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

动物发育过程中不同细胞类型的产生受细胞信号级联和转录因子活性之间的相互作用调节。E蛋白转录因子在脊椎动物中由三个基因位点编码：E2 A（Tcf 3）、HEB（Tcf 12）和E2- 2（Tcf 4）。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的基因座产生典型（Can）和替代（Alt）E蛋白，而E2 A仅以典型形式存在。我们的研究表明，HEBAlt和HEBCan在T细胞发育中发挥不同的作用，但对E-Alt蛋白的了解很少。在上一次NSERC发现资助期间，我们发现E-Alt蛋白的独特Alt结构域包含最佳功能所需的酪氨酸磷酸化基序，揭示了E蛋白和信号通路之间相互作用的新节点。我们还发现E-Alt蛋白存在于缺乏T细胞的动物中。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计算机筛选，并在前脂肪细胞，神经祖细胞和肌肉前体中发现它们，表明HEBAlt在这些细胞类型的发育中发挥作用。我们假设，E-Alt蛋白可能能够启动E蛋白反应基因的表达，同时保持它们在检查，直到收到分化信号。更令人兴奋的是，我们发现不同谱系的“主调节因子”与Alt启动子结合，这表明E-Alt mRNA可以作为谱系特异性基因早期波的一部分被诱导。在这里，我们建议确定和表征的E-Alt蛋白的相互作用的合作伙伴，评估的Alt域在肌肉，神经和脂肪细胞谱系的要求，并建立E-Alt结合DNA和染色质的可及性之间的关系。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，然后进行质谱测序并通过蛋白质印迹验证伴侣。在目标2中，我们将从敲除（功能丧失）或诱导型转基因小鼠（功能获得）中分离前体，并在体外评估它们的分化。目标3将专注于获取ChIP-seq和RNA-seq数据，以确定不同谱系前体中E-Alt结合的位点，这些位点的染色质构型以及这些位点的mRNA表达状态。由于我们之前的研究，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其在免疫系统外的作用。这些研究将导致对谱系特化和分化的多步骤过程如何发生的新理解，并可能提供发育生物学的新范式。

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.