Transcription factors play an important role in tissue-specific gene regulation during development. The basic
helix-loop-helix (bHLH) transcription factors TWIST1 and TWIST2 are critical regulators of cell fate and
differentiation during vertebrate development. These factors are mutated in several diseases that lead to
craniofacial, digit, and other defects making it important to understand how these bHLH factors function and
the underlying causes associated with their disease phenotypes. bHLH proteins form dimers with their HLH
domains and bind to DNA sequences called E boxes with the basic DNA binding domains of the dimer
partners. Although much is known about DNA binding and dimer formation, very little is known about the
cooperation between the two domains for proper bHLH function. A glutamic acid in the DNA binding domain is
predicted to play a critical role based on three pieces of evidence. There is a lack of amino acid variation at
that position in bHLH protein alignments, bHLH crystal structures predict the glutamic acid makes direct
contacts with DNA, and patients with amino acid substitutions in either TWIST1 or TWIST2 in that residue have
disease phenotypes. The experiments in this proposal are designed to test the hypothesis that missense
mutations of the conserved glutamic acid residue in the Twist DNA binding domain alter E-box regulated
transcription due to disrupted protein-protein interactions.
The simple nematode, Caenorhabditis elegans, is an excellent model genetic system for elucidating Twist
function because the organism contains only one Twist-related protein, HLH-8, and HLH-8’s partner protein
and downstream target genes are conserved with human TWIST1. Even though this organism is an
invertebrate and can’t have craniofacial or digit defects, the conserved Twist pathway suggests that aspects of
the cellular mechanisms will be conserved as well. In Aim 1, missense mutations in HLH-8 that mimic human
disease alleles in the conserved glutamic acid will be characterized in C. elegans. The goal will be to
understand the specific cellular defects of the mutants. Preliminary studies indicate that some of the mutants
can still bind DNA to turn on target genes. These mutants will be the focus of the research in the second aim.
In Aim 2, the C. elegans mutants will be used in modifier genetic screens designed to identify new proteins that
cooperate with HLH-8 and, by analogy, may be potential therapeutic targets for Twist-related human diseases.
The projects in the proposed work are suitable for students so the research will have a profound impact on the
education of undergraduate and graduate students. The broad goal of this research is to elucidate HLH-8
function and target gene regulation. Due to the relatedness between the human and C. elegans Twist proteins,
information learned will be relevant to understanding human TWIST1 and TWIST2 and disease pathology.