Establishing CUX1 as a determinant of hematopoietic stem cell fate

确定 CUX1 作为造血干细胞命运的决定因素

• 批准号: 10558905
• 负责人: Megan McNerney
• 金额: $ 47.49万
• 依托单位: UNIVERSITY OF CHICAGO
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-12-01 至 2026-11-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10558905
• 关键词: Acute leukemia; Address; Anemia; Binding; Biological Assay; Blood; Cell surface; Cells; Chromatin; Chromatin Remodeling Factor; DNA; Data; Defect; Development; Developmental Biology; Developmental Gene; Disease; Dysmyelopoietic Syndromes; Enhancers; Epigenetic Process; Erythroid; Exhibits; Family; Fluorochrome; Gene Expression; Gene Expression Regulation; Genes; Genetic Transcription; Genomic approach; Goals; Hematology; Hematopoiesis; Hematopoietic; Hematopoietic stem cells; Heterogeneity; Human; Intervention; Investigation; Knowledge; Lead; Length; Lymphoid; Measures; Megakaryocytes; Methodology; Modeling; Molecular; Monosomy 7; Multipotent Stem Cells; Mus; Mutation; Myelopoiesis; Nucleosomes; Pathogenesis; Patients; Prevalence; Process; Production; Prognosis; Proliferating; Proteins; Public Health; Quality of life; Recurrence; Reporter; Reporting; Role; SMARCE1 gene; Series; Somatic Mutation; Tertiary Protein Structure; Testing; Therapeutic; Therapeutic Intervention; Transcriptional Regulation; Tumor Suppressor Proteins; Up-Regulation; Work; cell behavior; chromatin protein; chromatin remodeling; chromosome 7q loss; cytopenia; dosage; experimental study; functional genomics; hematopoietic stem cell differentiation; hematopoietic stem cell fate; homeodomain; improved; in vivo; knock-down; loss of function mutation; novel; novel therapeutics; progenitor; progression risk; recruit; self-renewal; single cell analysis; stem; stem cell function; stem cells; transcription factor; transcriptome; tumor

PROJECT SUMMARY A longstanding question in developmental biology is how stem cells commit to a certain lineage. While cell surface markers correlate with hematopoietic stem cell (HSC) functions, less is known regarding transcriptional regulators that drive stem cell behavior. CUX1 encodes a highly conserved homeodomain-containing transcription factor. Deletions and inactivating mutations of CUX1 are recurrent in clonal hematopoiesis of indeterminate potential and myelodysplastic syndrome, and CUX1 loss is associated with cytopenias, lineage skewing, and a poor-prognosis in disease states. Typically, only one copy of CUX1 is inactivated, indicating that loss of CUX1 impacts hematopoietic development in a haploinsufficient manner. In line with this, we previously reported a dosage-dependent role for CUX1 in hematopoiesis. How CUX1 levels regulate hematopoietic stem and progenitor cell (HSPC) fate, with respect to proliferation, self-renewal, and lineage choice remains a major gap in knowledge. The overall objective of the current proposal is to determine the molecular mechanism by which CUX1 dosage regulates HSPC fate. To address this, we have now generated a novel CUX1-reporter mouse to measure CUX1 protein levels at the single-cell level during development. In preliminary data, we demonstrate that CUX1 is expressed highly in HSPCs while exhibiting heterogeneity within stem and progenitor compartments. We show that CUX1 levels correlate with hematopoietic stem cell (HSC) activity, with CUX1dim HSCs demonstrating higher multilineage, long-term hematopoiesis compared to CUX1bright HSCs. We show that CUX1 interacts with the SWI/SNF chromatin remodeling complex to regulate chromatin accessibility in primary human HSPCs. Thus, our central hypothesis is that CUX1 regulates HSC fate via dosage-dependent opening of lineage-specific enhancers through recruitment of SWI/SNF. We test this hypothesis with two Specific Aims. Aim 1: Overall hypothesis – CUX1 upregulation is necessary for HSC differentiation. In a series of in vivo experiments in mice and complementary assays with primary human cells, we will correlate CUX1 protein levels with HSC functions, including proliferation, self-renewal, and differentiation. We will perturb CUX1 levels to assess how CUX1 dosage impacts these functions. Aim 2: Overall hypothesis – CUX1 upregulation drives enhancer priming for differentiation. To determine how CUX1 impacts chromatin remodeling and enhancer activation, we will leverage cutting-edge functional genomics approaches and single-cell methodologies in HSCs using our CUX1-reporter and CUX1-knockdown mice and primary human HSPCs. Accomplishing the proposed studies will elucidate the critical epigenetic role for dosage-sensitive CUX1 transcriptional regulation in hematopoietic development. This work is critical to achieve our long-term goal of identifying therapeutic interventions for patients with CUX1 haploinsufficiency.

项目摘要 发育生物学的一个长期问题是干细胞如何致力于某种谱系。而细胞 表面标记与造血干细胞（HSC）功能相关，关于转录的知之甚少 驱动干细胞行为的调节器。 CUX1编码一个高度组成的含同源域的构成 转录因子。 Cux1的删除和灭活突变是在克隆造血的复发 不确定的电位和骨髓增生综合征，Cux1损失与细胞质，谱系有关 偏斜，疾病状态下的预测较差。通常，只有一个Cux1副本被灭活，表明 CUX1的丧失会以一种单倍体的方式影响造血发育。因此，我们 先前报道了Cux1在造血中的剂量依赖性作用。 CUX1水平如何调节 关于增殖，自我更新和谱系 选择仍然是知识的主要差距。当前建议的总体目的是确定 CUX1剂量调节HSPC命运的分子机制。为了解决这个问题，我们现在已经生成 一种新型的Cux1 reporter小鼠，可在发育过程中测量单细胞水平的CUX1蛋白水平。在 初步数据，我们证明CUX1在HSPC中表现出很高的表现，同时表现出异质性 在茎和祖细胞室内。我们表明CUX1水平与造血干细胞相关 （HSC）活性，与Cux1DIM HSC相比，Cux1DIM HSC表现出较高的多曲霉，长期造血作用 Cux1bright HSC。我们表明CUX1与SWI/SNF染色质重塑复合物相互作用以调节 原代人HSPC中的染色质可及性。这就是我们的中心假设是CUX1调​​节HSC 通过招募SWI/SNF的谱系特异性增强子的命运依赖性开放。我们测试 该假设具有两个具体的目标。 AIM 1：总体假设 - HSC需要CUX1上调 分化。在一系列在小鼠中的体内实验中，并用原代人类细胞完成测定 我们将将CUX1蛋白水平与HSC功能相关联，包括增殖，自我更新和 分化。我们将扰动CUX1水平，以评估CUX1剂量如何影响这些功能。目标2： 总体假设 - CUX1上调驱动增强子启动以进行分化。确定cux1的方式 影响染色质重塑和增强剂激活，我们将利用尖端的功能基因组学 使用我们的Cux1-Reporter和Cux1-Knockdown小鼠的HSC中的方法和单细胞方法 主要人类HSPC。完成拟议的研究将阐明 造血性发育中剂量敏感的CUX1转录调节。这项工作对 实现我们的长期目标，即确定CUX1单倍不足患者的治疗干预措施。