LSD1 demethylates repressive histone marks to promote androgen-receptor-dependent transcription Eric Metzger 1 , Melanie Wissmann 1 *, Na Yin 1 *, Judith M. Mu ¨ller 1 , Robert Schneider 2 , Antoine H. F. M. Peters 3 , Thomas Gu ¨nther 1 , Reinhard Buettner 4 & Roland Schu ¨le 1 Gene regulation in eukaryotes requires the coordinate interaction of chromatin-modulating proteins with specific transcription factors such as the androgen receptor 1 . Gene activation and repression is specifically regulated by histone methylation status at distinct lysine residues 2 . Here we show that lysine-specific demethylase 1 (LSD1; also known as BHC110) 3 co-localizes with the androgen receptor in normal human prostate and prostate tumour. LSD1 interacts with androgen receptor in vitro and in vivo, and stimulates androgen-receptor-dependent transcrip- tion. Conversely, knockdown of LSD1 protein levels abrogates androgen-induced transcriptional activation and cell prolifer- ation. Chromatin immunoprecipitation analyses demonstrate that androgen receptor and LSD1 form chromatin-associated complexes in a ligand-dependent manner. LSD1 relieves repres- sive histone marks by demethylation of histone H3 at lysine 9 (H3-K9), thereby leading to de-repression of androgen receptor target genes. Furthermore, we identify pargyline as an inhibitor of LSD1. Pargyline blocks demethylation of H3-K9 by LSD1 and consequently androgen-receptor-dependent transcription. Thus, modulation of LSD1 activity offers a new strategy to regulate androgen receptor functions. Here, we link demethylation of a repressive histone mark with androgen-receptor-dependent gene activation, thus providing a mechanism by which demethylases control specific gene expression. Transcriptional regulation by nuclear receptors such as androgen receptor (AR) involves interaction with multiple factors that act in both a sequential and combinatorial manner to reorganize chroma- tin 1 . Central to this dynamic organization is the modification of core histones. The amino-terminal tails of histones are subject to various covalent modifications such as acetylation, phosphorylation, ubiqui- tination and methylation by specific chromatin-modifying enzymes 2 . Histone methylation at specific lysine residues is linked to both transcriptional repression and activation 2 . We isolated LSD1 (ref. 3) by searching for new AR-interacting proteins. Endogenous LSD1 and AR associate specifically in vivo in androgen-sensitive tissues such as testis (Fig. 1a). LSD1 contains a centrally located SWIRM domain, which functions as a putative protein–protein interaction motif, and a carboxy-terminal amine oxidase domain that harbours the demethylase activity 3 (Fig. 1b). As shown in glutathione S-transferase (GST) pull-down analyses, full- length LSD1, as well as the SWIRM domain (LSD1 175–246) and the amine oxidase domain (LSD1 247–852) alone, associate with the N terminus (NTD), the DNA-binding domain (DBD) and the ligand- binding domain (LBD) of AR (Fig. 1b). In contrast, the N terminus of LSD1 (LSD1 1–174) does not interact with AR. Furthermore, neither LSD1 nor the LSD1 mutants associate with GST, GST–Nix1, GST–RORb or GST–ERb-NTD, thus demonstrating specificity of interaction with AR. To examine the expression pattern of LSD1, we performed north- ern blot analyses. LSD1 messenger RNA is ubiquitously expressed in human and murine fetal and adult tissue (Fig. 2a and data not shown) as a transcript of 3.3 kilobases (Supplementary Fig. S2a). To investigate LSD1 localization in normal prostate and prostate tumours, we used immunohistochemical analyses of 100 prostate cancer biopsies on tissue microarrays. As shown exemplarily in Fig. 2b, LSD1 is detected in the epithelium of normal prostate and in tumour cells. Importantly, these cells also express AR (Fig. 2b), showing that LSD1 and AR co-localize. The nuclear co-localization of LSD1 and AR was verified further in human LNCaP prostate tumour cells (Supplementary Fig. S2b). Taken together, our data LETTERS Figure 1 | LSD1 interacts with AR in vivo and in vitro. a, AR co- immunoprecipitates with LSD1. Extracts from mouse testis were immunoprecipitated (IP) with anti-LSD1 or anti-cyclin A antibodies and rabbit IgG as control. Western blots were decorated with anti-AR and anti- LSD1 antibodies. b, GST pull-down assays were performed with labelled LSD1 or LSD1 mutants and the corresponding bacterially expressed GST– AR fusion proteins. GST, GST–Nix1, GST–RORb and GST–ERb-NTD proteins were used as controls. aa, amino acids; NTD, N-terminal domain; DBD, DNA-binding domain; LBD, ligand-binding domain. 1 Universita ¨ts-Frauenklinik und Zentrum fu ¨r Klinische Forschung, Klinikum der Universita ¨t Freiburg, Breisacherstrasse 66, 79106 Freiburg, Germany. 2 Max-Planck-Institut fu ¨r Immunbiologie, Stu ¨beweg 51, 79108 Freiburg, Germany. 3 Friedrich Miescher Institute for Biomedical Research, Novartis Research Foundation, Maulbeerstrasse 66, 4058 Basel, Switzerland. 4 Institut fu ¨r Pathologie, Universita¨tsklinikum Bonn, Sigmund-Freud-Strasse 25, 53127 Bonn, Germany. *These authors contributed equally to this work. doi:10.1038/nature04020 1 © 2005 Nature Publishing Group