Differential control of gene expression by specific E protein dimers

后生动物的发育过程受转录因子和信号分子的相互作用调控。在脊椎动物中，大多数转录因子属于多基因家族，具有共享的蛋白质基序和独特的结构域，赋予它们调节不同基因的能力。其中最重要的是E蛋白家族，在脊椎动物基因组中由三个基因座编码：E2 A，HEB和E2-2。E蛋白作为一种专性二聚体，在发育、生理功能、细胞周期和细胞死亡等基因调控中发挥重要作用。然而，值得注意的是，不同的E蛋白二聚体的具体作用几乎是未知的。E蛋白功能的另一个重要方面是迄今为止被忽视的不同E蛋白形式的存在。HEB和E2-2基因座各自编码一种典型形式（HEBCan或E2-2Can）和一种替代形式（HEBAlt或E2-2Alt），而E2 A编码两种典型形式，E47和E12。我发现并首次描述了HEBAlt，我的实验室开创了替代E蛋白功能的研究。在这里，我们将解决不同的E蛋白二聚体如何调节基因表达的基本问题，以及二聚体的形成和功能如何通过细胞内信号调节。由于我们的专业知识和我们开发的多种工具，我们将使用前T细胞作为我们的模型系统。在目的1中，我们将通过在用HEBAlt、HEBCan和/或E2 A的表位标记形式转染的细胞中进行免疫共沉淀研究来定义在前T细胞的背景下形成的二聚体的组成。我们将使用这些通路的抑制剂，通过在T细胞前体中操作的不同信号通路来评估对二聚体形成的影响。这些实验将揭示前T细胞可用的二聚体的景观，以及它如何响应导致E蛋白翻译后修饰的信号事件而发生变化。在目标2中，我们将确定前T细胞中不同E蛋白二聚体的不同基因靶点。为了实现这一目标，我们将使用具有HEB、E2 A或两者的条件性敲除等位基因的小鼠，与携带诱导型Cre-ER转基因的小鼠交配。将用他莫昔芬处理在0 P9-DL 1共培养物中产生的前T细胞以诱导HEB、E2 A或两者的缺失，并通过RNA测序进行全转录组分析。我们将评估哪些二聚体调节这些基因的表达使用强制二聚体，其中不同的E蛋白伴侣通过灵活的系绳相互连接。这些研究将揭示不同的E蛋白二聚体的背景下，前T celles.In目的3，我们将确定不同的E蛋白彼此之间的相对亲和力和它们的拮抗剂，Id因子，使用FLIM（荧光实时成像显微镜）-FRET（荧光共振能量转移）。我们将产生编码E蛋白或Id蛋白的表达构建体，其与荧光分子如Venus、mTurquoise和mCherry融合。将E蛋白或Id蛋白融合构建体的组合转染到HEB和E2 A缺陷细胞中，并通过荧光显微镜测量FRET供体Venus随时间的寿命减少。我们将用信号传导抑制剂干扰细胞，如目的1中所述，并评估对二聚体形成和亚细胞定位的影响。总的来说，这些方法将揭示HEBAlt，HEBCan和E2 A如何合作或竞争调节控制细胞命运和功能的基因程序。

Metazoan developmental processes are regulated by the interplay between transcription factors and signaling molecules. In vertebrates, most transcription factors belong to multigene families with shared protein motifs coupled with unique domains that endow them with the ability to regulate different genes. One of the most important of these is the E protein family, encoded in the vertebrate genome by three gene loci: E2A, HEB, and E2-2. E proteins, which act as obligate dimers, are instrumental in the regulation of genes involved in development, physiological function, cell cycle, and cell death. Remarkably, however, the specific roles of different E protein dimers are virtually unknown. Another critical aspect of E protein function that has been largely overlooked thus far is the existence of different E protein forms. The HEB and E2-2 gene loci each encode a canonical form (HEBCan or E2-2Can) and an alternative form (HEBAlt or E2-2Alt), whereas E2A encodes two canonical forms, E47 and E12. I discovered and first described HEBAlt, and my laboratory has pioneered studies of alternative E protein function. Here, we will address the fundamental questions of how distinct E protein dimers regulate gene expression, and how dimer formation and function are modulated by intracellular signaling. Due to our expertise and the multiple tools that we have developed, we will use pre-T cells as our model system. In Aim 1, we will define the compositions of the dimers that form in the context of pre-T cells by conducting co-immunoprecipitation studies in cells transfected with epitope-tagged versions of HEBAlt, HEBCan, and/or E2A. We will evaluate the impact on dimer formation by distinct signaling pathways that operate in T cell precursors using inhibitors of those pathways. These experiments will reveal the landscape of dimers available to pre-T cells, and how it shifts in response to signaling events that lead to post-translational modifications of E proteins.In Aim 2, we will identify distinct gene targets of different E protein dimers in pre-T cells. To achieve this aim we will use mice that have conditional knockout alleles of HEB, E2A, or both, bred to mice that carry the inducible Cre-ER transgene. Pre-T cells generated in OP9-DL1 co-cultures will be treated with tamoxifen to induce deletion of HEB, E2A, or both, and whole transcriptome analysis will be performed by RNA sequencing. We will evaluate which dimers regulate the expression of these genes using forced dimers, in which distinct E protein partners are linked to each other by flexible tethers. These studies will reveal the genes that are regulated by distinct E protein dimers in the context of pre-T cells.In Aim 3, we will determine the relative affinities of different E proteins for each other and for their antagonists, the Id factors, using FLIM (fluorescence live imaging microscopy)-FRET (fluorescence resonance energy transfer). We will generate expression constructs encoding E proteins or Id proteins fused to fluorescent molecules such as Venus, mTurquoise, and mCherry. Combinations of E protein or Id protein fusion constructs will be transfected into HEB and E2A deficient cells, and the lifetime decrease of the FRET donor Venus will be measured over time by fluorescence microscopy. We will perturb the cells with signaling inhibitors as in Aim 1 and evaluate the impact on dimer formation and subcellular localization. Collectively, these approaches will reveal how HEBAlt, HEBCan, and E2A cooperate or compete to regulate gene programs that control cell fate and function.