Mechanisms of Homeodomain Transcriptional Specificity

项目摘要 同源结构域（HD）蛋白包含调节许多转录因子（TF）的大家族， 动物发展的各个方面。例如，Hox样（HoxL）和Nkx样（NKL）HD蛋白的成员 调节从胚胎前后轴（A-P）的模式到指定 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 在体外结合大量重叠的富含AT的DNA序列的HD。这些发现提供了经典的TF特异性 悖论：具有高度相似的体外DNA结合活性的TF如何获得足够的体内特异性， 确保不同细胞类型中基因程序的准确调控？为了解决这个矛盾，我的实验室 重点是定义HD TF如何通过在顺式- 监管模块。我们的初步和已发表的数据显示，HoxL和NKL TF的成员在以下方面不同： 它们在DNA上形成同源和异源二聚体TF复合物的能力。例如，我们意外地发现， Gsx/Ind转录因子，在从果蝇到哺乳动物的动物中指定神经元细胞的命运， 当与DNA结合为单体与同二聚体时表达。相比之下，Abdominal-A（Abd-A）Hox TF在果蝇腹部指定了不同的细胞命运，它不以同源二聚体的形式结合DNA，而是 与其他三种HD蛋白协同结合DNA：牙外体（Exd）、同胸（Hth）和Engrailed （En）.这些数据支持HD TF通过结合不同的靶向和调节特异性来实现靶向和调节特异性的假设。 富含AT的DNA位点的组合作为单体、协同同源二聚体或协同异源二聚体。测试 基于这一假设，我们提出了两个目标：在Aim 1中，我们提出确定HD单体与同型二聚体 结合影响靶基因的结合和调节。为了实现这一目标，我们将（1）系统地定义 HoxL和NKL HD以同源二聚体的形式协同结合DNA;（2）评估每种HD对DNA的调节潜力。 细胞培养试验中单体与二聚体位点的关系;（3）确定Ind同二聚体的作用机制和功能 利用结构生物学和转基因报告基因对果蝇成神经细胞基因表达的影响， CUT&RUN和RNA-seq测定。在Aim 2中，我们建议定义Hox异源二聚体伴侣的选择如何影响Hox异源二聚体伴侣的选择。 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 合作的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异源二聚化结构域在果蝇胚胎基因激活和抑制试验中的作用; (3)确定体内结合基序和由Abd-A调控的靶基因，重点是鉴定异源二聚体 使用CUT&RUN和RNA-seq测定结合事件。由于所研究的TF和生物过程高度 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 关系到人类健康和发展。

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.