MOUSE FERRITIN-H SUBUNIT GENE - FUNCTIONAL-ANALYSIS OF THE PROMOTER AND IDENTIFICATION OF AN UPSTREAM REGULATORY ELEMENT ACTIVE IN ERYTHYROID CELLS

Ferritin synthesis is regulated at the translational level by iron, bu t it is likely that transcriptional regulation of H and L genes is res ponsible for tissue-specific distribution of H and L mRNAs. In order t o define the regions important for transcriptional regulation of the m ouse ferritin H gene, we have linked the promoter, including the trans cription start site, and 5 kilobases of upstream sequence to a reporte r gene (human growth hormone). This construct and a series of 5' delet ion mutants have been used to transfect erythroid (K562, mouse erythro leukemia (MEL)) and hepatoma (HepG(2)) cell lines. Measurement of grow th hormone in the culture medium and analysis of ferritin-growth hormo ne transcripts by a ribonuclease protection assay revealed that a 140- base pair minimal promoter is sufficient to confer a high level of exp ression to the reporter gene in both cell types. In addition, a 180-ba se pair fragment, lying 4.5 kilobases upstream of the ferritin transcr iption start site, functions like an inducible enhancer during N,N'-he xamethylene-bis-acetamide-induced differentiation of MEL cells. A perf ect match to a consensus binding motif to the erythroid transcription factor NF-E2 is present in this regulatory element, but the mutant NF- E2 enhancer retains the inducible activity in stably transfected MEL c ells, and the results from gel retardation assays suggest that protein -DNA complexes that form in vitro between the ferritin enhancer and ME L nuclear extracts do not contain NF-E2. Thus, nuclear factors that me diate inducibility of the ferritin enhancer remain to be identified.