Function of bridging molecules in enhancer-promoter communication during erythropoieis

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Transcription factor GATA-1 is known to interact with various factors and form different protein complexes which may play distinct roles during erythropoiesis. GATA-1 gene itself is one of the candidates for GATA-1 target genes, while molecular basis of a putative GATA-1 complex regulating GATA-1 expression is not well understood.In this study, we used the zebrafish system to elucidate regulatory mechanisms of the GATA-1 gene. To this end, we isolated and analyzed zebrafish GATA-1 genomic DNA, resulting in a finding of a novel intron that was unknown in the previous analysis. This intron corresponds to the first intron of other vertebrate GATA-1 genes. GFP reporter analyses revealed that this intron and a distal double GATA motif in the regulatory region are important for the regulation of zebrafish GATA-1 gene expression. To examine whether GATA-1 regulates its own gene expression, we microinjected into embryos a GFP reporter gene linked to the GATA-1 gene regulatory region and the GATA-1 mRNA successively.Surprisingly, ectopic expression of the reporter gene was induced at the site of GATA-1 overexpression and was dependent on the distal dotuble GATA motif. Functional domain analyses using transgenic fish lines harboring the GATA-1-GFP reporter construct revealed that both the amino- and carboxyl-zinc finger domains of GATA-1, hence the intact GATA-1 function, are required for the ectopic GFP expression. These results provide the first in vivo evidence that GATA-1 gene expression undergoes positive autoregulation.