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J. Biol. Chem., Vol. 281, Issue 46, 35289-35295, November 17, 2006
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Dipartimento di Genetica e Microbiologia, Università di Pavia, via Ferrata 1, 27100 Pavia, Italy, Dipartimento di Biologia e Genetica per le Scienze Mediche, Università di Milano, via Viotti 3/5, 20133 Milano, Italy, and ¶Laboratory of Biochemistry, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands
Human lysine-specific demethylase (LSD1) is a chromatin-modifying enzyme that specifically removes methyl groups from mono- and dimethylated Lys4 of histone H3 (H3-K4). We used a combination of in vivo and in vitro experiments to characterize the substrate specificity and recognition by LSD1. Biochemical assays on histone peptides show that essentially all epigenetic modifications on the 21 N-terminal amino acids of histone H3 cause a significant reduction in enzymatic activity. Replacement of Lys4 with Arg greatly enhances binding affinity, and a histone peptide incorporating this mutation has a strong inhibitory power. Conversely, a peptide bearing a trimethylated Lys4 is only a weak inhibitor of the enzyme. Rapid kinetics measurements evidence that the enzyme is efficiently reoxidized by molecular oxygen with a second-order rate constant of 9.6 x 103 M-1 s-1, and that the presence of the reaction product does not greatly influence the rate of flavin reoxidation. In vivo experiments provide a correlation between the in vitro inhibitory properties of the tested peptides and their ability of affecting endogenous LSD1 activity. Our results show that epigenetic modifications on histone H3 need to be removed before Lys4 demethylation can efficiently occur. The complex formed by LSD1 with histone deacetylases 1/2 may function as a "double-blade razor" that first eliminates the acetyl groups from acetylated Lys residues and then removes the methyl group from Lys4. We suggest that after H3-K4 demethylation, LSD1 recruits the forthcoming chromatin remodelers leading to the introduction of gene repression marks.
Received for publication, August 4, 2006 , and in revised form, September 6, 2006.
* This work supported in part by the Italian Ministry of Science (PRIN and FIRB programs), the Italian Association for Cancer Research, and Fondazione Cariplo. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 Both authors contributed equally to this work.
2 Supported by a Young Investigator Fellowship from "Collegio Ghislieri", Pavia.
3 To whom correspondence should be addressed. Tel.: 39-0382-985534; Fax: 39-0382-528496; E-mail: mattevi{at}ipvgen.unipv.it.
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