AbstractThe E. coli AlkB protein reverses methylation damage from DNA and RNA by oxidativedemethylation, a reaction dependent on Fe(II) and 2-oxoglutarate. Based on sequencesimilarity, eight human AlkB homologues, denoted hABH1-8, have been identified. hABH2and hABH3 possess repair activities similar to that of AlkB. While hABH2 is preferentiallyactive on double-stranded DNA substrates, hABH3 is also active on RNA and prefers singlestrandedsubstrates. The functions of hABH1 and hABH4-8 are not yet discovered. Theseproteins have been suggested to function in regulation of gene expression, by demethylationof 5-methylcytosine on DNA or methylated lysine or arginine residues on histones.Here, the function of hABH4 has been investigated. The enzymatic activity of hABH4towards 5-methylcytosine was tested in vitro and in vivo. No hABH4-mediated demethylationwas observed. Two proteins identified as interactants of hABH4 in by the Yeast Two-Hybridmethod, may indicate a role of hABH4 transcriptional regulation. GST pull-down studieswere performed to verify these interactions, but a conclusion could not be made because ofhigh background. Further study is needed for determination of the function of hABH4.