A chromatin-based timer controlling T-cell development

项目总结 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，涉及多梳抑制系统及其相关的组蛋白H3 赖氨酸27三甲基化(H3K27me3)修饰，对于正确的细胞谱系规范至关重要，并且是 经常在癌症中中断；然而，尽管在许多系统中做了很多工作，但仍然不清楚到底是如何 这些组蛋白修饰控制着基因的表达，以及它们的破坏如何导致恶性肿瘤。这些 问题仍然没有答案，因为我们缺乏在活细胞中跟踪表观遗传过程的方法。我们 最近开发了一个报告系统来分析Bcl11b激活的表观遗传控制，Bcl11b是一种必不可少的 T细胞谱系承诺的转录因子。最终测试Bcl11b的激活是否受 顺式表观遗传机制作用于单个基因座，我们产生了一只小鼠，其中两个Bcl11b基因座被标记为 不同的荧光蛋白(Ng等人2018年)。通过跟踪这些小鼠的祖先，我们发现有两个 Bcl11b等位基因在同一细胞中独立启动，一个等位基因通常在几天前启动 又一个。这项工作表明，表观遗传开关独立地作用于两个Bcl11b基因座，调节 基因激活和T细胞承诺的动态。在这里，在这项提议中，我们试图澄清 控制这一世系承诺转换的表观遗传机制，并确定其中断对 白血病启动期。我们将检验这一假设，即抑制性H3K27me3修饰支持关键控制 Bcl11b激活的时间点，扰乱这一过程可能会推迟谱系承诺，并导致白血病。 为此，我们首先定义H3K27me3丢失在控制Bcl11b激活中的作用(目标1)。为了做到这一点，我们 将干扰Bcl11b基因座上的H3K27me3修饰，并测量对基因座激活动力学的影响。 在这些检测中，双色Bcl11b报告菌株提供了一个强大的工具来可视化表观遗传控制 活细胞中的机制。接下来，我们将确定转录因子如何启动H3K27me3丢失和 基因激活(目标2)。为此，我们将扰乱Bcl11b上的候选TF和顺式调控区域 并确定其对H3K27me3状态和基因表达的综合影响。最后，我们将确定 表观遗传机制中断导致的分化延迟是否会导致白血病的发生 (目标3)。为此，我们将确定延迟的Bcl11b激活是否会减慢T细胞的速度 发育，以及这种发育减慢是否会加速小鼠模型中T-ALL的发生。 由于多梳机制在不同的哺乳动物发育过程中发挥作用，而且由于干扰 这些机制可能是恶性肿瘤的主要驱动因素，我们的发现可能会广泛影响不同的领域。

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.