Mechanisms of Homeodomain Transcriptional Specificity

项目摘要 同源结构域 (HD) 蛋白包含一个转录因子 (TF) 大家族，可调节多种转录因子 动物发育的各个方面。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）的模式到指定的过程 不同器官系统内个体细胞的命运。有趣的是，HoxL 和 NKL 蛋白具有高度相似性 HD 在体外结合大量重叠的富含 AT 的 DNA 序列。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 如何在体内获得足够的特异性 确保不同细胞类型中遗传程序的准确调控？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协同 TF 复合物来实现体内特异性 监管模块。我们的初步和公布的数据表明，HoxL 和 NKL TF 的成员在以下方面有所不同： 它们在 DNA 上形成同二聚体和异二聚体 TF 复合物的能力。例如，我们意外地发现 Gsx/Ind TF 指定从果蝇到哺乳动物等动物的神经细胞命运，对基因进行差异调节 当作为单体与同源二聚体结合到 DNA 时的表达。相比之下，腹部-A (Abd-A) Hox TF 指定果蝇腹部不同的细胞命运，它不会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：Extradenticle (Exd)、Homothorax (Hth) 和 Engrailed （恩）。这些数据支持这样的假设：HD TF 通过结合不同的特异性来实现目标和监管特异性。 富含 AT 的 DNA 位点以单体、协同同二聚体或协同异二聚体的形式组合。测试 对于这个假设，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区别 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）系统地定义哪些 HoxL 和 NKL HD 以同型二聚体的形式协同结合 DNA； (2) 评估每个 HD 的监管潜力 细胞培养测定中的单体与二聚体位点； (3)定义Ind同型二聚体的机制和功能 使用结构生物学和转基因报告基因对果蝇神经母细胞基因表达的形成， CUT&RUN 和 RNA-seq 检测。在 Aim2 中，我们建议定义如何选择 Hox 异二聚体伴侣 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 协同 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测定中的作用； (3)定义Abd-A调控的体内结合基序和靶基因，重点鉴定异二聚体 使用 CUT&RUN 和 RNA-seq 检测进行结合事件。由于研究的转录因子和生物过程高度 在果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.