Positive and negative regulation of the human heme oxygenase-1 gene expression in cultured cells.

To elucidate the regulation of the human heme oxygenase-1 (hHO-1) gene expression, we assessed approximately 4 kb of the 5'-flanking region of the hHO-1 gene for basal promoter activity and sequenced approximately 2 kb of the 5'-flanking region. A series of deletion mutants of the 5'-flanking region linked to the luciferase gene was constructed. Basal level expression of these constructs was tested in HepG2 human hepatoma cells and HeLa cervical cancer cells. By measuring luciferase activity, which was transiently expressed in the transfected cells, we found a positive regulatory region at position -1976 to -1655 bp. This region functions in HepG2 cells but not in HeLa cells. A negative regulatory region was also found at position -981 to -412 bp that functions in both HepG2 cells and HeLa cells.     

Isolation and characterization of the QM promoter.

This report describes the isolation, sequencing and preliminary characterization of the first 1 kb of the 5'-regulatory region of the human QM gene. This region and the 5' -half of the transcribed region of the QM gene are enriched for C and G nucleotides with no bias against CpG dinucleotides--indicative of a CpG island. Several consensus GC boxes are present within the sequence. Most are clustered at the distal end, with one site present in the proximal 200 bp of the promoter. Electrophoretic mobility shift experiments and luciferase assays done in insect cells transfected with an Sp1 expression construct suggest that most of these sites can bind Sp1 or a closely related factor. In addition, the promoter is shown to be responsive to cAMP via a response element (CRE) in the proximal promoter. Studies with 5'-end and internal deletion mutants suggest that elements in the distal promoter exert their positive effect through interactions with a proximal element(s). Candidate proximal elements include the proximal GC box and a 43 bp region between a KpnI site (at -182) and a Smal site (at -139).     

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

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.