A GATA Box in the GATA-1 Gene Hematopoietic Enhancer Is a Critical Element in the Network of GATA Factors and Sites That Regulate This Gene

Abstract

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 orientation-independent 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 endogenousGATA-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-1gene 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.

ACKNOWLEDGMENTS

We thank N. Kajiwara, N. Kaneko, Y. Kikuchi, N. Kasai, K.-C. Lim, J. Ohta, H. Motohashi, F. Sugiyama, N. Suzuki, and K. Yagami for help and discussion.

This work was supported in part by Grants-in-Aids from the Ministry of Education, Science, Sports and Culture, Core Research for Evolutional Science and Technology (CREST), the Japanese Society for Promotion of Sciences (JSPS), and NIH.