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

基本信息

批准号：
10401753
负责人：
CARMEN LYDIA CADILLA
金额：
$ 37.5万
依托单位：
UNIVERSITY OF PUERTO RICO MED SCIENCES
依托单位国家：
美国
项目类别：
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/10401753
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

项目摘要

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.

项目概要头部和面部的发育是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传导网络分子与蛋白质一起赋予细胞极性和细胞间相互作用。颅面部异常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) 在人类发展。 bHLH 转录因子 Twist 亚家族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。基本 HLH (bHLH) 亚家族成员可以充当阻遏蛋白或激活剂，取决于它们的二聚伙伴。拟议的长期目标工作是确定 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 TWIST1/2 基因组影响颅面发育的相互作用。 TWIST1 突变已被证实引起 Saethre-Chotzen (SCS)、Robinow-Sorauf (RSS)、Sweeney-Cox (SwCS) 综合征和颅缝早闭-1 (CRS1)，而 TWIST2 突变则导致 Setleis (SS)、Barber Say (BSS) 和 Ablepharon Macrostomia (AMS) 综合征，均为遗传性影响头部和面部结构发育的疾病。突变影响高度保守的谷氨酸（TWIST2 和 TWIST1 中的 E75 和 E117，分别）位于bHLH蛋白的碱性区域，该区域负责核苷酸 I 类和 II 类组中的结合会导致最严重的综合征。 E75Q 和 E75A 突变被认为会改变 TWIST2 的 DNA 结合活性，导致显性负效应和功能获得效应。在具体目标 1 中，我们将确定 TWIST1/2 和所选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治疗圆二色性、X射线晶体学等。在具体目标2中，我们将确定 TWIST1 和 TWIST2 复合物（作为同源二聚体或以 E12 作为伴侣的异二聚体）。我们将使用体内 (ChIP) 和体外 (SELEX) DNA 结合测定与 DNA 测序相结合以确定 DNA 结合特异性这些复合物的作用以及特定组蛋白修饰（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目标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 TWIST2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 TWIST2 DNA 结合域发生错义突变导致 DNA 结合改变。将进行生物信息学分析，以便预测野生型和突变蛋白之间基因靶标的变化。该项目将有助于我们了解遗传变异如何影响正常颅面 TWIST1 和 TWIST1 突变引起的颅面疾病分子水平上的TWIST2。