Studying MT regulation in L. rubellus, the red earthworm, suggests novel activation mechanisms

Metallothioneins (MTs) are multifunctional proteins which are of major importance in a variety of stress related cellular responses. The majority of studies on the transcriptional regulation of MTs have, to date, been performed in vertebrate species and have revealed a common activation mechanism, namely the binding of the metal transcription factor 1 (MTF-1) to metal responsive elements (MREs) located on the promotor region of MT genes. This present study provides first insights into the regulation of earthworm MTs by means of L. rubellus and the Cd-inducible wMT2 isoform. Electric mobility shift assay (EMSA) was applied to investigate whether MREs are functional binding sites and therefore involved in basal and Cd-induced wMT2 activation. DNase I Footprinting was used to evaluate putative protein binding sites other than MREs on the wMT2 promotor region. EMSA (encompassing three probes, each including one of the MRE binding sites of the wMT2 promotor region) uncovered the presence of a Zn-dependent shift in the cytosolic protein fraction which was independent of sample origin (including control and Cd-exposed earthworms). Subsequent DNase I Footprinting revealed that the MRE flanking regions (rather than the MREs themselves) are the putative transcriptional activator sites. Moreover, the latter method identified further functional transcription factor binding regions. These results indicate that the mechanisms of MTF-1 binding to MREs to activate MT transcription in vertebrates might not be universally applicable to invertebrates and would confirm the fact that neither the L. rubellus nor the nematode C. elegans genome contains MTF-1 homologs. Ongoing experiments aim to identify the transcription factors involved in basal and/or metal-induced transcriptional activation of the earthworm wMT2 isoform. Revealing the transcriptional activation mechanism of the earthworm MT will advance and update invertebrate MT research and improve our understanding of metal detoxification processes.