Functional characterization of the two alternative promoters of human p45 NF-E2 Gene

The transcription factor NF-E2, a heterodimeric protein complex composed of p45 and small Maf family proteins, is considered crucial for the proper differentiation of erythrocytes and megakaryocytes in vivo. Expression of the p45 subunit is restricted to selected blood lineages, including erythroid and megakaryocytic lineage cells. Here we report results of studies aimed to understand the regulatory mechanisms controlling p45 gene expression in erythroid cells. Human p45 mRNAs have the two alternative isoforms, aNF-E2 and fNF-E2, and these isoforms are transcribed from the alternative promoters. We investigated lineage-specific expression of both NF-E2 isomers by RT-PCR analysis. To examine the expression of both NF-E2 isomers in primary erythroid and megakaryocytic cells, CD34+ cells isolated from human cord blood were induced to unilineage erythroid or megakaryocytic differentiation in liquid suspension culture. fNF-E2 mRNA was found to be more abundant in erythroid cells than megakaryocytic cells, whereas aNF-E2 was predominantly expressed in megakaryocytic cells. Although both isomers were abundantly expressed in human erythroid-megakaryocytic cell lines, megakaryocytic maturation with loss of erythroid phenotype induced by phorbol 12-myristate 13-acetate (PMA) resulted in exclusive down regulation of fNF-E2. A functional analysis of fNF-E2 promoter showed that the promoter is active only in erythroid-megakaryocytic cells and that the double GATA site in the proximal region is necessary for its efficient activity. These results suggest that NF-E2 resides downstream of the GATA proteins in the transcription factor hierarchy that governs the differentiation of erythroid lineage cells.