Dysregulated transcriptional and post-translational control of DNA methyltransferases in cancer

R E V I E W

Open Access

Dysregulated transcriptional and post-translational

control of DNA methyltransferases in cancer

Ruo-Kai Lin

and Yi-Ching Wang

Abstract

Cancer is a leading cause of death worldwide. Aberrant promoter hypermethylation of CpG islands associated with

tumor suppressor genes can lead to transcriptional silencing and result in tumorigenesis. DNA methyltransferases

(DNMTs) are the enzymes responsible for DNA methylation and have been reported to be over-expressed in various

cancers. This review highlights the current status of transcriptional and post-translational regulation of the DNMT

expression and activity with a focus on dysregulation involved in tumorigenesis. The transcriptional up-regulation of

DNMT gene expression can be induced by Ras-c-Jun signaling pathway, Sp1 and Sp3 zinc finger proteins and virus

oncoproteins. Transcriptional repression on DNMT genes has also been reported for p53, RB and FOXO3a transcriptional

regulators and corepressors. In addition, the low expressions of microRNAs 29 family, 143, 148a and 152 are associated

with DNMTs overexpression in various cancers. Several important post-translational modifications including acetylation

and phosphorylation have been reported to mediate protein stability and activity of the DNMTs especially DNMT1. In

this review, we also discuss drugs targeting DNMT protein expression and activation for therapeutic strategy against

cancer.

Keywords: DNA methyltransferase, Cancer, Tumor suppressor gene, Transcription, Post-translational modifications

Introduction

Cancer is a leading cause of death worldwide, accounting

for 8.2 million deaths in 2012 [1]. The process of

tumori-genesis needs to be initiated and promoted by molecular

abnormalities including oncogenes activation and tumor

suppressor genes (TSGs) inactivation [2]. Methylation

of CpG islands is one of the epigenetic modifications

in mammalian genome that modulates gene expression

without changes in the DNA sequence [2]. Aberrant

promoter hypermethylation of CpG islands associated

with TSGs can lead to transcriptional silencing and result

in tumorigenesis. DNA methylation is frequently not

restricted to a single CpG island but affects multiple

independent loci, reflective of a widespread deregulation

of DNA methylation pattern in different types of tumors

[3,4]. Development of genome-wide high-throughput

technologies has facilitated the identification of global

DNA methylation pattern [5,6]. For example, genomic

screening of 98 different primary human tumors has

revealed that on an average there exist about 600 aberrantly

methylated CpG islands in each tumor [7]. In addition,

an increase of methylation variability may contribute

to tumor heterogeneity [8]. Collectively, dysregulation

of DNA methylation is apparently one of the major

barriers to effective cancer diagnosis and treatment in

different types of cancer.

Epigenetic disorders give rise to several significant

human diseases including various cancers, neuron disorder,

psychosis, and cardiovascular diseases, many of which are

associated with altered expression and activity of DNA

methyltransferases (DNMTs) [9-13]. DNMTs are the

en-zymes responsible for DNA methylation through transfer

of methyl group to cytosine residue of CpGs [2]. Five types

of DNMTs have been identified, viz. DNMT1, 2, 3A, 3B,

and 3L. DNMT1 comprises a large N-terminal domain

with regulatory function and a smaller C-terminal catalytic

domain [14]. The regulatory domain harbors different

motifs and is involved in the intracellular delivery and

regulation of catalytic activity of DNMT1. DNMT1 has

been shown to prefer hemimethylated over

unmethy-lated DNA 30- to 40-fold in vitro [15-17]. It is referred to

as a

“maintenance” methyltransferase and is the primary

* Correspondence:[email protected]

2

Department of Pharmacology and Institute of Basic Medical Sciences, National Cheng Kung University, No.1, University Road, Tainan 70101, Taiwan Full list of author information is available at the end of the article

© 2014 Lin and Wang; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

enzyme responsible for copying the methylation patterns

after DNA replication. DNMT1 localizes to replication

foci and interacts with PCNA, a processivity factor for

DNA replication complex [18]. However, evidences show

that DNMT1 may also work together with DNMT3A

and DNMT3B in de novo methyltransferase activity in

certain genome in both embryonic cells and differentiated

somatic cells [19,20]. Many interacting proteins have been

reported to bind to their N-terminal region by

biochem-ical interaction assay [14]. For example, DNMT1 directly

interacts with histone modifying enzymes such as histone

H3K9 methyltransferase SUV39H1, histone H3K27

meth-yltransferase EZH2, and histone deacytelase HDAC1 and

HDAC2 [14,21]. DNMT1 also interacts with

methyl-CpG-binding proteins such as MBD2, MBD3 and MeCP2 and

with the heterochromatin binding protein HP1 [14].

Notably, DNMT1, DNMT3A, and DNMT3B are

over-expressed in a coordinate manner in most tumor tissues

and at a significantly higher level in cancer than in

non-tumorous tissues [22-24]. The mechanism underlying

DNMTs overexpression is worthy of comprehensive

discus-sion. Delineating mechanisms of DNMTs overexpression

will provide more information and strategies to remedy the

altered epigenetic states. It will offer more exciting

oppor-tunities that can reactivate epigenetically silenced TSGs and

critical anti-cancer pathways [25].

Transcriptional regulation of

DNMT gene expression

The earlier study on transcriptional regulation of DNMT

mediated by Ras-c-Jun signaling pathway provided a

mo-lecular explanation for the role of DNMT1 to

carcinogen-esis [26,27]. The expressions of DNMT1, DNMT3A and

DNMT3B genes are also controlled by Sp1 and Sp3 zinc

finger proteins [28,29]. Wilms' tumour 1 protein has been

shown to directly transactivate DNMT3A expression [30].

Homeobox B3 can bind to and activate DNMT3B gene

[31]. In addition to transcription factors, several important

transcriptional repressors have been reported to suppress

the DNMT1, DNMT3A and DNMT3B gene expression,

including p53, RB and FOXO3a (Table 1 and Figure 1).

The major findings are described below.

The p53-mediated regulation of

DNMT genes

The tumor suppressor gene p53 encodes a transcription

factor that mediates many downstream effects such as

growth arrest and apoptosis through activation or

repres-sion of its target genes [46]. However, the p53 gene is a

frequent target of missense mutation rendering it unable

to recognize the p53 consensus binding sites therefore loss

of transcriptional function of p53 in cancers [47].

Se-quencing analyses reveal that point mutation and small

intragenic deletion/insertion of p53 gene are significantly

associated with DNMT1 protein overexpression [32]. A

study shows that deletion of p53 in the HCT116 human

colon carcinoma cell line results in increase of DNMT1

mRNA and protein [48]. Wild-type p53 decreases DNMT1

promoter activity and expression level through the exon 1

region (−19 to +317) of DNMT1 promoter, which contains

p53 putative binding sites, in lung cancer cells [32,48].

In addition, wild-type p53 protein has been shown to

negatively regulate DNMT1 expression by forming a

complex with Sp1 protein and chromatin modifiers on

the DNMT1 promoter [32]. Low level of exogenous Sp1

expression enhances the repressive activity of endogenous

p53 on the DNMT1 promoter, whereas a high level of Sp1

expression upregulates DNMT1 gene expression level in

A549 (p53 wild-type) cells. In H1299 (p53 null) cells,

exogenous Sp1 induces DNMT1 expression in a

dose-dependent manner. A high level of Sp1, via its

COOH-terminal domain, induces interaction between p53 and

MDM2, resulting in degradation of p53 by

MDM2-mediated ubiquitination [32]. Clinical data from 102 lung

cancer patients indicated that overexpression of DNMT1

is significantly associated with p53 mutation and high

expression of Sp1 protein. In addition, patients with

over-expression of both DNMT1 and Sp1 proteins show poor

prognosis [32]. Cell and clinical data provide evidence

that deregulation of DNMT1 is associated with gain of

transcriptional activation of Sp1 and/or loss of repression

of p53. DNMT1 overexpression is involved in epigenetic

alterations of multiple TSGs that ultimately leads to lung

tumorigenesis and poor prognosis [32].

Some reports have also shown that Sp1 and Sp3 increase

the activity of DNMT1, DNMT3A, and DNMT3B

pro-moters by physical binding to their propro-moters in mouse

NIH3T3 cells or human embryonic kidney 293T cells

[28,29]. p53 is shown to suppress the promoter activity

and mRNA/protein expression of DNMT3A through

binding to its promoter and the suppression can be

atten-uated by knockdown of p53 [33]. Whether overexpression

of DNMT3A and DNMT3B resulted from the loss of

transcriptional regulation of p53/Sp1 warrants further

in-vestigation. It is justifiable to propose that overexpression

of DNMTs is associated with the gain of transcriptional

activation of Sp1 and/or the loss of repression of p53

(Figure 1A).

The RB-mediated regulation of

DNMT genes

The RB (retinoblastoma) protein is a tumor suppressor,

which plays a pivotal role in the negative control of the

cell cycle and in tumor progression [49]. The RB protein

represses gene transcription, required for transition from

G1 to S phase, by directly binding to the transactivation

domain of E2F and by binding to the promoter of the

target genes as a complex with E2F [50]. RB also represses

transcription by remodeling chromatin structure through

interaction with proteins such as HP1, SWI/SNF, HDAC1

and SUV39H1, which are involved in DNA methylation,

nucleosome remodeling, histone deacetylation and histone

methylation, respectively [51-54].

The mouse and human DNMT1 promoters are found to

contain E2F binding sites that are required for RB/E2F

regulation in wtPrE (wild-type prostate epithelial cell line)

[34]. DNMT1 is negatively regulated by E2F-RB-HDAC

pathway in mouse NIH3T3 embryonic fibroblast, monkey

COS-7 kidney cell, and saos-2 human osteosarcoma cell

lines [35]. In addition, DNMT1 mRNA can be diminished

by overexpression of RB protein in saos-2 cells and are

induced by deletion of RB gene in wtPrE cells [34,35].

RB also suppresses DNMT3A promoter activity and

mRNA/protein expression through binding with E2F1

protein to the DNMT3A promoter [36]. Repression of

DNMT3A by RB leads to the decrease of methylation

level globally and TSG specifically, such as RARβ, FHIT,

and RASSF1A genes [36]. Together, these data suggest that

RB is a transcriptional repressor of DNMT1 and DNMT3A

genes (Figure 1B).

The FOXO3a-mediated regulation of

DNMT genes

Forkhead O transcription factor 3a (FOXO3a) belongs to

a large protein family of transcriptional regulators

charac-terized by a conserved DNA-binding domain termed the

“forkhead-box” [55]. To date, many reports indicate a

tumor suppressor role for FOXO3a. For example, ectopic

overexpression of FOXO3a significantly impairs tumor

growth in cell and xenograft models in breast cancer and

promotes apoptosis in leukemia and prostate cancer cells

[56,57]. In addition, restrained transcriptional activity of

FOXO3a in cancer cells results in promoting angiogenesis

and tumor progression [58-60]. FOXO3a has been shown

to transcriptionally up-regulate apoptotic-related gene

such as p27kip [61], Bim [62], and Fas ligand [63]. In

contrast, FOXO3a could transcriptionally repress

micro-RNA21, which suppresses the expression of Fas ligand

[64]. Of note, the gene deletion of FOXO3a is found in

early-stage lung adenocarcinoma in smokers and tobacco

carcinogen-initiated lung tumors in mice [37,65].

Restor-ation of FOXO3a in FOXO3a-deficient lung cancer cells

increases the cell apoptosis response to nicotine-derived

nitrosamino ketone-mediated DNA damage [66]. The

last-mentioned two studies implicate that loss of FOXO3a

may contribute to lung cancer pathogenesis.

We recently showed that FOXO3a negatively regulates

DNMT3B promoter activity by interacting with the binding

element FOXO3a (+166 ~ +173) of DNMT3B promoter

[67]. Ectopically overexpressed FOXO3a or combined

treatment with doxorubicin to induce FOXO3a nuclear

accumulation leads to further binding at the distal FOXO3a

Table 1 Transcriptional regulation of

DNMT promoter activity and/or mRNA expression

Pathways Mechanisms DNMTs mRNA/promoters References

Down regulation

p53 p53/Sp1 transcriptional repression DNMT1/3A/3B [32]

RB/E2F RB/E2F transcriptional repression DNMT1/3A [33-36]

FOXO3a Transcriptional repression DNMT3B [37]

Up regulation

Ras/AP-1 AP1 transcriptional activation DNMT1 [26,27,38]

Sp1 Transcriptional activation DNMT1/3A/3B [28,29,32]

Sp3 Transcriptional activation DNMT1/3A/3B [28,29]

E2F Transcriptional activation DNMT1 [34,39]

ERK Unknow DNMT1/3A [40]

17β-estradiol ER-dependent transcription activation DNMT3B [41]

Homeobox B3 Promoter binding DNMT3B [31]

Wilms' tumour 1 Transcriptional activation DNMT3A [30]

Viruse induction

LMP1 Activation of JNK/AP-1 pathway DNMT1 [42]

BKV Tag and E1a pRB/E2F pathway DNMT1 [43]

HBx Promoter transcriptional activator DNMT1/3A [44]

HBx Promoter transcriptional repression DNMT3b [44]

HIV-1 Through transcription factor AP-1 DNMT1 [45]

LMP1: latent membrane protein 1.

BKV Tag and E1a: Human polyomavirus BKV large T antigen and adenovirus E1a. HBx: Hepatitis B virus X protein.

site (−249 ~ −242). Abundant FOXO3a represses DNMT3B

promoter by establishing a repressed chromatin structure,

while knockdown of FOXO3a results in an open chromatin

structure and high DNMT3B mRNA and protein

expres-sion. Importantly, enforced abundant nuclear accumulation

of FOXO3a could decrease expression of DNMT3B with

synergistic inhibition of tumor growth and decrease in

methylation status on TSGs in human lung tumor

xeno-graft specimens [67]. It is plausible that FOXO3a binds to

the FOXO3a DNA element of the DNMT3B promoter to

repress DNMT3B expression (Figure 1C).

Transcriptional deregulation of

DNMT genes by MDM2

overexpression

p53 protein is known to be degraded in cytoplasm by

ubiquitin-mediated proteasomal degradation pathway

modulated by MDM2 [68]. MDM2, an E3 ubiquitin ligase,

also physically interacts with RB and FOXO3a

result-ing in degradation of RB and FOXO3a proteins [69,70].

Overexpression of MDM2 has been demonstrated in

many human cancers [36,71]. In addition, oncogenic

ERK phosphorylates FOXO3a at Ser

, Ser

, and

Ser

thereby enhancing the interaction with MDM2

and results in promoting degradation of FOXO3a [69].

Therefore, we hypothesized that MDM2 plays a critical

role in regulating the DNMT genes by synergistically

destabilizing p53, RB and FOXO3a proteins. To test

this hypothesis we analyzed the relationship of MDM2

protein with p53, RB, FOXO3a and DNMT proteins in

lung cancer cell, xenograft and patient models.

Dra-matic induction of DNMT3A and DNMT3B expression

by ectopic overexpression MDM2 suggests a negative

A

D

B

C

Figure 1 Transcriptional regulation onDNMT gene expression. (A) p53 transcriptionally suppresses DNMTs through binding with Sp1 protein to the DNMT1, 3A and 3B promoters. (B) RB transcriptionally suppresses DNMT1/3A through binding with E2F1 protein to the DNMT1 and 3A promoters. (C) FOXO3a binds to the FOXO3a DNA element of the DNMT3B promoter to repress DNMT3B transcription. (D) Clinically, overexpressed MDM2 dramatically induces DNMT1, DNMT3A, and DNMT3B expression by negative control over p53, RB and FOXO3a leading to methylation of multiple TSGs and tumorigenesis.

control of MDM2 over RB and FOXO3a [36,67]. Note

that treatment with the MDM2 inhibitor, Nutlin-3,

sig-nificantly reduces DNMT3A and DNMT3B expression

and methylation of TSGs, as well as tumor growth in vivo

[36,67]. Clinically, MDM2 overexpression inversely

corre-lates with expression of p53, RB and FOXO3a proteins in

tumor tissues from lung cancer patients. Importantly, a

sub-group of patient with gene expression signature of

DNMTs high, p53/RB/FOXO3a low, and MDM2 high

ex-pression profile correlating with poor survival [33,36,67].

This defined signature may serve as a prognostic marker

in lung cancer patients whose genomic DNA may exert

promoter hypermethylation in multiple TSGs (Figure 1D).

The microRNA-mediated regulation of DNMTs

MicroRNAs (miRs) are small, noncoding RNAs that

regulate expression of many genes. Recent studies

sug-gest that abnormal expressions of miRs are involved in

pathogenesis of different types of human cancers [72].

Previous reports have shown that expression profiles

of miRs in lung cancer are different from normal lung.

The miR-29 family (29a, 29b, and 29c) has intriguing

complementarities to the 3'-UTRs of DNM3A and

DNMT3B [73]. The expression of miR-29s is inversely

correlated to DNMT3A and DNMT3B in lung cancer

tissues, and miR-29s directly target the 3'-UTRs of

both DNMT3A and DNMT3B. The enforced expression

of miR-29s in lung cancer cell lines restores normal

pat-terns of DNA methylation. The miR-29s further induces

re-expression of methylation-silenced TSGs, such as FHIT

and WWOX, and inhibits tumorigenicity in vitro and

in vivo [73]. Enforced miR-29b expression in acute

mye-loid leukemia cells also results in marked reduction in the

expression of DNMT1, DNMT3A, and DNMT3B and

ultimately to re-expression of p15

and ESR1 via

pro-moter DNA hypomethylation [74]. Of note, an inverse

correlation between miR-29c expression and DNMT3A

and DNMT3B protein expression has been reported in

melanomas [75].

In addition to miR-29s, ectopic expression of

miRNA-148a in lung cancer cell lines also results in a significant

reduction in the expression of DNMT1 [76]. Using

lucifer-ase reporter assay, DNMT1 mRNA was found to be a

tar-get of miR-148b and miR-152 [77]. Antagomir-mediated

knock-down and re-expression of miRs assays support

that miR-148b, miR-29c, and miR-26b down-regulate

DNMT3B gene in breast cancer cells [78]. Furthermore,

overexpression of miR-148b and -152 in pancreatic cancer

cell lines decreases DNMT1 expression, restores normal

DNA methylation patterns and induces re-expression of

TSGs, like BNIP3 and SPARC [77]. It is to be noted that

miR-143 was reported to directly target DNMT3A. In

colorectal cancer tissues, the miR-143 expression was

observed to be inversely correlated with DNMT3A mRNA

and protein expression [79]. Specifically, miR-1741,

miR-16c, miR-222 and miR-1632 are found to influence

expression of DNMT3A or DNMT3B, possibly through

their 3′-UTR post-transcriptional regulation [80]. Table 2

summarizes the regulation of DNMTs by miRs.

Post-translational modification of DNMT proteins

Several important post-translational modification including

acetylation and phosphorylation have been reported to

mediate protein stability and activity of the DNMTs

es-pecially DNMT1 (Figures 2 and 3). The major findings

are described below.

Acetylation-mediated DNMT stability and activity

DNMT1 has been shown to be destabilized by

acetylation-mediated ubiquitination. DNMT1 physically interacts with

acetyltransferase Tip60, herpesvirus-associated ubiquitin

specific protease (HAUSP), ubiquitin-like containing PHD

and RING finger domains 1 (UHRF1), HDAC1 and PCNA

on chromatin [81]. Tip60 promotes acetylation of DNMT1,

which triggers ubiquitination by the E3 ligase UHRF1,

thereby targeting DNMT1 for proteasomal degradation

during late S phase [82]. Conversely, HAUSP and

HDAC1 protect DNMT1 from degradation through

deubiquitination and deacetylation, respectively [81]

(Figure 2A). In addition, the pleiotropic regulator of G

protein signaling (RGS) family member RGS6 facilitates

Tip60-mediated degradation of DNMT1 [83]. RGS6 may

serve as a scaffold of Tip60, DNMT1 and

Dnmt1-associated protein (DMAP1) to facilitate Tip60 acetylation

of DNMT1 and subsequent DNMT1 polyubiquitylation

and degradation [83] (Figure 2B). In contrast, the histone

deacetylase SIRT1 physically associates with DNMT1 and

deacetylates acetylated DNMT1 in vitro and in vivo [84].

Using mass spectrometry analysis, 12 new acetylated lysine

sites are identified in DNMT1 [85]. Deacetylation of

dif-ferent lysines on DNMT1 by SIRT1 has difdif-ferent effects

on the functions of DNMT1. For example, deacetylation

of Lys1349 and Lys1415 by SIRT1 in the catalytic domain

of DNMT1 enhances the methyltransferase activity of

DNMT1 (Figure 2C). Collectively, these findings suggest

that deacetylation of the identified acetylated lysine sites

in DNMT1 may be involved in the impaired activity of

DNMT1.

Phosphorylation-mediated DNMT stability and activity

Lin et al. found that the tobacco-specific nitrosamine

4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)

in-creases DNMT1 protein expression and activity [86].

Strong nuclear staining of DNMT1 protein in lung

tumor tissues is significantly associated with smoking

status of lung cancer patients [22,86]. Cigarette smoking

is a dominant risk factor for lung cancer. Among the

multiple components of tobacco smoke, 20 carcinogens

Table 2 Regulation of DNMT expression by miRNAs

Pathway Regulated regions DNMTs Cancer types References

miR-16c 3'-UTRs DNMT3B in vitro [80]

miR-26b ND DNMT3B breast cancer [78]

miR-29a 3'-UTRs DNMT3A/3B lung cancer [73]

miR-29b 3'-UTRs DNMT1/3A/3B lung, ALL and melanomas [73,74]

miR-29c 3'-UTRs DNMT3A/3B breast, lung and melanomas [73,75,78]

miR-143 3'-UTRs DNMT3A colorectal [79]

miR-148a ND DNMT1 lung and pancreas [76,77]

miR-148b ND DNMT3B breast cancer [78]

miR-152 ND DNMT1 pancreas [77]

miR-222 3'-UTRs DNMT3B in vitro [80]

miR-1632 3'-UTRs DNMT3B in vitro [80]

miR-1741 3'-UTRs DNMT3A in vitro [80]

ND: non-determined.

ALL: Acute lymphoblastic leukemia.

Figure 2 Acetylation-mediated DNMT stability and activity. (A) Tip60 promotes acetylation of DNMT1, which triggers ubiquitination by the E3 ligase UHRF1, thereby targeting DNMT1 for proteasomal degradation. (B) RGS6 serves as a scaffold to facilitate Tip60 acetylation of DNMT1 and subsequent DNMT1 degradation. (C) Deacetylation of Lys1349 and Lys1415 by SIRT1 in the catalytic domain of DNMT1 enhances the methyltransferase activity of DNMT1.

convincingly cause lung tumors in laboratory animals or

humans. Of these, NNK is likely to play a major role

because NNK has been shown to induce lung tumor in

rats, mice and hamsters [87]. In addition, exposure of

NNK not only causes gene mutation, but also stimulates

the promoter hypermethylation on multiple TSGs in

bladder, liver, and lung cancers, including FHIT,

RASSF1A, and p16

, DAPK1, RUNX3, RARβ and

SFRP genes [88-93]. Notably, NNK stimulates the AKT,

NF-κB, EGFR, and ERK1/2 signal pathways resulting in

increased cell proliferation and survival [94-96]. It has

previously been shown that AKT inactivates GSK3β

Ser/Thr kinase, which phosphorylates its substrate protein

and recruits the E3-ubiqutin ligase

βTrCP, leading to

substrate degradation [97,98]. In addition, stabilization

of DNMT1 protein is regulated by inhibiting

GSK3β-mediated phosphorylation and proteasomal degradation

of DNMT1 upon activation of T cell receptor signaling

[99]. Lin et al. showed that GSK3β interacts with

DNMT1 to phosphorylate DNMT1 at Ser410 and Ser414

and promotes binding of DNMT1 by

βTrCP leading to

proteasomal degradation of DNMT1. They also

demon-strated that DNMT1 contains a domain, ESGXXS, similar

to the conserved binding motif DSGXXS of

βTrCP [86].

This study suggests that NNK activates AKT, then inhibits

GSK3β/βTrCP–mediated protein degradation, leading to

DNMT1 protein accumulation [86]. In addition, DNMT1/

βTrCP interaction could be interrupted by treating cells

with NNK. NNK also induces

βTrCP translocation to the

cytoplasm by interacting with phosphorylated

heteroge-neous nuclear ribonucleoprotein U (hnRNP-U) shuttling

protein mediated by AKT. Therefore, NNK exposure

re-sults in DNMT1 nuclear accumulation and

hypermethyla-tion of the promoters of multiple TSGs [86]. Figure 3A

shows the phosphorylation-mediated DNMT stability

con-trol induced by the cigarette carcinogen NNK.

In addition to Ser410 and Ser414 of DNMT1, recent

studies have suggested that the enzymatic activity of

DNMT1 is possibly modulated by phosphorylation of

Ser/Thr residues located in the N-terminal domain of

the enzyme [100-102]. Protein kinase C (PKC)

α, βI,

βII, δ, γ, η, ζ and μ preferentially phosphorylate the

N-terminal domain of human DNMT1 [102].

Phosphoryl-ation of DNMT1 by PKCζ reduces its methyltransferase

activity in vitro [102] (Figure 3B). In addition,

phosphoryl-ation of DNMT1 at Ser154 by CDKs, including CDK1, 2

and 5, is important to enhance enzymatic activity and

pro-tein stability of DNMT1 [100] (Figure 3C). AKT and PKC

Figure 3 Phosphorylation-mediated DNMT stability and activity. (A) Cigarette carcinogen NNK activates AKT, inhibits GSK3β-mediated DNMT1 phosphorylation at Ser410 and Ser414, thereby leading to DNMT1 protein accumulation. (B) Phosphorylation of DNMT1 by PKCζ reduces its methyltransferase activity. (C) Phosphorylation of DNMT1 at Ser154 by CDK1, 2 and 5 enhances enzymatic activity and protein stability of DNMT1. (D) Phosphorylation of DNMT1 at Ser127 and/or Ser143 decreases its interaction with UHRF1 ubiquitin-like protein and renders DNMT1 less efficient to catalyze the DNA methyltransferase activity at the replication fork.

are capable of phosphorylating DNMT1 at the residues

Ser127/143 and Ser127, respectively [101].

Phosphoryl-ation of the DNMT1 at Ser127 and/or Ser143 decreases

the capacity of the protein to interact with PCNA and

UHRF1 proteins and renders DNMT1 less efficient to

catalyze methylation [101] (Figure 3D). Interestingly,

phosphorylation of DNMT1 at Ser143 by AKT1 interferes

with the methylation of Lys142 by SET7, a known histone

methyltransferase involved in proteasome-mediated

deg-radation of DNMT1 [103].

The impact of viruses on the regulation of

DNMT

genes

Several viruses have been reported to increase DNMTs

expression (Table 1). Epstein-Barr virus (EBV) is closely

associated with human malignancies, including

nasopha-ryngeal carcinoma, Burkitt's lymphoma, T-cell lymphoma,

gastric carcinoma [104,105]. Epigenetic regulation of EBV

plays a central role in viral latency and viral-associated

carcinogenesis [105]. EBV latent membrane protein 1

(LMP1) activates cellular DNMTs, resulting in

hyperme-thylation and silencing of E-cadherin. LMP1-mediated

DNMT1 activation involves JNK but not NF-κB and p38

mitogen-activated protein kinases [42]. The EBV

onco-gene product LMP1, induces promoter hypermethylation

of RARβ2 via up-regulation of DNMT1, DNMT3A, and

DNMT3B proteins, leading to decrease in RARβ2

expres-sion in nasopharyngeal carcinoma cell lines [43]. Human

polyomavirus BKV large T antigen and adenovirus E1a

also strongly increase DNMT1 expression. Mutation of

the E2F sites within the DNMT1 promoter dramatically

abrogates transcriptional activation, suggesting that BKV

viral induction of DNMT1 may be through modulation of

pRB/E2F pathway [39].

The hepatitis B virus (HBV) X protein (HBx) plays a

key role in the molecular pathogenesis of HBV-related

hepatocellular carcinoma. HBx expression increases total

DNMT activities and selectively promotes regional

hyper-methylation of specific TSGs, including RASSF1A, GSTP1,

and CDKN2B, in pHBx-transfected cells [44]. Another

study shows that enforced HBx suppresses RASSF1A

possibly via induction of DNMT1 and DNMT3B

ex-pression [106].

Human immunodeficiency virus type 1 (HIV-1) also has

been reported to induce DNMT1 through the responsive

element residing in the

−1634 to +71 of DNMT1

pro-moter [45]. The increase in expression of DNMT1 and

overall genomic methylation as well as hypermethylation

of the p16

gene are found when infected with HIV-1

in Hut 78 lymphoid cells [107]. HIV infection of human

regulatory T cells down-regulates FOXP3 expression

me-diated by increasing DNMT3B levels and DNA

methyla-tion in the FOXP3 gene [108]. Therefore, the ability of

increased DNMT activity to downregulate the expression

of critical genes may be one of the mechanisms for

dys-function of T cells in HIV-1-infected individuals.

Concluding remark

DNMTs are the enzymes which catalyze the CpG DNA

methylation and have been reported to be over-expressed

in various cancers. The mechanisms of DNMT

over-expression are worthy of investigation. The transcriptional

up-regulation on DNMT gene expression can be induced

by Ras-c-Jun signaling pathway, Sp1 and Sp3 zinc finger

proteins, wilms' tumour 1, homeobox B3 and various

hu-man viruses. Loss of transcriptional repression control on

DNMT genes has also been reported. For example, p53

transcriptionally suppresses DNMTs through binding with

Sp1 protein to the DNMT promoters. RB transcriptionally

suppresses DNMT1/3A through binding with E2F1

pro-tein to the DNMT1 and 3A promoters. FOXO3a binds to

the FOXO3a DNA element of the DNMT3B promoter to

repress DNMT3B transcription. In addition, overexpressed

MDM2 may induce DNMT1, DNMT3A, and DNMT3B

expression by negative control over p53, RB and FOXO3a.

Low expressions of some miRs such as miR-29s, miR-143,

miR-148a and miR-152 are associated with DNMT

overexpression in various cancers. Several important

post-translational modification including acetylation

and phosphorylation have been reported to affect

pro-tein stability and activity of the DNMTs especially

DNMT1. Therefore, drugs targeting DNMT protein

in-activation and depletion, such as MDM2, AKT and

CDKs inhibitors may prove to be a good therapeutic

strategy for cancer treatment. Combined treatment with

the known DNMT inhibitors such as decitabine could

be a potential therapeutic strategy through epigenetic

modulation warranting further investigation in cancer

treatment.

Abbreviations

DNMT:DNA methyltransferase; FOXO3a: forkhead O transcription factor 3a; HBx: hepatitis B virus X protein; HAUSP: herpesvirus-associated ubiquitin specific protease; hnRNP-U: heterogeneous nuclear ribonucleoprotein U; LMP1: latent membrane protein 1; miR: microRNA; NNK: nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone; RB: retinoblastoma; RGS: regulator of G protein signaling; TSG: tumor suppressor gene; UHRF1: ubiquitin-like containing PHD and RING finger domains 1.

Competing interests

The authors declare that they have no competing interests.

Authors’ contributions

RKL and YCW wrote the review. Both authors read and approved the final manuscript.

Acknowledgements

This work was supported by Taiwan National Science Council grant 100-2320-B-038-002 to RKL; and Taiwan National Science Council grant 102-2627-B-006-010 and Taiwan Ministry of Health and Welfare grant 103-TDU-PB-211-133005 to YCW.

Author details

1

Graduate Institute of Pharmacognosy, Taipei Medical University, 250 Wu-Hsing Street, Taipei 110, Taiwan.2_{Department of Pharmacology and}

Institute of Basic Medical Sciences, National Cheng Kung University, No.1, University Road, Tainan 70101, Taiwan.3_{Program for the Clinical Drug}

Discovery from Botanical Herbs, Taipei Medical University, 250 Wu-Hsing Street, Taipei 110, Taiwan.4_{Program for Clinical Pharmacogenomics and}

Pharmacoproteomics, Taipei Medical University, 250 Wu-Hsing Street, Taipei 110, Taiwan.

Received: 21 May 2014 Accepted: 1 July 2014 Published: 19 August 2014

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doi:10.1186/2045-3701-4-46

Cite this article as: Lin and Wang: Dysregulated transcriptional and post-translational control of DNA methyltransferases in cancer. Cell & Bioscience 2014 4:46.

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