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Abstract:
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The <italic>hlh-8</italic> gene encodes a basic helix-loop-helix transcription factor called Twist that is involved in mesoderm development and the morphogenesis of enteric and sex muscles in <italic>C. elegans</italic>. Twist binds to the canonical binding site CANNTG referred to as an E box. The <italic>hlh-8</italic> gene is composed of 5 exons with a 2 kb intron after the first exon. Because the function of <italic>hlh-8</italic> is dependent on the gene being expressed in the appropriate cell type, it is important to understand how the expression of the <italic>hlh-8</italic> gene is regulated. Typically, elements in the promoter of a gene determine the regulation of gene expression in different tissues. However, only elements that control expression in a subset of Twist containing tissues have been discovered in the promoter. Therefore, additional elements must exist elsewhere and a hypothesis was developed that regulatory elements are present in the large first intron. This study explores the <italic>hlh-8</italic> intron using a construct that contains a basal promoter and can be activated to express GFP in a variety of tissues by juxtaposition to a tissue-specific enhancer. Constructs containing portions of intron 1 reveal two regions, with a single E box each, which are sufficient to drive expression of <italic>gfp</italic> in a subset of tissues that express Twist. Furthermore, expression of <italic>gfp</italic> is lost when both E boxes are disrupted. My hypothesis is that these E boxes are important for <italic>hlh-8</italic> autoregulation. Results from expressing these reporters in <italic>hlh-8</italic> null mutants and examining Twist binding to the E boxes by <italic>in vitro</italic> gel shift analysis support this hypothesis. Additionally, this study characterizes a mutant with a large deletion (646 bp) in the first intron of <italic>hlh-8</italic>. Altogether, results from this study lead to an understanding of tissue-specific regulation of <italic>hlh-8</italic>. Intron elements appear to control expression in differentiated tissues, whereas it has been shown previously that other factors regulate expression in undifferentiated cells. Moreover, since there is homology between <italic>C. elegans</italic> and human Twist proteins, understanding the regulation of <italic>hlh-8</italic> will elucidate the control of expression for the gene that encodes for the human Twist protein. |