Signal-dependent control of cell differentiation by E protein transcription factors

在动物发育过程中，不同类型细胞的产生受到细胞信号级联和转录因子活性相互作用的调节。E蛋白转录因子在脊椎动物中由三个基因座编码：E2a(Tcf3)、Heb(Tcf12)和E2-2(Tcf4)。E蛋白作为二聚体与自身、彼此或其他基本螺旋--环--螺旋(BHLH)因子一起发挥作用。我们和其他人已经发现，编码Heb和E2-2的基因座同时产生规范(Can)和替代(Alt)E蛋白，而E2a只以规范形式存在。我们的研究表明，HEBAlt和HEBCan在T细胞发育中发挥着不同的作用，但除此之外，人们对E-Alt蛋白知之甚少。在上一次NSERC发现拨款期间，我们发现E-Alt蛋白独特的Alt结构域包含一个最佳功能所需的酪氨酸磷酸化基序，揭示了E蛋白与信号通路之间相互作用的新节点。我们还发现，在缺乏T细胞的动物中存在E-Alt蛋白。这一发现促使我们对Alt外显子附近的开放染色质标记进行了电子筛查，并在前脂肪细胞、神经前体细胞和肌肉前体细胞中发现了它们，这表明HEBAlt在这些细胞类型的发展中发挥了作用。我们假设，E-Alt蛋白可能能够启动E-蛋白反应基因的表达，同时抑制它们，直到收到分化信号。更令人兴奋的是，我们发现不同谱系的“主调节器”与Alt启动子结合，这表明E-Alt mRNAs可以作为早期谱系特异性基因的一部分被诱导。在这里，我们建议识别和表征E-Alt蛋白的相互作用伙伴，评估肌肉、神经和脂肪细胞系对Alt结构域的需求，并建立E-Alt结合DNA和染色质可及性之间的关系。在目标1中，我们将使用表达HA标记的HEBAlt WT或突变蛋白的细胞进行共沉淀，然后进行质谱测序和Western blots验证合作伙伴。在目标2中，我们将从基因敲除(功能丧失)或可诱导的转基因小鼠(功能获得)中分离前体细胞，并在体外评估它们的分化。目标3将集中于获取CHIP-SEQ和RNA-SEQ数据，以确定E-Alt在不同谱系的前体中的结合位置、这些位置上的染色质构型以及这些座位的mRNA表达状态。由于我们以前的研究，我们有丰富的工具和跟踪记录来研究HEBAlt，我们还组建了一个具有不同专业知识的合作者团队来研究它在免疫系统之外的作用。这些研究将对谱系指定和分化的多步骤过程有一个新的理解，并可能为发育生物学提供一个新的范式。

The generation of different cell types during animal development is regulated by the interplay between cell signaling cascades and transcription factor activity. E protein transcription factors  are encoded in vertebrates by three gene loci: E2A (Tcf3), HEB (Tcf12) and E2--2 (Tcf4). E proteins function as dimers with themselves, each other, or other basic helix--loop--helix (bHLH) factors. We and others have found that the loci encoding HEB and E2-2 generate both canonical (Can) and alternative (Alt) E proteins, whereas E2A exists only in the canonical form. Our studies have shown that HEBAlt and HEBCan play distinct roles in T cell development, but otherwise very little is known about E-Alt proteins. During the last NSERC Discovery grant, we discovered that the unique Alt domain of E--Alt proteins contains a tyrosine phosphorylation motif that is required for optimal function, revealing a new node of interaction between E proteins and signaling pathways. We also discovered that E--Alt proteins are present in animals that lack T cells. This discovery prompted us to conduct an in--silico screen for open chromatin marks near the Alt exon, and found them in pre-adipocytes, neural progenitors, and muscle precursors, suggesting a role for HEBAlt in development of these cell types. We hypothesized that E--Alt proteins may be able to prime E-protein responsive genes for expression while holding them in check until a differentiation signal is received. Even more exciting, we have discovered that "master regulators" of distinct lineages bind to the Alt promoters, suggesting that E--Alt mRNAs can be induced as part of an early wave of lineage--specific genes. Here we propose to identify and characterize interaction partners of E--Alt proteins, evaluate the requirements for the Alt domain in muscle, neural, and adipocyte lineages, and establish the relationship between E--Alt binding of DNA and chromatin accessibility. In Aim 1, we will use cells expressing HA-tagged HEBAlt WT or mutant proteins to perform co-precipitation, followed by mass spectrometry sequencing and verification of partners by Western blots. In Aim 2, we will isolate precursors from knockout (loss of function) or inducible transgenic mice (gain of function) and evaluate their differentiation in vitro. Aim 3 will focus on acquiring ChIP-seq and RNA-seq data to determine the sites of E-Alt binding in the precursors of different lineages, the chromatin configuration at those sites, and the status of mRNA expression from those loci. Due to our prior studies, we have abundant tools and the track record to study HEBAlt, and we have also assembled a team of collaborators with diverse expertise to investigate its role outside the immune system. These studies will lead to a new understanding of how the multi-step process of lineage specification and differentiation occurs, and may provide a new paradigm in developmental biology.