Inhibition of Tartrate-Resistant Acid Phosphatase Gene Expression by Hemin and Protoporphyrin IX. Identification of a Hemin-Responsive Inhibitor of Transcription

Abstract

Tartrate-resistant acid phosphatase (TRAP) is an iron-containing protein encoded by the same gene that codes for uteroferrin, a placental iron transport protein. In human peripheral mononuclear cells, TRAP expression is inhibited by both hemin (ferric protoporphyrin IX) and protoporphyrin IX. Nuclear run-on assays confirmed that this inhibition occurs at the level of gene transcription. Previous studies with mTRAP deletion mutants showed that the hemin effect was dependent on repressor activity in the mTRAP 5'-flanking region at -1846 bp to -1240 bp relative to ATG (Reddy et al, J Bone Mineral Res 10:601, 1995). We now report that gel shift assays showed a DNA binding protein in nuclear extracts of hemin-treated cells termed hemin response element binding protein (HREBP). Additional studies have localized the HREBP binding region in the mTRAP 5'-flanking DNA to a 27-bp sequence at -1815 to -1789 bp relative to ATG. A tandem repeat sequence, GAGGC;GAGGC, contained within this DNA segment, was shown to be involved in binding of HREBP. Highly homologous sequences are present in the 5'-flanking region of the hTRAP gene. Binding of HREBP to the mTRAP DNA sequence was inhibited by anti-HAP1 antibodies, indicating homology between the hemin-responsive factor and the yeast heme-dependent transcription factor, HAP1. A 607-bp segment of the mTRAP 5'-flanking region containing the candidate hemin response element and surrounding sequences conferred hemin regulation on the viral SV40 promoter. Southwestern blotting experiments probing nuclear extracts of hemin-treated U937 cells with the 27-bp binding sequence showed two protein bands at 37 and 133 kD representing candidate HREBPs. A GENINFO search showed several other mammalian genes with tandem GAGGC motifs in noncoding regions, providing the possibility that additional genes may also be regulated by hemin at the level of transcription. These studies provide the first description of a novel iron/hemin-responsive transcriptional regulatory mechanism in mammalian cells.