Key nodes in the T-cell developmental gene regulatory network

描述（由申请人提供）：该项目是利用最新的进展，可以帮助打破打开基因网络电路，介导T细胞发育的关键阶段。T细胞发育是从多能干细胞的生理池开始的细胞命运选择机制的模型。哺乳动物T细胞发育允许对谱系定型过程进行高度精细的解剖，因为在该系统中，人们可以高纯度地分离出具有渐进程度的发育潜力限制的中间前体。优秀的T细胞系分化体外系统使整个谱系选择过程能够以开放的、实验可及的方式发生。特异性基因敲除实验还鉴定了T细胞特化所需的一组转录因子，包括加塔-3和E蛋白。基于在短期转录因子扰动实验中测得的基因调控效应，我们已经发表了一个临时的T细胞发育基因网络模型。然而，为了解释承诺过程的性质，网络中需要三个函数，而这些函数的代理人是未知的。 这个建议是基于两个非常近期的进展，我们预测现在将有可能占网络中的关键功能。这些使我们能够看到，该过程由两个可分离的阶段组成，第一阶段由从干细胞前体遗传的调节因子主导，第二阶段由T细胞相关因子集主导。首先，我们发现Bcl 11b是长期寻找的T细胞特异性负调控成分，需要将细胞从1期转变为2期。bcl 11b仅在第1阶段结束时开启，然后需要允许承诺并关闭第2阶段的干细胞相关调控基因。第二，我们已经产生了一个主要的资源：在整个T细胞特化过程的五个阶段中对转录组和全基因组表观遗传标记变化的全面调查。因此，我们可以完成所有的基因动态调节T细胞的承诺，确定候选人几乎所有的顺式调控元件，积极的监管变化发生，并与特定的转录因子结合位点的监管状态标记预测功能。 我们假设Bcl 11 b表达是第1阶段T细胞调节因子第一波成功激活的读数，当过渡到第2阶段时，先前表达的因子包括加塔-3和E蛋白，然后重新聚焦其功能，打开新的T细胞特异性基因。使用Bcl 11b作为分裂T细胞定型过程的楔子，我们将首先确定在第1阶段完成时打开Bcl 11b本身的顺式和反式元件。在正常和Bcl 11 b突变细胞中使用ChIPseq作图，我们还将确定E蛋白和加塔-3的靶位点结合和相关组蛋白修饰如何从1期转变为2期。然后，我们将使用功能的获得和丧失来测试E蛋白和加塔-3的1期或2期相互作用伴侣如何导致其部署的特定转变。

DESCRIPTION (provided by applicant): This project is to exploit recent advances that can help break open the gene network circuitry that mediates a pivotal stage in T cell development. T cell development is a model for the mechanism of cell fate choice starting from a physiological pool of multipotent stem cells. Mammalian T-cell development permits highly refined dissection of the process of lineage commitment, because in this system one can isolate intermediate precursors with progressive degrees of developmental potential restriction in high purity. Excellent in vitro systems for T-lineage differentiation enable the whole lineage choice process to occur in an open, experimentally accessible way. Specific gene knockout experiments have also identified a group of transcription factors needed for T-cell specification, including GATA-3 and E proteins. Based on gene regulatory effects measured in short-term transcription factor perturbation experiments, we have published a provisional gene network model for T-cell development. However, to explain the properties of the commitment process, three functions were needed in the network for which the agents were not known. This proposal is based on two very recent advances which we predict will now make it possible to account for the crucial functions in the network. These enable us to see that the process consists of two separable phases, the first dominated by regulatory factors inherited from the stem-cell precursor, the second dominated by a T-cell associated factor set. First we found that Bcl11b is the long-sought T-cell specific negative regulatory component that is needed to shift cells from phase 1 to phase 2. Bcl11b is turned on only as phase 1 ends, then needed to allow commitment and to turn off the stem-cell associated regulatory genes for phase 2. Second, we have generated a major resource: a full survey of transcriptome and genome-wide epigenetic marking changes across five stages throughout the T-cell specification process. We can thus complete identification of all genes dynamically regulated during T-cell commitment, identify candidates for nearly all cis-regulatory elements where active regulatory change occurs, and correlate regulatory status marks with sites for specific transcription factor binding to predict function. We hypothesize that Bcl11b expression is a readout for successful activation of a first wave of T-cell regulators in phase 1, and that when transition to phase 2 occurs, previously expressed factors including GATA-3 and E proteins then refocus their functions to turn on new T-cell specific genes. Using Bcl11b as a wedge to split the T-cell commitment process, we will first identify the cis- and trans-elements that turn Bcl11b itself on at the completion of phase 1. Using ChIPseq mapping in normal and Bcl11b mutant cells, we will also determine how the target site binding and associated histone modifications of E proteins and GATA-3 may shift from phase 1 to phase 2. We will then use gain and loss of function to test how phase 1 or phase 2 interaction partners of E proteins and GATA-3 can cause specific shifts in their deployment.