Mechanisms of TWIST bHLH Transcription Factors Binding to Functional Target Regions

点击翻译按钮获取中文摘要

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.