Functional analysis of the Caenorhabditis elegans transcription factor,elt-1, in yeast

The Caenorhabditis elegans GATA transcription factor, elt-1, was cloned because the GATA recognition sequence was found upstream of the vitellogenin genes. This sequence had been shown to be required for activation of the vit genes in the intestines of adult hermaphrodites. Therefore, it was hoped that study of ELT-1 would provide a better understanding of the mechanism of tissue-, time-, and sex-specific gene regulation during worm development.;elt-1 is a single copy gene and specifies a 1.75 kb mRNA that is expressed at its highest level in the germ line of both sexes. For further functional analysis of the ELT-1 protein, yeast was used as an in vivo assay. ELT-1 was expressed under the control of the GAL1 promoter by inserting either the entire elt-1 cDNA or various deletion fragments of this cDNA into a yeast expression plasmid. LacZ driven by the CYC1 promoter lacking an UAS was used as a reporter gene by inserting an oligonucleotide containing the presumptive elt-1 recognition site at the deleted UAS site.;Deletion analysis of ELT-1 indicates that the single C-terminal zinc finger and the adjacent basic region are sufficient for DNA-binding. In addition, two transactivation domains were identified. Combination of deletion analysis of the protein and mutational analysis of the activation site indicated that while the C-terminal zinc finger was sufficient for recognition of GATA, the presence of the upstream finger was also required for recognition of GATC and for excluding binding to sites with non-optimal sequences surrounding the core.;My findings demonstrated that ELT-1 is a transactivator, a positive autoregulator, and it recognizes both GATA and GATC core sequences. The best recognition site I tested was the sequence containing both GATA and GATC, found in the major sperm protein (msp) gene promoter region. These findings, combined with the observation that elt-1 is expressed at high levels in the male germline suggest that ELT-1 may serve as the activator of the abundantly expressed msp genes.