A chromatin-based timer controlling T-cell development

基于染色质的定时器控制 T 细胞发育

• 批准号: 10323024
• 负责人: Hao Yuan Kueh
• 金额: $ 38.27万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-01-01 至 2024-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10323024
• 关键词: Alleles; Binding; Biological Assay; Biology; Blood; Cell Differentiation process; Cell Lineage; Cells; Chemicals; Chromatin; Color; Development; Developmental Gene; Developmental Process; Disease; Enhancers; Environment; Enzymes; Epigenetic Process; Etiology; Event; Excision; Gene Activation; Gene Expression; Genes; Genome; Goals; Hematopoietic stem cells; Histone H3; Homeostasis; In Vitro; Instruction; Lead; Lysine; Malignant - descriptor; Malignant Neoplasms; Measures; Methods; Methylation; Modeling; Modification; Mus; Mutation; Nucleic Acid Regulatory Sequences; Oncogenic; Polycomb; Process; Proteins; Regulation; Regulator Genes; Reporter; Repression; Role; Signal Transduction; Specific qualifier value; System; T-Cell Development; T-Lymphocyte; Testing; Thymus Gland; Time; Untranslated RNA; Work; base; cancer type; cell type; gain of function mutation; genomic locus; histone modification; insight; leukemia; mouse model; notch protein; progenitor; recruit; response; stem cells; tool; transcription factor; tumorigenesis

PROJECT SUMMARY During multicellular development, cells establish and maintain stable identities by activating lineage-specifying genes. Epigenetic mechanisms, involving the polycomb repressive system and its associated histone H3 lysine 27 tri-methylation (H3K27me3) modification, are critical for proper cell lineage specification, and are frequently disrupted in cancer; however, despite much work in many systems, it remains unclear how exactly these histone modifications control gene expression, and how their disruption drives malignancy. These questions remain unanswered, because we lack methods to follow epigenetic processes in living cells. We recently developed a reporter system to analyze epigenetic control in the activation of Bcl11b, an essential transcription factor for T-cell lineage commitment. To definitively test whether Bcl11b activation is controlled by cis-epigenetic mechanisms acting at single loci, we generated a mouse, where two Bcl11b loci are tagged with different fluorescent proteins (Ng et al. 2018). By following progenitors from these mice, we found that two Bcl11b alleles turn on independently in the same cell, with one allele often turning on multiple days before another. This work demonstrates that an epigenetic switch, acting independently on two Bcl11b loci, regulates the dynamics of gene activation and T-cell commitment. Here, in this proposal, we seek to elucidate the epigenetic mechanism controlling this lineage commitment switch, and determine impact of its disruption for leukemia initiation. We will test the hypothesis that repressive H3K27me3 modifications uphold a key control point for Bcl11b activation, and that disrupting this process can delay lineage commitment and drive leukemia. To do so, we will first define the role for H3K27me3 loss in controlling Bcl11b activation (Aim 1). To do so, we will perturb H3K27me3 modifications on the Bcl11b locus, and measure effects on locus activation dynamics. In these assays, the dual-color Bcl11b reporter strain provides a powerful tool to visualize control by epigenetic mechanisms in living cells. Next, we will determine how transcription factors work initiate H3K27me3 loss and gene activation (Aim 2). To do so, we will perturb candidate TFs and cis-regulatory regions on the Bcl11b locus, and determine resultant effects on H3K27me3 states and gene expression. Finally, we will determine whether delays in differentiation, caused by disruptions of epigenetic mechanisms, drive leukemia initiation (Aim 3). To do so, we will determine whether delayed Bcl11b activation slows down the pace of T-cell development, and whether this developmental slowdown can accelerate the onset of T-ALL in a mouse model. As polycomb mechanisms operate in diverse mammalian developmental processes, and because disruption of these mechanisms may be a major driver of malignancy, our findings could broadly impact diverse fields.

项目摘要 在多细胞发育过程中，细胞通过激活谱系指定来建立和维持稳定的身份 基因。表观遗传机制，涉及polycomb抑制系统及其相关组蛋白H3 赖氨酸27三甲基化（H3K27me3）修饰，对于适当的细胞谱系规范至关重要，并且是 经常在癌症中破坏；但是，尽管在许多系统中进行了很多工作，但尚不清楚如何确切 这些组蛋白修饰控制基因表达，以及它们的破坏如何驱动恶性肿瘤。这些 问题仍然没有得到答复，因为我们缺乏遵循活细胞表观遗传过程的方法。我们 最近开发了一个记者系统，用于分析Bcl11b激活中的表观遗传控制，这是必不可少的 T细胞谱系承诺的转录因子。确定测试BCL11B激活是否由 作用在单位基因座的顺式景观机制，我们生成了一个鼠标，其中两个Bcl11b基因座用 不同的荧光蛋白（Ng等，2018）。通过跟随这些小鼠的祖细胞，我们发现两个 Bcl11b等位基因在同一单元格中独立打开，一个等位基因经常在几天之前打开 其他。这项工作表明，在两个Bcl11b基因座上独立起作用的表观遗传转换可调节 基因激活和T细胞承诺的动力学。在此提案中，我们试图阐明 控制这种谱系承诺开关的表观遗传机制，并确定其破坏的影响 白血病启动。我们将检验以下假设：抑制性H3K27ME3修改维护一个关键控制 BCL11B激活的点，并且破坏此过程可以延迟谱系承诺并驱动白血病。 为此，我们将首先定义H3K27ME3损失在控制BCL11B激活中的作用（AIM 1）。为此，我们 将在Bcl11b基因座上考虑到H3K27ME3修改，并测量对基因座激活动力学的影响。 在这些测定中，双色BCL11B报告菌株提供了一种强大的工具，可以通过表观遗传来可视化控制 活细胞中的机制。接下来，我们将确定转录因子的工作原理启动H3K27me3损失和 基因激活（AIM 2）。为此，我们将在BCL11b上扰动候选TFS和顺式调节区域 基因座，并确定对H3K27me3状态和基因表达的结果影响。最后，我们将确定 是否延迟差异是由表观遗传机制中断引起的，驱动白血病的启动 （目标3）。为此，我们将确定延迟Bcl11b激活是否会减慢T细胞的速度 发展以及这种发展放缓是否可以加速鼠标模型中T-ALL的发作。 随着多肉机制在各种哺乳动物的发育过程中运行，并且因为破坏了 这些机制可能是恶性肿瘤的主要驱动力，我们的发现可能会广泛影响各种​​领域。