Activity of a C. elegans GATA transcription factor, ELT-1, expressed in yeast.
The GATA motif (WGATAR) is found in the promoter regions of numerous Caenorhabditis elegans genes, including two intestine-specific genes, vit-2 and ges-1, in which it has been shown to be required for promoter function. The protein ELT-1, encoded by a single-copy gene homologous to the GATA family of vertebrate transcription factors, is potentially capable of interacting with this element. In order to determine whether ELT-1 is a transcriptional activator that recognizes this sequence, we have expressed it under the control of the GAL1 promoter in yeast. lacZ driven by the CYC1 promoter lacking an upstream activation sequence (UAS) but containing GATA sequences was used as a reporter. beta-Galactosidase was expressed upon induction only when GATA sequences were present, and expression was increased dramatically by additional binding sites. Deletion analysis demonstrated that the C terminus, containing only one of the two zinc fingers, is sufficient for activation. In addition, the DNA-binding domain and two transactivation regions were identified by fusing these isolated domains to previously defined domains of heterologous transcription factors. While most single base alterations in the GATA core sequence eliminated activity, an A to C change in position four, creating a GATC core, was found to increase activity significantly. The deleted ELT-1 protein containing only the C-terminal Zn finger was sufficient for activation in response to GATA, but both fingers were required for activation at GATC. A variety of sites with non-optimal sequences surrounding the GATA core also were found to be excluded better by the protein containing both Zn fingers. Furthermore, a fusion protein containing the entire ELT-1 DNA binding domain fused to the VP16 activation domain was found to have an even greater preference for the GATC core, as well as the optimal flanking bases. We conclude that, although ELT-1 having only its C-terminal finger is capable of activation in response to the WGATAR site, the presence of the upstream finger supplies additional base specificity.
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D017173:Caenorhabditis elegans / Q000235:genetics*; D029742:Caenorhabditis elegans Proteins; D003574:Cytochrome c Group / Q000235:genetics; D045304:Cytochromes c; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D050980:GATA Transcription Factors; D015966:Gene Expression Regulation, Fungal; D005800:Genes, Fungal; D015801:Helminth Proteins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012045:Regulatory Sequences, Nucleic Acid; D012441:Saccharomyces cerevisiae / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins; D013379:Substrate Specificity; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism*; D016335:Zinc Fingers / Q000235:genetics*
C014059:CYC1 protein, S cerevisiae; D029742:Caenorhabditis elegans Proteins; D003574:Cytochrome c Group; D004268:DNA-Binding Proteins; C096869:ELT-1 protein, C elegans; D050980:GATA Transcription Factors; D015801:Helminth Proteins; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D015534:Trans-Activators; D045304:Cytochromes c; D004247:DNA