Structural and Functional Analysis of the Rainbow Trout (Oncorhyncus mykiss) Metallothionein-A Gene

Abstract

In the present study, the distal part of the 5'-flanking region of the rainbow trout metallothionein-A promoter was sequenced in order to identify cis-acting regulatory elements. Analysis of this sequence combined with that previously reported for the 5'-flanking region directly proximal to the start of transcription revealed several putative regulatory sequences. In total, six metal-responsive elements (MREs) were identified; these sequences were organised into two clusters, one containing two copies of MRE and located close to the predicted TATA box sequence, and a second consisting of four MREs and lying 500-700 bp upstream from the start of transcription. In addition, the 5'-flanking region contained sequences sharing high similarity with the activator protein 1 consensus sequence as well as one nuclear-factor-interleukin-6-responsive element. Functional analysis of the promoter was performed by introducing deletion mutants of the 5'-flanking region into the vector pGL-2, directly upstream from the luciferase reporter gene. Both MRE clusters were needed for maximal metal inducibility in both rainbow trout hepatoma (RTH-149) and human hepatoblastoma (Hep G2) cell lines. Furthermore, the distal region was found to be functional in promoting gene transcription following exposure of RTH-149 cells to hydrogen peroxide.

Recombinational micro-evolution of functionally different metallothionein promoter alleles from Orchesella cincta

“…This may be explained by the deletion of the MRE-a in this allele as follows. During exposure to Cd and paraquat, trans -activation in non-mammalian metallothionein promoters can occur by interaction of an enhancer (containing MREs and/or ARE) with the MRE-a and other proximal MREs [43,63,66-69]. The induction of pmt C by Cd is low and completely abolished by paraquat possibly due to the incapability of trans -activation between the MRE-a (lacking in the C allele) and the distal enhancer, directly or in combination with CCAAT/enhancer binding proteins [70,71].…”

Differential regulation of the rainbow trout (Oncorhynchus mykiss) MT-A gene by nuclear factor interleukin-6 and activator protein-1

“…In the present study we aimed to characterize the regulatory role of the rtMT-A enhancer region in response to oxidative stress and tumor promotion. Previous identification and functional analysis of distal elements on the rtMT-A promoter have revealed that a cluster including 4 AP1 elements and a single NF-IL-6 element may be involved in free radical inducibility [7]. While free radical regulation of mammalian MT genes seem to be mediated via USF/ARE and MRE cis-acting elements [20], teleost MT genes may be regulated via conserved clusters of cis-acting elements sharing high homology to the NF-IL6 and the AP1 consensus core sequences [7,11,12].…”

“…The role of metallothionein (MT) as a free radical scavenger has been well documented in vitro [3], in cell lines [4], and at the organism level [5,6]. Furthermore, it has been shown that MT is induced by ROS inducing agents such as paraquat (PQ), hydrogen peroxide (H 2 O 2 ) and glutathione depleting agents such as diethylmaleate [1,4,7]. In addition, Phorbol-12-myristate-13-acetate (PMA) is a potent tumor promoter shown to induce MT expression via activator protein 1 (AP1) cis-acting elements in mammals [8-10].…”

“…Sequencing of several teleost MT enhancer regions have revealed the presence of distally located nuclear factor interleukin 6 (NF-IL6) and AP1 elements, inferring that these elements are involved in a conserved function [7,11-13]. The transcription factor NF-IL6 is activated by the cytokine IL-6 in response to NF-κB activation.…”

“…Functional analysis of the rtMT-A promoter has indicated that free radicals regulate the rtMT-A gene via a region containing multiple copies of AP1 elements and one NF-IL6 element [7]. In the rtMT-A gene there is a distal region of the promoter that contain AP1 elements with a potential to regulate MT gene expression.…”