Interactions of the zinc-regulated factor (ZiRF1) with the mouse metallothionein Ia promoter

Biochem J. 1997 Apr 1;323 ( Pt 1)(Pt 1):79-85. doi: 10.1042/bj3230079.

Abstract

A mouse cDNA clone, M96, encoding a metal-regulating-element (MRE)-binding protein, was analysed for its ability to act as a metal-regulated transcription factor. The metal depletion of a glutathione S-transferase (GST)-M96 fusion protein showed that Zn2+ ions modulate the MRE-binding activity, suggesting that the M96-encoded protein is a Zn2+-regulated factor (ZiRF1). The methylation interference assay showed the specific interactions of ZiRF1 with the MRE, MREd/c, present on the mouse metallothionein Ia promoter. Point mutations of the MREd/c nullified the metal-regulatory properties of this region. In mouse L-cell nuclear extracts, mobility-shift assays revealed a Zn2+-dependent MRE-binding complex (MBC) with DNA-recognition properties similar to those of ZiRF1. Antibodies raised against purified GST-ZiRF1 were able to specifically recognize MBC in Western-blot analyses. Competition analysis of MRE-binding proteins from mouse NIH3T3 cells with oligonucleotide matching the binding sites for SP1 and MTF1 confirmed that both the basal SP1 and the metal-regulated MBC/ZiRF1 interact with the MREd/c region. The significance of mutual interactions with the metal-responsive promoter regions of either metal-regulated or basal transcription factors is discussed.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • 3T3 Cells
  • Animals
  • Base Sequence
  • DNA / metabolism
  • DNA Methylation
  • DNA-Binding Proteins
  • Electrophoresis, Polyacrylamide Gel
  • L Cells
  • Metallothionein / genetics*
  • Mice
  • Molecular Sequence Data
  • Point Mutation
  • Promoter Regions, Genetic*
  • Sp1 Transcription Factor / metabolism
  • Transcription Factor MTF-1
  • Transcription Factors / genetics*
  • Transcription Factors / metabolism*
  • Zinc / metabolism*

Substances

  • DNA-Binding Proteins
  • Sp1 Transcription Factor
  • Transcription Factors
  • DNA
  • Metallothionein
  • Zinc