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 头部和面部的发育是人类发育过程中最复杂的事件之一。 胚胎发育，需要转录因子和信号网络 分子与赋予细胞极性和细胞-细胞相互作用的蛋白质一起。 颅面畸形是出生缺陷中最常见的发现之一。 螺旋-环-螺旋（HLH）家族的转录因子（TF）在转录过程中具有重要作用。 人类发展bHLH TF的Twist亚家族中的突变导致遗传性 影响脊椎动物中胚层衍生物形成的疾病 胚胎发生碱性HLH（bHLH）亚家族成员可作为阻遏物或 活化剂，这取决于它们的二聚化伴侣。拟议的长期目标 我们的工作是确定TWIST 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TWIST 1/2基因组 影响颅面发育的相互作用TWIST 1中的突变已被证明 导致Saethre-Chotzen（SCS）、Robinow-Sorauf（RSS）、Sweeney-Cox（SwCS） Syndrome和Craniosynostosis-1（CRS 1），而TWIST 2的突变导致Setleis (SS)，Barber Say（BSS）和Ablepharon Macrostomia（AMS）Syndrome，所有遗传 影响头部和面部结构发育的疾病。的突变 影响高度保守的谷氨酸（TWIST 2和TWIST 1中的E75和E117， bHLH蛋白的碱性区，负责核苷酸的合成， 在I类和II类组中结合，引起最严重的综合征。E75 Q和 已经提出E75 A突变改变TWIST 2的DNA结合活性， 导致显性负效应和功能获得效应。具体目标1： 确定TWIST 1/2和在细胞中发现的选定突变蛋白的结合亲和力。 通过EMSA、生物层干涉测量和结构研究等方法对患者进行研究， 圆二色性、X射线晶体学等。在具体目标2中，我们将确定 TWIST 1和TWIST 2复合物的DNA-序列特异性（作为同源二聚体或 以E12作为配偶体的异二聚体）。我们将使用体内（ChIP）和体外（SELEX）DNA 结合试验与DNA测序相结合，以确定DNA结合特异性 这些复合物的作用和特定的组蛋白修饰（激活和 失活标记）和染色质结构（使用ATAC-Seq）。生物信息学分析 将进行以解释野生型和野生型之间基因靶点的变化 突变蛋白，并确定用于调节基因的TWIST结合位点序列 目的基因的表达。通过这种方法，我们将确定 TWIST 2结合位点用于调节靶基因的基因表达，因为存在 已发表的证据表明TWIST 2的DNA结合结构域中的错义突变 导致DNA结合改变。将进行生物信息学分析，以 预测野生型和突变蛋白之间的基因靶点变化。该项目将 有助于我们理解遗传变异如何影响正常颅面 发展和由TWIST 1突变引起的颅面疾病， 分子水平上的TWIST 2。