THE UNIVERSE OF DAPK

Transcription control of DAPK

Natalya Benderska • Regine Schneider-Stock

Published online: 12 November 2013

� Springer Science+Business Media New York 2013

Abstract Imbalanced cell death is a common phenome-

non in many human diseases, including cancer. DAPK0sessential function is in promoting apoptosis. DAPK inter-

acts with stress-induced receptors through its death domain

to initiate an apoptosis cascade. In addition, DAPK phos-

phorylates multiple cytosolic substrates and can mediate

transfer of signaling pathways to the effector caspases. A

series of studies demonstrated that, depending on stimuli,

DAPK expression is regulated on both the transcriptional

and posttranscriptional levels. Silencing of DAPK due to

hypermethylation of its promoter was reported in many

types of cancer. STAT3 and p52-NFkB transcription fac-

tors have been shown to down-regulate DAPK expression.

In contrast, p53, C/EBP-b and Smad transcription factors

bind to their specific response elements within the DAPK

promoter and induce its transcription. Post-transcription-

ally, DAPK undergoes alternative splicing, which results in

the production of two functionally different isoforms.

Moreover, miRNA 103 and miRNA 107 recently were

shown to inhibit DAPK in colorectal cancer. Here we

summarize our recent knowledge about transcriptional

regulation of DAPK expression.

Keywords DAPK � Transcription factor �Methylation � Cancer � Apoptosis

Introduction

Death-associated protein kinase (DAPK) is a serine/threo-

nine protein kinase which performs diverse functions in the

cell. DAPK’s major role is cytoskeletal reorganization

under cytokine stimuli (such as IFN-c [1, 2], CD95 (Fas),

TNF-a and TGF-b [3, 4]) and induction of cell death.

Recent studies have revealed that the apparatus of DAPK

gene transcription is not controlled through a simple switch

‘‘on/off’’ at the promoter, but the factors and mechanisms

involved in transcription are subjected to the regulation at

different levels. Among them, promoter methylation,

phosphorylation by other kinases and autophosphorylation,

also protein–protein interactions influencing DAPK protein

stabilization play an important role in the expression of

DAPK. In this review we provide an overview of all

aspects of DAPK0s transcriptional regulation from direct

transcription factor mediated effects, through miRNAs and

splicing processes that create DAPK isoforms with differ-

ent cellular functions. Understanding DAPK’s role in

transcriptional regulation may lead to the discovery of

novel therapeutics to combat cancer and inflammation-

associated diseases.

Transcriptional regulation

Epigenetic regulation via 50-UTR

Hypermethylation of CpG islands in a promoter region is

an epigenetic marker of inactivation of a cell0s guardian

molecules, such as tumor suppressors. DNA methylation

acts through the covalent addition of a methyl moiety to the

cytosine residue of a CpG dinucleotide by the DNA

methyltransferase (DNMT) family of proteins [5]. CpG

N. Benderska � R. Schneider-Stock (&)

Experimental Tumorpathology, Institute of Pathology,

Friedrich-Alexander- University of Erlangen-Nuremberg,

Universitatstrasse 22, 91054 Erlangen, Germany

e-mail: regine.schneider-stock@uk-erlangen.de

123

Apoptosis (2014) 19:298–305

DOI 10.1007/s10495-013-0931-6

islands have been defined for sequences greater than

200 bp in length, with a GC content greater than 50 % and

an observed-to-expected CpG ratio of greater than or equal

to 0.6 [6]. CpG islands are present in the promoter regions

of approximately 40 % of the genes in the mammalian

genome [7, 8]. Methylation of these CpG islands is thought

to play a direct role in the control of gene transcription,

genomic imprinting [9], X-chromosome inactivation [10]

and in tumorigenesis [11]. For the transcriptional outcome,

it means that transcription-regulated proteins are no longer

able to bind to the DNA sequences, resulting in gene

silencing.

Different CpG-island-prediction algorithms (CpG Island

Finder; CpG Island Searcher and UCSC Genome browser)

identified one CpG island (around 600 bp) overlapping

with the start site of DAPK (Fig. 1a). However, four other

CpG islands upstream from the start site for the minimum

200 bp length parameter have also been mapped (‘‘CpG

islands Finder’’). Moreover, the database predicted 14 CpG

islands distributed along the transcript downstream from

the first exon. It was reported that the DAPK gene lacks a

TATA box within the core promoter, but that it contains a

number of other positive regulators (Sp1, AP2-binding

sites, E box, CAAT box, consensus binding sites for

NFkB, AP1, E2F) located within 1,500 bp of the transla-

tion start site in exon 2 [12]. Further investigation of the

DAPK gene and bisulfite sequencing of a 659 bp fragment

(-1,411 to -752) that included exon 1 revealed an addi-

tional promoter in lung and breast tumor cells [12]. Pulling

and co-authors identified dual promoter regulation of

DAPK in lung and breast tumor cells. They have reported

about the presence of dominant promoter 1 (-2,533 to

-1,025) which exhibits 40–50 % higher reporter activity

than the moderate promoter 2 (-969 to -1) (Fig. 1b). The

three most common transcription factors identified were

CP2, Sp1 and MFZ. Mutation of the CP2-binding site

(-1,184) had a dramatic effect, reducing promoter activity

[65 % among three breast cancer and twelve lung cancer

cell lines. The 90 bp region from -176 to -86 bp with

highest reporter activity includes the three most common

transcription factors: HNF3B, MZF (myeloid zinc finger)

and NF-kB. This region was shown to contribute to the

transcription of promoter 2. Mutation of the HNF3B-

binding site reduced luciferase activity by 60–70 %, while

Fig. 1 Transcriptional regulation of DAPK expression. a CpG

islands of human DAPK promoter region (-10,000 bp) predicted

by CpG islands Finder database. DAPK promoter sequence is taken

from http://www.mybioinfo.info and submitted to the CpG islands

finder algorithm (http://dbcat.cgm.ntu.edu.tw). DNA sequence input

labeled in white color. Single CpG sites are marked in yellow. CpG

island regions indicated in blue color and the most dense CpG island

colored in red. b Schematic representation of repression of DAPK

transcription via methylation of CpG islands within the DAPK pro-

moter region (-2,500 to 0 bp). c, d Experimentally validated regu-

lators of DAPK expression. c Transcription factors acting as a

positive regulators of DAPK expression. d Transcription factors and

cofactors suppressing DAPK expression

Apoptosis (2014) 19:298–305 299

123

mutation in the MZF or NF-kB-binding sites reduced

activity by 25 %. Moreover, prevalence in methylation of

promoter 2 was found in breast tumors whereas in lung

carcinoma cells the promoter 1 site is most affected,

indicating tissue-specific differences in transcript silencing

[12].

Numerous studies have demonstrated the shutdown of

DAPK transcription by promoter hypermethylation in

cancer (Fig. 2). The data published by different research

groups vary even for the same type of tumor. The number

of patients involved in the studies, DNA taken from dif-

fering tumor regions as well as tumor grades and the

method of analysis remarkably contribute to the discrep-

ancy. It was observed that DAPK methylation correlates

with the progression of disease and inflammation. For

example, the frequency of DAPK methylation in ulcerative

colitis-associated carcinoma with high inflammatory

background is relatively low (27.6 %) compared to non-

neoplastic ulcerative colitis mucosa (48.3 %) [13], which is

in agreement with up-regulation of DAPK protein expres-

sion in ulcerative colitis-associated carcinoma tissue as

shown by Chakilam et al. [14]. Otherwise, DAPK promoter

methylation frequency in sporadic colorectal carcinoma

was 57.4 %. Mittag et al. [15] co-authors reported DAPK

methylation as an early event in colorectal tumorigenesis

and suggested two major switches: first, one between

normal mucosa (25 %) and low grade intraepithelial neo-

plasia (57.6 %) in T1 colorectal tumors, and a second one

between low grade and high grade intraepithelial neoplasia

(81,8 %). The authors suggested that DAPK loss in initial

stages may be an important step in abrogating apoptosis

and could be a precondition for further accumulation of

various genetic aberrations.

In most cancer types, normal, non-malignant tissues

displayed no or a very low frequency of DAPK promoter

methylation. An exception applies to thyroid cancer in

which the level of DAPK methylation was 65 % versus

71 % in normal tissues [16].

DAPK silencing occurs also in B cell lymphomas [17,

18]. Using cell sorting analysis, in normal individuals the

Califano team identified the presence of DAPK aberrant

methylation in an IgM- subpopulation of B-cells (1–6 %)

versus T-cells, monocytes, or neutrophils, which were

below 0.6 % [19]. This phenomenon could explain the

development of B-cell malignancies as arising from a

subpopulation of the IgM- cells and suggests DAPK as a

promising tumor biomarker.

The correlation of DAPK aberrant promoter methyla-

tion with clinical data of patients has yielded significant

outcomes. First, cancer cells with aberrant DAPK pro-

moter methylation may be less sensitive to radiochemo-

therapy because the induction of apoptosis is an important

mechanism for various anticancer agents as well as irra-

diation [20–22]. Hypermethylation of the DAPK promoter

has been associated with poor prognosis in lung cancer

patients [22, 23]. Loss of DAPK expression predicted

reduced survival and recurrence in breast cancer patients

[24]. Lymph node progression and DAPK methylation

were found to be correlated with advanced disease stages

of head and neck cancer [25]. Thus, loss of DAPK may

drive tumor progression and aggressiveness in most of the

cancers [12].

Fig. 2 Aberrant methylation of DAPK promoter in different tumor

types. A color graph indicates the percentage of the DAPK promoter

methylation identified by different studies. The minimal percentage of

the methylation frequency or single study on certain tumor type is

represented in blue. The maximal value of the methylation frequency

for the given tumor reported in the literature is marked in red

300 Apoptosis (2014) 19:298–305

123

Interestingly, the DAPK expression could be restored by

treating the cells with the Dnmt inhibitor 5-azacytidine

(Vidaza) [26] or 5-aza-20-deoxycytidine (Decitabine) [12]

in an 1–2 lM concentration range. These drugs are cur-

rently in clinical trials for selected types of malignancies.

The mechanism, discovered by Puto and Reed [26], dem-

onstrated an involvement of Daxx and RelB transcription

factors. RelB is a member of the NF-kB family that plays a

crucial role in regulating the immune system response, cell

differentiation and apoptosis. When both factors are pres-

ent, Daxx interacts with RelB bound to its target (including

DAPK), recruits Dnmtases and induces CpG hypermethy-

lation of RelB target gene promoters, subsequently induc-

ing gene inactivation.

Blockage of DAPK due to mutation is very rare in most

cancers [27], except in some B cell chronic lymphocytic

leukemias, where its expression is down-regulated by a

single polymorphism [28].

Positive DAPK regulators

p53

Martoriati and co-authors [29] have identified DAPK

among the direct target genes of p53. DAPK mRNA and

protein level are induced in response to DNA damage (UV-

irradiation, doxorubicin treatment and gamma-irradiation)

and are correlated with p53 activation in both normal and

tumor cells. EMSA experiments coupled with Chromatin-

IP revealed nine p53-binding sites upstream of the first

exon or within the first intron of DAPK and confirmed a

recruitment of p53 to this sequence. The highest activity

among these had a responsible element overlapping with

the start codon (Fig. 1c).

C/EBP-b

C/EBP-b transcription factor is yet another regulator of

DAPK function. The C/EBPs is a member of a superfamily

constituted of CREB, Fos, Jun/AP-1, ATF and Maf/Nrf.

The subfamily of C/EBP consists of six proteins, which

play a role in a number of biological responses, including

energy metabolism, tissue differentiation [30], fat storage

[31, 32], hematopoiesis [33], and immune response [34].

Among these proteins, C/EBP-b uniquely responds to a

number of extracellular and intracellular signals to mediate

various cellular functions [35, 36]. Mutation analysis of

this complex revealed two motifs—a promoter-proximal

CRE/ATF-binding site (core sequence GACG) and a distal

CBS site (core sequence TGGG), through which C/EBP-bregulates DAPK (Fig. 1c). Interestingly, C/EBP-b binds to

the CBS of the DAPK promoter constitutively, but to the

CRE/ATF sequence only under IFN-c stimulation [37]. It

was shown that IFN-c-induced ERK-dependent phosphor-

ylation of C/EBP-b permits its association with CRE/ATF.

From the other hand, it was shown that downstream kinase

of ERK, RSK, phosphorylates DAPK on Ser289, which

leads to an inhibition of its apoptotic activity [38]. Con-

versely, ERK may control DAPK by phosphorylating

DAPK at Ser735 leading to an increased catalytic activity

and triggering further DAPK-ERK interaction through their

death domains finally promoting apoptosis [39].

SMAD

DAPK mediates an early response in cells that undergo

apoptosis in response to TGF-b [4]. TGF family members

are dimeric ligands that, under stress conditions, bind to

pairs of membrane receptor serine/threonine kinases

(receptor types I and II), inducing the formation of a het-

ero-tetrameric receptor complex. Phosphorylation of the

regulatory region or GS domain by the type II receptor

creates a repeated pS-X-pS motif that serves as a docking

site for receptor-regulated Smad proteins (RSmad). The

resulting Smad complex accumulates in the nucleus where

it incorporates different DNA-binding cofactors and serves

as a transcription factor [40]. Sequence screening of DAPK

promoters identified the existence of a TGF-b-responsive

motif in the -705 to -352 promoter region [4]. This

region contains four copies of the Smad-binding elements

and two copies of the acute myeloid leukemia (AML)

family of transcription factors (Fig. 1c). Since it is known

that Smad3 and Smad4 cooperate with AML transcription

factors to activate transcription, it is likely that TGF-b-

induced transcription of the DAPK promoter is mediated

by cooperation of Smad proteins with AML transcription

factors. Smad-mediated activation leads to rapid, approxi-

mately eight-fold induction of DAPK mRNA already 8 h

after TGF-b treatment and subsequently to an increase in

the level of DAPK protein in human Hep3B hepatocellular

carcinoma cells [4].

Negative regulators of DAPK

STAT3

DAPK possesses cyto-protective capability during chronic

inflammation [13]. TNF induces a dual signaling: pro-

inflammatory IL-6/STAT3 and anti-inflammatory DAPK-

mediated pathways. Recently, it has been shown that

DAPK is a novel repressive transcriptional target of

STAT3. STAT3-enriched regions were found in the DAPK

promoter sequence: region 1 (-1,471 to -1,821), con-

taining five STAT3 binding motifs and region 2 (-351 to

-631), containing three STAT3 responsive elements

(Fig. 1d). EMSA and Chromatin-IP demonstrated that

Apoptosis (2014) 19:298–305 301

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TNF-activated phospho-STAT3 translocated to the

nucleus, where its DNA binding activity to the DAPK

promoter is enhanced. However, over-expression of DAPK

acts further as negative regulator of STAT3 that attenuates

its activity by altering protein–protein interaction via either

the masking of the STAT3 nuclear localization signal to

impede its nuclear translocation or by preventing the access

to the upstream kinase JAK and the subsequent dimeriza-

tion of STAT3 [14]. The important implication of this

study that DAPK and STAT3 negatively regulate each

other to promote their own expression/activation in order to

neutralize the TNF signaling was found in normal intesti-

nal cells. This finding opens a new therapeutic perspective

for DAPK in the treatment of patients suffered from

ulcerative colitis and ulcerative colitis-associated car-

cinoma.

Flt3ITD/p52NF-kB

The existence of a Flt3-JNK1-cJun pathway in human

AML cells has been shown by Shanmugam et al. [41].

Since c-Jun is known to drive expression of DAPK [42]

and AML is a highly aggressive disease showing apoptosis

resistance, the authors suggested that the DAPK promoter

is under severe repression. Chromatin-IP assays revealed a

strong p52NF-KB binding to the DAPK promoter together

with histone deacetylase 2 (HDAC2) and HDAC6 in the

tyrosine kinase (Flt3) internal tandem duplication (ITD).

More precisely, mitogen activated protein kinase kinase

kinase 7 (TAK1) activates p52NF-KB, binds at the tandem

NF-kB and CRE sites of the DAPK locus (-134 bp) and

recruits transcriptional repressors belonging to the HDAC

family (Fig. 1d).

Post-transcriptional regulation of DAPK

Alternative splicing

Alternative splicing is a post-transcriptional regulation

mechanism that allows generating of more than one mes-

senger RNA from a single gene [43]. However, alternative

splicing depends not only on the interaction of splicing

factors with pre-mRNA enhancers or silencers but also on

the coupling between transcription and splicing [44]. First,

splicing often integrates an alternative promoter usage

which may affect the final splicing product. Second,

alternative splicing can produce isoforms which will be

degraded via a nonsense mediated decay mechanism;

however, the transcription level will remain unchanged.

An example of alternative promoter usage has been

described above of this chapter, although the mechanism

regulating such a choice remains undiscovered. It is known

that alternative splicing is tissue-specific, cell cycle- and

cell stress-responsive and that it depends on the develop-

mental stage of an individual [45–47].

Jin and colleagues found that DAPK has two alternatively

spliced isoforms—alpha and beta [48]. The last exon 26 of

pre-mRNA contains a retained intron sequence in alpha

isoform, which is skipped in DAPK beta. As a result, a new

stop-codon is introduced downstream from the intron

retention (Fig. 3a). Therefore, human DAPK beta extends

for ten amino acids (12 amino acids in the mouse isoform) at

the C-terminus. It is important to note that the two differing

isoforms have antagonistic functions: DAPK alpha is the

pro-apoptotic form, whereas DAPK beta has cytoprotective

properties and attenuates TNF-induced apoptosis [49].

miRNA regulation

miRNAs belong to the family of small, noncoding RNA

molecules that inhibit translation or induce mRNA decay

through binding to the 30-UTR of their target RNAs [50,

51]. Recently Chen and co-authors demonstrated that

DAPK is a direct target of two miRNAs: miR-103 and

miR-107, which have identical seed sequences. These

miRNAs suppress the expression of DAPK by targeting its

30-UTR. The authors observed a positive correlation

between miR-103/107 and the metastasis potential of

colorectal cancer cells. This tendency was confirmed in a

cohort of colorectal patients, indicating that metastasation

and poor survival were associated with the high-expression

signature of miR 103/107 and subsequently a loss of DAPK

function [52]. Interestingly, bioinformatics approaches

Fig. 3 Post-transcriptional regulation of DAPK expression.

a Schematic representation of human DAPK alpha and DAPK beta

isoforms produced from a single DAPK pre-mRNA by alternative

splicing. b List of predicted miRNAs bound to the 3‘UTR of DAPK

mRNA identified by Targetscan database (http://www.targetscan.org)

302 Apoptosis (2014) 19:298–305

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have predicted that 30-UTR sequence of DAPK carries a

number of binding sites of other potential miRNAs

(Fig. 3b). In-depth investigations in this field are under way

and will produce new findings about miRNA-mediated

posttranscriptional regulation of DAPK during tumor-

igenesis.

Conclusion

The interest in DAPK signaling and functions has signifi-

cantly increased during the past decade. This is facilitated

by the broad spectrum of DAPK0s functions and its role in

neoplastic processes. In this article, we have summarized

recent studies describing control mechanisms of DAPK

expression at the transcriptional and posttranscriptional

levels.

There are only limited studies characterizing how

DAPK is regulated transcriptionally. However there are a

lot of putative binding sites for transcription factors in the

DAPK promoter and their complex network have to be

analyzed in future work. DAPK is tightly regulated

depending on the signaling pathway the cell undergoes

with specific stimuli. Tumor cells employ more devious

mechanisms aimed at inactivating DAPK expression and,

subsequently, at the elimination or reduction of apoptosis.

There are still many open questions regarding transcrip-

tional control mechanisms of DAPK. So it is not under-

stood how external signals lead to the production of the

DAPK beta isoform and which are the responsible splicing

factors. We do not know anything about the DAPK beta

interactome and its functional role under external and

internal signals or about the occurrence of this splice iso-

forms in human diseases and cancer. Finally, a better

understanding on transcriptional control of DAPK and its

regulation under different pathological conditions should

help to define key instruments for curing cancer.

Acknowledgments The work in RSS0s Lab is supported by Deut-

sche Forschungsgemeinschaft grants (SCHN477-9-2 to R.SS.),

Manfred-Stolte Stiftung (38736003, 38736005, 38736007 to R.SS.)

and Interdisciplinary Centre for Clinical Research (IZKF-D18 to

R.SS).

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