GATA-1 binding sites in exon 1 direct erythroid-specific transcription of PPOX

Abstract

We investigated erythroid-specific expression of the human PPOX gene. This gene encodes protoporphyrinogen oxidase, which is involved in synthesizing heme for red blood cells and heme as a cofactor for the respiratory cytochromes. In vitro luciferase transfection assays in human uninduced and hemin induced erythroleukemic K562 cells showed that the presence of exon 1 increased promoter activity fourfold as compared to reporter constructs lacking this exon. This transcriptional regulation was mediated by two GATA-1 sites in exon 1. Electrophoretic mobility shift and chromatin immunoprecipitation assays demonstrated that both GATA sites were able to bind GATA-1 in vitro and in vivo. Exon 1 did not affect promoter activity in human hepatoma HepG2 cells and U937 monocytic cells but its presence decreased promoter activity in HeLa human cervical carcinoma cells. We conclude that the GATA-1 binding sites in exon 1 constitute key regulatory elements in differential expression of PPOX in erythroid and non-erythroid cells.