秘的白血病，染色质结合蛋白 压抑HSC自我更新，为剖析基础监管网络提供了有吸引力的模型。 该提议的目的是阐明正常自我更新与异常之间的机械联系 通过解剖PHF6如何调节由钥匙造血TFS结合的增强子的增殖。 我们的体内研究表明，PHF6造血基因敲除专门增加了HSC自我更新 留下下游造血症，在很大程度上不受干扰。试验实验表明本构hoxa9 表达与PHF6损失合作，导致快速致命的祖细胞扩张。因此，我们已经确定了 PHF6损失的强烈对比的稳态和HOXA9驱动的表型，为理想的系统提供了 研究HSC自我更新如何在异常增殖中选择。我们的初步数据表明，PHF6绑定 和压抑增强剂由TFS Runx1，pu.1，irf8共占据。因此，我们已经确定了机械 PHF6活性的基础（与关键造血TFS的染色质共占占用率）。 我们假设PHF6抑制了HSC的自我更新和异常的髓样祖细胞扩张 调节由Runx1，PU.1和IRF8绑定的增强子的常见机制。 具体目标1：我们将确定PHF6在抑制HSC自我更新和髓样祖细胞中的作用 通过确定PHF6损失是否加速HOXA9驱动的髓样祖细胞的扩展，在体内扩张， R274Q突变是否消除了HSC和髓样祖细胞中的PHF6功能，以及PHF6损失是否损失 激活runx1/pu.1/irf8结合的增强剂。 AIM 1中的实验将提高我们对 HSC生物学通过将自我更新与通过下游的核心监管电路联系起来。 PHF6。 具体目标2：我们将通过确定是否是否确定PHF6活性的机制 runx1/pu.1/irf8募集PHF6至染色质，R274Q突变是否消除了PHF6染色质结合， PHF6是否招募了其他复合物来抑制增强剂活性。 AIM 2中的实验将 阐明从关键TF募集PHF6到染色质的事件的顺序，到下游 PHF6对增强子功能的影响，因此对基因表达的影响。 这些研究（如果成功）将确定与HSC自我更新与异常相关的疾病的机制 通过PHF6与造血TF一起对增强子的调节，祖细胞扩展。这 在我们理解HSC自我更新的表观遗传调节方面，将是一个重要的进步。