Establishing CUX1 as a determinant of hematopoietic stem cell fate

项目摘要 在发育生物学中，一个长期存在的问题是干细胞如何形成特定的谱系。而细胞 表面标记与造血干细胞（HSC）功能相关，但对转录因子的了解较少。 调节器驱动干细胞的行为。CUX 1编码一个高度保守的含有同源结构域的 转录因子CUX 1的缺失和失活突变在造血干细胞的克隆性造血中是经常发生的。 不确定的潜力和骨髓增生异常综合征，CUX 1丢失与血细胞减少，谱系 偏差和疾病状态的不良预后。通常，只有一个CUX 1拷贝失活，表明 CUX 1的缺失以单倍不足的方式影响造血发育。为此，我们 先前报道了CUX 1在造血中的剂量依赖性作用。CUX 1水平如何调节 造血干细胞和祖细胞（HSPC）在增殖、自我更新和谱系方面命运 选择仍然是知识上的一个主要差距。本提案的总体目标是确定 CUX 1剂量调节HSPC命运的分子机制。为了解决这个问题，我们现在已经生成了 一种新的CUX 1报告小鼠，用于在发育过程中在单细胞水平上测量CUX 1蛋白水平。在 初步数据表明，CUX 1在HSPCs中高度表达，同时表现出异质性， 在茎和祖细胞间室中。我们发现CUX 1水平与造血干细胞相关， (HSC)活性，与CUX 1dim HSC相比，CUX 1dim HSC表现出更高的多系长期造血功能。 CUX1bright HSC。我们发现，CUX 1与SWI/SNF染色质重塑复合物相互作用， 原代人HSPC中的染色质可及性。因此，我们的中心假设是CUX 1调节HSC 通过SWI/SNF的募集通过谱系特异性增强子的剂量依赖性开放的命运。我们测试 这个假设有两个具体的目的。目的1：总体假设-CUX 1上调对于HSC是必要的 分化在一系列小鼠体内实验和用原代人细胞进行的补充测定中， 我们将CUX 1蛋白水平与HSC功能，包括增殖，自我更新， 分化我们将干扰CUX 1水平，以评估CUX 1剂量如何影响这些功能。目标二： 总体假设-CUX 1上调驱动增强子引发分化。要确定CUX 1 影响染色质重塑和增强子激活，我们将利用尖端的功能基因组学 使用我们的CUX 1-报告基因和CUX 1-敲低小鼠在HSC中的方法和单细胞方法， 原代人HSPCs。完成拟议的研究将阐明关键的表观遗传作用， 造血发育中剂量敏感的CUX 1转录调控。这项工作对于 实现我们为CUX 1单倍不足患者确定治疗干预措施的长期目标。

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.