Báo cáo khoa học: New roles of flavoproteins in molecular cell biology: Histone demethylase LSD1 and chromatin pot

MINIREVIEW New roles of flavoproteins in molecular cell biology: Histone demethylase LSD1 and chromatin Federico Forneris 1 , Elena Battaglioli 2 , Andrea Mattevi 1 and Claudia Binda 1 1 Dipartimento di Genetica e Microbiologia, Universita ` di Pavia, Italy 2 Dipartimento di Biologia e Genetica per le Scienze Mediche, Universita ` di Milano, Italy Introduction In eukaryotic cells, DNA is packaged within the com- plex and organized structure of chromatin. The basic unit of chromatin is the nucleosome, which is a com- pact core of four histones (H2A, H2B, H3 and H4, each one present in two copies forming an octamer) surrounded by a 147 bp stretch of DNA [1]. The histone N-terminal tails bear a number of sites for post-translational modifications that have a direct role in modulating gene expression. The term ‘epigenetics’ refers to the mechanisms that regulate gene transcrip- tion through the read-out of these covalent modifica- tions on the histone tail. This epigenetic control is achieved either by simply modulating the accessibility of DNA or by recognition of histone post-translational modifications through specific protein domains. In par- ticular, the lysine residues of histone tails are subject to both acetylation and methylation, and the meaning of such epigenetic marks depends on the site where they occur. Histone lysine acetylation is often associ- ated with gene activation. In some cases, this condition Keywords amine oxidase domain; chromatin; epigenetics; enzyme; flavin; gene expression; histone demethylation; hydrogen peroxide; protein complex; Rossmann fold Correspondence C. Binda, Dipartimento di Genetica e Microbiologia, Universita ` di Pavia, Via Ferrata 1, 27100 Pavia, Italy Fax: +39 382 528496 Tel: +39 382 985534 E-mail: binda@ipvgen.unipv.it Website: http://www.unipv.it/biocry (Received 6 January 2009, revised 21 May 2009, accepted 22 May 2009) doi:10.1111/j.1742-4658.2009.07142.x Lysine-specific demethylase 1 (LSD1) is an enzyme that removes methyl groups from mono- and dimethylated Lys4 of histone H3, a post-transla- tional modification associated with gene activation. Human LSD1 was the first histone demethylase to be discovered and this enzymatic activity is conserved among eukaryotes. LSD1 has been identified in a number of chromatin-remodeling complexes that control gene transcription and its demethylase activity has also been linked to pathological processes includ- ing tumorigenesis. The 852-residue sequence of LSD1 comprises an amine oxidase domain which identifies a family of enzymes that catalyze the FAD-dependent oxidation of amine substrates ranging from amino acids to aromatic neurotransmitters. Among these proteins, LSD1 is peculiar in that it acts on a protein substrate in the nuclear environment of chromatin- remodeling complexes. This functional divergence occurred during evolu- tion from the eubacteria to eukaryotes by acquisition of additional domains such as the SWIRM domain. The N-terminal part of LSD1, predicted to be disordered, contains linear motifs that might represent functional sites responsible for the association of this enzyme with a variety of transcriptional protein complexes. LSD1 shares structural features with other flavin amine oxidases, including the overall fold of the amine oxidase domain region and details in the active site that are relevant for amine substrate oxidation. Abbreviations AOD, amine oxidase domain; LSD1, lysine-specific demethylase 1; MAO, monoamine oxidase; PAO, polyamine oxidase; REST, repressor element 1-silencing transcription. 4304 FEBS Journal 276 (2009) 4304–4312 ª 2009 The Authors Journal compilation ª 2009 FEBS results from the neutralizing effect of acetyl groups on the lysine positive charge, which weakens the electro- static interaction between the histone tail and the DNA phosphate moiety, with a resulting loosening of the nucleosome compactness [2]. In addition, lysine acetylation at particular sites on the histone tail is spe- cifically recognized by chromatin-interacting proteins such as the bromodomains [3]. Histone lysine methyla- tion may signal either the activation or repression of gene expression depending on the site of methylation [4]. These histone post-translational modifications rep- resent platforms for the binding of protein modules that recruit or instruct effector proteins regulating transcriptional activity [5]. Although histone lysine methylation has been known for decades, it was not until 2004 that the first human histone demethylase lysine-specific demethylase 1 (LSD1) was identified [6,7]. This led to the discovery of other human histone demethylases, the so-called JmjC domain-containing proteins, which revolution- ized the concept of histone methylation as a dynami- cally regulated process, rather than a permanent epigenetic mark [8]. Moreover, orthologs of LSD1 in Caenorhabditis elegans, Arabidopsis thaliana and Drosophila melanogaster were identified and extensively investigated [9–11]. The ever-growing number of chromatin-remodeling enzymes led scientists in the field to create an organized nomenclature [12]. Accord- ing to these guidelines, histone lysine demethylases are named K-demethylases and the LSD1-like enzymes are named K-demethylase 1 proteins. This minireview focuses on LSD1 without involving its orthologs or JmjC demethylases; the acronym LSD1 is adopted for consistency with previous publications on the same subject. LSD1 specifically removes one or two methyl groups from Lys4 of histone H3 via a FAD-depen- dent reaction (Fig. 1). LSD1 activity can be mea- sured in vitro on free histones and on a peptide of at least 21 amino acids corresponding to the N-ter- minal tail of histone H3 [6]. LSD1 is not active on trimethylated Lys4, which is consistent with the fla- vin-catalyzed amine oxidation reaction that requires a lone pair of electrons on the lysine amino group. It has been shown that the presence of a second post-translational modification on the same histone H3 peptide dramatically reduces LSD1 activity on methylated Lys4 [13]. This demonstrates that LSD1 is capable of reading the histone code and suggests a timing scheme for gene repression in which this enzyme removes the last activation mark [14,15]. The crystal structure of the protein in complex with a histone H3 peptide (Fig. 2) provides an explanation for this highly specific recognition mechanism, which is accomplished through an intricate network of electrostatic interactions between a long stretch of the histone tail and the active site residues [16,17]. This structural feature also accounts for the inhibi- tory effect of an unmodified histone peptide on LSD1 activity and suggests that the affinity of this enzyme for the histone tail may have a role in docking LSD1-associated co-factors to the nucleo- some. LSD1 was originally identified as part of the multi- protein repressor complex coordinated by the repressor element 1-silencing transcription (REST, also known as the neuron-restrictive silencing factor) factor in the regulation of neuronal genes [18]. Methylation on Lys4 Fig. 1. FAD-dependent histone demethylation reaction catalyzed by LSD1. The figure shows the one-letter code amino acid sequence of the histone H3 tail corresponding to the peptide used for biochemical and structural studies. The enzyme is specific for Lys4, which is highlighted in red. R stands for either H or CH 3 , because LSD1 is active on both mono- and dimethylated Lys4 in H3 peptides. The three-ring oxidized flavin is shown in yellow, whereas the reduced form is colorless, consistent with the spectroscopic properties of the FAD coenzyme. The LSD1-catalyzed reaction produces an imine intermediate that is hydrolyzed to the demethy- lated product and formaldehyde. Reduced flavin is reoxidized by molecular oxygen with the concomitant production of hydrogen peroxide. F. Forneris et al. Flavin-dependent histone demethylation FEBS Journal 276 (2009) 4304–4312 ª 2009 The Authors Journal compilation ª 2009 FEBS 4305 of histone H3 is a gene-activation mark and LSD1 activity contributes to transcriptional repression of neuronal genes by removing this post-translational modification. LSD1 interacts with CoREST, a co-repressor protein that binds REST and recruits other histone-modifying enzymes such as histone deacetylases 1 ⁄ 2. The function of the LSD1–CoREST– histone deacetylase subcomplex in transcriptional repression events is not limited to REST-regulated neuronal genes, but can be extended to other contexts such as hematopoietic differentiation [19] and the telo- merase reverse transcriptase genes [20]. Moreover, a very recent study showed that, in C. elegans, patterns of dimethylated Lys4 of histone H3 serve as epigenetic memory to maintain the transcriptional program dur- ing cell division, and LSD1 was shown to be essential in resetting this memory in the germline in order to ensure pluripotency [21]. In parallel, a role for LSD1 in gene activation was originally discovered in the reg- ulation of androgen receptor target genes [22] and was also identified in other hormone-dependent transcrip- tional programs [23]. LSD1 is suggested to promote these gene activation events by acting on methylated Lys9 of histone H3, an epigenetic mark associated with gene repression. However, biochemical and structural studies on the recombinant enzyme showed that LSD1 is not active on H3 peptides methylated at Lys9 [6,7] and it features a highly specific substrate-recognition mechanism [14,16]. Unquestionably, the involvement of LSD1 in an ever-growing number of transcriptional protein complexes emphasizes the central role of this flavoenzyme in the chromatin environment. LSD1 activity has also been linked to tumorigenesis events [24], which highlights this protein as a potential drug target. The aim of this minireview is to dissect LSD1 domain organization in light of its role in transcrip- tional regulation. The minireview focuses on the amine oxidase moiety of this protein which is responsible for the catalytic activity on methylated Lys4 of histone H3 and will involve other flavin-dependent amine oxidases that are evolutionary related to LSD1. LSD1 is a flavin-dependent amine oxidase Amine oxidations are widespread reactions in nature. Flavin-dependent amine oxidases are enzymes that catalyze oxidative cleavage of the C–N bond by two- electron reduction of the FAD coenzyme, which pro- duces an imine intermediate that is then hydrolyzed nonenzymatically [25] (Fig. 1). Reduced FAD can be reoxidized by molecular oxygen, which generates hydrogen peroxide and makes the enzyme available for a new catalytic cycle [26]. The overall globular struc- ture of these enzymes was defined as an amine oxidase domain (AOD; Figs 3 and 4) whose genes are present from eubacteria to eukaryotes [27]. The structural par- adigm of these proteins is the Rossmann-fold topology [28], a dinucleotide-binding motif that is shared by other (nonamine oxidizing) enzymes. Typically, the AOD structure consists of two subdomains, a FAD- binding moiety characterized by the Rossmann-fold topology and a substrate-binding domain. Evolution has ‘modeled’ the structure of these AOD enzymes to fit different amine substrates and cellular landscapes. LSD1 was the subject of a dali search [29] to investigate structural affinities with other proteins and the outcome of this analysis is summarized in Table 1. The highest score is found with polyamine oxidases (PAOs), such as maize PAO [30] (Fig. 3A) and yeast FMS1 [31], which are characterized by the typical AOD two-domain fold. Fungal monoamine Fig. 2. Ribbon representation of the LSD1-CoREST crystal struc- ture in complex with a histone H3 peptide (PDB code 2v1d) [16]. The crystals were grown using the recombinant forms of both proteins comprising residues 157–852 and 305–482 for LSD1 and CoREST, respectively. The LSD1 molecule is in light blue (labels for the different domains of LSD1 structure are indicated in light blue font), whereas CoREST is in cyan. The histone H3 peptide (resi- dues 1–16 of the N-terminal tail) is in magenta (the two ends of the peptide, residues 1 and 16, are labeled accordingly). The FAD cofactor is in yellow ball-and-stick representation. Figure 2 was generated with MOLSCRIPT [54]. Flavin-dependent histone demethylation F. Forneris et al. 4306 FEBS Journal 276 (2009) 4304–4312 ª 2009 The Authors Journal compilation ª 2009 FEBS A B Fig. 3. Comparative structural analysis of flavin amine oxidases. (A) Overall folding topology of Zea mays PAO (PDB code 1b5q), Aspergillus niger MAO N (PDB code 2vvm), human MAO B (PDB code 1gos) and human LSD1 (PDB code 2v1d; with respect to Fig. 2, the picture includes only the LSD1 molecule). The protein molecules are drawn with the FAD cofactor in the same orienta- tion (i.e. the Rossmann-fold is at the top of the structure). In all ribbon diagrams, the AOD is colored blue and the FAD molecule is in a yellow ball-and-stick representation. The N- and C-terminus of each structure are labeled as ‘N’ and ‘C’, respectively. In each structure, the cavity representing the substrate-binding site is drawn as a gray surface. The overall structure of human MAO B is representative of mammalian monoamine oxidases and it has the same folding topology as human MAO A [38]. The MAO B C-terminal segment responsible for anchoring the protein to the outer mitochon- drial membrane is depicted in green. Fungal monoamine oxidase (MAO N) is not bound to the membrane and does not have the C-terminal helix that in mammalian MAO A and MAO B represents the transmembrane domain. In LSD1, the Tower domain that interrupts the sequence of the AOD is colored green, whereas the SWIRM domain is in red. The cavities were calculated using VOIDOO [55]. The volumes of the cavities are 601, 313, 637 and 1736 A ˚ 3 for PAO, MAO N, MAO B and LSD1, respectively. (B) Over- view of the active site surrounding the flavin cofactor in each of the four structures reported in (A). The structures are rotated 90° anti-clockwise with respect to their orientation in (A). The flavin and the active site residues are shown in a ball-and-stick representation, whereas the conserved water molecule H-bonded with the flavin N5 atom is represented as a cyan sphere. The substrate-binding site cavity is drawn as semi-transparent gray surface. Figure 3 was generated with BOBSCRIPT [56]. F. Forneris et al. Flavin-dependent histone demethylation FEBS Journal 276 (2009) 4304–4312 ª 2009 The Authors Journal compilation ª 2009 FEBS 4307 oxidase MAO N [32] is a soluble enzyme supposed to be located in the peroxisome and has an overall globu- lar structure similar to that of PAOs (Fig. 3A). During evolution, its mammalian homologs, the monoamine oxidases MAO A and MAO B, acquired an additional C-terminal segment which folds into an a helix that anchors these enzymes to the outer mitochondrial membrane (Fig. 3A) [33,34]. The rationale for such cel- lular localization relative to the role of MAO A and B in neurotransmitter metabolism remains unknown. A fascinating aspect of LSD1 is that this enzyme has an even higher level of complexity because its AOD sequence comprises an insertion of 100 amino acids folded into a helix–turn–helix motif (Tower domain) [35] and preceded by a segment of 200 residues that includes a SWIRM domain and a putative unstruc- tured region (Figs 3A and 4). The Tower domain pro- trudes from the globular AOD structure and provides the interface for CoREST binding (Fig. 2). Interest- ingly, the Tower domain originates on the surface of the AOD structure that corresponds (with respect to the position of the FAD coenzyme) to the area where the transmembrane helix branches off in MAO A and B (Fig. 3A). The SWIRM domain is a motif com- monly found in chromatin enzymes and is responsible for protein–protein and DNA–protein interactions [36]. The name of this domain derives from its original identification in the proteins SWI3, Rsc8 and Moira, which are ATP-dependent chromatin remodeling enzymes. The N-terminal part of LSD1 is predicted to be a disordered region and is discussed in the next section. The active site of all these amine oxidases is nor- mally located in the proximity of the flavin ring of the FAD cofactor and extends along the substrate-binding domain. Despite the similar overall folding topology in the AOD enzymes, there is significant variability in the size and shape of their substrate-binding sites, which Fig. 4. ELM analysis [47] of the full-length LSD1 sequence (1–852 residues). The first line shows the domain organization as identified by the CD search tools [57]. LSD1 domains are shown as bars and the color code is the same as in Fig. 3A, i.e. the AOD is in blue (interrupted by the sequence corresponding to the Tower domain that is in green in Fig 3A), whereas the SWIRM domain is in red. The second line reports the intrinsic protein disorder, domain and globularity prediction of the sequence as determined by GLOBPLOT analysis [58]: disordered regions are brown, low-complexity segments are yellow, globular regions are green. This analysis is in agreement with the domain organiza- tion in the first line. The third line shows a summary of the identified linear motifs (after filtering processing taking in account the accessibi- lity of the putative binding ⁄ modification sites; represented as black bars) which may represent functional sites (see text). This analysis has been carried out considering the detectable linear motifs for a human protein located in the nuclear cell compartment to improve the signal- to-noise ratio of the bioinformatics results. Table 1. Overview of the structural and functional properties of flavin-dependent amine oxidases. Lysine-specific demethylase 1 (LSD1) is compared with other amine oxidase domain (AOD) enzymes of known 3D structure. The analysis is based on a DALI search [29] carried out by using the LSD1 structure (PDB code 2v1d) and the table reports the identified AOD homologs together with the sequence identity and root mean square deviation (rmsd, in A ˚ ) of atomic positions with respect to LSD1. Sequence identity values refer to residues belonging to the AOD of each enzyme. PAO, polyamine oxidase [30]; FMS1, yeast polyamine oxidase [31]; MAO, monoamine oxidase [32–34]; LAAO, L-amino acid oxidase [37]; GOX, glycine oxidase [48]; MSOX, monomeric sarcosine oxidase [49]. Enzyme Sequence identity (%) rmsd Substrate Biological role LSD1 (human) – – methylated Lys4 of histone H3 tail gene expression regulation PAO (maize) 26 2.8 polyamines polyamines metabolism FMS1 (yeast) 26 2.7 polyamines vitamin B5 biosynthesis MAO A (human) 20 2.9 aromatic amines neurotransmitters metabolism MAO B (human) 20 3.0 aromatic amines neurotransmitters metabolism MAO N (fungal) 19 3.0 aromatic amines amine metabolism (peroxisome) LAAO (snake) 19 4.0 L-amino acids amino acids metabolism (venom toxin?) GOX (bacterial) 14 4.0 glycine, sarcosine amino acids metabolism MSOX (bacterial) 10 4.3 sarcosine amino acids metabolism Flavin-dependent histone demethylation F. Forneris et al. 4308 FEBS Journal 276 (2009) 4304–4312 ª 2009 The Authors Journal compilation ª 2009 FEBS have been evolutionarily adapted to the enzyme func- tion and type of substrate. PAO is outstanding in this regard because it is endowed with a long tunnel suited to the aliphatic chain of polyamines (Fig. 3) [30] and its yeast homolog FMS1 displays similar features [31]. In l-amino acid oxidase a 25 A ˚ -long funnel provides access to the active site [37], whereas in MAO A, MAO B and MAO N the aromatic substrates bind in a hydrophobic cavity that, in MAO B (Fig. 3A), has a bipartite nature and is shaped by a loop that provides access to the enzyme active site [38]. In LSD1, the sub- strate-binding site is represented by a large funnel that originates from the flavin and opens wide towards the outside to accommodate the histone substrate (Fig. 3A) [35,39]. In contrast to other histone-modify- ing enzymes, the mechanism of histone tail recognition by LSD1 is complex and highly specific. LSD1 enzymatic activity can be assayed using peptides corre- sponding to the 21 N-terminal amino acids of histone H3 [7]. The structure of the enzyme in complex with a histone peptide revealed that the histone tail adopts a folded conformation when bound to the enzyme and creeps deep into the funnel cavity, establishing a net- work of interactions with the active site residues [16]. These specific interactions act together to fix the his- tone tail in the correct register, which positions Lys4 (the site of the demethylation reaction) in front of the flavin cofactor. The co-repressor CoREST, which sta- bilizes LSD1 and enhances its enzymatic activity on histone peptides, is essential for the in vitro demethyla- tion of nucleosomal particles by LSD1 [40,41]. As shown in Fig. 2, CoREST embraces the LSD1 Tower domain and binds in the proximity of the funnel open- ing where the histone peptide C-terminus is located (i.e. where the globular part of the entire histone is likely to lie). Despite differences in the shape of the substrate- binding site, the details of the active sites of flavin amine oxidases are strikingly conserved (Fig. 3B). In all enzymes listed in Table 1 (except for glycine oxi- dase), there is a lysine residue on top of the flavin, bridged to the N5 atom of the coenzyme via a water molecule [25], which is proposed to have a role in the enzymatic activity. Indeed, mutagenesis experiments have shown that in LSD1, replacement of Lys661 with Ala produced an inactive enzyme [35]. Very recently, it was shown that mutation of this residue in monomeric sarcosine oxidase dramatically reduced the enzymatic turnover rate and oxygen reactivity, providing defini- tive evidence for this lysine as the site of oxygen acti- vation [42]. Another conserved feature in the structure of the AOD active site is the so-called ‘aromatic cage’ [25]. The substrate binds in front of the FAD cofactor (either the re or the si face depending on the enzyme), which has alongside one or two aromatic amino acids that form a sort of cage together with the flavin ring. In PAO and in MAO A, MAO B, MAO N the cage is composed of a Tyr–Phe and Tyr–Tyr couple, respec- tively. Mutagenesis experiments in MAO B demon- strated that these aromatic residues may have a steric role in substrate binding and in increasing the nucleo- philicity of the substrate amine moiety [43]. In LSD1 one of these aromatic residues is conserved, with the other being replaced by a Thr residue (Fig. 3B). Although the role of Tyr761 in the histone demethyla- tion reaction has not been clarified, it might be involved in recognition of the methylated Lys4 amino group. Multidomain organization of LSD1 and its enzymatic activity in the chromatin context Genes coding for LSD1 ⁄ K-demethylase 1 proteins exist only in eukaryotes and have undergone distinct evolutionary development in plants and animals [27]. In contrast to AOD enzymes, which are amine-meta- bolizing catalysts generally functioning by themselves, LSD1 oxidizes the C–N bond of methylated histone lysines within the frame of the chromatin matrix. Table 2 compares the enzymatic activities of those fla- vin amine oxidases for which a thorough biochemical characterization has been reported. The reactivity with oxygen is high, consistent with the fact that, in the case of oxidases, reoxidation of FAD is not the rate- limiting step, whereas the enzymatic activity responsi- ble for substrate oxidation is relatively low for LSD1 compared with that of the chemically similar amine Table 2. Enzymatic activity and oxygen reactivity of flavin-depen- dent amine oxidases. A selection of enzymes among those listed in Table 1 is reported. Values in the k cat column refer to the turnover rate at the steady-state measured with the substrate in parenthe- ses. The rate of reoxidation by molecular oxygen is expressed as second-order rate constant. LSD1, lysine-specific demethylase 1; MAO, monoamine oxidase; PAO, polyamine oxidase; MSOX, monomeric sarcosine oxidase; GOX, glycine oxidase. Enzyme k cat (min )1 ) Second-order rate constant (M )1 Æs )1 ) Ref. LSD1 (human) 3.4 (histone peptide) 9.6 · 10 3 [14] MAO B (human) 600 (benzylamine) 5.5 · 10 3 [50] PAO (maize) 5280 (spermidine) 5.0 · 10 6 [51] MSOX (bacterial) 7030 (sarcosine) 2.8 · 10 5 [42,52] GOX (bacterial) 242 (glycine) 3.0 · 10 4 [53] F. Forneris et al. Flavin-dependent histone demethylation FEBS Journal 276 (2009) 4304–4312 ª 2009 The Authors Journal compilation ª 2009 FEBS 4309 oxidations of other AOD proteins. However, it is note- worthy that the rates of the histone demethylation reaction carried out by enzymes of the JmjC class are even lower [44]. The catalytic activity of these histone- modifying enzymes needs to be evaluated taking into account that they participate in finely tuned cellular processes in which the cascade of events is regulated by a number of protein factors. In this regard, we observed that the presence of the co-repressor CoREST increases in vitro LSD1 activity by approxi- mately two-fold [16] and it has been reported that CoREST is essential for LSD1-mediated demethylation in intact nucleosomes [40,41]. So far, LSD1 is unique among the AOD proteins in catalyzing an amine oxi- dation reaction in the chromatin environment and its demethylase activity has to be pondered in the context of multiprotein transcriptional complexes. LSD1 is a multidomain protein and its involvement in many diverse gene-expression programs [45] is strictly related to its domain organization (Fig. 4). In addition to the SWIRM domain, whose role in the interaction with chromatin proteins is well known, the N-terminal part of LSD1 is of interest. This 200- residue segment is predicted to be disordered and, indeed, all structural studies to date have focused on the SWIRM–AOD portion of the enzyme. However, in recent years, the idea has emerged that, contrary to the traditional view that protein function is necessarily determined by a stable 3D structure, disordered regions may represent flexible modules that fold upon binding to their biological target [46]. In order to investigate the putative disordered 200-residue segment of LSD1 by bioinformatics tools, we used the ELM database [47] which contains a collection of linear motifs corresponding to consensus sequences of known functional meaning. These short sequences may provide sites for post-translational modifications or docking modules that are recognized by specific, well-characterized protein domains. In this way, it is possible to identify potential functional sites in a puta- tive disordered region and to formulate hypotheses regarding its biological role, which may suggest exper- imental investigations. It is important to note that the updated version of ELM produces results that are ‘fil- tered’ by additional information such as cell compart- ment, phylogeny and structural data, and this provides more reliable outcomes from a biological viewpoint. Figure 4 shows an overview of the results of ELM analysis on LSD1, which was restricted to the human cell nucleus compartment. The first two lines indicate that the globular and structured regions of LSD1 are located in the SWIRM–AOD part of the protein, as predicted by other tools and confirmed by the crystal structure [35]. Several linear motifs are identified as putative functional sites in the N-terminal low-complexity region of LSD1, others between the SWIRM and the AOD and two additional hits are found at the C-terminus (whose final 20 residues are not visible in the crystal structure). Some of these motifs are supposed to undergo Ser ⁄ Thr phosphory- lation by well-characterized kinases (e.g. cyclin- dependent kinases), whereas others are predicted to be sites of other post-translational modifications such as sumoylation and ubiquitination, which are recognized by specific domains in protein–protein interaction mechanisms. These predictions need to be validated using experimental studies, but they suggest that this N-terminal putative disordered domain may have a role in providing the enzyme with the necessary flexi- bility to target different chromatin proteins and to adapt LSD1 enzymatic activity to distinct gene tran- scription events. Conclusions The globular domain responsible for the flavin-depen- dent amine oxidase activity is a highly conserved mod- ule used in nature to oxidize amine substrates of different types and participating in distinct biological processes. LSD1 is a remarkable example because it exerts its oxidase activity on a complex substrate such as the histone tail in the eukaryotic cell nucleus, and because it is one of the first examples of the insertion of a protein–protein interaction module (the Tower domain) into a highly conserved functional domain. Evolution has provided LSD1 with the necessary complexity to catalyze a basic chemical reaction in the context of gene transcription regulation. It will be a challenge for future studies to understand how LSD1 fulfils its multiple roles in these complexes mechanisms. Acknowledgements Work in our laboratory was supported by grants from MIUR (COFIN06), the Italian Association for Cancer Research, and ‘Fondazione Cariplo’. References 1 Luger K, Maeder AW, Richmond RK, Sargent DF & Richmond TJ (1997) Crystal structure of the nucleosome core particle at 2.8 A ˚ resolution. Nature 389, 251–260. 2 Davie JR & Chadee DN (1998) Regulation and regulatory parameters of histone modifications. J Cell Biochem Suppl 30–31, 203–213. Flavin-dependent histone demethylation F. Forneris et al. 4310 FEBS Journal 276 (2009) 4304–4312 ª 2009 The Authors Journal compilation ª 2009 FEBS 3 Mujtaba S, Zeng L & Zhou MM (2007) Structure and acetyl–lysine recognition of the bromodomain. Onco- gene 26, 5521–5527. 4 Cheng X & Zhang X (2007) Structural dynamics of protein lysine methylation and demethylation. Mutat Res 618, 102–115. 5 Taverna SD, Li H, Ruthenburg AJ, Allis CD & Patel DJ (2007) How chromatin-binding modules interpret histone modifications: lessons from professional pocket pickers. Nat Struct Mol Biol 14, 1025–1040. 6 Shi Y, Lan F, Matson C, Mulligan P, Whetstine JR, Cole PA, Casero RA & Shi Y (2004) Histone demethy- lation mediated by the nuclear amine oxidase homolog LSD1. Cell 119, 941–953. 7 Forneris F, Binda C, Vanoni MA, Mattevi A & Battaglioli E (2005) Histone demethylation catalysed by LSD1 is a flavin-dependent oxidative process. FEBS Lett 579, 2203–2207. 8 Anand R & Marmorstein R (2007) Structure and mech- anism of lysine-specific demethylase enzymes. J Biol Chem 282, 35425–35429. 9 Smialowska A & Baumeister R (2006) Presenilin func- tion in Caenorhabditis elegans. Neurodegener dis 3, 227– 232. 10 Spedaletti V, Polticelli F, Capodaglio V, Schinina ` ME, Stano P, Federico R & Tavladoraki P (2008) Character- ization of a lysine-specific histone demethylase from Arabidopsis thaliana. Biochemistry 47, 4936–4947. 11 Rudolph T, Yonezawa M, Lein S, Heidrich K, Kubicek S, Scha ¨ fer C, Phalke S, Walther M, Schmidt A, Jenuwein T et al. (2007) Heterochromatin formation in Drosophila is initiated through active removal of H3K4 methylation by the LSD1 homolog SU(VAR)3-3. Mol Cell 26, 103–115. 12 Allis CD, Berger SL, Cote J, Dent S, Jenuwien T, Kouzarides T, Pillus L, Reinberg D, Shi Y, Shiekhattar R et al. (2007) New nomenclature for chromatin-modi- fying enzymes. Cell 131, 633–636. 13 Forneris F, Binda C, Vanoni MA, Battaglioli E & Mattevi A (2005) Human histone demethylase LSD1 reads the histone code. J Biol Chem 280, 41360–41365. 14 Forneris F, Binda C, Dall’aglio A, Fraaije MW, Battaglioli E & Mattevi A (2006) A highly specific mechanism of histone H3–K4 recognition by histone demethylase LSD1. J Biol Chem 281, 35289–35295. 15 Lee MG, Wynder C, Bochar DA, Hakimi MA, Cooch N & Shiekhattar R (2006) Functional interplay between histone demethylase and deacetylase enzymes. Mol Cell Biol 26, 6395–6402. 16 Forneris F, Binda C, Adamo A, Battaglioli E & Mattevi A (2007) Structural basis of LSD1-CoREST selectivity in histone H3 recognition. J Biol Chem 282, 20070–20074. 17 Yang M, Culhane JC, Szewczuk LM, Gocke CB, Brautigam CA, Tomchick DR, Machius M, Cole PA & Yu H (2007) Structural basis of histone demethylation by LSD1 revealed by suicide inactivation. Nat Struct Mol Biol 14, 535–539. 18 Ooi L & Wood IC (2007) Chromatin crosstalk in devel- opment and disease: lessons from REST. Nat Rev Genet 8, 544–554. 19 Saleque S, Kim J, Rooke HM & Orkin SH (2007) Epigenetic regulation of hematopoietic differentiation by Gfi-1 and Gfi-1b is mediated by the cofactors CoREST and LSD1. Mol Cell 27, 562–572. 20 Zhu Q, Liu C, Ge Z, Fang X, Zhang X, Stra ˚ a ˚ tK, Bjo ¨ rkholm M & Xu D (2008) Lysine-specific demethy- lase 1 (LSD1) is required for the transcriptional repres- sion of the telomerase reverse transcriptase (hTERT) gene. PLoS ONE 1, e1446. 21 Katz DJ, Edwards TM, Reinke V & Kelly WG (2009) A C. elegans LSD1 demethylase contributes to germline immortality by reprogramming epigenetic memory. Cell 137, 308–320. 22 Metzger E, Wissmann M, Yin N, Mu ¨ ller JM, Schneider R, Peters AH, Gu ¨ nther T, Buettner R & Schu ¨ le R (2005) LSD1 demethylates repressive histone marks to promote androgen-receptor-dependent transcription. Nature 437, 436–439. 23 Wang J, Scully K, Zhu X, Cai L, Zhang J, Prefontaine GG, Krones A, Ohgi KA, Zhu P, Garcia-Bassets I et al. (2007) Opposing LSD1 complexes function in developmental gene activation and repression pro- grammes. Nature 446, 882–887. 24 Wang GG, Allis CD & Chi P (2007) Chromatin remod- eling and cancer, part I: covalent histone modifications. Trends Mol Med 13, 363–372. 25 Binda C, Mattevi A & Edmondson DE (2002) Structure– function relationships in flavoenzyme-dependent amine oxidations: a comparison of polyamine oxidase and monoamine oxidase. J Biol Chem 277, 23973–23976. 26 Mattevi A (2006) To be or not to be an oxidase: chal- lenging the oxygen reactivity of flavoenzymes. Trends Biochem Sci 31, 276–283. 27 Zhou X & Ma H (2008) Evolutionary history of histone demethylase families: distinct evolutionary patterns sug- gest functional divergence. BMC Evol Biol 8, 294. 28 Rossmann MG, Moras D & Olsen KW (1974) Chemical and biological evolution of nucleotide-binding protein. Nature 250, 194–199. 29 Holm L, Ka ¨ a ¨ ria ¨ inen S, Rosenstro ¨ m P & Schenkel A (2008) Searching protein structure databases with Dali- Lite v.3. Bioinformatics 24, 2780–2781. 30 Binda C, Coda A, Angelini R, Federico R, Ascenzi P & Mattevi A (1999) A 30 A ˚ long U-shaped catalytic tunnel in the crystal structure of polyamine oxidase. Structure 7, 265–276. 31 Huang Q, Liu Q & Hao Q (2005) Crystal structures of Fms1 and its complex with spermine reveal substrate specificity. J Mol Biol 348, 951–959. F. Forneris et al. Flavin-dependent histone demethylation FEBS Journal 276 (2009) 4304–4312 ª 2009 The Authors Journal compilation ª 2009 FEBS 4311 32 Atkin KE, Reiss R, Koehler V, Bailey KR, Hart S, Turkenburg JP, Turner NJ, Brzozowski AM & Grogan G (2008) The structure of monoamine oxidase from Aspergillus niger provides a molecular context for improvements in activity obtained by directed evolution. J Mol Biol 384, 1218–1231. 33 Binda C, Newton-Vinson P, Hubalek F, Edmondson DE & Mattevi A (2002) Structure of human mono- amine oxidase B, a drug target for the treatment of neurological disorders. Nat Struct Biol 9, 22–26. 34 De Colibus L, Li M, Binda C, Lustig A, Edmondson DE & Mattevi A (2005) Three-dimensional structure of human monoamine oxidase A (MAO A): relation to the structures of rat MAO A and human MAO B. Proc Natl Acad Sci USA 102, 12684–12689. 35 Stavropoulos P, Blobel G & Hoelz A (2006) Crystal structure and mechanism of human lysine-specific demethylase-1. Nat Struct Mol Biol 13, 626–632. 36 Da G, Lenkart J, Zhao K, Shiekhattar R, Cairns BR & Marmorstein R (2006) Structure and function of the SWIRM domain, a conserved protein module found in chromatin regulatory complexes. Proc Natl Acad Sci USA 103, 2057–2062. 37 Pawelek PD, Cheah J, Coulombe R, Macheroux P, Ghisla S & Vrielink A (2000) The structure of l-amino acid oxidase reveals the substrate trajectory into an enantiomerically conserved active site. EMBO J 19, 4204–4215. 38 Edmondson DE, Binda C, Wang J, Upadhyay AK & Mattevi A (2009) Molecular and mechanistic properties of the membrane-bound mitochondrial monoamine oxidases. Biochemistry 48, 4220–4230. 39 Yang M, Gocke CB, Luo X, Borek D, Tomchick DR, Machius M, Otwinowski Z & Yu H (2006) Structural basis for CoREST-dependent demethylation of nucleo- somes by the human LSD1 histone demethylase. Mol Cell 23, 377–387. 40 Shi YJ, Matson C, Lan F, Iwase S, Baba T & Shi Y (2005) Regulation of LSD1 histone demethylase activity by its associated factors. Mol Cell 19, 857–864. 41 Lee MG, Wynder C, Cooch N & Shiekhattar R (2005) An essential role for CoREST in nucleosomal histone 3 lysine 4 demethylation. Nature 437, 432–435. 42 Zhao G, Bruckner RC & Jorns MS (2008) Identification of the oxygen activation site in monomeric sarcosine oxidase: role of Lys265 in catalysis. Biochemistry 47, 9124–9135. 43 Li M, Binda C, Mattevi A & Edmondson DE (2006) Functional role of the ‘aromatic cage’ in human mono- amine oxidase B: structures and catalytic properties of Tyr435 mutant proteins. Biochemistry 45, 4775–4784. 44 Smith BC & Denu JM (2009) Chemical mechanisms of histone lysine and arginine modifications. Biochim Bio- phys Acta 1789, 45–57. 45 Forneris F, Binda C, Battaglioli E & Mattevi A (2008) LSD1: oxidative chemistry for multifaceted functions in chromatin regulation. Trends Biochem Sci 33, 181–189. 46 Dyson HJ & Wright PE (2005) Intrinsically unstruc- tured proteins and their functions. Nat Rev Mol Cell Biol 6, 197–208. 47 Puntervoll P, Linding R, Gemu ¨ nd C, Chabanis- Davidson S, Mattingsdal M, Cameron S, Martin DMA, Ausiello G, Brannetti B, Costantini A et al. (2003) ELM server: a new resource for investigating short functional sites in modular eukaryotic proteins. Nucleic Acids Res 31 , 3625–3630. 48 Mo ¨ rtl M, Diederichs K, Welte W, Molla G, Motteran L, Andriolo G, Pilone MS & Pollegioni L (2004) Struc- ture–function correlation in glycine oxidase from Bacil- lus subtilis . J Biol Chem 279, 29718–29727. 49 Trickey P, Wagner MA, Jorns MS & Mathews FS (1999) Monomeric sarcosine oxidase: structure of a covalently flavinylated amine oxidizing enzyme. Struc- ture 7, 331–345. 50 Tan AK & Ramsay RR (1993) Substrate-specific enhancement of the oxidative half-reaction of mono- amine oxidase. Biochemistry 32, 2137–2143. 51 Bellelli A, Angelini R, Laurenzi M & Federico R (1997) Transient kinetics of polyamine oxidase from Zea mays L. Arch Biochem Biophys 343, 146–148. 52 Wagner MA & Jorns MS (2000) Monomeric sarcosine oxidase: 2. Kinetic studies with sarcosine, alternate sub- strates, and a substrate analogue. Biochemistry 39, 8825–8829. 53 Molla G, Motteran L, Job V, Pilone MS & Pollegioni L (2006) Kinetic mechanisms of glycine oxidase from Bacillus subtilis. Eur J Biochem 270, 1474–1482. 54 Kraulis PJJ (1991) MOLSCRIPT: a program to pro- duce both detailed and schematic plots of protein struc- tures. Appl Crystallogr 24, 946–950. 55 Kleywegt GJ & Jones TA (1994) Detection, delineation, measurement and display of cavities in macromolecular structures. Acta Crystallogr 50, 178–185. 56 Esnouf RM (1999) Further additions to molScript version 1.4, including reading and contouring of elec- tron-density maps. Acta Crystallogr 55, 938–940. 57 Marchler-Bauer A & Bryant SH (2004) CD-Search: pro- tein domain annotations on the fly. Nucleic Acids Res 32, W327–W331. 58 Linding R, Russell RB, Neduva V & Gibson TJ (2003) GlobPlot: exploring protein sequences for globularity and disorder. Nucleic Acids Res 31, 3701–3708. Flavin-dependent histone demethylation F. Forneris et al. 4312 FEBS Journal 276 (2009) 4304–4312 ª 2009 The Authors Journal compilation ª 2009 FEBS . MINIREVIEW New roles of flavoproteins in molecular cell biology: Histone demethylase LSD1 and chromatin Federico Forneris 1 , Elena Battaglioli 2 , Andrea. branches off in MAO A and B (Fig. 3A). The SWIRM domain is a motif com- monly found in chromatin enzymes and is responsible for protein–protein and DNA–protein