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:
Website: />(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’.
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