Regulation of globin gene transcription by heme in erythroleukemia cells: analysis of putative heme regulatory motifs in the p45 NF-E2 transcription factor.

The function of the NF-E2 transcription factor, a p45/small Maf heterodimer, was analyzed in the erythroleukemia cell lines MEL and CB3. In contrast to MEL cells, CB3 cells are null for p45 and thus express only extremely low levels of adult globin transcripts upon induction by agents promoting erythroid differentiation. We investigated the response of erythroleukemia cells to hemin treatment. Hemin rapidly induces beta-globin gene transcript levels in MEL cells, but not in CB3 cells. Stable expression of the large p45 NF-E2 subunit in CB3 cells restores hemin mediated beta-globin gene transcription, suggesting that the presence of a functional NF-E2 is required for strong induction of beta-globin mRNA levels by hemin in erythroleukemia cells. We performed mutagenesis of two potential heme-regulatory motifs (HRMs) in p45 NF-E2 and found that the mutated versions are expressed and can still recognize a NF-E2 DNA binding element. In addition, we showed that p45 NF-E2 HRM mutants are able to restore beta-globin gene transcription in CB3 cells upon induction by hemin. Our results suggest that globin gene activation by heme appears to be independent of the putative HRMs in the p45 subunit of the NF-E2 transcription factor.