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

果蝇系列同源结构发育中细胞类型特异性转录因子活性的控制机制

• 批准号: 10679757
• 负责人: Ross Munce
• 金额: $ 4.77万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-11-03 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10679757
• 关键词: ATAC-seq; Animals; Binding; Biological Assay; C-terminal binding protein; Cells; Chest; Chromatin; Chromatin Structure; Co-Immunoprecipitations; DNA; DNA Binding; Development; Distal; Drosophila genus; Ectopic Expression; Enhancers; Ensure; Enzymes; Gene Expression Profile; Genes; Genetic Transcription; Genome; Goals; Histones; Human Pathology; Individual; Malignant Neoplasms; Mediating; Methods; Modeling; Modification; Morphogenesis; Morphology; Mutation; Population; Positioning Attribute; Post-Translational Protein Processing; Process; Reading; Regulation; Regulator Genes; Regulatory Element; Repression; Research; Role; Series; Specific qualifier value; Stereotyping; Structure; Techniques; Testing; Tissues; Wing; Work; Zinc Fingers; appendage; cell fate specification; cell type; chromatin modification; chromatin remodeling; developmental disease; experimental study; gain of function; gene regulatory network; gene repression; genetic corepressor; genome-wide; genomic locus; insight; interest; loss of function; mutant; novel; programs; protein protein interaction; recruit; segregation; transcription factor

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.

项目摘要/摘要 为了确保适当的形态发生和细胞命运fi阳离子，动物必须产生高度刻板的空间 以及基因表达的时间模式。为此，转录因子(Tf)结合dna调节。 在特定细胞类型中激活或抑制转录的元件，如增强剂 发育阶段。许多转录因子在动物体内的不同阶段表达于多种细胞类型。 发展。这些转录因子在不同的ff细胞类型中调节不同的ff基因调控网络(GRN)，从而允许 单个Tf指定多个细胞命运。尽管进行了数十年的研究，但目前仍不清楚单个的TF 能够在不同的ff细胞类型中执行不同的功能。这种转移因子的一个例子是果蝇种群转移因子 超双胸(UBX)。通过结合数以千计的增强子来识别UBX种fiES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是高度的 Modifi在沿着近端-远端(PD)轴相对于序列同源T2的所有位置，包括 体壁、铰链和附肢本身的形态变化。所有这些变化最终都必须 是由于Ubx活动。像UBX这样的TF在GRN中实施变化的主要机制是通过 染色质结构的Modifi阳离子。这在很大程度上是通过TF与染色质重塑的相互作用来调节的 酶，导致组蛋白翻译后Modifi阳离子和染色质可及性的变化 靶向基因组基因座。我的提案概述了一系列鉴定细胞类型的实验--特殊的fic分子 构成细胞类型基础的相互作用--特殊的fic-Ubx结合、染色质修饰活动和转录 监管活动。我们实验室的最新证据表明，Ubx染色质重塑活动 在T3中沿PD轴空间分离。在我的研究策略的目标1中，我将检验以下假设 UBX的主要功能是在中间位置促进染色质状态更加开放 PD轴或沿PD轴的近端和远端位置的更封闭的染色质状态。我将使用 我们实验室开发了一种名为SpyChIP的新技术，用于检测这些不同人群的Ubx结合 T3中沿PD轴方向分布的细胞。我还将在这些人群中执行atac-seq，以确定 Ubx结合下游的染色质可及性。这些实验将提供关于TF如何 结合导致在不同的细胞群体中ff不同的染色质景观。在目标2中，我将测试 假设锌finger Tf Teashirt(Teashirt)介导近端区域的UBX抑制活动 对T3的影响。在这个目标中，我描述了获得和功能损失的实验，以表征TSH在 调节UBX抑制活动。我还将进行蛋白质-蛋白质相互作用实验，以确定 UBX与促甲状腺激素和其他辅调节因子以一种细胞类型特有的fic方式相互作用。总体而言，这些实验将 阐明单个转铁蛋白调节不同细胞群中不同ff的GRN的机制 在整个动物发育过程中产生多种类型的细胞。