Nuclear Factor-1 and Metal Transcription Factor-1 Synergistically Activate the Mouse Metallothionein-1 Gene in Response to Metal Ions

Abstract

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. Metallothioneins (MTs)3 are small metal-binding stress proteins grouped into four classes, MT-1 through MT-4 (1, 2).MTs have been identified in a wide range of species and are present in various tissues and cell types from yeast to human. In mice, MT-1 and MT-2 are ubiquitous and coordinately expressed in all tissues, whereas MT-3 is mainly expressed in the brain (3) and in the organs of the reproductive system (4), and MT-4 is restricted to stratified squamous epithelia (5). MTs have no enzymatic function but appear to play important roles in metal ion homeostasis, as an active donor of zinc to other sites within the cell, in detoxification of toxic metals, and in protection against oxidative damage, ionizing radiation, and xenobiotics (1, 2).MT genes are inducible at the transcription level by hormones, cytokines, and a variety of stress conditions that include exposure to transition metal ions, UV irradiation, hypoxia, and reactive oxygen species (2). Metals are the most general and potent of these inducers. Metal activation of MT gene transcription depends on the presence of regulatory DNA sequences termed metal regulatory elements (MREs) and involves metal-responsive transcription factor-1 (MTF-1) interacting with the MREs in a zinc-dependent manner (6, 7). MTF-1 is also involved in the response to hypoxia (8), reactive oxygen species (9), and amino acid starvation (10)...

The role of nuclear factor I-C in tooth and bone development

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