Stress response plasticity of Cd-metallothionein genes

The scope of the present proposal is first, to identify and characterize transcription factors and their DNA binding elements involved in stress-dependent transcriptional regulation of the Cd-Metallothionein gene of Helix pomatia; and second, to test the hypothesis that intronic repeat cassettes of this gene can function as regulatory units upon stress-dependent transcription. In most organisms, reaction to environmental stress is induced through specific signalling pathways, leading to the transcription of genes and their products involved in coping with the adverse effects of stressors. Thereby, transcriptional induction is often regulated by activation and binding of stress-dependent transcription factors to distinct DNA recognition elements of stress-specific target genes. Interesting examples for such induction mechanisms have been shown to exist in Metallothioneins (MTs). These proteins and their genes play a crucial role in the stress response reaction of many organisms, being involved, among others, in metal homeostasis and detoxification, radical scavenging, as well as many other stress-related functions. Unfortunately, however, most of this research has so far been focussed on a few vertebrate and invertebrate model organisms, where the most important transcription factor involved in stress-dependent induction of MT genes is the so-called Metal Responsive Transcription Factor-1 (MTF-1). In contrast, there is only limited knowledge available about the mechanisms of stress-dependent transcriptional regulation for MTs of non-model organisms or of species from animal taxa which are only distantly related to vertebrates. Gastropods (snails), for example, belong to the superphylum of the so-called Lophotrochozoa, for which no model organisms do exist so far. In our previous work we could show that the Cadmium-Metallothionein (Cd-MT) genes of terrestrial gastropods (e.g. Helix pomatia and Cantareus aspersus) contain in their promoters and in some intronic regions various binding elements for stress-specific transcription factors, sometimes occurring in highly ordered repeat structures, supposed to be involved in plasticity and regulation of stress response of gastropods towards environmental stressors. The main goal of the present proposal is, therefore, to test the stress- dependent activation of the Cd-MT gene of these two snail species after exposure to metallic and non-metallic environmental stressors. First, stress-dependent binding and identification of so far unknown transcription factors to the proximal promoter region of these genes will be studied by applying DNase I footprinting on cytosolic and nuclear midgut gland extracts of differently stressed snails, complemented by Electrophoretic Mobility Shift Assays (EMSA) and magnetic bead-based fishing and subsequent analysis of unknown transcription factors binding to generated functional DNA recognition elements deduced from footprinting results. In addition, transcription factor identification will be enhanced by applying a commercial yeast one-hybrid screening assay, where short DNA binding elements are used as "baits" in yeast cells cotransformed with a cDNA library of stressed snails. Second, intronic repeat cassettes of transcription factor binding sites will be tested for stress-related functionality by inserting these cassettes into luciferase reporter constructs driven by the snail Cd-MT promoters, transfected transiently into Drosophila melanogaster SL2 and mouse (Mus musculus) NIH 3T3 cell cultures. It is expected that the results obtained from these studies will help to better understand the mechanisms of stress- dependent transcriptional regulation of MT genes in gastropods, and to appreciate the adaptation strategies of these animals in coping with environmental stressors in their habitats. Moreover, the outcome of the present study is thought to contribute to a more critical use of MTs as biomarkers in environmental monitoring.

Pulmonate snails belong to the mollusk class of Gastropoda, having adapted from marine habitats to life on land. The successful adaptation of pulmonate snails to terrestrial conditions is based, among others, on their stress tolerance and their capacity to resist to fluctuating microclimatic conditions and a rapidly changing physico-chemical environment, as typically encountered in terrestrial habitats. In doing so, snails rely on a network of stress-responsive genes, whose activities account for cellular protection, stress resistance and repair. Of particular importance in this concern is the maintenance of intracellular metal homeostasis. Consequently, terrestrial helicid snails possess a Cd-specific metallothionein gene (CdMT) encoding for a protein that selectively binds and detoxifies harmful Cd2+ ions. In vertebrates, MT genes can be up-regulated by a number of stressors, so as to enable the corresponding MT protein not only to detoxify and regulate metal ions, but also to take over other intracellular protective tasks. Unfortunately, very little was so far known about mechanisms of MT gene regulation in Gastropoda.The main goal of the present research grant was, therefore, to study the activation of the CdMT gene in the Roman snail and other related helicids in response to metallic and non-metallic stressors. It appeared that the CdMT gene of terrestrial helicid snails is mainly induced by Cd exposure; in addition, there is a weak induction of the gene by certain kinds of physiological stressors, such as desiccation. Overall, the transcriptional activity of pulmonate CdMT genes turned out to be subjected to a high degree of individual variability depending, among others, on the physiological nutrition state, the daily cycle of activity and last but not least the developmental stage (embryonic / adult) of the respective snail species.The Roman snail CdMT accommodates in its promoter region, like most other MT genes, a number of conserved sequences known as Metal Responsive Elements (MREs). We could show in our studies by means of DNase I footprinting combined with an EMSA (Electrophoretic Mobility Shift Assay) approach that all four MREs in the promoter region of the Roman snail CdMT gene are indeed functional. There was only little difference, however, in MRE activation between control and Cd-exposed individuals, which indicates that the responsible mechanism of activation may be of low specificity. Our most recent studies based on transcriptomic and genomic analyses of different gastropod species suggest that at least some of them possess an MTF-1-like gene that is apparently an orthologue of the vertebrate MTF-1. So far, however, the role of this gene in MT induction of gastropods is not sufficiently clear, and we were not able yet to demonstrate its functionality. Moreover, the presence of this gene was not yet confirmed in Helix pomatia and related helicid species. Our current work will hopefully clarify these still open questions. Our findings confirm the usefulness and importance of studying molecular mechanisms of stress resistance across different animal phyla in a comparative approach, and especially in animal species that are not considered as classical model organisms.