Abstract
We have analyzed the human (h) metallothionein (MT)-IG proximal promoter region (-174 to +5) using a TATA box mutation (TATCA) and four trinucleotide mutants of the proximal MREa. Transient transfection of HepG2 cells was complemented by in vitro transcription with rat liver nuclear extracts. In both systems, mutations of the TATA box and conserved core of metal responsive element (MRE)a were detrimental to hMT-IG promoter activity suggesting that both elements make significant contributions to hMT-IG transcription. Although MRE binding factors were active in vitro, further metal activation of MT promoter activity was accomplished only by in vivo metal treatment rather than addition of zinc in vitro. Southwestern blotting identified nuclear proteins in rat liver and HepG2 cells which physically interact with MREa in a zinc-dependent manner and could be responsible for MREa function in each system. In addition, the functional effects of the TATCA mutation correlate with altered physical interaction with TATA box-binding protein as observed using DNase I protection.
Highlights
We have analyzed the human (h) metallothionein (MT)-IG proximal promoter region (؊174 to ؉5) using a TATA box mutation (TATCA) and four trinucleotide mutants of the proximal MREa
Effect of MREa and TATA Box Mutations on MT-IG Promoter Activity in Hep G2 Cells—The position and sequence of MREa suggest that it has an important role in MT-IG promoter activity
MREa is adjacent to the TATA box and synthetic metal responsive element (MRE) function is known to be modulated by distance from the TATA box and the gene origin of the TATA box used as the minimal promoter [8, 9]
Summary
We have analyzed the human (h) metallothionein (MT)-IG proximal promoter region (؊174 to ؉5) using a TATA box mutation (TATCA) and four trinucleotide mutants of the proximal MREa. Metallothioneins (MTs) are low molecular mass (6 –7 kDa), cysteine-rich, metal-binding proteins which are thought to be important for trace metal homeostasis, as with zinc and copper, and detoxification, as with cadmium [1] Consistent with these putative functions, MTs are transcriptionally induced by these metals, mediated by multiple copies of metal-responsive elements (MREs) in the MT gene 5Ј-regulatory regions [2]. The promoters of the five hMT-I isoform genes are comparatively simple [3, 6] only containing MREs and GC boxes, which are possible binding sites for the SP1 transcription factor [3] Because of this simplicity, the hMT-I promoters are excellent candidates to study metal-regulated gene expression without complication by other inducible non-MRE elements as are found in hMT-IIA. The hMT-I promoters display remarkable homology of sequence and MRE organization [3], these genes exhibit cell type-specific patterns of expression as well as distinct levels of basal and metal-induced transcription within one cell type [3, 4]
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