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

Metal activation of metallothionein gene transcription depends mainly on th e presence of regulatory DNA sequences termed metal-regulatory elements (MR Es) and involves MRE-binding transcription factor-1 (MTF-I) interacting wit h the MREs in a Zn2+-dependent manner. We previously identified and charact erized a nuclear protein, termed metal element protein-1 (MEP-I), specifica lly binding with high affinity to MRE elements. The precise relationship be tween 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 charac terize the binding properties of both proteins. Electrophoretic mobility-sh ift assays showed that MTF-1, produced in COS cells, produces a slower-migr ating band compared with that obtained with purified MEP-1. Using an anti-M TF-1 antibody, we showed that both the MTF-1-MRE and the MEP-1-MRE complexe s are supershifted by an anti-MTF-l antibody, thus demonstrating that MEP-1 is antigenically related to MTF-1. RNase protection analyses carried out w ith 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 pr oteolytic fragment of MTF-1. MTF-I DNA-binding activity was rapidly activat ed in vivo by Zn2+ ions but not by Cd2+, UV irradiation or PMA, and occurre d on ice as well as at 21 DC. In control and Zn2+-treated cell extracts, DN A-binding activity was not enhanced in vitro following the addition of exog enous Zn2+ or a preincubation at 37 degreesC. However, recombinant MTF-1 pr oduced in vitro required Zn2+ activation for DNA binding. Interestingly, tr eatment of nuclear extracts with calf intestine phosphatase completely abro gated MTF-1 DNA-binding activity, thus suggesting that phosphorylation is i nvolved in the regulation of MTF-1 activity.