Metal-responsive elements act as positive modulators of human metallothionein-IIA enhancer activity.

Abstract

The human metallothionein IIA (hMT-IIA) gene contains two enhancer elements whose activity is induced by heavy-metal ions such as Cd2+. To determine the nature of the relationship between the metal-responsive elements and the element(s) responsible for the basal activity of the enhancers, the basal-level enhancer element(s), the hMT-IIA enhancers were subjected to mutational analysis. We show that deletion of the metal-responsive elements had no effect on the basal activity of the enhancer but prevented further induction by Cd2 . On the other hand, replacement of the basal-level enhancer element with linker DNA led to inactivation of the enhancer both before and after treatment with Cd2+. Therefore, the metal-responsive elements seems to act as a positive modulator of enhancer function in the presence of heavy-metal ions. In addition to the two enhancers, the hMT-IIA promoter contained one other element, the GC box, required for its basal expression. Unlike deletion of the basal-level enhancer element, replacement of the GC box with linker DNA had no effect on the ability of the promoter to be induced by Cd2+.Metallothionein (MT) genes offer an attractive system for studying the control of gene expression in animal cells. These genes, which form a multigene family, are expressed in essentially every cell type, and their transcription can be further induced in response to a variety of exogenous stimuli, including heavy-metal ions (e.g., Cd, Zn, and Hg), glucocorticoid hormones, interferon, and interleukin 1. MT gene expression is also stimulated by exposure of cells to tumor promoters, inhibitors of protein and DNA synthesis, and ionizing radiation (see reference 14 for a review). To understand the mechanisms that regulate the basal and induced expression of these genes, it is essential to characterize both the cis-and trans-acting genetic elements involved in MT expression.Previously we have shown that cis elements important for basal and induced expression of the human MT IA (hMT-IA) and hMT-IIA genes are present in their 5'-flanking regions (15,22). Deletion analysis of the hMT-IIA control region defined two distinct regulatory elements that can independently mediate induction of the gene by heavy-metal ions and glucocorticoids. Two copies of the metal-responsive element (MRE) were originally detected, yet a single copy was found to be sufficient for induction. The glucocorticoidresponsive element (GRE) was shown to coincide with the DNA-binding site for the glucocorticoid hormone receptor, a trans regulator of gene expression (16). We also determined the approximate location of an upstream promoter element, centered around nucleotides -80 to -100, which is important for basal-level expression (16,17). Similar elements were found to control both the basal and metal-induced transcription of the mouse MT-I (mMT-I) gene (3,28 The hM-IIA gene is unique in its much higher levels of both basal and induced expression compared with each of the multiple "MT-I-like" genes (11,22). Recently it was found that the high bas...

Negative Regulatory Role of Sp1 in Metal Responsive Element-mediated Transcriptional Activation

“…In the hMT-IIA gene promoter, an Sp1 site located between MREa and MREb is known to be essential for promoter activity, both in vivo and in vitro (35,46). These observations together with our present results suggest that Sp1 could act either positively or negatively, depending on its binding sites within the same promoter.…”

“…These findings strongly suggest that Sp1 could act as a negative regulator by competing with the positive regulator MTF-1 for binding to particular MREs. Although the Sp1 recognition site was originally regarded as a positive regulatory element (23,26,35), reports suggesting their negative regulatory roles have recently been published. Several DNA elements identified as those responsible for negative gene regulation contain GC-rich motifs, and the binding of Sp1 or closely related proteins to them is likely to be involved in down-regulation (34, 36 -40).…”

Impaired Ionizing Radiation-induced Activation of a Nuclear Signal Essential for Phosphorylation of c-Jun by Dually Phosphorylated c-Jun Amino-terminal Kinases in Ataxia Telangiectasia Fibroblasts

“…AP1 mediates transcriptional activation by interacting with its DNA target sequence, TGA(C/G)TCA, which is known as the TPA response element (51)(52)(53)(54)(55)(56). Subsequently, many other promoter and enhancer regions of genes have been shown to contain AP1 binding sites.…”

Inhibitory Effects of Heavy Metals on Transcription Factor Sp1.

“…The results are shown in Fig. 1 Sp1 is known to activate transcription via the GC-box sequence located between MREa and MREb of the hMT-IIA gene 14,15) . We then examined how Cd 2+ and Zn 2+ affect the binding of Sp1 to this positive element, in comparison with MREb.…”