Investigating the control of hlh-8 gene expression
The hlh-8 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 C. elegans. Twist binds to the canonical binding site CANNTG referred to as an E box. The hlh-8 gene is composed of 5 exons with a 2 kb intron after the first exon. Because the function of hlh-8 is dependent on the gene being expressed in the appropriate cell type, it is important to understand how the expression of the hlh-8 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 hlh-8 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 gfp in a subset of tissues that express Twist. Furthermore, expression of gfp is lost when both E boxes are disrupted. My hypothesis is that these E boxes are important for hlh-8 autoregulation. Results from expressing these reporters in hlh-8 null mutants and examining Twist binding to the E boxes by in vitro gel shift analysis support this hypothesis. Additionally, this study characterizes a mutant with a large deletion (646 bp) in the first intron of hlh-8. Altogether, results from this study lead to an understanding of tissue-specific regulation of hlh-8. 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 C. elegans and human Twist proteins, understanding the regulation of hlh-8 will elucidate the control of expression for the gene that encodes for the human Twist protein.
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