A chromatin-based timer controlling T-cell development

项目摘要 在多细胞发育过程中，细胞通过激活谱系特异性来建立和维持稳定的身份。 基因.表观遗传机制，涉及polycomb抑制系统及其相关的组蛋白H3 赖氨酸27三甲基化（H3 K27 me 3）修饰对于适当的细胞谱系特化至关重要， 在癌症中经常被破坏;然而，尽管在许多系统中进行了大量工作，但仍不清楚 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 问题仍然没有答案，因为我们缺乏跟踪活细胞中表观遗传过程的方法。我们 最近开发了一个报告系统，以分析表观遗传控制的激活Bcl 11b，一个重要的 T细胞谱系定型的转录因子。为了明确检测Bcl 11b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Bcl 11b基因座被标记为 不同的荧光蛋白（Ng et al. 2018）。通过跟踪这些小鼠的祖细胞，我们发现， bcl 11b等位基因在同一细胞中独立开启，一个等位基因通常在多天前开启。 另这项工作表明，一个表观遗传开关，独立作用于两个Bcl 11b基因座， 基因激活和T细胞定型的动力学。在此，在本提案中，我们寻求阐明 表观遗传机制控制这种谱系承诺开关，并确定其中断的影响， 白血病启动。我们将检验抑制性H3 K27 me 3修饰维持关键控制的假设， Bcl 11b激活的点，破坏这一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H3 K27 me 3丢失在控制Bcl 11b激活中的作用（目的1）。为此，我们 将扰乱Bcl 11b基因座上的H3 K27 me 3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Bcl 11b报告菌株提供了一个强大的工具，通过表观遗传控制可视化 活细胞中的机制。接下来，我们将确定转录因子如何启动H3 K27 me 3丢失， 基因激活（Aim 2）。为此，我们将干扰Bcl 11b上的候选TF和顺式调节区域， 基因座，并确定对H3 K27 me 3状态和基因表达的结果影响。最后，我们将确定 表观遗传机制的破坏引起的分化延迟是否驱动白血病的发生 (Aim 3）。为此，我们将确定延迟的Bcl 11b激活是否会减缓T细胞增殖的速度。 发育，以及这种发育放缓是否会加速小鼠模型中T-ALL的发作。 由于多梳机制在不同的哺乳动物发育过程中起作用， 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各个领域。

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.