Mechanisms controlling cell type-specific transcription factor activity in the development of serially homologous structures in Drosophila

项目概要/摘要 为了确保正确的形态发生和细胞命运的具体化，动物必须产生高度定型的空间 和基因表达的时间模式。为此，转录因子（TF）结合DNA调节因子， 在特定的细胞类型中激活或抑制转录的元件，例如增强子， 发育阶段许多TF在动物的多个阶段在几种细胞类型中表达 发展这些转录因子调节不同细胞类型中的不同基因调控网络（GRNs）， 单个TF指定多个细胞命运。尽管几十年的研究，仍然不清楚个别TF是如何 能够在不同的细胞类型中执行不同的功能。这种TF的一个例子是果蝇Hox TF 超双胸（Ubx）。Ubx通过结合数千个增强子来特异性地识别第三胸（T3）节段身份， 调节数百个基因，修改基态第二胸段（T2）GRN。T3高度 相对于连续同源T2，在沿着近端-远端（PD）轴的所有位置修改了艾德，包括 体壁、铰链和附属物的形态变化。所有这些变化最终都必须 这是由于UBX的活动。诸如Ubx之类的TF在GRN中实施更改的主要机制是通过 染色质结构的修饰。这在很大程度上是由TF与染色质重塑的相互作用介导的 酶，导致组蛋白翻译后修饰和染色质可及性的变化， 靶向基因座。我的建议概述了一系列实验，以确定细胞类型特异性分子， 细胞类型特异性Ubx结合、染色质修饰活性和转录活性的基础相互作用 监管活动。我们实验室最近的证据表明，Ubx染色质重塑活性是 在T3中沿PD轴空间分离沿着。在我的研究策略的目标1中，我将检验以下假设： ubx的功能主要是促进沿着中间位置的更开放的染色质状态 PD轴或沿PD轴沿着近端和远端位置的更封闭的染色质状态。我会用 我们实验室开发了一种名为SpyChIP的新技术，用于测定Ubx在这些不同人群中的结合。 细胞沿着PD轴在T3。我还将在这些人群中进行ATAC-seq，以确定 Ubx结合下游的染色质可及性。这些实验将提供有关TF如何 结合导致不同细胞群体中不同的染色质景观。在目标2中，我将测试 锌指TF Teashirt（Teashirt）介导近端结构域中Ubx抑制活性的假设 的T3。在这个目标中，我描述了增益和损失的功能实验，以表征促甲状腺激素的作用， 调节Ubx的抑制活性我还将进行蛋白质相互作用实验，以确定是否 Ubx以细胞类型特异性的方式与Tsh和其他辅助调节因子相互作用。总的来说，这些实验将 阐明单个TF调节不同细胞群体中不同GRNs的机制 在动物的整个发育过程中产生多种细胞类型。

Project summary/abstract To ensure proper morphogenesis and cell fate specification, animals must generate highly stereotyped spatial and temporal patterns of gene expression. To this end, transcription factors (TFs) bind DNA regulatory elements such as enhancers to activate or repress transcription in particular cell types at particular developmental stages. Many TFs are expressed in several cell types at multiple stages throughout animal development. These TFs modulate different gene regulatory networks (GRNs) in different cell types, allowing a single TF to specify multiple cell fates. Despite decades of research, it remains unclear how individual TFs are able to perform distinct functions in different cell types. One example of such a TF is the Drosophila Hox TF Ultrabithorax (Ubx). Ubx specifies third thoracic (T3) segmental identity by binding thousands of enhancers to regulate hundreds of genes, modifying the ground-state second thoracic segment (T2) GRN. T3 is highly modified at all positions along the proximal-distal (PD) axis relative to the serially homologous T2, including morphological changes to the body wall, hinge, and appendage proper. All of these changes must ultimately be due to Ubx activity. A primary mechanism by which TFs such as Ubx enact changes in GRNs is through modification of chromatin structure. This is largely mediated by TF interaction with chromatin remodeling enzymes, leading to histone post-translational modifications and changes in chromatin accessibility at targeted genomic loci. My proposal outlines a series of experiments to identify cell type-specific molecular interactions that underlie cell type-specific Ubx binding, chromatin-modifying activities, and transcriptional regulatory activities. Recent evidence from our lab suggests that Ubx chromatin remodeling activity is spatially segregated along the PD axis in T3. In Aim 1 of my Research Strategy, I will test the hypothesis that Ubx functions predominantly to either promote a more open chromatin state in intermediate positions along the PD axis or a more closed chromatin state in proximal and distal positions along the PD axis. I will use a novel technique developed in our lab called SpyChIP to assay Ubx binding in these distinct populations of cells along the PD axis in T3. I will also perform ATAC-seq in these populations to identify changes in chromatin accessibility downstream of Ubx binding. These experiments will provide information as to how TF binding leads to differential chromatin landscapes in different populations of cells. In Aim 2, I will test the hypothesis that the zinc finger TF Teashirt (Teashirt) mediates Ubx repressive activity in the proximal domain of T3. In this Aim, I describe both gain- and loss-of-function experiments to characterize the role of Tsh in regulating Ubx repressive activity. I will also perform protein-protein interaction experiments to determine if Ubx interacts with Tsh and other coregulators in a cell type-specific manner. Overall, these experiments will elucidate the mechanisms by which a single TF modulates different GRNs in different populations of cells throughout animal development to give rise to multiple cell types.