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. We showed that MREa and MREd are the main elements controlling mouse Mt-1 gene induction by hypoxia. Transfection experiments in Mtf-1-null cells showed that MTF-1 is essential for induction by hypoxia. Chromatin immunoprecipitation analysis showed that MTF-1 DNA-binding activity was strongly enhanced in the presence of zinc but not by hypoxia. Notably, hypoxia inducible factor- (HIF) 1α was recruited to the Mt-1 promoter in response to hypoxia but not to zinc. MTF-1 activation was inhibited by PKC, JNK, and PI3K inhibitors and by the electron transport chain inhibitors rotenone and myxothiazol, but not by the antioxidant N-acetylcysteine. We showed that prolyl-hydroxylase inhibitors can activate MTF-1, but this activation requires the presence of HIF-1α. Finally, HIF-dependent transcription is enhanced in the presence of MTF-1 and induction of an MRE promoter is stimulated by HIF-1α, thus indicating cooperation between these 2 factors. However, coimmunoprecipitation experiments did not suggest direct interaction between MTF-1 and HIF-1α.

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. This technique was used in our laboratory to visualize the metal regulatory consensus sequence-binding protein MTF-1 in L cell crude nuclear extracts.

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. Transfection experiments showed that loss of the two NF1 sites decreased metal-induced MT promoter activity by 55-70% in transiently transfected cells and almost completely abrogated metal and tert-butylhydroquinone (tBHQ) induction in stably transfected cells. Similarly, expression of an inactive NF1 protein strongly inhibited MT-1 promoter activity. Using synthetic promoters containing NF1 and MRE sites fused to a minimal MT promoter, we showed that these NF1 sites did not confer metal induction but enhanced metal-induced promoter activity. Chromatin immunoprecipitation assays confirmed that NF1 binds to the mouse MT-1 promoter in vivo and showed that NF1 binding is zinc-inducible. In addition, zinc-induced NF1 DNA binding was MTF-1-dependent. Taken together, these studies show that NF1 acts synergistically with MTF-1 to activate the mouse MT-1 promoter in response to metal ions and tert-butylhydroquinone and contributes to maximal activation of the gene.