Drosophila dSet2 functions in H3-K36 methylation and is required for development Marianne Stabell a , Jan Larsson b , Reidunn B. Aalen a , Andrew Lambertsson a, * a Institute of Molecular Biosciences, University of Oslo, P.O. Box 1041 Blindern, NO-0316 Oslo, Norway b UCMP, Umea ˚ University, SE-901 87 Umea ˚ , Sweden Received 5 May 2007 Available online 4 June 2007 Abstract Lysine methylation has important functions in biological processes that range from heterochromatin formation to transcription reg- ulation. Here, we demonstrate that Drosophila dSet2 encodes a developmentally essential histone H3 lysine 36 (K36) methyltransferase. Larvae subjected to RNA interference-mediated (RNAi) suppression of dSet2 lack dSet2 expression and H3-K36 methylation, indicating that dSet2 is the sole enzyme responsible for this modification in Drosophila melanogaster. dSet2 RNAi blocks puparium formation and adult development, and causes partial (blister) separation of the dorsal and ventral wing epithelia, defects suggesting a failure of the ecdy- sone-controlled genetic program. A transheterozygous EcR null mutation/dSet2 RNAi combination produces a complete (balloon) sep- aration of the wing surfaces, revealing a genetic interaction between EcR and dSet2. Using immunoprecipitation, we demonstrate that dSet2 associates with the hyperphosphorylated form of RNA polymerase II (RNAPII). Ó 2007 Elsevier Inc. All rights reserved. Keywords: H3-K36 methyltransferase; Drosophila development; RNAi; EcR; Nuclear receptors; RNAPII Eukaryotic DNA is wrapped around histone octamers, composed of two molecules each of histones H2A, H2B, H3, and H4, resulting in a tightly packaged DNA in the nucleus. The histones, particularly their tails, are subject to covalent modifications, e.g. acetylation, methylation, phosphorylation, and ubiquitination. Recent results show that the methylation of lysine and arginine residues regu- lates a number of diverse cellular functions such as tran- scriptional repression and activation, heterochromatin formation, X-inactivation, and polycomb-mediated gene silencing [1]. The enzymes involved in histone lysine meth- ylation are the evolutionary conserved chromatin-modify- ing SET [Su(var)3-9, Enhancer of zeste, Trithorax] domain histone lysine methyltransferases (HKMTs) [2].A characteristic signature of this class of methyltransferases is the presence of the 130-amino-acid SET domain [3], which is crucial for catalytic activity but also requires adja- cent cysteine-rich domains [4], although their presence in some cases have shown to be dispensable [5]. While methylation of lysines 9 (K9) and 27 (K27) of his- tone H3 appears to be associated with condensed chroma- tin, methylation of lysines 4 (K4), 36 (K36), and 79 (K79) of H3 are hallmarks of euchromatic regions [1]. More recently, studies have revealed an unexpected role for his- tone methylation in the process of transcription elongation by RNA polymerase II (RNAPII). Thus, the yeast histone methyltransferases Set1p and Set2p, which methylate H3 lysine 4 (K4) and lysine 36 (K36), respectively, have been found to be associated with RNAPII during the transcrip- tion elongation cycle [6,7]. While the precise function of these enzyme associations with RNAPII is still unclear, it is believed that K4 and K36 methylation function in the elongation process at different stages of the transcription elongation cycle [8,9]. It has recently been shown that Set2p interacts with the C-terminal domain (CTD) of RNAPII via a novel domain, the Set2 Rpb1-interacting (SRI) domain [6,10]. The association of SET2 with elongating 0006-291X/$ - see front matter Ó 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2007.05.189 * Corresponding author. Fax: +47 22854726. E-mail address: g.a.lambertsson@imbv.uio.no (A. Lambertsson). www.elsevier.com/locate/ybbrc Biochemical and Biophysical Research Communications 359 (2007) 784–789