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 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.
Larochelle
O
O
Centre de recherche en cancérologie de l'Université Laval, CHUQ, Hôtel-Dieu de Québec, 11, côte du Palais, Québec, QC, Canada.
Stewart
G
G
Moffatt
P
P
Tremblay
V
V
Séguin
C
C
D000818:Animals; D001483:Base Sequence; D002850:Chromatography, Gel; D017931:DNA Primers; D004268:DNA-Binding Proteins; D051379:Mice; D010766:Phosphorylation; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014157:Transcription Factors / Q000302:isolation & purification / Q000378:metabolism*; D000097809:Transcription Factor MTF-1
D017931:DNA Primers; D004268:DNA-Binding Proteins; D014157:Transcription Factors; D000097809:Transcription Factor MTF-1