Verification of the in vivo activity of three distinct cis ‐acting elements within the Gata1 gene promoter‐proximal enhancer in mice

The transcription factor GATA 1 is essential for erythroid and megakaryocytic cell differentiation. G ata1 h ematopoietic r egulatory d omain ( G1HRD ) has been shown to recapitulate endogenous Gata1 gene expression in transgenic mouse assays in vivo . G1HRD contains a promoter‐proximal enhancer composed of a GATA ‐palindrome motif, four CP 2‐binding sites and two CACCC boxes. We prepared transgenic reporter mouse lines in which green fluorescent protein and β‐galactosidase expression are driven by wild‐type G1HRD (as a positive control) and the G1HRD harboring mutations within these cis ‐acting elements (as the experimental conditions), respectively. Exploiting this transgenic dual reporter ( TDR ) assay, we show here that in definitive erythropoiesis, G1HRD activity was markedly affected by individual mutations in the GATA ‐palindrome motif and the CACCC boxes. Mutation of CP 2‐binding sites also moderately decreased G1HRD activity. The combined mutation of the CP 2‐binding sites and the GATA ‐palindrome motif resulted in complete loss of G1HRD activity. In contrast, in primitive erythroid cells, individual mutations of each element did not affect G1HRD activity; G1HRD activity was abolished only when these three mutations were combined. These results thus show that all three elements independently and cooperatively contribute to G1HRD activity in vivo in definitive erythropoiesis, although these are contributing redundantly to primitive erythropoiesis.