Mechanisms of Homeodomain Transcriptional Specificity

同源域转录特异性的机制

• 批准号: 10455727
• 负责人: BRIAN GEBELEIN
• 金额: $ 44.64万
• 依托单位: CINCINNATI CHILDRENS HOSP MED CTR
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-04-01 至 2024-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10455727
• 关键词: Abdomen; Address; Animals; Anterior; Architecture; Binding; Binding Sites; Biochemical; Bioinformatics; Biological Assay; Biological Process; Cell Culture Techniques; Cells; Complex; DNA; DNA Binding; DNA Sequence; Data; Development; Drosophila genus; Drosophila melanogaster; Embryo; Ensure; Event; Family; Family member; Gene Activation; Gene Expression; Genes; Genetic; Genetic Transcription; Genomics; Goals; Health; Heterodimerization; Homeodomain Proteins; Homo; Human; Human Development; In Vitro; Individual; Logic; Luciferases; Mammalian Cell; Mammals; Mediating; Neurons; Organism; Organogenesis; Outcome; Output; Pattern; Physiological; Process; Proteins; Publishing; Regulation; Regulator Genes; Reporter; Repression; Research Proposals; Role; Site; Specific qualifier value; Specificity; System; Testing; To specify; Transcription Repressor; Transgenic Organisms; body system; cell type; comparative; dimer; fly; gene interaction; gene repression; homeodomain; in vivo; insight; leukemia; member; molecular domain; monomer; neuroblast; novel; programs; structural biology; transcription factor; transcriptome sequencing

PROJECT ABSTRACT Homeodomain (HD) proteins comprise a large family of transcription factors (TFs) that regulate numerous aspects of animal development. For example, members of the Hox-like (HoxL) and Nkx-like (NKL) HD proteins regulate processes ranging from patterning of the anterior-posterior axis (A-P) of the embryo to specifying individual cell fates within different organ systems. Intriguingly, the HoxL and NKL proteins have highly similar HDs that bind largely overlapping AT-rich DNA sequences in vitro. These findings provide a classic TF specificity paradox: How do TFs with highly similar in vitro DNA binding activities achieve sufficient in vivo specificity to ensure the accurate regulation of genetic programs in different cell types? To address this paradox, my lab is focused on defining how HD TFs achieve in vivo specificity by forming cooperative TF complexes on cis- regulatory modules. Our preliminary and published data reveal that members of the HoxL and NKL TFs differ in their ability to form homo- and heterodimer TF complexes on DNA. For instance, we unexpectedly found that the Gsx/Ind TFs, which specify neuronal cell fates in animals from flies to mammals, differentially regulate gene expression when bound to DNA as monomers versus homodimers. In contrast, the Abdominal-A (Abd-A) Hox TF, which specifies distinct cell fates in the Drosophila abdomen, does not bind DNA as a homodimer, but instead cooperatively binds DNA with three other HD proteins: Extradenticle (Exd), Homothorax (Hth), and Engrailed (En). These data support the hypothesis that HD TFs achieve target and regulatory specificity by binding distinct combinations of AT-rich DNA sites as monomers, cooperative homodimers, or cooperative heterodimers. To test this hypothesis, we propose two aims: In Aim1, we propose to determine how HD monomer versus homodimer binding impacts target gene binding and regulation. To achieve this goal, we will (1) systematically define which HoxL and NKL HDs cooperatively bind DNA as homodimers; (2) assess the regulatory potential of each HD on monomer vs dimer sites in cell culture assays; and (3) define the mechanism and function of Ind homodimer formation on Drosophila neuroblast gene expression using structural biology and transgenic reporter, CUT&RUN, and RNA-seq assays. In Aim2, we propose to define how the choice of Hox heterodimer partner impacts the DNA binding and regulatory specificity of the Abd-A Hox TF. To achieve this goal, we will (1) define the DNA motifs and molecular domains required for cooperative Abd-A/Hth and Abd-A/En complexes; (2) test the role of Abd-A heterodimerization domains in gene activation and repression assays in the Drosophila embryo; (3) define the in vivo binding motifs and target genes regulated by Abd-A with a focus on identifying heterodimer binding events using CUT&RUN and RNA-seq assays. Since the TFs and biological processes studied are highly conserved between flies and mammals, we are optimistic our studies will uncover gene regulatory mechanisms relevant to human health and development.

项目摘要 同源域（HD）蛋白包括大量调节众多转录因子（TFS）的蛋白质 动物发展的各个方面。例如，hox状（HOXL）和NKX类（NKL）HD蛋白的成员 调节从胚胎的前轴（A-P）的图案到指定的过程 不同器官系统中的单个细胞命运。有趣的是，HOXL和NKL蛋白具有高度相似的 在体外结合了很大程度上重叠的富含富含的DNA序列的HDS。这些发现提供了经典的TF特异性 悖论：高度相似的体外DNA结合活性的TF如何获得足够的体内特异性 确保对不同细胞类型的遗传程序进行准确调节？为了解决这个悖论，我的实验室是 专注于定义HD TF如何通过在顺式上形成合作TF复合物来实现体内特异性 监管模块。我们的初步和发布的数据表明，HOXL和NKL TF的成员在 它们在DNA上形成同二聚体TF复合物的能力。例如，我们意外地发现 GSX/IND TFS（指定从蝇到哺乳动物的动物中的神经元细胞命运）差异调节基因 作为单体与同型二聚体结合到DNA时的表达。相反，腹部A（ABD-A）HOX TF指定果蝇腹部不同细胞的命运，不是将DNA作为同型二聚体结合，而是结合 与其他三种HD蛋白合作结合DNA：内部（EXD），Hyothorax（HTH）和插入 （en）。这些数据支持HD TFS通过结合不同的假设实现目标和调节特异性 作为单体，合作同二聚体或合作异二聚体的富含DNA位点的组合。测试 这个假设，我们提出了两个目标：在AIM1中，我们建议确定HD单体与同型二聚体的方式 结合影响靶基因结合和调节。为了实现这一目标，我们将（1）系统地定义哪个 HOXL和NKL HDS合作将DNA作为同型二聚体结合； （2）评估每个高清的调节潜力 细胞培养分析中的单体与二聚体位点； （3）定义IND同型二聚体的机理和功能 使用结构生物学和转基因报告基因果蝇神经细胞基因表达形成， 剪切和RNA-seq分析。在AIM2中，我们建议定义HOX HETRODIMER合作伙伴的选择 影响ABD-A HOX TF的DNA结合和调节特异性。为了实现这一目标，我们将（1）定义 合作ABD-A/HTH和ABD-A/EN复合物所需的DNA基序和分子结构域； （2）测试 ABD-A异二聚化结构域在果蝇胚胎中基因激活和抑制测定中的作用； （3）定义由ABD-A调节的体内结合基序和靶基因，重点是识别异二聚体 使用剪切和RNA-seq分析结合事件。由于所研究的TF和生物过程很高 在苍蝇和哺乳动物之间保守的，我们很乐观，我们的研究将发现基因调节机制 与人类健康和发展有关。