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

在多细胞生物体中，细胞特异性基因表达是分化细胞功能的基础。是什么将基因表达限制在特定的细胞类型上，目前还不清楚。转录因子通过结合基因附近的DNA序列来调节基因转录，这种基因被称为启动子序列。通常情况下，表达一个基因需要多种因素的组合。通常，这些因子是大转录因子家族的成员，它们本身具有有限的DNA结合选择性。本项目研究转录因子之间的蛋白质-蛋白质相互作用在调节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之间的相互作用，碱性-螺旋-环-螺旋(BHLH)和同源结构域(HD)因子。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，我们将研究前阿片黑素皮质素(POMC)启动子的调控。该启动子调节脑垂体促肾上腺皮质激素细胞的细胞特异性表达。促肾上腺皮质激素细胞是脑下垂体的六个内分泌细胞之一，每个细胞都分泌一种独特的激素。POMC启动子的一个区域被称为“微型增强子”，它与bHLH异源二聚体E47/NeuroD1和垂体HD因子Pitx1结合。BHLH和HD结构域在没有DNA的情况下相互作用。此外，POMC启动子还与促肾上腺皮质激素限制性T盒结构域蛋白T-PIT结合。这项研究将检验这一假设，即转录因子相互作用和微型增强子序列之间的互补性有助于POMC的细胞特异性激活。目的1是确定当与小增强剂结合时，溶液中的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以及小角X射线和中子散射的组合来开发POMC微型增强剂的蛋白质和DNA相互作用的结构模型。目的4将通过在蛋白质界面产生突变来检验模型的有效性以及蛋白质-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”之间的相互作用。Bhlh和hd因子在分化和发育中起着关键作用，然而，即使在同一个细胞内，个别家庭成员的区别特征仍然难以捉摸。这项研究将作为进一步研究启动子结构和通过进一步相互作用稳定转录因子复合体之间关系的基础。蛋白质结晶学和小角散射的结合(与橡树岭国家实验室合作)有望促进更大的多蛋白质复合体的结构研究。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。生物化学和数学、科学与技术教育系联合开设了一个名为生物学研究的新课程，将让教育硕士学生参与研究。这门课将利用研究项目激发教学的科学性，目标是示范探究式教学方法。课程参与者将发展分子生物学的实验室技能，并应用这些技能产生突变，以测试微型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。将提供设备供教师借用。该项目将为生物化学本科生参与研究提供背景。教育部的一名研究助理将帮助实施该项目的教育目标，并充当教师和学校的联络人。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.