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之间的相互作用的调节。在脊椎动物中，大多数转录因子属于多基因家族，具有共同的蛋白质基序和独特的结构域，使它们具有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位点编码：E2a、Heb和E2-2。E蛋白作为专性二聚体，在调控与发育、生理功能、细胞周期和细胞死亡相关的基因方面起着重要作用。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。到目前为止，E蛋白功能的另一个关键方面是存在不同的E蛋白形式，这在很大程度上被忽视了。Heb和E2-2基因座编码一种标准型(HEBCan或E2-2Can)和一种替代形式(HEBAlt或E2-2Alt)，而E2a编码两种标准型，E47和E12。我发现并首次描述了HEBAlt，我的实验室开创了替代E蛋白功能的先河。在这里，我们将讨论不同的E蛋白二聚体如何调控基因表达，以及细胞内信号如何调节二聚体的形成和功能等基本问题。 由于我们的专业知识和我们开发的多种工具，我们将使用Pre-T细胞作为我们的模型系统。在目标1中，我们将通过在转染有表位标记的HEBAlt、HEBCan和/或E2A的细胞中进行免疫共沉淀研究，确定在前T细胞背景下形成的二聚体的组成。我们将评估不同的信号通路对二聚体形成的影响，这些信号通路使用这些通路的抑制剂作用于T细胞前体。这些实验将揭示T前细胞可用的二聚体的格局，以及它如何随着导致E蛋白翻译后修饰的信号事件而改变。 在目标2中，我们将确定不同E蛋白二聚体在前T细胞中的不同基因靶点。为了实现这一目标，我们将使用具有Heb、E2a或两者的条件基因敲除等位基因的小鼠，与携带可诱导的Cre-ER转基因的小鼠杂交。在OP9-DL1共培养中产生的Pre-T细胞将用他莫昔芬处理以诱导Heb、E2a或两者的缺失，并将通过RNA测序进行整个转录组分析。我们将使用强制二聚体来评估哪些二聚体调节这些基因的表达，在强迫二聚体中，不同的E蛋白配对通过灵活的系链相互连接。这些研究将揭示在前T细胞中受不同E蛋白二聚体调控的基因。 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)来确定不同E蛋白之间以及它们的拮抗剂ID因子之间的相对亲和力。我们将产生编码E蛋白或ID蛋白的表达构建体，这些蛋白与荧光分子如Venus、m绿松石和mCherry融合。E蛋白或ID蛋白融合构建体的组合将被导入Heb和E2a缺陷细胞，FRET供体金星的寿命缩短将通过荧光显微镜来测量。我们将像在目标1中那样用信号抑制物干扰细胞，并评估其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.