Transcription factor GATA-1 is required for the terminal differentiation of both the primitive and definitive erythroid cell lineages, and yet the regulatory mechanisms of GATA-1 itself are not well understood. To clarify how the GATA-1 gene is transcriptionally controlled in vivo, presumptive regulatory regions of the gene were tested by fusion to a reporter gene and then examined in transgenic mice. We found that a transcriptional control element located between ؊3.9 and ؊2.6 kb 5 to the erythroid first exon serves as an activating element and that this sequence alone is sufficient to recapitulate the expression of GATA-1 (but uniquely in primitive erythroid cells). Addition of sequences from the GATA-1 first intron to this upstream element provides a necessary and sufficient condition for complete recapitulation of GATA-1 expression in both primitive and definitive erythroid cells. The first intron element does not possess intrinsic transcriptional activation potential when linked to the GATA-1 gene promoter but rather requires the upstream activating element for its activity. These experiments show that GATA-1 gene expression is regulated by discrete transcriptional control elements during definitive and primitive erythropoiesis: The 5 element displays properties anticipated for a primitive erythroid cell-specific activating element, and the novel element within the GATA-1 first intron specifically augments this activity in definitive erythroid cells.

A region located at kbp ؊3.9 to ؊2.6 5 to the first hematopoietic exon of the GATA-1 gene is necessary to recapitulate gene expression in both the primitive and definitive erythroid lineages. In transfection analyses, this region activated reporter gene expression from an artificial promoter in a position-and orientationindependent manner, indicating that the region functions as the GATA-1 gene hematopoietic enhancer (G1HE). However, when analyzed in transgenic embryos in vivo, G1HE activity was orientation dependent and also required the presence of the endogenous GATA-1 gene hematopoietic promoter. To define the boundaries of G1HE, a series of deletion constructs were prepared and tested in transfection and transgenic mice analyses. We show that G1HE contains a 149-bp core region which is critical for GATA-1 gene expression in both primitive and definitive erythroid cells but that expression in megakaryocytes requires the core plus additional sequences from G1HE. This core region contains one GATA, one GAT, and two E boxes. Mutational analyses revealed that only the GATA box is critical for gene-regulatory activity. Importantly, G1HE was active in SCL ؊/؊ embryos. These results thus demonstrate the presence of a critical network of GATA factors and GATA binding sites that controls the expression of this gene.

Background:  The GATA‐1haematopoietic enhancer (G1HE), located between 3.9 and 2.6 kb 5′ to the haematopoietic first exon, is essential for GATA‐1 gene transcription in erythroid cells. However, G1HE is not sufficient to confer tissue specificity on the GATA‐1 gene in vivo, indicating that additional regulatory sequences are necessary. Results: We demonstrate here that two other upstream promoter elements containing a double GATA motif or two CACCC boxes are also indispensable for reporter gene expression in erythroid cells in the transgenic mouse. The combination of these three cis‐acting regions was sufficient for reporter expression in primitive erythroid cells, as demonstrated by linking the elements together into a 659 bp artificial (GdC) minigene. The minigene activated the transcription of a reporter gene from either the endogenous or an exogenous thymidine kinase promoter, retaining cell type‐specificity. The addition of a 320 bp fragment in the first intron to the GdC minigene sustained reporter expression in the definitive stage. Moreover, a line of transgenic mouse that expressed GATA‐1 cDNA under the control of the complete 979 bp minigene rescued GATA‐1 germ line mutant mice from embryonic lethality. Conclusions: A combination of four distinct sequence motifs co‐operatively serve as a fundamental functional unit for GATA‐1 erythroid transcription in vivo.