Transcription factors play an important role in tissue-specific gene regulation during development. Twist is a basic helix-loop-helix (bHLH) transcription factor that functions in the mesoderm, or middle embryonic germ layer. Humans with Twist mutations have craniofacial and digit defects characteristic of Saethre-Chotzen syndrome. Caenorhabditis elegans is an excellent model organism for elucidating Twist function due to its simplicity, availability of molecular tools, and powerful genetics. The information learned in C. elegans about CeTwist function will be relevant to humans and human disease because CeTwist shares homology with human Twist and multiple CeTwist target genes identified to date are homologous to genes that also cause craniofacial disorders when mutated in humans. bHLH proteins function as dimers. The only dimer partner identified for CeTwist is CeE/DA, which is the homolog of vertebrate E proteins and Drosophila Daughterless (DA). The work in this proposal is designed to uncover the unique functions of CeTwist homodimers and CeTwist/CeE/DA heterodimers in the mesoderm. In the first specific aim, we will use gfp reporters to examine the mesoderm development and CeTwist target gene expression both in CeE/DA mutant animals as well as in animals expressing physically-linked CeTwist dimers that are predicted to behave as forced homodimers in vivo. These experiments are expected to reveal the role that CeE/DA and CeTwist/CeE/DA heterodimers play in the mesoderm as well as to confirm a predicted role for CeTwist homodimers. In the second specific aim, we will examine a promoter paradigm that we recently developed for understanding elements that regulate tissue-specific expression of a model CeTwist target gene, arg-1. bHLH proteins bind to E boxes that are defined as CANNTG, with N being any nucleotide. The minimal arg-1 promoter has 3 E boxes that we have shown by site-directed mutagenesis are uniquely required for gene expression in multiple tissues. The contribution of each E box as well as the intervening sequences between the E boxes will be examined using gfp reporters. Then, the E boxes will be tested in vivo for expression in the presence of excess CeTwist or CeTwist and CeE/DA to determine whether homodimers or heterodimers are working through the individual E boxes. Because the projects described here are suitable for students, this work will also have a profound impact on the education of undergraduate and graduate students. The goal of this work is to examine CeTwist function and target gene regulation. Due to the relatedness between the human and C. elegans Twist proteins, information we learn in our simple model system will be relevant to understanding human Twist and provide insight into diseases caused by defective Twist. 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. ? ? ?
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