Direct visualization of the elt-2 gut-specific GATA factor binding to a target promoter inside the living Caenorhabditis elegans embryo.
In analyzing the transcriptional networks that regulate development, one ideally would like to determine whether a particular transcription factor binds directly to a candidate target promoter inside the living embryo. Properties of the Caenorhabditis elegans elt-2 gene, which encodes a gut-specific GATA factor, have allowed us to develop such a method. We previously have shown, by means of ectopic expression studies, that elt-2 regulates its own promoter. To test whether this autoregulation is direct, we fused green fluorescent protein (GFP) close to the C terminus of elt-2 in a construct that contains the full elt-2 promoter and the full elt-2 zinc finger DNA binding domain; the construct is expressed correctly (i.e., only in the gut lineage) and is able to rescue the lethality of an elt-2 null mutant. Multicopy transgenic arrays of this rescuing elt-2::GFP construct were integrated into the genome and transgenic embryos were examined when the developing gut has 4-8 cells; the majority of these embryonic gut nuclei show two discrete intense foci of fluorescence. We interpret these fluorescent foci as the result of ELT-2::GFP binding directly to its own promoter within nuclei of the developing gut lineage. Numerous control experiments, both genetic and biochemical, all support this conclusion and support the specificity of the binding. The approach should be applicable to studying other transcription factors binding target promoters, all within the living C. elegans embryo.
Fukushige
T
T
Department of Biochemistry, University of Calgary, Calgary, Alberta, Canada T2N 4N1.
Hendzel
M J
MJ
Bazett-Jones
D P
DP
McGhee
J D
JD
D000818:Animals; D030801:Animals, Genetically Modified; D017173:Caenorhabditis elegans / Q000196:embryology*; D029742:Caenorhabditis elegans Proteins; D002467:Cell Nucleus / Q000378:metabolism; D005260:Female; D050980:GATA Transcription Factors; D049452:Green Fluorescent Proteins; D007413:Intestinal Mucosa / Q000378:metabolism; D007422:Intestines / Q000196:embryology; D008164:Luminescent Proteins / Q000378:metabolism; D008297:Male; D018715:Microscopy, Video / Q000379:methods*; D008957:Models, Genetic; D010957:Plasmids / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*
D029742:Caenorhabditis elegans Proteins; C094134:ELT-2 protein, C elegans; D050980:GATA Transcription Factors; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D049452:Green Fluorescent Proteins