Control of Neural Stem Cell Identity by Tafs and Trf2

 描述（由申请人提供）：构成大脑的绝大多数神经元来源于神经干细胞（NSC）。像其他干细胞一样，神经干细胞必须平衡其定义，但似乎相反，自我更新的功能和最终分化为神经元或神经胶质的能力。这种微妙的平衡是如何在神经发生过程中实现的仍然不清楚。该项目的长期目标是了解体内NSC身份的转录机制。这需要识别NSC决定簇，并从机制上了解它们如何单独发挥作用，以及它们如何整合到转录网络中，从而在多次细胞分裂中保持其稳健性。虽然编码表观遗传调节因子或经典转录因子的NSC身份基因近年来引起了人们的广泛关注，但我们未发表的工作和其他人的工作越来越多地指出了一般转录因子在干细胞身份中的高度选择性作用。特别是，我们已经确定了一个独特的子集TATA盒结合蛋白（TBP）相关因子（TAFs）和TBP parasites，TRF 2，但不是TBP本身，作为新的NSC身份基因。由于TAF最初是通过与TBP的结合而被鉴定的，因此它们通常被认为是一般的转录因子，并被认为是大部分转录所需的。相比之下，我们的初步研究表明，一个独特的TAFs（NSC-TAFs）和TRF 2的子集是选择性地需要在体内的NSC属性。NSC-TAF或TRF 2的敲低导致NSC细胞核中的过早分化、细胞周期进展缺陷和促分化因子Prospero/Prox 1的异位积累。此外，虽然TAF 7的NSC突变体增殖不良且过早分化，但Taf 7双突变体NSC产生大的NSC克隆，其主要由表达NSC标志物Asense的细胞组成。这表明去除Prospero恢复了TAF 7突变体NSC的干细胞特性，并且Prospero对TAF 7具有上位性。我们的中心假设是NSC-TAFs和TRF 2形成一种新的复合物，该复合物选择性地与具有共享核心启动子结构的NSC-expressed gene的一个新子集的表达相关，并且是该新子集的表达所必需的。为了测试这一点，我们建议利用最近的，复杂的转录本和染色质分析技术，在果蝇探测控制NSC身份的NSC-TAFs和TRF 2的分子基础。具体而言，在目标1中，我们将纯化NSC并进行RNA-seq以鉴定需要NSC-TAF和TRF 2用于NSC表达的转录物。在目标2中，我们将分析NSC-TAF和TRF 2在体内NSCs中的基因组结合位点，并尝试使用免疫共沉淀和邻近连接测定来鉴定NSC-TAF-TRF 2复合物。总的来说，我们的工作将使我们能够立足于通过NSC-TAFs和TRF 2控制NSC身份的分子机制。重要的是，TAF突变与多种人类神经系统疾病有关，我们希望我们提出的研究将阐明疾病的发病机制，为TAF相关神经系统疾病的建模提供信息，并指导基于NSCs的治疗。

 DESCRIPTION (provided by applicant): The vast majority of the neurons comprising the brain are derived from Neural Stem Cells (NSCs). Like other stem cells, NSCs must balance their defining, yet seemingly opposing, features of self-renewal and the ability to terminally differentiate into neurons or glia. How this exquisite balance is achieved during neurogenesis remains unclear. The long-term goal of the proposed project is to understand the transcriptional mechanisms that govern NSC identity in vivo. This requires identifying NSC determinants and gaining mechanistic insight into how they function individually and how they are integrated within a transcriptional network that maintains its robustness over multiple cell divisions. While NSC identity genes that encode epigenetic regulators or classical transcription factors have garnered much attention in recent years, our unpublished work and that of others increasingly points to a highly selective role of general transcription factors in stem cell identity. In particlar, we have identified a unique subset of TATA-box binding protein (TBP)-associated factors (TAFs) and a TBP paralog, TRF2, but not TBP itself, as novel NSC identity genes. Because TAFs were first identified by virtue of their association with TBP, they are often regarded as general transcription factors and assumed to be required for the bulk of transcription. In contrast, our preliminary studies have shown that a unique subset of TAFs (NSC-TAFs) and TRF2 are selectively required for NSC properties in vivo. Knockdown of NSC-TAFs or TRF2 leads to premature differentiation, cell cycle progression defects, and ectopic accumulation of the pro- differentiation factor Prospero/Prox1 in the NSC nucleus. In addition, while NSCs mutant for TAF7 proliferate poorly and prematurely differentiate, prospero, Taf7 double mutant NSCs generate large NSC clones that are mostly composed of cells expressing the NSC marker Asense. This indicates that removing prospero restores stem cell properties to TAF7 mutant NSCs and that Prospero is epistatic to TAF7. Our central hypothesis is that NSC-TAFs and TRF2 form a novel complex that selectively associates with, and is required for, the expression of a novel subset of NSC-expressed genes with shared core promoter architecture. To test this, we propose to leverage recent, sophisticated transcript and chromatin profiling techniques available in Drosophila to probe the molecular basis of control of NSC identity by NSC-TAFs and TRF2. Specifically, in Aim 1 we will purify NSCs and perform RNA-seq to identify transcripts that require both NSC-TAFs and TRF2 for NSC expression. In Aim 2 we will profile the genomic binding sites of NSC-TAFs and TRF2 in NSCs in vivo and attempt to identify a NSC-TAF-TRF2 complex, using both co-immunoprecipitation and proximity ligation assays. Overall, our work will allow us to gain a foothold on the molecular mechanisms that control NSC identity through NSC-TAFs and TRF2. Importantly, TAF mutations have been linked to diverse human neurological disorders and we expect our proposed research will shed light on disease pathogenesis, inform modeling of TAF-linked neurological disorders, and guide NSC-based therapies.