Differential control of gene expression by specific E protein dimers

后生动物的发育过程受到转录因子和信号分子之间相互作用的调节。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。其中最重要的蛋白家族之一是 E 蛋白家族，由三个基因位点在脊椎动物基因组中编码：E2A、HEB 和 E2-2。 E 蛋白作为专性二聚体，有助于调节涉及发育、生理功能、细胞周期和细胞死亡的基因。然而值得注意的是，不同 E 蛋白二聚体的具体作用实际上是未知的。 E 蛋白功能的另一个重要方面迄今为止一直被忽视，那就是不同 E 蛋白形式的存在。 HEB 和 E2-2 基因位点各自编码一种规范形式（HEBCan 或 E2-2Can）和一种替代形式（HEBAlt 或 E2-2Alt），而 E2A 编码两种规范形式：E47 和 E12。我发现并首先描述了 HEBAlt，我的实验室开创了替代 E 蛋白功能的研究。在这里，我们将解决以下基本问题：不同的 E 蛋白二聚体如何调节基因表达，以及二聚体的形成和功能如何通过细胞内信号传导进行调节。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。在目标 1 中，我们将通过在用表位标记版本的 HEBAlt、HEBCan 和/或 E2A 转染的细胞中进行免疫共沉淀研究来定义在前 T 细胞背景下形成的二聚体的组成。我们将使用这些途径的抑制剂来评估在 T 细胞前体中起作用的不同信号途径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概况，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，我们将识别前 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、E2A 或两者的条件性敲除等位基因的小鼠，这些小鼠与携带可诱导的 Cre-ER 转基因的小鼠交配。 OP9-DL1 共培养物中产生的前 T 细胞将用他莫昔芬处理，以诱导 HEB、E2A 或两者的删除，并通过 RNA 测序进行全转录组分析。我们将评估哪些二聚体使用强制二聚体调节这些基因的表达，其中不同的 E 蛋白伴侣通过灵活的绳索相互连接。这些研究将揭示前 T 细胞中受不同 E 蛋白二聚体调节的基因。在目标 3 中，我们将使用 FLIM（荧光实时成像显微镜）-FRET（荧光共振能量转移）确定不同 E 蛋白彼此之间及其拮抗剂 Id 因子的相对亲和力。我们将生成编码与荧光分子（例如 Venus、mTurquoise 和 mCherry）融合的 E 蛋白或 Id 蛋白的表达构建体。 E蛋白或Id蛋白融合构建体的组合将被转染到HEB和E2A缺陷细胞中，并且FRET供体Venus的寿命随时间的减少将通过荧光显微镜测量。我们将像目标 1 一样用信号抑制剂扰乱细胞，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 E2A 如何合作或竞争来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.