A Highly Specific Mechanism of Histone H3-K4 Recognition by Histone Demethylase LSD1

Annachiara Dall'Aglio,Marco W. Fraaije,Federico Forneris,Elena Battaglioli,Andrea Mattevi,Claudia Binda

doi:10.1074/jbc.m607411200

Annachiara Dall'Aglio, Marco W. Fraaije

Open Access

https://doi.org/10.1074/jbc.m607411200

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Abstract

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 9.6x10(3) 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.

Highlights

EXPERIMENTAL PROCEDURESProtein Preparation and Steady-state Kinetics Measurements—All chemicals were purchased from Sigma unless specified
States by removing methyl groups from lysine residues on histone N-terminal tails
Within the recognized segment of 21 amino acids, essential for productive binding, we found that both post-translational modifications and conformational properties of the histone H3 tail are important factors in substrate recognition and Lys4 demethylation

Summary

EXPERIMENTAL PROCEDURES

Protein Preparation and Steady-state Kinetics Measurements—All chemicals were purchased from Sigma unless specified. Stopped Flow Kinetics of the Oxidative Half-reaction— Stopped flow kinetic experiments were performed using a SX17MV stopped-flow instrument equipped with a diode array detector (Applied Photophysics, Leatherhead, UK). For these experiments, the enzyme was artificially reduced by preparing a solution of 10 ␮M enzyme in 50 mM HEPES, pH 7.5, containing benzyl viologen (1.0 ␮M), and xanthine (400 ␮M) in a total volume of 1.2 ml. The enzyme was artificially reduced by preparing a solution of 10 ␮M enzyme in 50 mM HEPES, pH 7.5, containing benzyl viologen (1.0 ␮M), and xanthine (400 ␮M) in a total volume of 1.2 ml After flushing this enzyme solution with nitrogen, enzyme reduction was initiated by adding 0.2– 0.5 nM xanthine oxidase resulting in full reduction of LSD1 within 30 min. The antibodies used were anti-LSD1 ab17721 (Abcam) and anti-␣/␤-tubulin (Cell Signaling)

RESULTS

Peptide kcata

No inhibition Weak

DISCUSSION