metallothionein and 1-10-phenanthroline

The metal-dependent activation of metallothionein (MT) genes requires the interaction of positive trans-activators (MRFs) with metal-regulatory (MRE) regions of MT promoters. In this report, we examined the role of transition metals in modulating the MRE-binding activities of two different MRE-binding proteins: the metal-regulated factor ZiRF1 and the basal factor SP1. We showed the ability of both proteins to interact with a similar sequence specificity with the cognate target site (MRE-S) of another known MRE-binding protein, mMTF1. We next evaluated the role of metal ions in modulating the MRE-binding activity of recombinant ZiRF1 and basal SP1 proteins by measuring the effect of different metal chelators on DNA interaction. We observed a dose-dependent inhibition of the GST-ZiRF1/MRE-binding activity using three different metal chelators: EDTA, 1,10 PHE and TPEN. Interestingly, EDTA treatment failed to inhibit the recombinant SP1 MRE-binding activity while the effect of 1,10 PHE was comparable to that obtained analyzing 1,10 PHE-treated GST-ZiRF1. The MRE-binding complexes detected in cell extracts showed a response to metal chelator treatment very similar to that displayed by the recombinant ZiRF1 and SP1 proteins. The hypothesis of mutual interactions of both basal and metal-regulated transcription factors with the same metal-regulatory regions is discussed.

Topics: Animals; Base Sequence; Binding Sites; Cell Nucleus; Chelating Agents; DNA-Binding Proteins; Edetic Acid; Ethylenediamines; Gene Expression Regulation; Glutathione Transferase; L Cells; Metallothionein; Metals; Mice; Oligodeoxyribonucleotides; Phenanthrolines; Podophyllin; Podophyllotoxin; Promoter Regions, Genetic; Recombinant Fusion Proteins; Substrate Specificity; Transcription Factor MTF-1; Transcription Factors; Transfection

Metal regulatory elements (MREs) shared by metallothionein (MT) gene promoters are essential for metal induction of MT genes. MEP-1, a nuclear protein which binds to these elements has been purified from heavy metal-resistant mouse L cells using footprinting, Southwestern and UV cross-linking techniques to assay its binding activity. The purification scheme, starting from crude nuclear extracts, involved a combination of heparin-Sepharose and MRE-DNA affinity chromatography. The purified protein preparation showed a single polypeptide band of 108 kDa on polyacrylamide gel electrophoresis, and 2D-gel analyses revealed the presence of a protein species migrating as a single population of approximately 110 kDa. MEP-1 does not appear to be glycosylated since it eluted with the flow-through on a Wheat Germ Sepharose column. It was retained by a zinc-Chelating Sepharose column suggesting that amino acid residues (i.e., cysteine, histidine) which have an affinity for zinc ions are exposed on the protein surface. Binding studies with the purified protein indicated that it binds specifically to MRE sequences and that the binding can be abolished by a point mutation in the MRE core consensus sequence or by the addition of the chelating agent 1,10-phenanthroline. Binding activity can be restored by the addition of zinc ions to the chelated protein. These results suggest that MEP-1 is one of the major proteins interacting with MRE sequences.

Topics: Animals; DNA-Binding Proteins; L Cells; Metallothionein; Mice; Nuclear Proteins; Phenanthrolines; Regulatory Sequences, Nucleic Acid; Zinc