Hypoxia acts through multiple signaling pathways to induce metallothionein transactivation by the metal-responsive transcription factor-1 (MTF-1).

Metal-responsive transcription factor-1 (MTF-1) is essential for the induction of genes encoding metallothionein by metals and hypoxia. Here, we studied the mechanism controlling the activation of MTF-1 by hypoxia. Hypoxia activation of Mt gene transcription is dependent on the presence of metal regulatory elements (MREs) in the promoter of Mt genes.

Identification of sequence-specific DNA-binding proteins by southwestern blotting.

We describe a Southwestern blotting method for characterization of both DNA-binding proteins and their specific sites. Proteins are first separated on a sodium dodecyl sulfate (SDS) polyacrylamide gel, then renatured in SDS-free buffer and transferred by electroblotting to an immobilizing membrane, and detected by their ability to bind radiolabeled DNA. The protein(s) interacting with the labeled DNA is visualized by autoradiography.

Nuclear factor-1 and metal transcription factor-1 synergistically activate the mouse metallothionein-1 gene in response to metal ions.

Metal activation of metallothionein (MT) gene transcription is dependent on the presence of metal regulatory elements (MREs), which are present in five non-identical copies (MREa through MREe) in the promoter of the mouse MT-1 gene and on the capacity of metal transcription factor-1 (MTF-1) to bind to the MREs in the presence of zinc. We detected a protein, distinct from MTF-1, specifically binding to the MREc region. DNA binding competition experiments using synthetic oligonucleotides and specific anti-NF1 antibodies showed that this protein binds to an NF1 site overlapping the MREc element as well as to a second site upstream of the Sp1a site and corresponds to NF1 or a related protein.