trans-2-Phenylcyclopropylamine is a mechanism-based inactivator of the histone demethylase LSD1

The catalytic domain of the flavin-dependent human histone demethylase lysine-specific demethylase 1 (LSD1) belongs to the family of amine oxidases including polyamine oxidase and monoamine oxidase (MAO). We previously assessed monoamine oxidase inhibitors (MAOIs) for their ability to inhibit the reaction catalyzed by LSD1 [Lee, M. G., et al. (2006) Chem. Biol. 13, 563-567], demonstrating that trans-2-phenylcyclopropylamine (2-PCPA, tranylcypromine, Parnate) was the most potent with respect to LSD1. Here we show that 2-PCPA is a time-dependent, mechanism-based irreversible inhibitor of LSD1 with a KI of 242 microM and a kinact of 0.0106 s-1. 2-PCPA shows limited selectivity for human MAOs versus LSD1, with kinact/KI values only 16-fold and 2.4-fold higher for MAO B and MAO A, respectively. Profiles of LSD1 activity and inactivation by 2-PCPA as a function of pH are consistent with a mechanism of inactivation dependent upon enzyme catalysis. Mass spectrometry supports a role for FAD as the site of covalent modification by 2-PCPA. These results will provide a foundation for the design of cyclopropylamine-based inhibitors that are selective for LSD1 to probe its role in vivo.     

Human histone demethylase LSD1 reads the histone code

Human histone demethylase LSD1 is a flavin-dependent amine oxidase that catalyzes the specific removal of methyl groups from mono- and dimethylated Lys4 of histone H3. The N-terminal tail of H3 is subject to various covalent modifications, and a fundamental question in LSD1 biology is how these epigenetic marks affect the demethylase activity. We show that LSD1 does not have a strong preference for mono- or dimethylated Lys4 of H3. Substrate recognition is not confined to the residues neighboring Lys4, but it requires a sufficiently long peptide segment consisting of the N-terminal 20 amino acids of H3. Electrostatic interactions are an important factor in protein-substrate recognition, as indicated by the high sensitivity of Km to ionic strength. We have probed LSD1 for its ability to demethylate Lys4 in presence of a second modification on the same peptide substrate. Methylation of Lys9 does not affect enzyme catalysis. Conversely, Lys9 acetylation causes an almost 6-fold increase in the Km value, whereas phosphorylation of Ser10 totally abolishes activity. LSD1 is inhibited by a demethylated peptide with an inhibition constant of 1.8 microM, suggesting that LSD1 can bind to H3 independently of Lys4 methylation. LSD1 is a chromatin-modifying enzyme, which is able to read different epigenetic marks on the histone N-terminal tail and can serve as a docking module for the stabilization of the associated corepressor complex(es) on chromatin.     

Transrepressive function of TLX requires the histone demethylase LSD1

TLX is an orphan nuclear receptor (also called NR2E1) that regulates the expression of target genes by functioning as a constitutive transrepressor. The physiological significance of TLX in the cytodifferentiation of neural cells in the brain is known. However, the corepressors supporting the transrepressive function of TLX have yet to be identified. In this report, Y79 retinoblastoma cells were subjected to biochemical techniques to purify proteins that interact with TLX, and we identified LSD1 (also called KDM1), which appears to form a complex with CoREST and histone deacetylase 1. LSD1 interacted with TLX directly through its SWIRM and amine oxidase domains. LSD1 potentiated the transrepressive function of TLX through its histone demethylase activity as determined by a luciferase assay using a genomically integrated reporter gene. LSD1 and TLX were recruited to a TLX-binding site in the PTEN gene promoter, accompanied by the demethylation of H3K4me2 and deacetylation of H3. Knockdown of either TLX or LSD1 derepressed expression of the endogenous PTEN gene and inhibited cell proliferation of Y79 cells. Thus, the present study suggests that LSD1 is a prime corepressor for TLX.     

Regulation of LSD1 histone demethylase activity by its associated factors

LSD1 is a recently identified human lysine (K)-specific histone demethylase. LSD1 is associated with HDAC1/2; CoREST, a SANT domain-containing corepressor; and BHC80, a PHD domain-containing protein, among others. We show that CoREST endows LSD1 with the ability to demethylate nucleosomal substrates and that it protects LSD1 from proteasomal degradation in vivo. We find hyperacetylated nucleosomes less susceptible to CoREST/LSD1-mediated demethylation, suggesting that hypoacetylated nucleosomes may be the preferred physiological substrates. This raises the possibility that histone deacetylases and LSD1 may collaborate to generate a repressive chromatin environment. Consistent with this model, TSA treatment results in derepression of LSD1 target genes. While CoREST positively regulates LSD1 function, BHC80 inhibits CoREST/LSD1-mediated demethylation in vitro and may therefore confer negative regulation. Taken together, these findings suggest that LSD1-mediated histone demethylation is regulated dynamically in vivo. This is expected to have profound effects on gene expression under both physiological and pathological conditions.     

The histone LSD1 demethylase in stemness and cancer transcription programs

DNA and histone chromatin modifying enzymes play a crucial role in chromatin remodeling in several biological processes. Lysine-specific demethylase 1 (LSD1), the first identified histone demethylase, is a relevant player in the regulation of a broad spectrum of biological processes including development, cellular differentiation, embryonic pluripotency and cancer. Here, we review recent insights on the role of LSD1 activity in chromatin regulatory complexes, its functional role in the epigenetic changes during embryonic development, in the establishment and maintenance of stemness and during cancer progression.     

Solution structure of the SWIRM domain of human histone demethylase LSD1

SWIRM is an evolutionarily conserved domain involved in several chromatin-modifying complexes. Recently, the LSD1 protein, which bears a SWIRM domain, was found to be a demethylase for Lys4-methylated histone H3. Here, we report a solution structure of the SWIRM domain of human LSD1. It forms a compact fold composed of 6 alpha helices, in which a 20 amino acid long helix (alpha4) is surrounded by 5 other short helices. The SWIRM domain structure could be divided into the N-terminal part (alpha1-alpha3) and the C-terminal part (alpha4-alpha6), which are connected to each other by a salt bridge. While the N-terminal part forms a SWIRM-specific structure, the C-terminal part adopts a helix-turn-helix (HTH)-related fold. We discuss a model in which the SWIRM domain acts as an anchor site for a histone tail.     

A highly specific mechanism of histone H3-K4 recognition by histone demethylase LSD1

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.     

Histone demethylase LSD1 is a folate-binding protein

Methylation of lysine residues in histones has been known to serve a regulatory role in gene expression. Although enzymatic removal of the methyl groups was discovered as early as 1973, the enzymes responsible for their removal were isolated and their mechanism of action was described only recently. The first enzyme to show such activity was LSD1, a flavin-containing enzyme that removes the methyl groups from lysines 4 and 9 of histone 3 with the generation of formaldehyde from the methyl group. This reaction is similar to the previously described demethylation reactions conducted by the enzymes dimethylglycine dehydrogenase and sarcosine dehydrogenase, in which protein-bound tetrahydrofolate serves as an accepter of the formaldehyde that is generated. We now show that nuclear extracts of HeLa cells contain LSD1 that is associated with folate. Using the method of back-scattering interferometry, we have measured the binding of various forms of folate to both full-length LSD1 and a truncated form of LSD1 in free solution. The 6R,S form of the natural pentaglutamate form of tetrahydrofolate bound with the highest affinity (K(d) = 2.8 μM) to full-length LSD1. The fact that folate participates in the enzymatic demethylation of histones provides an opportunity for this micronutrient to play a role in the epigenetic control of gene expression.     

The growing structural and functional complexity of the LSD1/KDM1A histone demethylase

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Thermodynamic characterization of the binding interaction between the histone demethylase LSD1/KDM1 and CoREST

Flavin-dependent histone demethylases catalyze the posttranslational oxidative demethylation of mono- and dimethylated lysine residues, producing formaldehyde and hydrogen peroxide in addition to the corresponding demethylated protein. In vivo, histone demethylase LSD1 (KDM1; BCH110) is a component of the multiprotein complex that includes histone deacetylases (HDAC 1 and 2) and the scaffolding protein CoREST. Although little is known about the affinities of or the structural basis for the interaction between CoREST and HDACs, the structure of CoREST(286-482) bound to an α-helical coiled-coil tower domain within LSD1 has recently been reported. Given the significance of CoREST in directing demethylation to specific nucleosomal substrates, insight into the molecular basis of the interaction between CoREST and LSD1 may suggest a new means of inhibiting LSD1 activity by misdirecting the enzyme away from nucleosomal substrates. Toward this end, isothermal titration calorimetry studies were conducted to determine the affinity and thermodynamic parameters characterizing the binding interaction between LSD1 and CoREST(286-482). The proteins tightly interact in a 1:1 stoichiometry with a dissociation constant (K(d)) of 15.9 ± 2.07 nM, and their binding interaction is characterized by a favorable enthalpic contribution near room temperature with a smaller entropic penalty at pH 7.4. Additionally, one proton is transferred from the buffer to the heterodimeric complex at pH 7.4. From the temperature dependence of the enthalpy change of interaction, a constant-pressure heat capacity change (ΔC(p)) of the interaction was determined to be -0.80 ± 0.01 kcal mol(-1) K(-1). Notably, structure-driven truncation of CoREST revealed that the central binding determinant lies within the segment of residues 293-380, also known as the CoREST "linker" region, which is a central isolated helix that interacts with the LSD1 coiled-coil tower domain to create a triple-helical bundle. Thermodynamic parameters obtained from the binding between LSD1 and the linker region of CoREST are similar to those obtained from the interaction between LSD1 and CoREST(286-482). These results provide a framework for understanding the molecular basis of protein-protein interactions that govern nucleosomal demethylation.