Mechanisms of TWIST bHLH Transcription Factors Binding to Functional Target Regions

TWIST bHLH 转录因子与功能靶区结合的机制

• 批准号: 10089973
• 负责人: CARMEN LYDIA CADILLA
• 金额: $ 36.5万
• 依托单位: UNIVERSITY OF PUERTO RICO MED SCIENCES
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-07-01 至 2025-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10089973
• 关键词: ATAC-seq; Affect; Affinity; BHLH Protein; Barbering; Binding; Binding Sites; Bioinformatics; Biological Assay; Cell Communication; Cell Polarity; Cells; Chromatin Structure; Circular Dichroism; Complex; Congenital Abnormality; Craniofacial Abnormalities; Craniosynostosis; DNA; DNA Binding; DNA Binding Domain; DNA Sequence; DNA sequencing; DNA-Binding Proteins; Development; Dimerization; Dominant-Negative Mutation; EMSA; Electrophoretic Mobility Shift Assay; Embryonic Development; Event; Face; Family; Functional disorder; Gene Expression; Gene Mutation; Gene Targeting; Genes; Genetic Diseases; Genetic Variation; Genome; Genomics; Glutamates; Goals; Head; Helix-Turn-Helix Motifs; Human Development; In Vitro; Interferometry; Lead; Macrostomia; Methods; Missense Mutation; Molecular; Mutation; Nucleotides; Outcome; Pathogenicity; Pathway interactions; Patients; Property; Proteins; Publishing; Rare Diseases; Role; Setleis syndrome; Shapes; Signaling Molecule; Specificity; Structure; Syndrome; TWIST1 gene; Work; X-Ray Crystallography; craniofacial; craniofacial development; disease-causing mutation; experimental study; gain of function; histone modification; in vivo; member; method development; mutant; protein complex; protein function; three dimensional structure; transcription factor

PROJECT SUMMARY Development of the head and face constitutes one of the most complex events during embryonic development, requiring a network of transcription factors and signaling molecules together with proteins conferring cell polarity and cell-cell interactions. Craniofacial abnormalities are among the most common findings in birth defects. Transcription factors (TFs) of the helix-loop-helix (HLH) family have important roles during human development. Mutations in the Twist subfamily of bHLH TFs result in genetic disorders that impact the formation of mesodermal derivatives during vertebrate embryogenesis. The basic HLH (bHLH) subfamily members can act as repressors or activators, depending on their dimerization partner. The long-term goal of the proposed work is to determine the molecular mechanisms by which TWIST bHLH proteins decode genomic information, and how genetic variation modulates TWIST1/2-genome interactions that impact craniofacial development. Mutations in TWIST1 have been shown to cause the Saethre-Chotzen (SCS), Robinow-Sorauf (RSS), Sweeney-Cox (SwCS) Syndromes and Craniosynostosis-1 (CRS1), while mutations in TWIST2 cause Setleis (SS), Barber Say (BSS) and Ablepharon Macrostomia (AMS) Syndromes, all genetic disorders that impact the development of the head and facial structures. Mutations that affect a highly conserved Glutamate (E75 and E117 in TWIST2 and TWIST1, respectively) in the basic region of bHLH proteins, which is responsible for nucleotide binding in both class I and II groups, cause the most severe syndromes. The E75Q and E75A mutations have been suggested to alter the DNA-binding activity of TWIST2, leading to both dominant-negative and gain-of-function effects. In Specific Aim 1, we will determine the binding affinities of TWIST1/2 and selected mutant proteins found in patients by EMSAs, biolayer interferometry and structural studies via methods such as circular dichroism, X-ray Crystallography, etc. In Specific Aim 2, we will determine the DNA-sequence specificity of TWIST1 and TWIST2 complexes (as homodimers or heterodimers with E12 as partner). We will use in vivo (ChIP) and in vitro (SELEX) DNA binding assays combined with DNA sequencing to determine the DNA-binding specificity of these complexes and the role that specific histone modifications (both activating and inactivating marks) and chromatin structure (using ATAC-Seq). Bioinformatic analyses will be performed in order to interpret changes in gene targets between wild-type and mutant proteins and determine the TWIST binding site sequences used to regulate gene expression of target genes. With this approach we will determine the sequences of TWIST2 binding sites used to regulate gene expression of target genes, since there is published evidence that missense mutations in the DNA-binding domain of TWIST2 results in altered DNA-binding. Bioinformatics analyses will be performed in order to predict changes in gene targets between wild-type and mutant proteins. This project will contribute to our understanding of how genetic variation contributes to normal craniofacial development and to the craniofacial diseases caused by mutations in TWIST1 and TWIST2 at the molecular level.

项目摘要 头和脸的发展构成了最复杂的事件之一 胚胎开发，需要转录因子和信号的网络 分子与蛋白质赋予细胞极性和细胞 - 细胞相互作用的蛋白质。 颅面异常是先天缺陷中最常见的发现之一。 螺旋环螺旋（HLH）家族的转录因子（TFS）在 人类发展。 BHLH TFS的扭曲亚家族中的突变导致遗传 影响脊椎动物期间中胚层衍生物形成的疾病 胚胎发生。基本的HLH（BHLH）亚家族成员可以充当阻遏物或 激活剂，具体取决于其二聚合作伙伴。拟议的长期目标 工作是确定扭曲BHLH蛋白解码的分子机制 基因组信息以及遗传变异如何调节扭曲1/2基因组 影响颅面发展的相互作用。扭曲中的突变已显示 引起Saethre-Chotzen（SC），Robinow-Sorauf（RSS），Sweeney-Cox（SWC） 综合征和颅骨突变症1（CRS1），而Twist2中的突变导致setleis （SS），理发师说（BSS）和Ablepharon宏观（AMS）综合征，所有遗传 影响头部和面部结构发展的疾病。突变 影响高度保守的谷氨酸（Twist2和Twist1中的E75和E117， 在BHLH蛋白的基本区域中分别），该区域负责核苷酸 在I类和II组中结合，导致最严重的综合征。 E75Q和 已经提出E75a突变改变了Twist2的DNA结合活性， 导致占主导地位和功能的效果。在特定目标1中，我们将 确定Twist1/2的结合亲和力，并在 通过EMSA，Biolayer干涉法和结构研究的患者通过诸如 圆二色性，X射线晶体学等。在特定目标2中，我们将确定 Twist1和Twist2复合物的DNA序列特异性（作为同二聚体或 E12作为合作伙伴的异二聚体）。我们将使用体内（芯片）和体外（SELEX）DNA 结合测定与DNA测序结合以确定DNA结合特异性 这些复合物以及特定组蛋白修饰的作用（激活和 灭活标记）和染色质结构（使用ATAC-SEQ）。生物信息学分析 将执行以解释野生型和之间基因靶标的变化 突变蛋白并确定用于调节基因的扭曲结合位点序列 靶基因的表达。通过这种方法，我们将确定 Twist2结合位点用于调节靶基因的基因表达，因为有 发表的证据表明，扭曲的DNA结合域中的错义突变2 导致DNA结合改变。将进行生物信息学分析以进行 预测野生型和突变蛋白之间基因靶标的变化。这个项目将 有助于我们对遗传变异如何促进正常颅面的理解 发育和颅面疾病是由Twist1中突变引起的 分子水平的Twist2。