CAREER: Structural Studies of Basic-helix-loop-helix and Homeodomain Interactions that Elicit Cell-specific Gene Expression

细胞特异性基因表达是多细胞生物中分化细胞功能的基础。是什么限制了特定细胞类型的基因表达尚不清楚。转录因子通过结合基因附近的DNA序列（称为启动子序列）来调节基因转录。一般来说，表达一个基因需要多种因素的组合。这些因子通常是大型转录因子家族的成员，它们自身的DNA结合选择性有限。本项目研究转录因子之间的蛋白-蛋白相互作用在调控启动子识别和基因激活中的作用。该项目的重点是两个大的转录因子家族，基本螺旋-环-螺旋（bHLH）和同源结构域（HD）因子之间的相互作用。不同的bHLH和HD因子在许多不同的细胞类型中相互作用。作为一个模型系统，POMC启动子的调控将被研究。该启动子调节垂体促肾上腺皮质激素的细胞特异性表达。促肾上腺皮质激素是脑垂体的六个内分泌细胞之一，每个细胞分泌一种独特的激素。POMC启动子的一个区域，称为“微增强子”，结合bHLH异源二聚体E47/NeuroD1和垂体HD因子Pitx1。bHLH和HD结构域在没有DNA的情况下相互作用。此外，POMC启动子结合促皮质功能受限的T-box结构域蛋白T-pit。本研究将验证转录因子相互作用和微增强子序列之间的互补性有助于细胞特异性活化POMC的假设。目的1是确定当与微增强子结合时，溶液中因子之间是否保持相同的蛋白质-蛋白质相互作用。目标2和3将利用高分辨率晶体结构和小角度x射线和中子散射相结合，开发POMC微增强剂的蛋白质和DNA相互作用的结构模型。目的4将通过在蛋白质界面中产生突变来测试模型的有效性和蛋白质-蛋白质相互作用对DNA结合和转录激活的贡献。目前的研究将有助于我们理解转录因子与控制细胞特异性基因表达的“启动子密码”的相互作用。bHLH和HD因子在分化和发育中发挥关键作用，但个体家庭成员的区别特征仍然难以捉摸，即使在同一细胞中也是如此。该研究将为进一步研究启动子结构与转录因子复合物的相互作用之间的关系奠定基础。蛋白质晶体学和小角度散射（与橡树岭国家实验室合作）的结合有望促进更大的多蛋白质复合物的结构研究。这个项目的跨学科方法提供了一个很好的环境，让学生参与真实的研究，并促进基于探究的教学方法。生物化学系、数学系、科学与技术教育系联合开设了一门名为“作为探究的生物研究”的新课程，将吸引教育学硕士学生参与研究。本课程将以研究计画来激励科学内容的教学，目标是建立以探究为基础的教学模式。课程参与者将发展分子生物学的实验室技能，并应用这些技能来产生突变，以测试迷你增强子模型的有效性（研究计划的目标4）。与此同时，他们将面临理解和考虑研究假设的挑战。后续课程将支持教师将研究经验应用于他们的高中课堂。设备将提供给教师借用。该项目将为生物化学本科生参与研究提供一个背景。一名教育部研究助理将帮助实施该项目的教育目标，并作为教师和学校之间的联络人。教育和生物化学专业的少数民族学生将被鼓励参加。

Cell-specific gene expression is fundamental to the functions of differentiated cells in multi-cellular organisms. What restricts gene expression to specific cell types is not well understood. Transcription factors regulate gene transcription by binding DNA sequences near a gene, called the promoter sequence. Generally combinations of factors are required to express a gene. Often these factors are members of large transcription factor families with limited DNA binding selectivity in themselves. This project investigates the role of protein-protein interactions between transcription factors in regulating promoter recognition and gene activation. The project focuses on interactions between two large transcription factor families, basic-helix-loop-helix (bHLH) and homeodomain (HD) factors. Different bHLH and HD factors interact in a number of different cell types. As a model system, the regulation of the proopiomelanocortin (POMC) promoter will be studied. This promoter regulates cell-specific expression in pituitary corticotrophs. Corticotrophs are one of six endocrine cells of the pituitary, each secreting a unique hormone. A region of the POMC promoter, called a "minienhancer", binds the bHLH heterodimer, E47/NeuroD1, and the pituitary HD factor Pitx1. The bHLH and HD domains interact in the absence of DNA. In addition, the POMC promoter binds the corticotroph-restricted T-box domain protein T-pit. This research will test the hypothesis that complementarity between transcription factor interactions and the minienhancer sequence contributes to cell-specific activation of POMC. Aim 1 is to determine whether the same protein-protein interactions between factors in solution are maintained when bound to the minienhancers. Aim 2 and 3 will develop structural models of the protein and DNA interactions of the POMC minienhancer using a combination of high-resolution crystal structures and small angle X-ray and neutron scattering. Aim 4 will test the validity of the models and the contribution of the protein-protein interactions to DNA binding and transcriptional activation by generating mutations in protein interfaces.The current study will contribute to our understanding of transcription factor interactions to the "promoter code" that controls cell-specific gene expression. bHLH and HD factors play key roles in differentiation and development, yet distinguishing features of individual family members remain elusive, even within the same cell. This research will serve as the basis for future studies addressing the relationship between promoter architecture and stabilization of the transcription factor complexes by further interactions. The combination of protein crystallography and small angle scattering (in collaboration with Oak Ridge National Labs) promises to facilitate structural studies of larger multi-protein complexes. The interdisciplinary approach of this project provides an excellent context to involve students in authentic research, and promote inquiry-based teaching approaches. A new class entitled Biology Research as Inquiry, co-listed between the Departments of Biochemistry and Mathematics, Science & Technology Education, will engage education masters students in research. The class will use the research project to motivate teaching scientific content with the goal of modeling inquiry-based teaching approaches. Class participants will develop laboratory skills in molecular biology and apply those skills to generate mutations to test the validity of the minienhancer model (Aim 4 of the research plan). At the same time they will be challenged to understand and consider the research hypothesis. A follow-up class will support teachers in applying the research experience to their high school classrooms. Equipment will be made available for teachers to borrow. The project will provide a context for involving biochemistry undergraduates in research. A Department of Education research assistant will help implement the educational goals of the project and act as a liaison for teachers and schools. Minority students in the education and biochemistry programs will be encouraged to participate.