Identification and characterization of novel homodimer targets of CeTwist
Twist, a basic-helix-loop-helix (bHLH) transcription factor, functions in the mesoderm in many organisms. bHLH transcription factors dimerise with their helix-loop-helix region forming a heterodimer or a homodimer and the basic regions of the dimers bind to a consensus sequence known as an E box on their target genes. Previous evidence suggests a distinct role for CeTwist homodimers and heterodimers (with CeE/DA) in different mesoderm cells in the model organism C. elegans. We have already identified a number of CeTwist heterodimer targets. However, no CeTwist homodimer targets have been identified to date. The aim of this project is to discover and study such genes to understand bHLH dimer-specific transcriptional regulation. To find CeTwist homodimer targets, we overexpressed CeTwist by using an inducible heat shock promoter and found the potential target genes that are overexpressed using a global gene expression measurement technique called Affymetrix oligonucleotide microarrays. Nineteen genes from a list of overexpressed genes were prioritized based on higher microarray value. Using transcriptional GFP reporters, we examined the expression pattern of these genes. We were interested in a pattern that may partially overlap with the CeTwist expression pattern, and we found five genes in this category. Then GFP expression was studied in mutant animals of the hlh-2 (CeE/DA) and hlh-8 (CeTwist) genes for further validation. The results suggest that we found one CeTwist homodimer target, two targets of CeTwist and CeE/DA heterodimers and two more CeTwist targets that need further characterization. Next, we studied the promoter region of the new CeTwist targets by using 5'deletion reporter constructs. In one of the heterodimer targets, we found a previously identified E box playing a role in target gene expression whereas for the homodimer target, we identified a palindromic sequence CACGTG that plays an important role in expression. This interesting result is consistent with bHLH homodimers preferentially binding to palindromic sequences with identical half-sites for regulation. Moreover, we also found that two of the new CeTwist target genes identified in this study, have human homologues which are mutated in human diseases. Therefore, understanding the transcriptional regulation of these genes is important.
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