Transcriptional regulation of L.rubellus metallothionein

Metallothioneins (MTs) are multifunctional proteins playing a major role in detoxification of heavy metals and homeostasis of essential trace elements and occur throughout the animal kingdom. MT gene expression, studied in model organisms and mainly vertebrates, is mediated by the metal transcription factor 1 (MTF-1) which binds to metal-responsive elements (MRE, core sequence: TGCA/TCNC) located on the respective MT promotor regions. Beside D. melanogaster, no information on MT regulation in invertebrates is available so far. Interestingly, in C. elegans no MTF-1 could be elucidated, as well as in L. rubellus and in two species of terrestrial snails. In the present proposal, L. rubellus (the earthworm), will serve as study organism to elucidate putative transcription factors (TFs) which bind to wMT2, an MT isoform known to be inducible by Cd2+ and playing a role in detoxification processes. Nuclear and cytosolic extracts from control and Cd2+-treated earthworms will be used in DNaseI Footprinting Assay, which will elucidate functional protein binding sites on the wMT2 promotor region. EMSA studies will accordingly verify the respective promotor sequence for specific DNA-protein binding. By screening a cDNA expression library the respective transcription factor will be defined. The present proposal will reveal information about functional protein binding sites in the promotor region and a difference in control and Cd2+-exposed samples of L. rubellus wMT2. Using nuclear and cytosolic protein extracts as samples will furthermore give information on a putative translocation of the transcription factor from the cytosol to the nucleus. All of these studies will be performed in Stephen Stürzenbaum`s lab at King`s College in London. During the return phase at the University of Innsbruck, the results from L. rubellus can be used to screen genome databases for homolog transcription factors in C. elegans, L. gigantea, and if by that time available in B. glabrata, using a computational approach. Furthermore for terrestrial pulmonate snails, the study organisms we are working on at the University of Innsbruck, no genome sequence information is available. Degenerate primers can be used to find homolog TFs using a PCR approach. First information on MT induction within these species will be of major importance as basis for future projects in invertebrate MT research and will provide a basis for functional studies of MT regulation. It will moreover improve biomarker associated and toxicological studies and will refine the understanding of detoxification mechanisms in sentinel organisms.