Abstract

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 Shigeko Nishimura 1 , Satoru Takahashi 1 , Takashi Kuroha 1 , Naruyoshi Suwabe 1 , Toshiro Nagasawa 2 , Cecelia Trainor 3 , and Masayuki Yamamoto 1 , * Center for Tsukuba Advanced Research Alliance and Institutes of Basic Medical Sciences 1 and Clinical Medicine, 2 University of Tsukuba, Tsukuba 305-8577, Japan, and Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892 3 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 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.