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Metallothioneins (MTs) are multifunctional proteins occurring throughout the animal kingdom. While the expression and transcriptional regulation of MTs is well-studied in vertebrates, the mechanism of MT activation is still unknown for most invertebrates. Therefore, we examined wMT-2 gene regulation and expression patterns in Lumbricus rubellus and L. terrestris. Transcription levels, the occupation of DNA binding sites, the expression of putative transcriptional regulators, and promotor DNA methylation were determined. We found that wMT-2 expression does not follow a circadian pattern. However, Cd-induced wMT-2 induction was observed, and was, interestingly, suppressed by physical injury. Moreover, the promotor region that is responsible for the wMT-2 gene regulation was elucidated. ATF, a putative transcriptional regulator, showed increased phosphorylation upon Cd exposure, suggesting that it plays a major role in wMT-2 gene activation. The promotor methylation of wMT-2, on the other hand, is probably not involved in transcriptional regulation. Elucidating the regulatory mechanism of the earthworm MT gene activation might provide insights into the molecular coordination of the environmental stress response in invertebrates, and might also reveal a link to wound repair and, in a broader sense, to immunity. [ABSTRACT FROM AUTHOR]