Transcriptional regulation of mesoderm development

中胚层发育的转录调控

• 批准号: 9231603
• 负责人: ANN K CORSI
• 金额: $ 44.19万
• 依托单位: CATHOLIC UNIVERSITY OF AMERICA
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-09-05 至 2022-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9231603
• 关键词: Alleles; Amino Acid Substitution; Amino Acids; Animal Model; Animals; Award; BHLH Protein; Binding; Biological Assay; CRISPR/Cas technology; Caenorhabditis elegans; Cells; Cellular biology; Clinical; Crystallization; Cytology; DNA; DNA Binding; DNA Binding Domain; DNA Sequence; Defect; Development; Digit structure; Dimerization; Disease; Elements; Family; Fibroblast Growth Factor Receptors; Frameshift Mutation; Gene Expression; Gene Expression Regulation; Gene Targeting; Genes; Genetic Crosses; Genetic Models; Genetic Screening; Genetic Transcription; Glutamic Acid; Goals; Green Fluorescent Proteins; Hand; Helix-Turn-Helix Motifs; Homologous Gene; Human; Image; Invertebrates; Laboratories; Lead; Learning; Light; Limb structure; Mediating; Mesoderm; Missense Mutation; Mutate; Mutation; National Institute of Dental and Craniofacial Research; National Institute of Diabetes and Digestive and Kidney Diseases; Nematoda; Organism; Pathology; Pathway interactions; Patients; Phenotype; Play; Positioning Attribute; Proteins; Reagent; Reporter; Research; Role; Series; Structure; Students; Syndrome; System; TWIST1 gene; Technology; Testing; Therapeutic Intervention; Tissues; Transcriptional Activation; Transcriptional Regulation; Ubiquitin; United States National Institutes of Health; Variant; Vertebrates; Work; base; craniofacial; design; dimer; disease phenotype; experimental study; genetic manipulation; genome editing; graduate student; human disease; in vivo; insight; member; mutant; novel; null mutation; protein expression; protein function; protein protein interaction; targeted treatment; therapeutic target; transcription factor; undergraduate education; undergraduate student

PROJECT SUMMARY Transcription factors play an important role in tissue-specific gene regulation during development. The basic helix-loop-helix (bHLH) transcription factors TWIST1 and TWIST2 are critical regulators of cell fate and differentiation during vertebrate development. These factors are mutated in several diseases that lead to craniofacial, digit, and other defects making it important to understand how these bHLH factors function and the underlying causes associated with their disease phenotypes. bHLH proteins form dimers with their HLH domains and bind to DNA sequences called E boxes with the basic DNA binding domains of the dimer partners. Although much is known about DNA binding and dimer formation, very little is known about the cooperation between the two domains for proper bHLH function. A glutamic acid in the DNA binding domain is predicted to play a critical role based on three pieces of evidence. There is a lack of amino acid variation at that position in bHLH protein alignments, bHLH crystal structures predict the glutamic acid makes direct contacts with DNA, and patients with amino acid substitutions in either TWIST1 or TWIST2 in that residue have disease phenotypes. The experiments in this proposal are designed to test the hypothesis that missense mutations of the conserved glutamic acid residue in the Twist DNA binding domain alter E-box regulated transcription due to disrupted protein-protein interactions. The simple nematode, Caenorhabditis elegans, is an excellent model genetic system for elucidating Twist function because the organism contains only one Twist-related protein, HLH-8, and HLH-8’s partner protein and downstream target genes are conserved with human TWIST1. Even though this organism is an invertebrate and can’t have craniofacial or digit defects, the conserved Twist pathway suggests that aspects of the cellular mechanisms will be conserved as well. In Aim 1, missense mutations in HLH-8 that mimic human disease alleles in the conserved glutamic acid will be characterized in C. elegans. The goal will be to understand the specific cellular defects of the mutants. Preliminary studies indicate that some of the mutants can still bind DNA to turn on target genes. These mutants will be the focus of the research in the second aim. In Aim 2, the C. elegans mutants will be used in modifier genetic screens designed to identify new proteins that cooperate with HLH-8 and, by analogy, may be potential therapeutic targets for Twist-related human diseases. The projects in the proposed work are suitable for students so the research will have a profound impact on the education of undergraduate and graduate students. The broad goal of this research is to elucidate HLH-8 function and target gene regulation. Due to the relatedness between the human and C. elegans Twist proteins, information learned will be relevant to understanding human TWIST1 and TWIST2 and disease pathology.

项目摘要 转录因子在发育过程中的组织特异性基因调控中起重要作用。基本 螺旋-环-螺旋（bHLH）转录因子TWIST 1和TWIST 2是细胞命运的关键调节因子， 脊椎动物发育过程中的分化。这些因子在几种疾病中发生突变， 颅面、手指和其他缺陷使得了解这些bHLH因子的功能变得重要， 与其疾病表型相关的潜在原因。bHLH蛋白与其HLH形成二聚体 二聚体的基本DNA结合结构域与称为E盒的DNA序列结合 伙伴虽然对DNA结合和二聚体形成了解很多，但对DNA结合和二聚体形成的了解很少。 这两个结构域之间的合作，以适当的bHLH功能。DNA结合结构域中的谷氨酸是 根据三个证据预测它将发挥关键作用。缺乏氨基酸变异， 在bHLH蛋白比对中，bHLH晶体结构预测谷氨酸使直接 与DNA接触，并且在该残基中TWIST 1或TWIST 2中具有氨基酸取代的患者， 疾病表型这个提议中的实验是为了检验一个假设， Twist DNA结合结构域中保守的谷氨酸残基的突变改变了E-box调控的 由于蛋白质-蛋白质相互作用被破坏而导致转录。 秀丽隐杆线虫是研究Twist的理想模式遗传系统 因为生物体只含有一种Twist相关蛋白HLH-8和HLH-8的伴侣蛋白 下游靶基因与人TWIST 1保守。尽管这种生物是一种 无脊椎动物，不能有颅面或手指缺陷，保守的扭曲途径表明， 细胞机制也将被保存。在目标1中，HLH-8中模拟人类的错义突变， 保守谷氨酸中的疾病等位基因将在C.优雅的我们的目标是 了解突变体的特定细胞缺陷。初步研究表明一些突变体 仍然可以结合DNA来启动靶基因。这些突变体将是第二个目标的研究重点。 在目标2中，C.线虫突变体将用于修饰基因筛选，旨在鉴定新的蛋白质， 与HLH-8合作，并且通过类推，可能是Twist相关的人类疾病的潜在治疗靶标。 在拟议的工作项目是适合学生，所以研究将产生深远的影响， 本科生和研究生教育。本研究的主要目标是阐明HLH-8 功能和靶基因调控。由于人类与C.线虫扭曲蛋白， 所学到的信息将与理解人类TWIST 1和TWIST 2以及疾病病理学相关。

项目成果

Localized TWIST1 and TWIST2 basic domain substitutions cause four distinct human diseases that can be modeled in Caenorhabditis elegans.

• DOI: 10.1093/hmg/ddx107
• 发表时间: 2017-06-01
• 期刊: Human molecular genetics
• 影响因子: 3.5
• 作者: Kim S;Twigg SRF;Scanlon VA;Chandra A;Hansen TJ;Alsubait A;Fenwick AL;McGowan SJ;Lord H;Lester T;Sweeney E;Weber A;Cox H;Wilkie AOM;Golden A;Corsi AK
• 通讯作者: Corsi AK

Using Caenorhabditis elegans as a Model for Mechanistic Insights of Craniofacial Development.

• DOI: 10.1007/978-1-0716-1847-9_1
• 发表时间: 2022
• 期刊: Methods in molecular biology (Clifton, N.J.)