Mechanisms of Homeodomain Transcriptional Specificity

项目摘要 同源结构域(HD)蛋白由一个转录因子(TF)大家族组成，这些转录因子调控着许多 动物发育的各个方面。例如，Hox-like(HoxL)和Nkx-like(NKL)HD蛋白的成员 调节从胚胎的前后轴(A-P)的图案化到指定 单个细胞在不同的器官系统中的命运。有趣的是，HoxL和NKL蛋白高度相似 在体外结合大量重叠的富含AT的DNA序列的HDS。这些发现提供了典型的转铁蛋白特异性。 悖论：具有高度相似的体外DNA结合活性的转录因子如何在体内获得足够的特异性 确保对不同细胞类型的遗传程序进行准确调控？为了解决这个悖论，我的实验室 重点研究了HD转录因子是如何通过在顺式转铁蛋白上形成协同的转铁蛋白复合体来实现体内特异性的。 监管模块。我们的初步和公布的数据显示，HoxL和NKL TF的成员在 它们在DNA上形成同二聚体和异二聚体Tf复合体的能力。例如，我们出人意料地发现 GSX/Ind转录因子决定从苍蝇到哺乳动物等动物的神经细胞命运，对基因进行差异调控 当以单体而不是同源二聚体的形式与DNA结合时表达。相比之下，腹型A型(ABD-A)HOX 指定果蝇腹部不同细胞命运的Tf并不以同源二聚体的形式结合DNA，而是相反 DNA与其他三种HD蛋白协同结合：牙外环(EXD)、同胸(HTH)和增强 (恩)。这些数据支持这一假设，即HD转录因子通过结合不同的基因来实现靶向和调控特异性 作为单体、协作性同源二聚体或协作性异二聚体的富含AT的DNA位点的组合。为了测试 在这一假设中，我们提出了两个目标：在Aim1中，我们提出了确定HD单体与同源二聚体之间的关系 结合影响靶基因的结合和调控。为了实现这一目标，我们将(1)系统地定义 HoxL和NKL HD作为同源二聚体协同结合DNA；(2)评估每个HD在 单体与二聚体在细胞培养分析中的位置；以及(3)确定IND同源二聚体的机制和功能 利用结构生物学和转基因报告基因构建果蝇神经母细胞基因表达 Cut&Run和RNA-Seq分析。在AIM2中，我们建议定义HOX异二聚体伙伴的选择 影响Abd-A Hox Tf的DNA结合和调控特异性。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基因激活和抑制分析中的作用； (3)确定Abd-A调控的体内结合基序和靶基因，重点识别异源二聚体 结合事件使用切割和Run和RNA-seq分析。由于对转铁蛋白和生物过程的研究很高 在苍蝇和哺乳动物之间保守，我们乐观地认为我们的研究将揭示基因调控机制 与人类健康和发展有关。

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.