JMJD2A-D belong to the JmjC domain-containing family of histone demethylases. JMJD2D is the most structurally divergent JMJD2 protein as it lacks the PHD and Tudor domains present in JMJD2A-C. Here, we systematically analyzed the histone demethylase specificity of JMJD2 proteins in vivo. We found that JMJD2A and C demethylate tri- and dimethylated H3K9 and H3K36, whereas JMJD2D demethylates tri-, di-, and monomethylated H3K9. Enzymatic activity requires the N terminal JmjN domain. It also contributes to efficient nuclear localization together with the PHD and Tudor domains of JMJD2A and C. Furthermore, JMJD2 proteins form homomers, and JMJD2A and C, but not JMJD2D, can also heteromerize. Finally, we show that JNIJD2 proteins p repress or activate gene transcription. Altogether, our results reveal novel properties of and functional differences between JMJD2 proteins that may therefore have different effects on chromatin structure. (c) 2006 Elsevier Inc. All rights reserved.