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-N-methyl-lysine was first found in a bacterial flagel-lar protein in 1959 (REF. 1)and, 5 years later, this post-translational modification (PTM) was also identified inhistone proteins2. Biochemically, S-adenosyl-L-methionine(AdoMet) is the principal methyl donor in the methyl-transferase reaction and is the second most widely usedenzyme substrate following ATP3. This frequency indi-cates the importance of the methylation reaction in
various biochemical or metabolic pathways. Althoughthe physiological importance of protein lysine methyla-tion was unknown for many years, several protein lysinemethyltransferases (PKMTs) have now been identified,and their physiological significance, particularly inthe field of epigenetics, has begun to be elucidated48.The SET-domain proteins constitute a major group of
AdoMet-dependent PKMTs; nearly 50 human proteinsare categorized into this family, although not all of themhave known PKMT activity. In addition to the SET-domain proteins, several non-SET-domain proteins,including DOT1-like histone H3K79 methyltransferase(DOT1L), are also reported to have PKMT activity9.
Moreover, protein lysine methylation was thought tobe irreversible because the half-life of the histone meth-ylation was approximately equivalent to the half-life ofhistones themselves10. However, the first protein lysinedemethylase lysine-specific demethylase 1 (LSD1; alsoknown as KDM1A) was discovered in 2004 (REF. 11), andsubsequently the Jumonji C (JmjC)-domain-containing
family was reported to have protein demethylase activ-ity12. These findings indicate that protein lysine methyla-tion seems to be dynamically regulated.
Many reports have indicated that dysregulation ofPKMTs and protein lysine demethylases (PKDMs)has substantial roles in tumorigenesis4,13,14, and theseenzymes are considered to be important targets forthe development of anticancer therapy15,16. Functionalanalyses of PKMTs and PKDMs have been carried outprimarily in the context of their role in epigenetic regu-lation. For instance, the PKMT EZH2, which methyl-ates histone H3 at lysine 27 (H3K27), is a componentof Polycomb-repressive complexes and is overexpressedin various types of cancer17. Polycomb-repressive com-plexes play a fundamental part in controlling the expres-
sion of downstream genes, including cyclin-dependentkinase inhibitor 2A (CDKN2A), to promote cell cycleprogression in physiological and pathological condi-tions18. Similarly, several reports have demonstratedthat aberrations of other PKMTs and PKDMs promotemalignant transformation via histone methylation-dependent transcriptional regulation4,13,1921.
Beyond histones, the biological and physiologicalsignificance of non-histone lysine methylation in humantumorigenesis has recently begun to be explored2227.Accumulating evidence indicates that, similar to otherPTMs such as phosphorylation and acetylation, lysinemethylation may be important not only in epigenetic
Section of Hematology/
Oncology, Department of
Medicine, The University of
Chicago, 5841 S. Maryland
Avenue, MC 2115 Chicago,
Illinois 60637, USA.
Correspondence to R.H.
e-mail: rhamamoto@
medicine.bsd.uchicago.edu
doi:10.1038/nrc3884
S-adenosyl-L-methionine
(AdoMet).A moleculesynthesized from methionine
and ATP by methionine adeno-
syltransferase. The methylation
group attached to the
methionine sulphur atom in
AdoMet is chemically reactive.
This allows donation of this
group to an acceptor substrate
in transmethylation reactions.
Critical roles of non-histoneprotein lysine methylation inhuman tumorigenesisRyuji Hamamoto, Vassiliki Saloura and Yusuke Nakamura
Abstract | Several protein lysine methyltransferases and demethylases have been identified
to have critical roles in histone modification. A large body of evidence has indicated that
their dysregulation is involved in the development and progression of various diseases,including cancer, and these enzymes are now considered to be potential therapeutic targets.
Although most studies have focused on histone methylation, many reports have revealed
that these enzymes also regulate the methylation dynamics of non-histone proteins such as
p53, RB1 and STAT3 (signal transducer and activator of transcription 3), which have
important roles in human tumorigenesis. In this Review, we summarize the molecular
functions of protein lysine methylation and its involvement in human cancer, with a particular
focus on lysine methylation of non-histone proteins.
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Competitiveinhibition
Indirect effect
on othermodifications
Cytoplasm
Methylated-lysine-specificbinding proteins
Inhibition of
proteinproteininteractions
Inhibition ofpolyubiquitylation
Protein degradationby proteasomes
Transcriptionfactors
Methylationactivatestranscription
a Other protein modifications b Proteinprotein interactions c Protein stability
d e Promoter binding
K Me KMeMe
KMeMeUb
Ub UbUb Ub Ub
Ac
K SMe P
Chromodomain
KMeMe
UbUbUb
Ub Ub
HSP70
HSP70
HSP70
Subcellularlocalization
Me Me Me
AURKB
Activate
Nucleus
Promoter
Oxygenase
An enzyme of the
oxidoreductase class that
catalyses the incorporation of
both atoms of molecular
oxygen into the substrate.
energies and hydrogen bonding potential of the lysineside chains. Indeed, there are methyl lysine-binding pro-teins that have a special motif such as a chromodomainfor recognizing methylated lysine residues, called thearomatic cage. This aromatic cage comprises a collec-tion of aromatic protein residues, often accompanied byone or more neighbouring anionic residues34. The com-bination of favourable cation-, electrostatic and vander Waals interactions, as well as shape complementa-rity, provides methyl lysine-binding proteins with a highdegree of specificity for methylated lysine35. By contrast,lysine methylation of MAPK kinase kinase 2 (MAP3K2)inhibits its interaction with the serine/threonine proteinphosphatase 2A (PP2A) complex, which is a key negativeregulator of the MAPK pathway, implying that methyllysine can also block proteinprotein interactions33.
Protein stability.As lysine polyubiquitylation plays akey part in protein degradation through the ubiquitinproteasome pathway, lysine methylation may increasethe stability of proteins by preventing polyubiquityla-tion.Indeed, in Saccharomyces cerevisiae, lysine methyl-ated proteins show a significantly longer half-life thannon-methylated proteins. Moreover, 43% of methylatedlysine sites are predicted to be amenable to ubiquityla-tion, suggesting that methylated lysine residues mightblock the action of ubiquitin ligases36(FIG. 2c). However,the DCAF1DDB1CUL4 E3 ubiquitin ligase complexrecognizes monomethylated lysine and promotes poly-ubiquitylation of the other lysine residues on substrates
such as retinoic acid-related orphan nuclear receptor-(ROR)37, implying that lysine methylation may alsodestabilize target proteins through regulation of distantpolyubiquitylation.
Subcellular localization.Typically, a nuclear localiza-tion signal consists of one or more short sequences ofpositively charged lysine or arginine residues exposedon the protein surface38. As both lysine and arginineresidues are critical for the nuclear localization of pro-teins, one could speculate that methylation of lysine orarginine may affect subcellular localization. Indeed,some lysine methylated proteins such as heat shock pro-tein 70 (HSP70) and p53 are predominantly localizedin the nucleus, whereas unmodified versions of thesesame proteins are localized in both the cytoplasm andthe nucleus23,39(FIG. 2d)
Promoter binding.Lysine methylation also regulatesthe binding affinity of transcription factors for pro-moters, which changes the transcription levels of tar-get genes40(FIG. 2e). For example, lysine methylation ofp53 by the PKMT SETD7 and that of nuclear factor-B(NF-B) by the PKMT nuclear receptor-bindingSET domain-containing protein 1 (NSD1) markedlyincreased their promoter binding ability and activationof downstream genes39,41. In a structural modellinganalysis of RELA (a subunit of NF-B) in complex withDNA, two methylated lysine residues on RELA wereshown to interact with DNA through hydrophobic
Figure 2 | Molecular functions of lysine methylation in human tumorigenesis. The biological importance of lysine
methylation is primarily categorized into five groups. a | Lysine methylation affects other protein modifications directly
(through competitive inhibition) or indirectly.b | Lysine methylation regulates proteinprotein interactions. Methylated-
lysine-specific binding proteins have been reported, and these proteins contain motifs that specifically recognizemethylated lysine residues such as chromodomains and Tudor domains. Methylated lysine can also negatively regulate
some proteinprotein interactions. c | Lysine methylation competitively inhibits the polyubiquitylation of lysine residues
and stabilizes the protein. Additionally, lysine methylation also promotes the polyubiquitylation of other lysine residues on
the same protein, leading to protein destabilization.d | Subcellular localization is regulated by lysine methylation. For
example, methylated heat shock protein 70 (HSP70) proteins are predominantly localized in the nucleus, whereas
unmodified versions are predominantly localized in the cytoplasm. e | Lysine methylation regulates promoter binding
affinity of transcription factors, thereby changing transcription levels of target genes. Ac, acetyl group; AURKB, Aurora
kinase B; P, phosphate group; Me, methyl group; Ub, ubiquitin.
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Tudor domains
Protein domains originally
identified as a region of 50
amino acids found in the
Drosophila melanogaster
Tudor protein. The structurally
characterized Tudor domain in
human proteins recognizes
symmetrically dimethylated
arginine. This domain is also
reported as a methyl
lysine-binding protein module.
contacts42. This result reveals that increased hydropho-bicity of lysine residues by methylation can enhancethe promoter binding ability of transcription factors.
Non-histone PKMT and PKDM substrates
So far, nearly 20 non-histone substrates have been dis-covered for PKMTs and PKDMs (TABLE 1). Many PKMTsand PKDMs are localized in the nucleus, and severalnuclear proteins, including transcription factors, canserve as substrates of PKMTs and PKDMs. However,several PKMTs or PKDMs are also localized in the cyto-plasm, and cytoplasmic proteins are also reported tobe substrates of these enzymes. Below, we highlight thelysine methylation of non-histone proteins involved inseveral tumour-associated signalling pathways.
p53. p53 is one of the most important tumour sup-pressor genes involved in human tumorigenesis43.Chuikov et al.39demonstrated that SETD7 methyl-ates lysine 372 of p53 (p53K372) and that this meth-ylation enhances p53 stability and transcriptional
activity. K372-methylated p53 is restricted to thenucleus, although p53 is equally distributed betweenthe nuclear and cytosolic f ractions39. Subsequently,Huang et al.44 showed that the PKMT SET andMYND-domain containing 2 (SMYD2) monometh-ylates lysine 370 of p53 (p53K370); this lysine residueis located in the regulatory domain at the extremecarboxyl terminus of p53 (FIG. 3a). Knockdownof SMYD2 by small interfering RNA enhancesp53-mediated apoptosis in cancer cells44. In addition,SMYD2-dependent p53K370 methylation impairsthe expression of CDKN1A, an important down-stream target of p53, implying that SMYD2 repressesthe function of p53 through K370 monomethylation.Moreover, p53K372 methylation by SETD7 seemsto inhibit SMYD2-depedent p53K370 methylationthrough blocking the interaction between SMYD2and p53 (REFS 39,44)(FIG. 3a).
The function of monomethylation and dimethylationof p53 on lysine 370 seems to be different. For example,LSD1 was reported to demethylate p53K370 and repressp53 activity45(FIG. 3a). In this case, LSD1 preferentiallyreverses dimethylated p53K370 in cancer cells, althoughit can remove both monomethylation and dimethylationat lysine 370 in biochemical reactions in vitro45. p53K370dimethylation promotes the association of p53 with theco-activator p53-binding protein 1 (p53BP1) through
tandem Tudor domainsin p53BP1 (REF. 45), leading toincreased p53 function, including apoptosis induc-tion. These results indicate that monomethylation ofp53K370 is crucial for the repression of p53 activity,whereas dimethylation of p53K370 seems to activatep53. The complexity of methylation pathways in humancancer is highlighted by the fact that both SMYD2 andLSD1 affect the same lysine residue of p53, and thatboth of these enzymes can promote oncogenesis and areoverexpressed in various types of cancer22,46. It is plau-sible that their oncogenic actions on p53 may be syn-ergistic, although they could also have effects on othercancer-related pathways.
Furthermore, Shi et al.47reported that the proteinlysine methyltransferase SETD8 monomethylates p53at lysine 382 and that this methylation suppressesp53-dependent transcription activation in cancercells. Taken together, these results indicate that lysinemethylation is an important regulator of p53 function.
RB1 and E2F.RB1 is a key cell cycle regulator and tumoursuppressor, and is dysfunctional in several cancer types48.RB1 was identified as a binding partner for mitogeniconcoproteins49and was also discovered to be phosphoryl-ated in synchrony with the cell cycle50. Recently, we andothers reported that lysine methylation of RB1 seems tobe one of the substantial regulators of RB1 function and ispivotal for cell cycle regulation22,51,52. Methylation at lysine810 of RB1 by SMYD2 is likely to enhance phosphory-lation of RB1 at serines 807 and 811 (REF. 22). Additionally,although RB1 normally interacts with E2F (a family oftranscription factors that modulate important cellularevents, including cell cycle progression, apoptosis andDNA damage response53) to suppress transcription of
E2F target genes, lysine 810 methylation of RB1 acceler-ates E2F transcriptional activity through enhancement ofRB1 phosphorylation and promotes cell cycle progression(FIG. 3b). Furthermore, we identified that lysine 442 of pro-tein phosphatase 1, regulatory subunit 12A (PPP1R12A;also called MYPT1) in the myosin phosphatase holo-enzyme, which stimulates dephosphorylation of RB1, isa target for methylation and demethylation catalysed bySETD7 and LSD1, respectively24
(FIG. 3b). Demethylationof PPP1R12A by LSD1 enhances PPP1R12A polyubiq-uitylation, which increases the proteasome-mediateddegradation of PPP1R12A and therefore the amount ofphosphorylated RB1. Subsequently, released E2F acti-
vates transcription genes required for S phase, and cellcycle progression is enhanced24. Together, methylationand demethylation dynamics seems to play an impor-tant part in cell cycle progression through the regulationof RB1 activity.
Deregulated expression or activity of membersof the E2F family has also been detected in manyhuman cancers54. Kontaki et al.55demonstrated that inp53-deficient cancer cells, methylation of lysine 185 ofE2F1 (E2F1K185) by SETD7 inhibits acetylation andphosphorylation at distant positions and, in parallel,induces polyubiquitylation and degradation of E2F1.This process prevents E2F1 accumulation duringDNA damage and activation of its pro-apoptotic tar-
get gene TP73(FIG. 3b). Moreover, LSD1 demethylatesE2F1K185, which is important to maintain a substan-tial pool of unmethylated E2F1 in cancer cells, whichcan then be subjected to lysine acetyltransferase 2B(KAT2B)-mediated hyperacetylation, as well as check-point kinase 2 (CHEK2)-mediated phosphorylation,following DNA damage55(FIG. 3b). These results implythat in p53-deficient cancer cells, LSD1 and SETD7can influence DNA damage-induced cell death in amanner that is in contrast to the aforementionedp53-dependent apoptosis induction regulated byLSD1 and SETD7. These results suggest that antican-cer treatments that combine DNA-damaging agents
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Table 1 | Protein lysine methyltransferases and demethylases involved in human tumorigenesis and their substrates
Family Enzymename (alias)
Domain Subcellularlocalization
Substrate Cancer type Refs
Histone Non-histone
Lysine methyltransferases
Polycombcomplex
EZH2 (KMT6) SET, CXC andSANT
Nucleus*and
cytoplasm
H3K27 andH2BK120
RORand STAT3 AML, bladder cancer, breastcancer, CCC, CML, CRC,
glioblastoma, lymphoma,NSCLC, oesophageal cancer,osteosarcoma, SCLC and RCC
17,31,37,74
SMYDfamily
SMYD2(KMT3C)
SET Cytoplasm*and nucleus
H3K4 andH3K36
p53, RB1, PARP1,HSP90AB1 and ER
Bladder cancer, breastcancer, cervical cancer, CRC,HCC, head and neck cancer,lymphoma, oesophagealcancer, ovarian cancer,pancreatic cancer, prostatecancer, seminoma and skincancer
22,25,27,44,51,52,69,111113
SMYD3(KMT3E)
SET Cytoplasm*and nucleus
H3K4 andH4K5
VEGFR1 andMAP3K2
ACC, breast cancer, CCC,cervical cancer, CRC,HCC, lung cancer, MTC,oesophageal cancer,pancreatic and prostatecancer
4,13,26,33,114121
NSD family NSD1(KMT3B)
SET, PWWP,AWS, PHD,RING andPostSET
Nucleus*andchromosome
H3K36 NF-B AML, glioblastoma, lungcancer and multiple myeloma
122124
WHSC1(MMSET andNSD2)
SET, PWWP,AWS, PHD,RING andPostSET
Nucleus*,cytoplasmandchromosome
H3K36 Bladder cancer, breastcancer, CCC, CML, HCC,multiple myeloma, NSCLC,oesophageal cancerosteosarcoma, prostatecancer, RCC and SCLC
125129
WHSC1L1(NSD3)
SET, PWWP,AWS, PHD,RING and
PostSET
Nucleus*andchromosome
H3K36 Bladder cancer, breast cancer,lymphoma and SCLC
130132
SETDfamily
SETD1A(KMT2F)
SET, N-SET andPRM
Nucleus*andchromosome
H3K4 HSP70 Bladder cancer, CRC, HCC,lung cancer and RCC
23,133
SETD7(KMT7)
SET and MORN Nucleus*andchromosome
H3K4 PPP1R12A, p53,NF-B, E2F1, DNMT1and STAT3
Breast cancer and multiplemyeloma
24,39,55,66,73,134,135
SETD8(KMT5A)
SET Nucleus*andchromosome
H4K20 PCNA and p53 Bladder cancer, CML, HCC,NSCLC and SCLC
47,75
SUV39family
SUV39H2(KMT1B)
SET, PreSET,PostSET andchromodomain
Nucleus*andchromosome
H3K9 andH2AXK134
Bladder cancer, cervicalcancer, NSCLC, oesophagealcancer, osteosarcoma,prostate cancer and STT
32,136
EHMTfamily
EHMT2(KMT1C)
SET, PreSET,PostSET andANK
Nucleus*andchromosome
H3K9 C/EBP AML, bladder cancer, breastcancer, CCC, CML, NSCLC,oesophageal cancer, prostatecancer and SCLC
19,71,137,138
MLL family MLL2(KMT2D)
SET, PHD, RING,FYRN, FYRC andHMG
Nucleus* H3K4 Bladder cancer, breast cancer,CRC, lung cancer, melanomaand MLL
139
MLL3(KMT2C)
SET, PHD, RING,FYRN, FYRC,HMG, AT_hook,C1 and PostSET
Nucleus* H3K4 Breast cancer, glioblastoma,melanoma, MLL, oesophagealcancer, pancreatic cancer andstomach cancer
140142
DOT1Lfamily
DOT1L(KMT4)
AT_hook Nucleus* H3K79 MLL 143145
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Family Enzymename (alias)
Domain Subcellularlocalization
Substrate Cancer type Refs
Histone Non-histone
Lysine demethylases
LSD1family
LSD1(KDM1A)
FADbinding 2and SWIRM
Nucleus* H3K4 andH3K9
PPP1R12A, p53, ERand STAT3
Bladder cancer, CRC andSCLC
11,24,45,46
JMJDfamily
JMJD1A(KDM3A)
JmjC Nucleus*andcytoplasm
H3K9 Bladder cancer, CCC, HCC,NSCLC, osteosarcoma, SCLCand RCC
20,146,147
JMJD2A(KDM4A)
JmjC, JmjN, PHDand Tudor
Nucleus* H3K9 andH3K36
Bladder cancer, breast cancer,CML, NSCLC, oesophagealcancer, osteosarcoma, ovariancancer, prostate cancer, SCLC,stomach cancer and uterinecancer
148151
JMJD2B(KDM4B)
JmjC, JmjN, PHDand Tudor
Nucleus* H3K9 Bladder cancer, CRC, NSCLC,oesophageal cancer, SCLC,stomach cancer and RCC
21,152154
JMJD3(KDM6B)
JmjC Nucleus* H3K27 Glioblastoma 155
UTX UTX(KDM6A)
JmjC and TPR Nucleus* H3K27 Bladder cancer, breast cancer,CML, CRC, glioblastoma,multiple myeloma, NSCLC,oesophageal cancer,pancreatic cancer, RCC andSCLC and TALL
156159
JARIDfamily
JARID1B(KDM5B)
JmjC, JmjN,ARID, PHD,ZFC5HC2 andPLU-1
Nucleus* H3K4 AML, bladder cancer, breastcancer, cervix cancer, CML,CRC, melanoma, NSCLC,oesophageal cancer, SCLCand RCC
160164
ACC, adenocarcinoma; AML, acute myeloid leukaemia; CCC, cholangiocarcinoma; C/EBP, CCAAT/enhancer binding protein; CML, chronic myelogenousleukaemia; CRC, colorectal cancer; DNMT1, DNA (cytosine5)methyltransferase 1; DOT1L, DOT1like histone H3K79 methyltransferase; EHMT, euchromatichistone-lysine N-methyltransferase; ER, oestrogen receptor-; H3K27, histone H3 lysine 27; HCC, hepatocellular carcinoma; HSP, heat shock protein; JARID,
Jumonji, ATrich interactive domain; JMJD, Jumonji domaincontaining; KMT, lysineN-methyltransferase; LSD1, lysine-specific demethylase 1; MAP3K2, MAPKkinase kinase 2; MLL, mixedlineage leukaemia; MTC, medullary thyroid cancer; NFB, nuclear factor-B; NSCLC, nonsmall cell lung carcinoma; NSD, nuclearreceptorbinding SET domaincontaining; PARP1, poly(ADPribose) polymerase 1; PCNA, proliferating cell nuclear antigen; PPP1R12A, protein phosphatase 1,regulatory subunit 12A; RCC, renal cell carcinoma; ROR,retinoic acid-related orphan nuclear receptor-; SCLC, small cell lung carcinoma; SETD, SETdomain-containing; SMYD, SET and MYND-domain-containing; STAT3, signal transducer and activator of transcription 3; STT, soft tissue tumour; SUV39H,suppressor of variegation 39 homologue; T-ALL, T-cell acute lymphoblastic leukaemia; VEGFR1, vascular endothelial growth factor receptor 1.*The predominantsubcellular localization.
Table 1 (cont.) | Protein lysine methyltransferases and demethylases that are involved in human tumorigenesis and their substrates
and drugs modulating LSD1 or SETD7 activity mayhave inverse effects depending on the p53 status incancer cells.
Heat shock proteins.HSPs are overexpressed in a widerange of human cancers and have substantial roles intumour cell proliferation, differentiation, invasion,metastasis and recognition by the immune system56.
Increased expression of HSPs can also protect malignantcells from activation of pro-apoptotic signalling, and thismay underlie the role of HSPs in tumour progressionand resistance to treatment57. HSP70 and HSP90AB1undergo several PTMs, and we previously identified thatthey are also lysine methylated23,25.
HSP70 is a ubiquitous molecular chaperone thatfunctions in a myriad of biological processes, includingmodulation of polypeptide folding, protein degrada-tion, subcellular translocation of proteins across mem-branes and proteinprotein interactions58. We reportedthat dimethylation of HSP70 lysine 561 was markedlyincreased in cancer cells, and methylated HSP70 was
predominantly localized in the nucleus. This is in contrastto non-methylated HSP70, which is localized predomi-nantly in the cytoplasm23. The methylation of HSP70 iscatalysed by the PKMT SETD1A, which is overexpressedin various types of cancer23. The lysine 561 methylatedHSP70 preferentially binds to and activates Aurorakinase B in the nucleus, resulting in cell cycle progressionin cancer cells23(FIG. 2d). The importance of this modifica-
tion in cancer was confirmed by immunohistochemicalanalysis using an HSP70K571 dimethylation-specificantibody, which revealed positive nuclear staining in 354of 409 (86.6%) non-small cell lung carcinoma cases. Inaddition to lung cancer, this positive staining of lysine561 methylated HSP70 was commonly found in bladderand kidney cancer tissues, whereas no such staining wasobserved in non-neoplastic tissues.
HSP90 is an evolutionarily conserved molecularchaperone that participates in the stabilization and acti-
vation of more than 200 proteins, which are referred to asHSP90 clients. Many of the client proteins of HSP90 areessential for various cell signalling pathways, including
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a
b
c
Me Me
TA1 and TA2 PRR DBDL OD CTDp53
370 KSKKGQSTSRHKK 382
SMYD2
SMYD2
K370me1K370me2 toK370me1
SETD7
SETD7
SETD8
K372me1
p53 inactivation Inhibition of K370me1
K382me1
p53 inactivation
RB1
CDK4
High affinity
E2F1
K185
SMYD2-dependent RB1methylation activates E2F
Cell cycle progression
Regulation of E2F1-dependentapoptosis in p53-deficient cancer cells
p53
53BP1
Me
Me
Me
Me
Me
Me
Me
MeMe
p53
53BP1LSD1
LSD1
LSD1
SETD7 LSD1
VEGFR1
MAP3K2
TM
SMYD3
K260
PP2A
Inhibition ofPP2A interaction Activation of oncogenic
RAS signalling
IG-LD
PPP1R12A
PPP1R12AdephosphorylatesRB1
K442
CC
CTD
K810
Enhances RB1hyperphosphorylation
K831me2
K260me2or K260me3
ANK
Stabilizes PPP1R12A protein
Pocket A Pocket B
DBD CC MB TD
TK1 TK2
K831Activation ofkinase activity
PB1 Kinase domain
Figure 3 | Effects of lysine methylation on the pathways of p53, RB1 and protein kinases. a | Methylation of lysine
at residues 370, 372 and 382 in the carboxyterminal domain (CTD) of p53 has been identified. Lysine 370dimethylated p53
(p53K370me2) can promote the association of p53 with the coactivator p53binding protein 1 (53BP1) through tandem
Tudor domains in 53BP1. By contrast, lysine-specific demethylase 1 (LSD1) prevents the accumulation of p53K370me2 by
demethylating this site, thereby preventing the binding of 53BP1 to p53. Both LSD1 and the protein lysine
methyltransferase (PKMT) SET and MYND-domain containing 2 (SMYD2) produce p53K370me1 that results in the
inactivation of p53. Meanwhile, the PKMT SETD7 monomethylates K372 (K372me1), which inhibits K370me1 and activatesp53. SETD8 also inactivates p53 through monomethylation of K382 (K382me1). b | The RB1 pathway is regulated by lysine
methylation. Lysine methylation sites on RB1, E2F1 and protein phosphatase 1, regulatory subunit 12A (PPP1R12A) are
displayed. SETD7 monomethylates K442, which stabilizes PPP1R12A, and this methylation is reversibly demethylated by
LSD1. SMYD2 monomethylates K810 on RB1 and increases the affinity between cyclindependent kinase 4 (CDK4) and RB1,
which enhances hyperphosphorylation of RB1 and therefore activates E2F. SETD7 monomethylates K185 of E2F1
(E2F1K185me1), which prevents apoptotic functions of E2F1 in p53-deficient cells during DNA damage, and E2F1K185me1
is demethylated by LSD1, promoting E2F1 stability and apoptosis.c | K831 of vascular endothelial growth factor receptor 1
(VEGFR1) and K260 of MAPK kinase kinase 2 (MAP3K2) are methylated by SMYD3. VEGFR1K831 dimethylation by SMYD3
activates the kinase activity of VEGFR1. K260 of MAP3K2 is dimethylated and trimethylated by SMYD3, which activates
oncogenic RAS signalling through inhibition of the MAP3K2PP2A (protein phosphatase 2A) interaction. ANK, ankyrin
repeat; CC, coiledcoil domain; DBD, DNAbinding domain; IGLD, immunoglobulinlike domain; MB, markedbox;
OD, oligomerization domain; PB1, Phox and Bem1p; PRR, prolinerich region; TA, transcriptionactivation domain;
TD, transactivation domain; TK, tyrosine kinase domain; TM, transmembrane domain.
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Nitrosylation
Nitrosylation, specifically
S-nitrosylation, involves the
covalent incorporation of a
nitric oxide moiety into thiol
groups, to form S-nitrosothiol.
S-nitrosylation is a
physiologically important
post-translational modification
that affects a variety of
proteins involved in a number
of cellular processes.
adaptive responses to various stresses57,59. HSP90 andproteins called co-chaperones form the dynamic com-plex known as the HSP90 chaperone machinery60.Cancer cells use the HSP90 chaperone machinery to pro-tect an array of mutated and overexpressed oncoproteinsfrom misfolding and degradation. Therefore, HSP90 isrecognized as a critical facilitator of oncogene addictionand cancer cell survival61. HSP90 function is regulated by
various PTMs such as acetylation, phosphorylation andnitrosylation. We also reported methylation of lysines 531and 574 of HSP90AB1 by SMYD2. The lysine methyla-tion of HSP90AB1 is important for its homodimerizationand its interaction with stress-induced phosphoprotein 1(STIP1) and cell division cycle 37 (CDC37), which areco-chaperones of HSP90AB1 in human cancer cells,resulting in enhancement of cancer cell growth25.
Protein kinases (VEGFR1 and MAP3K2). Vascularendothelial growth factor receptor 1 (VEGFR1), a recep-tor tyrosine kinase, mediates signalling that is involvedin cell proliferation and angiogenesis62. We found that
SMYD3 methylates lysine 831 of VEGFR1, which islocated in the tyrosine kinase domain, and this meth-ylation enhances the kinase activity of VEGFR1 bothin vitroand in vivo26(FIG. 3c). VEGFR1 in cancer cellsis implicated in liver metastasis of colorectal cancer63,and exogenous expression of VEGFR1 enhances themigration and invasion of pancreatic cancer cells64.Hence, therapeutic approaches targeting VEGFR1 meth-ylation by SMYD3 can directly attenuate kinase activ-ity of VEGFR1 in cancer cells and benefit patients byinhibiting invasion and metastasis of cancer cells.
SMYD3 can methylate MAP3K2, a member of the ser-ine/threonine protein kinase family33. SMYD3-mediatedmethylation of MAP3K2 at lysine 260 activates the RASRAFMEKERK signalling module, and SMYD3 deple-tion synergizes with a MEK inhibitor to block RAS-driventumorigenesis33(FIG. 3c). A clinical implication of this find-ing is the identification of SMYD3 as a candidate thera-peutic target to treat pancreatic and lung cancers drivenby RAS, as well as potentially other RAS-driven tumours.The complete loss of SMYD3 function has no apparentphenotype in mice, implying that SMYD3 inhibitorswould have minimal adverse effects as chemotherapeuticagents33. Consequently, we could envisage a therapeuticstrategy comprising inhibitors of RAF or MEK (whichare already currently used for anticancer therapy) withan SMYD3 inhibitor, which could mitigate potential side
effects by lowering the overall dosage needed for eachagent and also limit the development of resistance.
Transcription factors (NF-B, ER, C/EBP andSTAT3).The NF-B transcription factor regulates multi-ple biological functions, including inflammation, immu-nity, cell proliferation and apoptosis65. SETD7 methylateslysine 37 of RELA in the nucleus, which enhances thepromoter binding affinity of RELA. This methylationseems to have a critical role in the induction of a sub-set of NF-B-dependent genes in response to tumournecrosis factor- (TNF) stimulation66. In addition, Luet al.41showed that NSD1 methylates lysines 218 and 221
of RELA and activates NF-B activity. Given that theK218A, K221A or combined K218A and K221A muta-tion of RELA substantially diminished its DNA-bindingability, it seems that methylation of several lysine residueson NF-B plays an important part in NF-B-dependenttranscriptional regulation through increasing its bind-ing to promoters of downstream genes. Interestingly,these two methylation sites of RELA are demethylatedby the PKDM F-box and leucine-rich repeat protein 11(FBXL11), and overexpression of FBXL11 inhibitsNF-B activation and retards the growth of HT29 coloncancer cells41(FIG. 4a). Furthermore, SETD6 methylatesRELA on lysine 310 (RELAK310), and this methyla-tion renders RELA inert and attenuates RELA-driventranscriptional programmes, including inflammatoryresponses in primary immune cells67. MonomethylatedRELAK310 is recognized by the ankyrin repeat ofeuchromatic histone-lysine N-methyltransferase 1(EHMT1), and this interaction stabilizes EHMT1 activ-ity. As EHMT1 works as a transcriptional repressorthrough generating monomethylated and dimethylated
H3K9 at euchromatin, the interaction of EHMT1 withRELA attenuates transcription of NF-B target genes(FIG. 4a). Taken together, NF-B activity is intricatelyregulated by lysine methylation of RELA in a positiveor negative manner depending on the methylation site.
Oestrogen receptor- (ER), a ligand-activatedtranscription factor involved in human breast cancer 68,is also a substrate of PKMTs. Upon oestrogen stimula-tion, ER recruits several co-regulators to the oestrogenresponse elements that modulate activation or repres-sion of target genes. SMYD2 methylates lysine 266 ofER (ERK266) both in cell-free biochemical assays andin breast cancer cells, and this SMYD2-mediated ERmethylation negatively regulates acetylation of lysines266 and 268. Given that acetylation of these lysine resi-dues promotes ER transcriptional activity in responseto the ER ligand 17-oestradiol through enhancing theDNA-binding activity of ER69, SMYD2-dependentlysine 266 methylation attenuates the chromatin recruit-ment of ER to prevent ER target gene activation underan oestrogen-depleted condition69. On oestrogen stim-ulation, ERK266 methylation is diminished, therebyenabling p300/CREB-binding protein (CBP) to acetylateER, which can activate ER target genes. Importantly,ERK266 is also demethylated by LSD1. These resultssuggest a model in which SMYD2 and LSD1 control thedynamics of ERK266 methylation and its crosstalk with
acetylation of lysines 266 and 268, thereby modulatingER functions in breast cancer cells69(FIG. 4b).
CCAAT/enhancer-binding protein- (C/EBP) is amember of the basic leucine zipper family of transcriptionregulators that regulates tissue-specific gene expression,proliferation and differentiation in numerous cell types.Increased C/EBP expression has been detected in breastcancer, ovarian tumours and colorectal tumours, andC/EBP is required for tumour progression in the epider-mis70. Pless et al.71demonstrated that lysine 39 of C/EBP(C/EBPK39), which is located in the amino-terminaltransactivation domain, is methylated by EHMT2, leadingto repression of C/EBP transcriptional activity (FIG. 4c).
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a b
cd
e f
SETD7
EHMT2
SETD6NSD1
EHMT1
PHF20
MeMe
Me
Me
Me Me Me
LSD1
LSD1
PP2A
PCNA PCNA_N PCNA_C
SETD8
StabilizesPCNA protein
Interaction
FEN1
High affinity
PARP1 DBD
Automodification domain
Catalytic domain
NLS
SMYD2
Enhances poly(ADP-ribose)formation aer oxidative stress
ER
Transcriptional
activity
DBD Hinge Ligand-binding
Agonistantagonist
distinction
K266
SMYD2
p300
K268
Prevents ERtargetgene activation underan oestrogen-depleted condition
RELA
C/EBP
RHD-n TA1TA2IPT_NF-B
K218
K221
K221
Me
K39
Me
K248
Me
K140
K310
Constitutiveactivation ofNF-B activity
K37
K180
Me
K528
Gene activation
in response toTNFstimulation
FBXL11
Highaffinity
Suppression of NF-B-regulated genes throughEHMT1-dependentH3K9 methylation
STAT3
K218me1; K221me2
SETD7
EZH2
Promotes STAT3activity in GSCs
K37me1 K310me1 K266me1; K266me2
K248me1
K140me2
K180me3
K528me1
Ac
DBD LZI II
TAs
Repressestranscriptionalactivity
NTD CC DBD LD
Negatively regulatestranscription ofsome specific genes
SH2 CTDY
Mechanistically, methylation of C/EBP by EHMT2 maycreate a new binding site for a repressive protein com-plex or enhance the interaction between EHMT2 andC/EBP, which promotes H3K9 methylation by EHMT2in the vicinity of C/EBP target genes.
Signal transducer and activator of transcription 3(STAT3) is a latent transcription factor that acts as anoncoproteinin several cancers72. Intriguingly, STAT3 is
dimethylated on lysine 140 by SETD7 when it is boundto the subset of promoters that it activates following itstyrosine phosphorylation, and this methylation is revers-ibly demethylated by LSD1 (REF. 73) (FIG. 4d). In this case,STAT3 is methylated in the nucleus and not in the cyto-sol, in particular only when it is part of a promoter-boundcomplex. Moreover, dimethylation only negatively affectssubsets of STAT3-activated genes, implying that lysine
Figure 4 | Effects of lysine methylation on transcription factors and nuclear proteins. a | Lysine 37 of RELA, a subunit
of nuclear factor-B (NF-B) , is monomethylated by SETD7, which activates NF-B-dependent genes in response totumour necrosis factor-(TNF) stimulation. Nuclear receptor-binding SET domain-containing protein 1 (NSD1)monomethylates K218 (K218me1) and dimethylates K221 (K221me2), resulting in constitutive NF-B activation throughinteraction with PHF20, which disrupts the recruitment of PP2A to RELA, and these sites are reversibly demethylated by
Fbox and leucinerich repeat protein 11 (FBXL11). SETD6 monomethylates K310 of RELA and suppresses
NF-B-regulated genes through euchromatic histone-lysineNmethyltransferase 1 (EHMT1)dependent H3K9methylation. b | SMYD2-mediated oestrogen receptor-(ER) methylation prevents ERtarget gene activation under anoestrogen-depleted condition, and lysine-specific demethylase 1 (LSD1) demethylates this site.c | K39 of CCAAT/
enhancer-binding protein-(C/EBP) is methylated by EHMT2, which represses C/EBPtranscriptional activity. d | K140 ofsignal transducer and activator of transcription 3 (STAT3) is dimethylated by SETD7, which negatively regulates the
transcription of various STAT3 target genes. STAT3K140me2 is reversibly demethylated by LSD1. EZH2 trimethylates K180
of STAT3, which promotes STAT3 activity in glioblastoma stemlike cells (GSCs). e | SETD8 monomethylates K248 of
proliferating cell nuclear antigen (PCNA), and this methylation stabilizes the PCNA protein and enhances the interaction
between PCNA and flap endonuclease 1 (FEN1).f| SMYD2 monomethylatesK528 of poly(ADPribose) polymerase 1
(PARP1) and enhances poly(ADPribose) formation after oxidative stress. CC, coiledcoil domain; CTD, carboxyterminal
domain; DBD, DNA-binding domain; IPT, immunoglobulin-plexin-transcription; LD, linker domain; LZ, leucine zipperdomain; NLS, nuclear localization signal; NTD, amino terminal domain; PCNA_C, PCNA Cterminal domain; PCNA_N,
PCNA Nterminal domain; PHF20, PHD finger protein 20; RHD, REL homology domain; SH2, Src homology 2 domain;
TA, transcriptionactivation domain; Y, conserved tyrosine domain.
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Okazaki fragments
Short, newly synthesized DNA
fragments that are formed on
the lagging template strand
during DNA replication. The
fragments are produced
because of the need for DNA
polymerase to always
synthesize in a 5-to-3
direction and are subsequently
ligated together to form a
continuous strand.
methylation may contribute to specific transcriptionalregulation. Furthermore, lysine 180 on STAT3 is also tri-methylated by EZH2. EZH2-dependent STAT3 methyla-tion activates STAT3 functions by increasing its tyrosinephosphorylation in glioblastoma stem-like cells, and thisEZH2STAT3 pathway seems to be one of the key signal-ling nodes in glioblastoma stem-like cells74. Given thatinhibition of EZH2 inhibits the expression of Polycombtarget genes and diminishes STAT3 activity, it might be apotential therapeutic strategy in glioblastoma.
Other nuclear proteins (PCNA and PARP1).We reportedpreviously that the PKMT SETD8 methylates lysine 248of proliferating cell nuclear antigen (PCNA), one of thekey regulators in DNA replication and cell cycle pro-gression75. Methylation of lysine 248 by SETD8 stabilizesPCNA proteins through inhibition of polyubiquityla-tion and substantially enhances the interaction betweenPCNA and the flap endonuclease FEN1 (REF. 75). Lossof PCNA methylation retards the maturation of Okazakifragmentsand slows DNA replication. Moreover, cells
expressing a PCNA mutant are unable to be methylatedand are more susceptible to hydrogen peroxide-inducedDNA damage75. There are increased levels of methyl-ated PCNA in cancer cells, and there is a correlationbetween the expression levels of SETD8 and PCNA inhuman cancer tissues75(FIG. 4e). PCNA has been widelyrecognized as a tumour marker for cancer progressionand poor patient prognosis because of its function incell proliferation76. Given this, SETD8-dependent PCNAmethylation is likely to promote tumorigenesis. Hence,inhibition of PCNA methylation by SETD8 might be arational approach to treat cancer.
Poly(ADP-ribose) polymerase 1 (PARP1) catalyses thetransfer of an ADP ribose unit from its substrate, NAD+,to protein acceptors such as histones and PARP1 itself.The pivotal roles of PARP1 in the DNA repair pathwayprompted researchers to investigate the effect of PARP1inhibition on DNA-damaging anticancer therapies.Indeed, inhibition of PARP1 was shown to enhance thecytotoxicity of DNA-damaging agents to cancer cells77. Weidentified that SMYD2 methylates lysine 528 of PARP1,which is located in the catalytic domain. Lysine 528-meth-ylated PARP1 shows increased poly(ADP-ribosyl)ationactivity after oxidative stress27(FIG. 4f). As SMYD2 deple-tion results in the reduction of PARP1 enzymatic activity,SMYD2 inhibition might increase the susceptibility ofcancer cells to DNA-damaging chemotherapy.
Somatic mutations of PKMTs and PKDMs
Many somatic mutations have been discovered inPKMTs and PKDMs through whole-genome sequenc-ing or exome sequencing of cancers (see Supplementaryinformation S1(table)). For example, the missense muta-tions Y641 and A677 in EZH2are frequently observedin diffuse large B cell lymphoma (DLBCL), and thesemutations significantly increase the methyltransferaseactivity of EZH2, resulting in enhanced proliferation ofcancer cells15. In addition, the E1099K missense muta-tion in the PKMT WolfHirschhorn syndrome candi-date 1-like 1 (WHSC1), which is found in 14% of t(12;21)
ETV6RUNX1-containing acute lymphoblastic leukae-mia cases, enhances the methyltransferase activity ofWHSC1 (REF. 78). It is crucial to consider these tumour-specific mutations for the development of anticancertreatment. Furthermore, somatic mutations in mixed-lineage leukaemia (MLL) lysine methyltransferase fam-ily members and in the PKDM UTX (also known asKDM6A) have frequently been reported in various typesof cancer7990, indicating the importance of these enzymesin human tumorigenesis. So far, the effect of dysregulationof PKMTs and PKDMs caused by somatic mutations hasonly been discussed in the context of their roles in epige-netic regulations through histone methylation or demeth-ylation. Thus, additional studies to clarify non-histonesubstrates and their effects in human cancer are needed.
Protein lysine methylation as a therapeutic target
Accumulating evidence clearly indicates that the inhibitionof PKMTs or PKDMs shows promise for the developmentof anticancer therapy. In 2005, Greiner et al.91identifiedthat chaetocin, the first described inhibitor of a PKMT,
specifically inhibits suppressor of variegation 39 homo-logue 1 (SUV39H1; also known as SU(VAR)3-9) bothin vitroandin vivo.
Subsequently, BIX-01294, which is an inhibitor of thePKMT EHMT2 (REF. 92), was shown to effectively suppressthe growth of cancer cell lines19(TABLE 2). Analysis of itscrystal structure shows that BIX-01294 lies in a loca-tion occupied by the histone H3 peptide of lysine 4 toarginine 8 (H3K4H3R8) that is next to the methylationsite of lysine 9 (REF. 93), implying that BIX-01294 occu-pies the substrate-binding site. This mode of action ismarkedly different from that of kinase inhibitors becausemost of the anticancer drugs targeting kinases discov-ered so far block phosphotransferase activity by com-peting with ATP94. In 2011, the novel EHMT2 inhibitorUNC0638 with a half-maximal inhibitory concentration(IC
50) value of
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effectively inhibits the proliferation of EZH2-mutantDLBCL cell lines and xenografts in mice15. The other isEPZ005687, which also targets Y641- and A677-mutatedEZH2 found in non-Hodgkin lymphoma97(TABLE 2).Recently, a new inhibitor (EPZ6438) specific for mutatedEZH2 has been reported that has superior potency andproperties to EPZ005687, including better oral bioavail-ability98(TABLE 2). A Phase I/II clinical trial of EPZ6438has begun for patients with non-Hodgkin lymphoma.By contrast, 10% of patients with myelodysplastic syn-drome (MDS) harbour loss-of-function mutations inEZH2 (REF. 99), and EZH2mutations in these patients areassociated with significantly worse prognosis, althoughthis is not due to transformation to acute myeloid leu-kaemia100. Patients with MDS without monosomy7/del(7q) chromosome anomalies also have reducedexpression of EZH2 in CD34+cells101, which implies thepossibility that EZH2 functions as a tumour suppressorin MDS, and similar results are also reported in myelo-proliferative neoplasm102. Along this line, patients withcolorectal cancer who have EZH2overexpression have a
good prognosis17. These results highlight that althoughthere is no doubt that it is an ideal therapeutic target,caution is necessary in developing anticancer therapiestargeting EZH2.
EPZ5676, a potent and selective aminonucleosideinhibitor of the PKMT DOT1L103,104, has also beenstudied in a Phase I clinical trial for the treatment ofpatients with acute leukaemia in which theMLLgenehas undergone rearrangement or tandem duplication(TABLE 2). The data derived from these clinical studiesare expected to provide important information regard-ing further possibilities and potential issues in thedevelopment of anticancer drugs targeting PKMTs.
With regard to PKDMs as candidates for anticancertherapy development, inhibitors targeting LSD1 havebeen actively studied. LSD1 is significantly overexpressedin human cancer, including small cell lung carcinoma,and has a crucial role in the growth of cancer cellsthrough the regulation of chromatin functions46. Becauseof the similarity in the catalytic and structural proper-ties, drugs targeting monoamine oxidase (MAO) werefirst investigated as LSD1 inhibitors. For example, tranyl-cypromine, an MAO inhibitor used as an antidepressantdrug, can inhibit LSD1 (REF. 105), and tranylcypromineor its analogues show antitumour activity when admin-istered alone or in combination with all-trans-retinoicacid in leukaemia models106. Given that anti-MAO drugs
are not specific to LSD1 and induce substantial toxicityin vivo, further refined LSD1-specific inhibitors haverecently been developed107. In particular, a Phase I clini-cal trial of GSK2879552, an LSD1-specific inhibitor, hasbeen initiated for patients with relapsed/refractory smallcell lung cancer (TABLE 2). Additionally, a Phase I clinicaltrial of the novel LSD1 inhibitor ORY-1001 has begun foracute myeloid leukaemia (TABLE 2).
As mentioned above, in addition to the LSD1 fam-ily proteins, JmjC-containing proteins also have PKDMactivity. In 2012, Kruidenier et al.108reported a selectiveJmjC H3K27 demethylase inhibitor. The authors indicatedthe relevance and tractability of demethylase inhibition
and that targeting the JmjC-containing proteins may havebroad therapeutic application108. Indeed, several JmjC-containing demethylases have been reported as candidatesfor anticancer therapy (TABLE 1), and inhibitors targetingthe Jumonji-type demethylase activity showed antitu-mour effects even though no drugs have yet been evalu-ated in a clinical trial109. Further investigation may reveala great potential of Jumonji-type demethylase inhibitorsas antitumour agents.
Conclusions and outlook
Although the first protein lysine methylation was dis-covered in 1959, its physiological significance remainedunknown for a long time. In the twenty-first century, thediscovery of several PKMTs and PKDMs markedly accel-erated a deeper understanding of the role of protein lysinemethylation in many biological processes, particularlyits vital role in epigenetics and also its involvement in
various human diseases, including cancer. In this regard,although it was difficult to identify methylation sitesof novel substrates, the progress of proteomic analysis
using mass spectrometry has enabled the identificationof methylation sites to become feasible both in vitroandin vivo. Consequently, lysine methylation is now widelyrecognized as a fundamental PTM. Pang et al.36reported45 high-confidence lysine methylation sites in 40 of 2,607(1.53%) S. cerevisiae proteins, even though their func-tions are mostly unknown. This result demonstrates thepossibility that many substrates of human PKMTs orPKDMs remain uncharacterized. Importantly, severalPKMTs or PKDMs are localized in both the cytoplasmand the nucleus; in particular, SMYD2 and SMYD3 pre-dominantly localize in the cytoplasm. In fact, a couple ofcytoplasmic proteins have been identified as substratesof SMYD2 or SMYD3 (REFS 25,26,33). These facts providehints that important cytoplasmic substrates of PKMTs orPKDMs remain to be identified. Historically, the researchon protein lysine methylation was started to analyse itsepigenetic functions, focusing on histone methylation,and there is no doubt as to its importance for epigeneticregulation. This is also confirmed by the fact that manyresearchers still use the terms histone methyltransferaseand histone demethylase for enzymes relevant to theprotein lysine methylation. Moreover, in addition to itsepigenetic function, further functional analyses mayunveil a wide range of functions of lysine methylationon non-histone proteins and its relevance to humancancer. In this regard, a large number of non-histone
proteins have been identified as substrates of histoneacetyltransferases and histone deacetylases (HDACs),and many of those are the products of oncogenes ortumour suppressor genes and are directly involved inhuman tumorigenesis110. As HDAC inhibitors are nowconsidered an emerging class of anticancer therapeutics,detailed examinations of the functions of non-histonesubstrates is essential to correctly understand the mecha-nism of actions of HDAC inhibitors110. For the successfuldevelopment of PKMT and PKDM inhibitors for cancertherapy, comprehensive functional analyses to identifycritical substrates of these enzymes in tumorigenesis arealso crucial.
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AcknowledgementsThe authors express great gratitude to the past and present
members of Y.N.s laboratory.
Competing interests statementThe authors declare competing interests: see Web version for
details.
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