Mechanisms of TWIST bHLH Transcription Factors Binding to Functional Target Regions

项目总结 头部和面部的发育是整个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 分子和蛋白质一起决定细胞的极性和细胞与细胞的相互作用。 头面部异常是出生缺陷最常见的发现之一。 螺旋-环-螺旋(HLH)家族的转录因子(TF)在细胞周期中发挥重要作用。 人的发展。BHLHTF的Twist亚家族突变导致遗传 影响脊椎动物中胚层衍生物形成的紊乱 胚胎发生。基本的hhh(Bhlh)亚家族成员可以作为阻遏因子或 激活剂，取决于它们的二聚化伙伴。建议的长期目标是 工作是确定扭曲的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双1/2基因组 影响头面部发育的相互作用。已发现Twist1基因突变 导致Saethre-Chotzen(SCS)、Robinow-Sorauf(RSS)、Sweeney-Cox(SWCS) 综合征和颅缝早闭-1(CRS1)，而TWIST2的突变导致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紊乱。突变 影响高度保守的谷氨酸(TWIST2和TWIST1中的E75和E117， 分别)，在bHLH蛋白的碱性区域，负责核苷酸 在I类和II类组中，绑定都会导致最严重的症状。E75Q和 E75A突变被认为改变了TWIST2的DNA结合活性， 导致显性负效应和功能增益效应。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 患者通过EMSA、生物分子层干涉测量和结构研究等方法 圆二色谱、X射线结晶学等。在具体目标2中，我们将确定 Twist1和TWIST2复合物的DNA序列特异性(作为同源二聚体或 以E12为伙伴的杂二聚体)。我们将使用体内(芯片)和体外(SELEX)DNA 结合DNA测序确定DNA结合的特异性 以及特定的组蛋白修饰(激活和 失活标记)和染色质结构(使用ATAC-Seq)。生物信息学分析 将用于解释野生型和野生型之间基因靶标的变化 突变蛋白并确定用于调控基因的扭转结合位点序列 目的基因的表达。使用这种方法，我们将确定以下序列 TWIST2结合位点用于调节靶基因的基因表达，因为有 已发表的证据表明TWIST2的DNA结合域的错义突变 导致DNA结合的改变。将进行生物信息学分析，以便 预测野生型和突变型蛋白之间的基因靶标变化。这个项目将 有助于我们理解基因变异是如何影响正常颅面的 Twist1和Twist1基因突变引起的颅面疾病的发生和发展 TWIST2在分子水平上。

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.