Mechanisms of TWIST bHLH Transcription Factors Binding to Functional Target Regions

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.

Grant Number: 5SC1GM139706-04
NIH Institute/Center: NIH
Principal Investigator: CARMEN CADILLA