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.
The goal of this project is to provide insight into the function of proteins that are defective in human syndromes characterized by craniofacial and limb defects. We have developed a system for examining how these proteins are regulating gene expression in the roundworm, which is a simple model genetic organism. We expect because roundworm and human proteins are related, the information we learn will be relevant to humans and human disease.
| Kim, Sharon; Twigg, Stephen R F; Scanlon, Victoria A et al. (2017) Localized TWIST1 and TWIST2 basic domain substitutions cause four distinct human diseases that can be modeled in Caenorhabditis elegans. Hum Mol Genet 26:2118-2132 |
| Philogene, Mary C; Small, Stephany G Meyers; Wang, Peng et al. (2012) Distinct Caenorhabditis elegans HLH-8/twist-containing dimers function in the mesoderm. Dev Dyn 241:481-92 |
| Meyers, Stephany G; Corsi, Ann K (2010) C. elegans twist gene expression in differentiated cell types is controlled by autoregulation through intron elements. Dev Biol 346:224-36 |
| McGovern, Marie; Voutev, Roumen; Maciejowski, John et al. (2009) A ""latent niche"" mechanism for tumor initiation. Proc Natl Acad Sci U S A 106:11617-22 |
