The trithorax-group protein Lid is a histone H3 trimethyl-Lys4 demethylase Nara Lee 1,2 , Junyu Zhang 3 , Robert J Klose 1,2 , Hediye Erdjument- Bromage 4 , Paul Tempst 4 , Richard S Jones 3 & Yi Zhang 1,2 Recent studies have demonstrated that histone methylation can be dynamically regulated through active demethylation. However, no demethylase specific to histone H3 trimethyl- Lys4 (H3K4me3) has been identified. Here we report that the Drosophila melanogaster protein ‘little imaginal discs’ (Lid), a JmjC domain–containing trithorax group protein, can demethylate H3K4me3. Consistent with its genetic classification, Lid positively regulates Hox gene expression in S2 cells. Histone methylation contributes to diverse biological processes 1 . Recent studies indicate that histone methylation, like other histone modifications, is dynamically regulated through active demethylation by two distinct classes of enzymes. The first class of histone demethy- lase, exemplified by LSD1, catalyzes demethylation of H3K4me2 and H3K4me1 in a flavin adenine dinucleotide (FAD)-dependent manner 2 . The second class of histone demethylase encompasses a large protein family 3 and uses a conserved JmjC domain to catalyze demethylation in an Fe(II)- and a-ketoglutarate–dependent hydroxylation reaction 4 . Several JmjC domain–containing proteins with specificity toward various methylation states of H3 Lys9 and Lys36 (H3K9 and H3K36) have been characterized 4–9 . However, no H3K4me3-specific demethylase has been identified thus far, leaving open the possibility that H3K4me3 might not be a reversible modification. To facilitate identification of novel histone demethylases, we per- formed a phylogenetic analysis of the JmjC domain–containing proteins in six model organisms 3 . This analysis allowed us to divide the JmjC domain–containing proteins into seven subfamilies on the basis of conservation in the JmjC domain and overall protein domain architecture 3 . Members from three of the seven subfamilies have been shown to encode active histone demethylases. Of the remaining four subfamilies, the JARID1 subfamily is of particular interest because members of this subfamily contain multiple conserved functional domains (Supplementary Fig. 1a online), some of which are reported to participate in transcriptional regulation 10,11 . Notably, the sole Size M F-Lid 400 300 200 Formaldehyde release (c.p.m.) 100 0 Lid H3K4me3 peptide (1–21) Relative intensity Me 14 Da 2,764.6 m/z 2,750.6 +Lid –Lid –+ H3K4 SET7 (kDa) 170 130 95 72 56 a c d e b –+ H3K4 SET7 Y245A –+ H3K9 DIM5 –+ H3K27 EZH2 –+ H3K36 SET2 –+ H3K79 DOT1 –+ H4K20 SUV4- 20 H1 H3K4me2 peptide (1–21) Relative intensity Substrate % after HDM assay Me 14 Da 2,863.2 0 me3 me2 me1 K4me3 substrate K4me2 substrate me0 20 40 60 80 100 m/z 2,849.2 +Lid –Lid Figure 1 Lid is a histone demethylase with specificity for H3K4me3. (a) Coomassie-stained SDS-PAGE gel of Flag-tagged Lid protein purified from baculovirus-infected Sf9 cells. M, marker; F-Lid, Flag-tagged Lid. (b) Recombinant Lid can demethylate substrates generated by a SET7 mutant. Various radiolabeled methylhistone substrates were generated and used in demethyla- tion assays with recombinant Lid. The release of radioactively labeled formaldehyde was used to measure enzymatic activity. Owing to steric constrains of the SET7 catalytic site, the wild- type SET7 can only monomethylate H3K4, whereas the Y245A mutant can di- and trimethy- late H3K4. (c,d) Lid demethylates both di- and trimethylated H3K4 peptides. Histone peptides (residues 1–21) containing either tri- (c) or dimethylated (d) H3K4 were subjected to demethylation reactions in the presence or absence of Lid followed by mass spectrometric analysis. (e) Quantification of the mass spectro- metry results in c and d. Relative percentage of peptide substrates at different methylation states after demethylation reaction is shown. Lid has similar efficiency in converting H3K4me3 to H3K4me2 and H3K4me2 to H3K4me1 in vitro. Received 21 December 2006; accepted 16 February 2007; published online 11 March 2007; doi:10.1038/nsmb1216 1 Howard Hughes Medical Institute and 2 Department of Biochemistry and Biophysics, Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, North Carolina 27599-7295, USA. 3 Department of Biological Sciences, Southern Methodist University, Dallas, Texas 75275, USA. 4 Molecular Biology Program, Memorial Sloan Kettering Cancer Center, 1275 York Avenue, New York, New York 10021, USA. Correspondence should be addressed to Y.Z. (yi_zhang@med.unc.edu). NATURE STRUCTURAL & MOLECULAR BIOLOGY VOLUME 14 NUMBER 4 APRIL 2007 341 BRIEF COMMUNICATIONS © 2007 Nature Publishing Group http://www.nature.com/nsmb