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 id eally would like to determine whether a particular transcription factor bin ds directly to a candidate target promoter inside the living embryo. Proper ties 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 fl uorescent protein (GFP) close to the C terminus of elt-2 in a construct tha t contains the full elt-2 promoter and the full elt-2 zinc finger DNA bindi ng domain; the construct is expressed correctly (i.e., only in the gut line age) 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 in to 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 r esult of ELT-2::CFP binding directly to its own promoter within nuclei of t he developing gut lineage. Numerous control experiments, both genetic and b iochemical, 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.