Administrative Supplement for R01-GM079428: Mechanisms of homeodomain transcriptional specificity

项目摘要 同源结构域（Homeodomain，HD）蛋白是一类转录因子家族，在动物体内调控多种生物学行为。 发展例如，Hox样（HoxL）和Nkx样（NKL）HD蛋白的成员调节以下过程： 从胚胎前后轴（A-P）的模式到指定不同器官内的单个细胞命运， 系统.有趣的是，HoxL和NKL蛋白具有高度相似的HD，其结合大量重叠的富含AT的DNA 体外序列。这些发现提供了一个经典的TF特异性悖论：具有高度相似的体外DNA的TF 结合活性达到足够的体内特异性，以确保在不同细胞中准确调节遗传程序， 类型？为了解决这个矛盾，我的实验室专注于定义HD TF如何通过形成 合作TF复合物的顺式调节模块。我们的初步和已发表的数据显示，HoxL成员 和NKL TF的不同之处在于它们在DNA上形成同源和异源二聚体TF复合物的能力。例如，我们出乎意料地 发现Gsx/Ind转录因子，在从苍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。相反，Abdominal-A（Abd-A）Hox TF， 在果蝇腹部指定不同的细胞命运，不作为同源二聚体结合DNA，而是协同结合 DNA与其他三种HD蛋白质：外齿（Exd），同胸（Hth）和Engrailed（En）。这些数据支持 假设HD TF通过结合富含AT的DNA位点的不同组合来实现靶向和调节特异性， 单体、协同同源二聚体或协同异源二聚体。为了验证这一假设，我们提出了两个目标：在目标1中， 我们建议确定HD单体与同二聚体结合如何影响靶基因结合和调节。到 为了实现这一目标，我们将（1）系统地定义哪些HoxL和NKL HD作为同源二聚体协同结合DNA; (2)在细胞培养试验中评估每种HD对单体与二聚体位点的调节潜力;以及（3）定义机制 用结构生物学和转基因技术研究Ind同源二聚体形成对果蝇成神经细胞基因表达的影响 报告基因、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DNA Abd-A/Hth和Abd-A/En复合物协同作用所需的基序和分子结构域;（2）测试Abd-A的作用 异源二聚化结构域在果蝇胚胎中的基因激活和抑制测定;（3）定义体内 Abd-A调控的结合基序和靶基因，重点是使用 CUT&RUN和RNA-seq测定。由于所研究的TF和生物学过程在果蝇和果蝇之间高度保守， 对于哺乳动物，我们乐观地认为，我们的研究将揭示与人类健康相关的基因调控机制， 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.