Dynamic Regulation of Histone Lysine Methylation by Demethylases

Molecular Cell

Review

Dynamic Regulation of HistoneLysine Methylation by Demethylases

Yang Shi1,* and Johnathan R. Whetstine1

1Department of Pathology, Harvard Medical School, 77 Avenue Louis Pasteur, Boston, MA 02115, USA*Correspondence: [email protected] 10.1016/j.molcel.2006.12.010

Recent studies demonstrated that histone methylation is not static but is dynamically regulated byhistone methyltransferases and the newly discovered histone demethylases. This review discussesthe chemical mechanisms for the known and potentially new classes of demethylases, the roles ofthese demethylases in chromatin and transcription, and their potential biological functions andconnections to human diseases.

A Brief History of Chromatin and HistoneMethylationIn eukaryotic cells, DNA is packaged into a nuclear struc-

ture called chromatin, which was first identified and

named by Walther Flemming in 1882 because of the re-

fractory nature and affinity for dyes (Flemming, 1882).

Flemming was the first to discover that chromosomes

are split into two identical halves during mitosis, which

provided important cytogenetic basis for Mendel’s rule

for heredity. Chromatin was found to contain both nucleic

acids and a series of acid-soluble proteins that were

termed ‘‘histone’’ by Albrecht Kossel (Kossel, 1911). The

histones and other chromosomal proteins are responsible

for the proper packaging of the DNA into the chromo-

somes. Through the use of electron microscopy and

crystallography, the basic building blocks of chromatin

were identified and referred to as nucleosomes (reviewed

in Olins and Olins [2003]). The nucleosome is composed of

146 base pairs of DNA wrapped around the histone

octamer (two copies each of H2A, H2B, H3, and H4) (Korn-

berg and Lorch, 1999). In the early 1960s, evidence was

accumulating that histone proteins were modified at the

posttranslational level by acetylation and methylation,

which may play a role in RNA synthesis (Allfrey et al.,

1964). Methylation was shown to occur on the 3-amino

group of lysine (K) (Murray, 1964) and the guanidino group

of arginine (R) (Paik and Kim, 1967, 1969) and was

catalyzed by enzymes using S-adenosyl-L-methionine

(SAM) as the methyl group donor (Kim and Paik, 1965;

Paik and Kim, 1971). However, it was not until the year

2000 that a landmark discovery uncovered the molecular

identity of the first histone methyltransferase (Rea et al.,

2000).

Histone methylation is now recognized as an important

modification linked to both transcriptional activation and

repression (Margueron et al., 2005). Histones contain

numerous lysine and arginine residues, of which many

are methylated in vivo (Zhang et al., 2003, 2004; Mar-

gueron et al., 2005). Six of the lysine residues, including

histones H3K4, -9, -27, -36, and -79 as well as histone

H4K20, have been studied extensively and linked to

chromatin and transcriptional regulation as well as DNA

damage response (Margueron et al., 2005; Martin and

Zhang, 2005). Lysine can be mono-, di-, and trimethylated

(Bannister and Kouzarides, 2004), while arginine can be

both monomethylated and symmetrically or asymmetri-

cally dimethylated (Bedford and Richard, 2005). The

numerous lysine and arginine residues on the histone

tails, in conjunction with the various methylation levels

that can be generated at each of these sites, provide

tremendous regulatory potentials for chromatin modifica-

tions.

Many of the covalent modifications that take place on

the histone tails are enzymatically reversible. For example,

phosphorylation and acetylation are reversed by phos-

phatases and deacetylases, respectively. This enzyme-

based reversibility makes sense because it provides the

cell with a means to respond quickly to changes through

rapid alteration in its gene expression programs. However,

unlike histone acetylation and phosphorylation, histone

methylation was considered static and enzymatically irre-

versible, a view that prevailed in the chromatin field for the

past 30 years. This dogma was based on early studies that

demonstrated comparable turnover rates of bulk histones

and the methyl groups on histone K and R residues in

mammalian cells (Byvoet et al., 1972; Thomas et al.,

1972). Interestingly, around the same time, a separate

study identified a low level of histone methyl group turn-

over (�2% per hour) (Borun et al., 1972). The same re-

search group also identified an enzymatic activity in rat

kidney extract that appeared to mediate demethylation;

however, the enzyme was never identified (Paik and

Kim, 1973). Given the importance of histone methylation,

these studies left a major question to be addressed, i.e.,

are there bona fide demethylases that reverse K and R

methylation? A recent study of a corepressor led to the

identification of the first histone demethylase (Shi et al.,

2004), thus changing our view of histone methylation

regulation and ushering in the identification of numerous

histone demethylases.

Molecular Cell 25, January 12, 2007 ª2007 Elsevier Inc. 1

mailto:[email protected]

Molecular Cell

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Histone Methylation Is Enzymatically ReversibleDiscovery of Lysine-Specific Demethylase 1

Over the past few years, several laboratories identified

a protein KIAA0601 (alias p110b, BHC110, NPAO) that

appeared to be a common component of multiple core-

pressor complexes (Hakimi et al., 2002; Humphrey et al.,

2001; Shi et al., 2003; Tong et al., 1998; You et al., 2001).

KIAA0601 has a SWIRM domain (Swi3p, Rsc8p, and

Moira), which has been found in a number of chromatin-

associated proteins (Aravind and Iyer, 2002), and a long

C-terminal domain with significant sequence homology

with flavin adenine dinucleodtide (FAD)-dependent amine

oxidases (Figure 1). The metabolic amine oxidases have

a range of substrates from small molecules to proteins,

raising the possibility that KIAA0601 may mediate an enzy-

matic reaction important for repression. The logical sub-

strates for KIAA0601 included spermine and spermidine,

which are components of chromatin (Casero, 1995), and/

or methylated proteins such as histones. Analysis of the

recombinant KIAA0601 protein allowed the investigators

to draw several important conclusions. First, KIAA0601 is

a lysine-specific demethylase (renamed LSD1 to reflect

this activity). Although the LSD1 sequence is most similar

to polyamine oxidases (Shi et al., 2004), subsequent anal-

ysis of the LSD1 and the maize polyamine oxidase crystal

structures provided insights into the substrate differences

between these enzymes. The maize polyamine oxidase is

shaped as a long tunnel to accommodate the long, linear

polyamine substrates (Binda et al., 2002). In contrast, the

LSD1 catalytic domain is wide and contains a large open-

ing that is more compatible with the side chains of the

histone tail rather than the small linear polyamines (Yang

et al., 2006). Second, the LSD1-mediated demethylation

reaction was surprisingly specific; i.e., recombinant

LSD1 demethylates lysine 4 of histone H3, but not other

methylated arginines or lysines (Figure 1; Shi et al.,

2004). The structural basis for the ability of LSD1 to

discriminate between methylated K and R and between

H3K4me and other methylated K residues on histone

remains an interesting and important question to be ad-

dressed in the future.

Chemical Mechanism of LSD1-Mediated Histone

Demethylation

Flavin-containing amine oxidases characterize oxidative

cleavage of the a-carbon bond of the substrate to form

an imine intermediate, which is hydrolyzed to form an al-

dehyde and amine. This reaction reduces cofactor FAD

to FADH2, which is then reoxidized by molecular oxygen,

producing H2O2. These chemical reactions have been

proposed to be compatible for the removal of methyl

groups from histone by unidentified FAD-dependent

enzymes (Bannister et al., 2002) and, specifically, for the

nuclear amine oxidase LSD1 (Figures 1 and 2; Shi et al.,

2004). In fact, the LSD1-mediated demethylation reac-

tions generated the predicted reaction products (un-

methylated histone peptides, formaldehyde, and H2O2)

that were readily detected by multiple approaches, includ-

ing mass spectrometry and formaldehyde dehydrogenase

2 Molecular Cell 25, January 12, 2007 ª2007 Elsevier Inc.

(FDH) assays (Shi et al., 2004). As illustrated in Figure 2, the

LSD1 demethylation reaction requires protonated nitrogen

in the substrate to form the imine intermediate, thus limiting

the substrates to mono- or dimethylated peptides, raising

the question of whether trimethylated lysine is reversible

by other classes of enzymes.

Regulation of LSD1 Activity and Substrate

Specificity

Several recent studies highlighted the importance of

protein-protein interactions in the control of LSD1 activity

and substrate specificity. The ability of LSD1 to demethy-

late H3-K4 on a nucleosome requires an interacting

corepressor, Co-REST (Figure 1; Lee et al., 2005; Shi

et al., 2005). The amine oxidase domain of LSD1 is disrup-

ted by an insertion, termed the ‘‘tower domain,’’ which

protrudes from the enzyme as seen in the 3D structure,

thereby reconstituting a contiguous catalytic domain

(Stavropoulos et al., 2006; Yang et al., 2006; Chen et al.,

2006a). The tower domain represents a surface for Co-

REST binding. Interestingly, not all LSD1-related proteins

have the tower domain, suggesting that other mecha-

nisms may be involved in regulating chromatin accessibil-

ity of these enzymes. The interaction between Co-REST

and LSD1 not only allows accessibility to nucleosomal

substrates (Lee et al., 2005; Shi et al., 2005) but also

stabilizes LSD1 (Shi et al., 2005). The LSD1/Co-REST

cocrystal structure showed that Co-REST makes multiple

contacts within the nucleosomes including a weak inter-

action with the DNA (Yang et al., 2006). The spatially sep-

arated, multivalent nucleosome binding may therefore

underlie the interaction between LSD1/Co-REST and the

nucleosome and may explain the Co-REST requirement

for LSD1 to access nucleosomal substrates (Yang et al.,

2006). Protein-protein interactions also appear to regulate

LSD1 substrate specificity. For example, LSD1 functioned

as an H3K9 demethylase and a transcriptional activator in

the presence of the androgen receptor (AR) (Metzger

et al., 2005). Although the structural basis for this observa-

tion remains to be investigated, this finding illustrated

a strategy whereby LSD1 can expand its substrate reper-

toire via protein-protein interactions (Figure 1). In addition

to protein-protein interactions, the surrounding modifi-

cations on the histone tails (e.g., H3 serine 10 phosphory-

lation) also affect LSD1 activity (Forneris et al., 2005),

providing another level of regulation.

Enzymatic Demethylation Is Essential for DynamicRegulation of Histone MethylationThe Fe(II) Dioxygenase Superfamily Contains

Histone Demethylases

The discovery of LSD1 demonstrates that histone methyl-

ation is enzymatically reversible and dynamically regu-

lated. It also raises an immediate question: how critical

is enzymatic demethylation in regulating histone methyla-

tion dynamics in general? This is an important issue

given that histone replacement plays an important role

in regulating histone methylation (Ahmad and Henikoff,

2002; Briggs et al., 2001; Johnson et al., 2004). In

Molecular Cell

Review

Figure 1. Schematic Diagrams of LSD1 Domains, 3D Structures, and Mechanism of ActionLSD1 domains are indicated by different colors. AOD stands for amine oxidase domain. The 3D structures are taken from two recentstudies (Stavropoulos et al., 2006; Yang et al., 2006). LSD1 alone demethylates H3K4me1/Me2 (Shi et al., 2004). Co-REST interacts with thetower/insert region of LSD1 (upper interaction). This interaction results in nucleosomal demethylation (Lee et al., 2005; Shi et al., 2005; Yanget al., 2006). The human androgen receptor (AR) has also been shown to interact with LSD1 and result in H3K9me1 and Me2 demethylation (Metzgeret al., 2005).

addition, histone methylation could also be removed by

protease-mediated histone tail clipping mechanism (Allis

et al., 1980). Lastly, a human peptidylarginine deiminase

(PAD4/PADI4) was shown to convert either non- or mono-

methylated arginine to citrulline, therefore effectively elim-

inating arginine from histone tails (Cuthbert et al., 2004;

Wang et al., 2004). Recently, a number of laboratories dis-

covered a large family of histone demethylases, indicating

that the demethylase-based mechanism is an important

means for dynamic regulation of histone methylation.

Demethylation Mediated by the JmjC

Domain-Containing Proteins

The LSD1 histone demethylase discovery suggests that

nuclear proteins with the ability to mediate oxidation of

N-methylated lysines would make excellent demethylase

candidates. Because lysines can be mono-, di-, and trime-

thylated and LSD1 only mediates mono- and didemethyla-

tion, it was likely that other oxidative mechanisms may

exist. Proteins that mediate oxidative demethylation by

radical attack involving Fe(II) and a-ketoglutarate-depen-

dent dioxygenases have been proposed and experimen-

tally tested (Kubicek and Jenuwein, 2004; Trewick et al.,

2005; Tsukada et al., 2006; Whetstine et al., 2006; Fodor

et al., 2006). This hypothesis was largely based on obser-

vations that ALKB dioxygenases were able to use oxidative

demethylation to remove alkylation from DNA (Falnes

et al., 2002; Kubicek and Jenuwein, 2004; Trewick et al.,

2002). The dioxygenases represented by the Jumonji C

(JmjC) domain containing proteins were the first group

suggested to function as histone demethylases (Trewick

et al., 2005). Jumonji is Japanese for ‘‘cruciform’’ and

was the name given to the transcription factor whose

ablation in mice resulted in neural plate deformation that

resembled a cruciform (Takeuchi et al., 1995). Two con-

served sequences have been noted in Jumonji and the

human proteins Smcx and RBP2 (Balciunas and Ronne,

2000; Clissold and Ponting, 2001; Takeuchi et al., 1995)

and were referred to as JmjN and JmjC based on their


Molecular Cell

Review

Figure 2. Chemical Mechanism for LSD1-Mediated DemethylationThe reaction mechanism, adapted from Shi et al. (2004), depicts LSD1 removing a methyl group from a dimethylated lysine residue, but the reactioncan proceed until an unmethylated lysine is generated.

relative locations to each other within the protein. The JmjC

domain was later found in more than 100 proteins from

bacteria to eukaryotes (Clissold and Ponting, 2001) and

was recently shown to be the catalytic domain for histone

demethylation (Tsukada et al., 2006). The JmjN domain is

found only in a subset of the Jmjc-containing proteins

and may play a role in the structural integrity of the enzyme

(Chen et al., 2006b).

Multiple Approaches Identified Numerous JmjC

Domain Proteins as Histone Demethylases

By using a classical biochemical approach, a recent study

identified and reported the first JmjC domain histone

demethylase, JHDM1/Fbx11 (JmjC domain-containing

histone demethylase 1, alias Fbx11), which reverses

mono- and dimethylation of histone H3K36, and demon-

strated that the JmjC domain was the catalytic moiety in-

volved in mediating the demethylation reaction (Tsukada

et al., 2006). JHDM1 also contains an F box domain, which

is a motif associated with E3 ligase functions (Bai et al.,

1996), suggesting a possible connection between histone

demethylation and ubiquitination. The biochemical ap-

proach, which tracks formaldehyde production in the

chromatography fractions to reflect histone demethylase

activity, also identified JHDM2, which mediates demethy-

lation of H3K9me2 (Yamane et al., 2006). These data were

consistent with the prediction that additional histone

demethylase may also use oxidative mechanisms to re-

move methyl groups in the form of formaldehyde (Shi

et al., 2004).

By using a candidate-based screen that focused on�50

different proteins with domains capable of oxidative re-

actions, including 28 human JmjC-containing proteins,

another recent study identified a new histone demethylase

subfamily, i.e., JMJD2, which consists of four members

(JMJD2A-D) (Katoh, 2004), capable of reversing lysine

trimethylation, specifically H3K9me3 and H3K36me3

(Figure 3; Whetstine et al., 2006). Similar candidate ap-

proaches and affinity purification scheme also led to the

identification of the JMJD2 family (Fodor et al., 2006; Klose

et al., 2006; Cloos et al., 2006). JMJD2A was previously

identified as a Rb-, HDAC1-, and N-CoR-interacting


protein and as a transcriptional repressor (Gray et al.,

2005; Zhang et al., 2005), while JMJD2C or GASC1 (gene

amplified in squamous cell carcinoma 1) was initially found

as an overexpressed gene in esophageal squamous carci-

noma (Yang et al., 2000). Since H3K9/K36me3 has been

implicated in transcriptional elongation and suppression

of inappropriate transcription within the body of the gene

(Carrozza et al., 2005; Joshi and Struhl, 2005; Keogh

et al., 2005; Morris et al., 2005; Vakoc et al., 2005), the

dual site specificity of JMJD2A/C may suggest a coordi-

nated role in regulating H3K9/K36 trimethylation for gene

repression. The identification of the JMJD2 family histone

demethylases provided several additional important in-

sights. First, in contrast to LSD1 and JHDM1/2, the JMJD2

family members displayed differential methyl group recog-

nition, i.e., they all mediated demethylation of trimethy-

lated lysine substrates H3K9me3 (Cloos et al., 2006; Fodor

et al., 2006; Klose et al., 2006; Whetstine et al., 2006).

Second, two members of this family, JMJD2A/JHDM3

and JMJD2C/GASC1, demethylated both H3K9me3 and

H3K36me3 (Klose et al., 2006; Whetstine et al., 2006),

thus revealing a potential link between these two modifica-

tions that was previously unsuspected. Third, JMJD2A/C

and JMJD2D generated different methylation states at

H3-K9 (H3K9me2 versus H3K9me1, respectively), sug-

gesting a potential fine tuning mechanism for histone

methylation regulation. Taken together, these studies

established the notion that demethylases represent an

important general mechanism to dynamically regulate

histone methylation.

Chemical Mechanism for JmjC-Mediated Histone

Demethylation

The chemical reactions catalyzed by the JmjC domain that

result in oxidative demethylation are shown in Figure 4. The

Fe(II) provides a resonance structure to the coordinated

molecular oxygen, resulting in Fe(III) and a superoxide

radical species that attacks the a-ketoglutarate and results

in the production of CO2 and an Fe(IV)-oxo intermediate

(Fe[IV] = O; Figure 4). The Fe(IV)-oxo intermediate removes

a hydrogen from the methyl group, creates a free radical,

and generates an Fe(III) hydroxide. Subsequently, the

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Figure 3. Phylogenic Analysis of the 28 Human JmjC-Containing ProteinsThe JmjC domain for each human member was analyzed with DRAWGRAM (Biology WorkBench 3.2-DRAWGRAM; Felsenstein, 1989), and diagram-matic representations of their associated protein domains are depicted. Some of the JmjC-containing genes have multiple splice variants, but thevariant with the JmjC domain or the one with the most domains recorded in Genbank is shown. The corresponding Gene ID is indicated, and proteinschematics are shown on the right.

free radical on the substrate attacks the iron (III) hydroxide

and becomes hydroxylated, resulting in a carbinolamine

that spontaneously releases formaldehyde. The biochem-

ical analysis of these proteins has identified the cofactors

and reaction products described above (Tsukada et al.,

2006; Yamane et al., 2006; Whetstine et al., 2006; Cloos

et al., 2006; Klose et al., 2006; Chen et al., 2006b). Unlike

LSD1, the JmjC domain demethylases do not require pro-

tonated nitrogen in the substrate; thus, they are capable of

demethylating not only mono- and dimethylated but also

trimethylated lysine residues (compare reactions in Fig-

ures 2 and 4). In this context, it is interesting to note that

JHDM1 and JHDM2 both demethylate only mono- and

dimethylated lysine, although they are chemically compat-

ible for the reversal of trimethylated lysine.

Structure of a JmjC Domain that Catalyzes Histone

Demethylation

The N-terminal domain of JMJD2A including the JmjN and

JmjC domains represents the first structure of a histone

demethylase catalytic core (Chen et al., 2006b). The cata-

lytic domain of JMJD2A has the classical ‘‘jelly roll’’ struc-

ture composed of antiparallel b strands conserved among

the 2-oxoglutarate (2-OG)-Fe(II)-dependent dioxygenases

(Dunwell et al., 2000; Gane et al., 1998). The iron is

chelated by His188, Glu190, and His 276, which are con-

served among all active enzymes of this class (Figure 4;

Clissold and Ponting, 2001). The JmjN domain has been

found to form extensive interactions with the catalytic

JmjC core and provide structural integrity (Chen et al.,

2006b). Structural analysis also identified a previously

unappreciated zinc finger motif located at the end of the

conventionally defined JmjC domain and the sequence

C-terminal to the JmjC domain, which was essential for

enzymatic function (Chen et al., 2006b). These structural

features identify a new subfamily of dioxygenases repre-

sented by JMJD2A. Consistent with this idea, the JMJD2A

catalytic core structure differs significantly from the

only other known JmjC domain structure of the aspara-

ginyl hydroxylase FIH (factor inhibiting hypoxia-inducible

factor) (Lancaster et al., 2004). The crystal structure and


Molecular Cell

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Figure 4. Chemical Mechanism for Demethylation Mediated by the JmjC ProteinsThis mechanism is based on the biochemical and structure/function data generated for JHDM1 (Tsukada et al., 2006) and the JMJD2 family (Whets-tine et al., 2006; Chen et al., 2006a) as well as the proposed chemical reactions for the HIF hydroxylase (Dann and Bruick, 2005) and TauD (Price et al.,2005). The amino acids responsible for coordinating the Fe(II) (red circle) and a-ketoglutarate are depicted in the JMJD2A catalytic core crystal struc-ture taken from Chen et al. (2006a). A schematic of the interaction and coordination of the molecular oxygen, a-ketoglutarate, and substrate is indi-cated as reaction step 1. An electron is transferred from the Fe(II) to the coordinated molecular oxygen, yielding a superoxide radical and Fe(III). Theradical attacks the carbonyl group (C2) in the a-ketoglutarate, which accepts an electron from the iron. Decarboxylation ensues, and succinate andCO2 are produced (step 3). During the split of molecular oxygen, a highly unstable Fe(IV)-oxo intermediate is generated. This oxoferryl group extractsa proton from the methylated lysine, forming an Fe(III) hydroxide that subsequently hydroxylates the radical on the methyl group (step 4), which formsa carbonyl group that will spontaneously demethylate (step 5). The reaction is then able to continue in the presence of molecular oxygen, Fe(II), anda-ketoglutarate.

site-directed mutagenesis also provided insights into the

degree of demethylation by identifying a potential methyl

group binding pocket and critical amino acid residues

associated with methyl group binding specificity (Chen

et al., 2006b). However, insights into the molecular basis

for the lysine specificity of these demethylases still await

enzyme-substrate cocrystal structures.


Do Demethylases Provide a Fine TuningMechanism for Histone Methylation?The degree of lysine methylation on nucleosomes and

their relative locations throughout the genome are associ-

ated with potentially different functional outcomes. For

instance, evidence is accumulating that H3K4me3 at the

promoter of a gene, but not H3K4me2, is directly involved

Molecular Cell

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in active transcription (Santos-Rosa et al., 2002; Ng et al.,

2003; Boa et al., 2003; Bernstein et al., 2002). While

H3K9me3 is linked to pericentric heterochromatin,

H3K9me1 and H3K9me2 are associated with silent do-

mains within the euchromatic regions (Lehnertz et al.,

2003; Peters et al., 2003). In addition, the increase of

H3K9me3 within the body of a gene has been linked to

active gene expression. These examples highlight the

functional significance of differentially methylated lysines,

their genomic position, and their degree of methylation,

emphasizing the relationship between the net accumula-

tion of these states and the formation of functionally

distinct chromatin domains. Thus, an important question

is how these differentially methylated states are generated

and maintained.

We speculate that both histone methyltransferases and

demethylases play a role in balancing methylation dynam-

ics (Figure 5). We further speculate that demethylases

may provide a mechanism for fine tuning histone methyl-

ation levels. Consistent with this, the trimethyl demethy-

lases JMJD2A and -2C predominantly convert H3K9me3

to H3K9me2, while JMJD2D reduces H3K9me3 to

H3K9me1 (Whetstine et al., 2006), indicating that these

related demethylases help generate differential methyla-

tion states at the same lysine residue. This conclusion is

supported by the identification of the methyl group bind-

ing pocket in JMJD2A and the ability to alter JMJD2A

methyl group recognition property through subtle muta-

genesis (Chen et al., 2006b). These findings strongly

suggest that the methyl group binding pocket plays an

important role in providing the discriminatory power for

these demethylases to function selectively on differentially

methylated lysine substrates.

The molecular mechanism of how these different meth-

ylation states help generate potentially different functional

chromatin states is incompletely understood. The current

model is that differentially methylated lysines may serve as

platforms for different chromatin modifying enzymes and

therefore may have significant functional implications.

For instance, the monomethylated H3K9, as a result of

the action of the JMJD2D, may function as the substrate

for the Suv39h methyltransferase, which is believed to

use H3K9me1 as a substrate for methylation (Peters

et al., 2001). To make the issue even more complicated,

recent findings suggest that the same methylation state

can also result in opposite functional outcomes depen-

dent on how this modification is ‘‘read’’ and interpreted in

a specific chromatin context. For instance, H3K4me3/2

can be recognized by BPTF and ING2 via the PHD domain

(plant homeodomain) (Shi et al., 2006; Wysocka et al.,

2006). However, because BPTF is part of the NURF chro-

matin-remodeling complex, recognition of H3K4me2/3 by

BPTF leads to transcriptional activation (Wysocka et al.,

2006). In contrast, transcriptional repression occurs in

the same H3K4me2/3 when bound by ING2, which is

part of a deacetylase corepressor complex (Shi et al.,

2006). These findings highlight the importance of the pro-

teins that read and ‘‘interpret’’ the methylation signals (see

the review in this issue of Molecular Cell by Ruthenburg

et al. [2007]).

Histone Demethylases, Biology, and HumanDiseasesThe patterns and levels of histone methylation have been

linked to many important biological processes such as

stem cell maintenance and differentiation (Boyer et al.,

2006; Bernstein et al., 2006; Lee et al., 2006), X inactiva-

tion (Plath et al., 2003), and DNA damage response

(Sanders et al., 2004). Thus, histone demethylases, which

play the opposite enzymatic role to that of methyltrans-

ferases, are expected to be involved in many of the

same processes. Investigation of the biological roles of

these newly identified histone demethylases is a largely

untapped area, but molecular studies have begun to sug-

gest potential roles of these proteins. For example, LSD1

and JHDM2 may play an important role in facilitating

AR-mediated transcriptional regulation (Metzger et al.,

2005; Yamane et al., 2006). Consistently, a recent report

suggests that increased LSD1 expression in prostate tu-

mors correlates significantly with relapse during therapy

(Metzger et al., 2006; Kahl et al., 2006).

Recent findings have implicated alteration in hetero-

chromatin formation as a contributing factor in cancer

development (Fraga and Esteller, 2005). Specifically,

a significant reduction of H3K9me3 and H4K20me3 levels,

which are hallmarks of heterochromatin, has been corre-

lated with tumorigenesis (Fraga et al., 2005; Pogribny

et al., 2006). Therefore, aberrant regulation of the deme-

thylases controlling these methylation marks could un-

doubtedly contribute to oncogenic potential. Available

evidence is consistent with such an expectation. For

instance, the JMJD2C/GASC1 gene is amplified in squa-

mous cell carcinoma (Yang et al., 2000) and regulates

cell proliferation (Cloos et al., 2006). Therefore, JMJD2C

may represent a novel target for chemotherapeutic agents

in the treatment of cancer, although its general require-

ment for cell proliferation may present significant chal-

lenges. A recent genetic screen identified two JmjC do-

main proteins as tumor suppressors (Suzuki et al., 2006),

including Fbx10, which has reported to be a H3K36me2-

specific demethylase (Tsukada et al., 2006). In addition,

the C. elegans JMJD2 homolog has been shown to influ-

ence genomic stability and germline apoptosis. The loss

of ceJMJD2 causes p53-dependent apoptosis, which

likely results from increased DNA double-strand breaks

(DSBs) in the germline. This finding provides a potentially

exciting link between H3K9/K36 methylation status and

DSB formation and suggests that coordinated demethyla-

tion may be critical for genomic maintenance (Whetstine

et al., 2006).

Mutations or translocations of histone methyltrans-

ferases have been directly linked to prostate, breast,

and hematopoietic cancers, emphasizing the importance

of histone methylation balance in vivo. For instance, the

misregulation of MLL (H3K4 methylase), hDOT-1L

(H3K79 methylase), and NSD-1 (H3K36 methylase) has


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Figure 5. Site and Methyl Group Specificities of Histone Methyltransferases and DemethylasesThe histone methyltransferases for various lysine residues are indicated on the left, and the corresponding demethylases are listed on the right. Themammalian counterparts are indicated for most of the known enzymes. The degree of methylation/demethylation is indicated with the arrows. Thearrow was determined by analyzing the literature for in vitro activity and/or genetic data from different species. The histone methyltransferases witha gray arrow and a dashed line have not been extensively analyzed for the degree of methylation. The white arrow indicates a lower-affinity methylationsubstrate or demethylation reaction. Due to size limitations, the primary references were omitted, but information about most of the methyltransferaseshas been reviewed in Lachner et al. (2003) and Dillon et al. (2005).

been linked to human hematopoietic cancers (Okada

et al., 2005; Jaju et al., 2001), which emphasizes the need

to maintain methylation balance at H3K4, -36, and -79.


Lastly, histone demethylases may also be involved in

other human diseases such as neurological disorders.

For instance, SMCX and PHF8 have both been implicated

Molecular Cell

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in X-linked mental retardation (Laumonnier et al., 2005;

Tzschach et al., 2006; Santos et al., 2006), and recent

studies suggest that they are both histone demethylases

(S. Iwase, F. Lan, P. Bayliss, L. de la Torre-Ubieta, M.

Huarte, H. Qi, J.R.W., A. Bonn, T.M. Roberts, and Y.S.,

unpublished data).

Are There More Histone Demethylases?The answer is most definitely yes. Considering that his-

tone lysine methylation is mediated by �50 methyl-

transferases (Jenuwein, 2006), it is likely that comparable

numbers and types of histone lysine and arginine deme-

thylases exist (Figure 5). The anticipation is that all three

methylation states of each and every methylated lysine

residue as well as methylated arginines on histone are

reversible by enzymes. While H3K4 can be methylated

by at least eight different methyltransferases including

MLL (Milne et al., 2002; Nakamura et al., 2002) and

SMYD3 (Hamamoto et al., 2004), only one demethylase,

LSD1, has been found to act at this site thus far. Since

LSD1 only acts on mono- and dimethylated H3K4 (Shi

et al., 2004), it is reasonable to expect that additional

H3K4-specific demethylases, especially H3K4 trimethyl-

specific demethylases, will be discovered. Indeed, recent

studies identified a family of H3K4 trimethyl-specific

demethylases that may function in various important bio-

logical processes (S. Iwase, F. Lan, P. Bayliss, L. de la

Torre-Ubieta, M. Huarte, H. Qi, J.R.W., A. Bonn, T.M. Rob-

erts, and Y.S., unpublished data).

What might these yet-unidentified demethylases be?

They may be members of the amine oxidase and JmjC

domain-containing protein families or other members of

the Fe(II) dioxygenase superfamily. Many members of

the non-JmjC domain-containing cupin family of proteins

are dioxygenases that use metallocenters like Fe(II) and

therefore are compatible for histone demethylation. New

demethylases may also use alternative enzymatic do-

mains/chemical reaction mechanisms as opposed to the

LSD1 and JmjC proteins. We predict these mechanisms

are likely to also involve oxidative reactions and a similar

release of formaldehyde as a result of demethylation.

One such group is the family of the Radical SAM proteins,

which consist of well over 600 proteins from prokaryotes

to eukaryotes (Sofia et al., 2001). This class of enzyme has

a conserved SAM binding domain and a cysteine-rich

region that binds an iron-sulfur cluster (Figure 6A; Frey,

2001; Sofia et al., 2001). Currently, not much is known

about this family of proteins, which is largely due to their

highly unstable nature in an oxygenated environment.

The idea of SAM radical proteins being histone deme-

thylase candidates was first put forward for Elp3, which

is a histone acetyltransferase (Chinenov, 2002). Recently,

the M. janaschii Elp3 has been shown to have a conserved

SAM binding site and a 4Fe-4S cluster and to generate

adenosine in the presence of SAM, which likely results

from generation of the 50-deoxyadenosine radical in

the presence of water (Paraskevopoulou et al., 2006).

Although Elp3 has not been shown to be a histone

demethylase, this enzyme and the many other uncharac-

terized Radical SAM proteins could use the proposed

chemistry to demethylate histones.

Specifically, a SAM radical enzyme could initiate deme-

thylation reactions by catalyzing the cleavage of SAM into

methionine and 50-deoxyadenosyl radical (Figure 6A). The

electron required for this reaction can be obtained from

the reduced form of a 4Fe-4S cluster within the enzyme

(Figure 6A). Two potential mechanisms could result in

the demethylation of either the lysine or arginine residues.

The first possibility would allow the 50-deoxyadenosyl

radical to abstract a hydrogen atom from one of the methyl

groups, resulting in 50-deoxyadenosine and a carbon-

centered radical that is stabilized by the one pair of elec-

trons on the nitrogen atom. The imine structure is formed

and the electron is either transferred to the iron-sulfur

center or another protein. A water molecule is added and

results in the carbinolamine, which releases formaldehyde

and an unmethylated lysine upon hydrolysis. This mecha-

nism would limit demethylation to mono- and dimethylated

lysine (Figure 6B). However, the second possibility would

allow the radical intermediate to abstract a hydrogen

atom from the substrate and add a hydroxyl moiety by

using water after electron transfer, generating a carbonyl

group that would undergo spontaneous demethylation

(Figure 6C). This mechanism would include trimethylated

residues as substrates; however, the initial radical and

subsequent cation may not be as stable. For this reason,

this enzyme class is more likely to be mono- or dimethyl

demethylases. However, it is also important to note that

Radical SAM proteins are suspected to be methyltrans-

ferases (Pierrel et al., 2004). Thus, proteins like Elp3 may

also be a unique class of histone methyltransferases.

Two additional classes of proteins, which are similar to

the Fe(II) dioxygenases, are also potential demethylases.

These include enzymes with two iron centers (e.g., ribonu-

cleotide reductases or methane monooxygenase like

proteins) that are able to hydroxylate a carbon backbone

through the use of oxygen and iron transition states. The

other class is the NADH/NADPH oxidoreductases, which

could potentially use a mechanism similar to that of

LSD1 to demethylate histones by cycling the NAD+/NADH

cofactor. Lastly, the dioxygenases (i.e., JmjC), the SAM

radical proteins and enzymes that contain two iron centers

discussed above, are also chemically compatible for

removal of methyl groups from DNA and RNA. It will be

interesting to determine whether any of these proteins

also participate in DNA and/or RNA demethylation.

Concluding RemarksThe field of demethylases has been moving forward at

a very fast pace. Since the 2004 discovery of the first

histone demethylase LSD1, a total of seven histone deme-

thylases have been documented in less than 2 years. The

identification of these enzymes provides new opportuni-

ties and challenges to understand histone methylation

and the complex nature of chromatin regulation. Given

the diverse roles histone methylation has been shown to


Molecular Cell

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Figure 6. Chemical Mechanism for Putative Demethylation Reaction Mediated by the Radical SAM Family of Enzymes(A) The 4Fe-4S center is depicted with SAM from the Radical SAM enzymes. When the substrate binds, the SAM and iron-sulfur center come in closeproximity and an electron is transferred from iron to SAM. The S-C-50 bond is then homolyitcally cleaved, forming the 50-deoxyadenosyl radical andmethionine.(B) A candidate mechanism for Radical SAM demethylation of di- or monomethylated lysines. The 50-deoxyadenosyl radical abstracts a hydrogenfrom the methyl residue, resulting in adenosine and a free radical on the methyl group. The carbon-centered radical is stabilized by one pair ofelectrons on the nitrogen atom. The imine structure forms, and the electron is transferred either to the iron-sulfur center or to another protein. A watermolecule is added and gives the carbinolamine, which releases formaldehyde and an unmethylated lysine upon hydrolysis.(C) A candidate mechanism for tridemethylation. The 50-deoxyadenosyl radical intermediate abstracts a hydrogen atom from the substrate and addsa hydroxyl moiety by using water after electron transfer. This generates a carbonyl group that would undergo spontaneous demethylation.


Molecular Cell

Review

play, histone demethylases are likely to also impact many

biological processes, including stem cell maintenance

and differentiation, genome integrity, X chromosome inac-

tivation and imprinting, cell cycle regulation, tissue devel-

opment, and differentiation, among others. The answers

lie in the analysis of these demethylases in multiple model

organisms such as yeast, C. elegans, Drosophila, zebra-

fish, and mice. In addition, the identification of genomic lo-

cations of these demethylases by chromatin immunopre-

cipitation will provide not only hints about their biological

roles but also tools to study the molecular mechanisms

that demethylases use to regulate chromatin structure

and gene transcription in vivo. It is likely that histones

are not the only substrates. The identification of nonhis-

tone substrates and investigation of their functional sig-

nificance will, therefore, further enhance our understanding

of the demethylases. Finally, several histone methylation

marks such as H3K27me3 and H4K20me1 have been im-

plicated in epigenetic regulation (Trojer and Reinberg,

2006). With the anticipation that demethylases exist for

most if not all methylated lysines on histone, we speculate

that expression and activities of demethylases are likely to

be highly regulated in order to ensure epigenetic integrity,

which is an exciting area of future investigations.

In sum, the discovery of the histone demethylases has

changed our view of histone methylation regulation and

has provided exciting new opportunities to understand

dynamic regulation of histone modifications. Future inves-

tigations of these enzymes will provide significant insight

into fundamental mechanisms that regulate chromatin

structure and transcription, which is expected to impact

normal biological processes and human diseases.

ACKNOWLEDGMENTS

We thank Grace Gill for helpful comments on the manuscript andSquire Booker for the advice on the various chemical mechanisms ofhistone demethylation. We apologize to those whose work was notcited due to space limitation. The work on histone demethylases inthe Shi lab is supported by grants from the National Institutes of Health(GM058012, GM071004, and NCI118487) as well as in part by a pilotgrant from Stewart Trust and from the Novartis Biomedical ResearchInstitute. J.R.W. was supported by the Ruth L. Kirschstein NationalService Award (GM 70095).

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Dynamic Regulation of Histone Lysine Methylation by Demethylases