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. 2001 Feb 1;353(Pt 3):591–601. doi: 10.1042/0264-6021:3530591

Characterization of the mouse metal-regulatory-element-binding proteins, metal element protein-1 and metal regulatory transcription factor-1.

O Larochelle 1, G Stewart 1, P Moffatt 1, V Tremblay 1, C Séguin 1
PMCID: PMC1221605  PMID: 11171056

Abstract

Metal activation of metallothionein gene transcription depends mainly on the presence of regulatory DNA sequences termed metal-regulatory elements (MREs) and involves MRE-binding transcription factor-1 (MTF-1) interacting with the MREs in a Zn(2+)-dependent manner. We previously identified and characterized a nuclear protein, termed metal element protein-1 (MEP-1), specifically binding with high affinity to MRE elements. The precise relationship between MTF-1 and MEP-1 was unclear, and to determine whether MEP-1 and MTF-1 were distinct protein species, we performed DNA binding analyses to characterize the binding properties of both proteins. Electrophoretic mobility-shift assays showed that MTF-1, produced in COS cells, produces a slower-migrating band compared with that obtained with purified MEP-1. Using an anti-MTF-1 antibody, we showed that both the MTF-1-MRE and the MEP-1-MRE complexes are supershifted by an anti-MTF-1 antibody, thus demonstrating that MEP-1 is antigenically related to MTF-1. RNase protection analyses carried out with RNA prepared from different tissues and cell lines failed to reveal the presence of MTF-1 splicing variants. This indicates that MEP-1 may be a proteolytic fragment of MTF-1. MTF-1 DNA-binding activity was rapidly activated in vivo by Zn(2+) ions but not by Cd(2+), UV irradiation or PMA, and occurred on ice as well as at 21 degrees C. In control and Zn(2+)-treated cell extracts, DNA-binding activity was not enhanced in vitro following the addition of exogenous Zn(2+) or a preincubation at 37 degrees C. However, recombinant MTF-1 produced in vitro required Zn(2+) activation for DNA binding. Interestingly, treatment of nuclear extracts with calf intestine phosphatase completely abrogated MTF-1 DNA-binding activity, thus suggesting that phosphorylation is involved in the regulation of MTF-1 activity.

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Selected References

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