Molecular Cell

Article

Long Noncoding RNA TARIDDirects Demethylation and Activationof the Tumor Suppressor TCF21 via GADD45AKhelifa Arab,1,2 Yoon Jung Park,2,13 Anders M. Lindroth,2,14 Andrea Schafer,1 Christopher Oakes,2 Dieter Weichenhan,2

Annekatrin Lukanova,3,4 Eva Lundin,4 Angela Risch,2,5 Michael Meister,5,6 Hendrik Dienemann,5,7 Gerhard Dyckhoff,8

Christel Herold-Mende,8,9 Ingrid Grummt,10,* Christof Niehrs,1,11,* and Christoph Plass2,12,*1Institute of Molecular Biology (IMB), 55128 Mainz, Germany2Division of Epigenomics and Cancer Risk Factors, German Cancer Research Center (DKFZ), 69120 Heidelberg, Germany3Division of Cancer Epidemiology, German Cancer Research Center (DKFZ), 69120 Heidelberg, Germany4Medical Biosciences, Department of Pathology, Umea University, 90185 Umea, Sweden5TranslationalLungResearchCenterHeidelberg (TLRC-H),Memberof theGermanCenter for LungResearch (DZL), 69120Heidelberg,Germany6Translational Research Unit, Thoraxklinik-Heidelberg gGmbH University of Heidelberg, 69120 Heidelberg, Germany7Department of Thoracic Surgery, Thoraxklinik-Heidelberg gGmbH, University of Heidelberg, 69120 Heidelberg, Germany8Department of Otorhinolaryngology, Head and Neck Surgery, University of Heidelberg, 69120 Heidelberg, Germany9Division of Neurosurgical Research, Department of Neurosurgery, University of Heidelberg, 69120 Heidelberg, Germany10Division of Molecular Biology of the Cell II, DKFZ-ZMBH Alliance, 69120 Heidelberg, Germany11Division of Molecular Embryology, DKFZ-ZMBH Alliance, 69120 Heidelberg, Germany12The German Cancer Consortium, 69120 Heidelberg, Germany13Present address: Department of Nutritional Science and Food Management, Ewha Womans University, 120-750 Seoul, Republic of Korea14Present address: Graduate School of Cancer Science and Policy, National Cancer Center, 410-769 Goyang-si, Republic of Korea

*Correspondence: i.grummt@dkfz.de (I.G.), c.niehrs@imb-mainz.de (C.N.), c.plass@dkfz.de (C.P.)

http://dx.doi.org/10.1016/j.molcel.2014.06.031

SUMMARY

DNAmethylation is a dynamic and reversible processthat governs gene expression during developmentand disease. Several examples of active DNAdemethylation have been documented, involvinggenome-wide and gene-specific DNAdemethylation.How demethylating enzymes are targeted to specificgenomic loci remains largely unknown.We show thatan antisense lncRNA, termed TARID (for TCF21antisense RNA inducing demethylation), activatesTCF21 expression by inducing promoter demethyla-tion. TARID interacts with both the TCF21 promoterand GADD45A (growth arrest and DNA-damage-inducible, alpha), a regulator of DNA demethylation.GADD45A in turn recruits thymine-DNA glycosylasefor base excision repair-mediated demethylationinvolvingoxidationof 5-methylcytosine to 5-hydroxy-methylcytosine in the TCF21 promoter by ten-eleventranslocation methylcytosine dioxygenase proteins.The results reveal a function of lncRNAs, serving asa genomic address label for GADD45A-mediateddemethylation of specific target genes.

INTRODUCTION

Long non-coding RNAs (lncRNAs) are key regulators of chromatin

structure, affecting epigenetic states and expression levels of

many target genes (Rinn and Chang, 2012; Ulitsky and Bartel,

604 Molecular Cell 55, 604–614, August 21, 2014 ª2014 Elsevier Inc.

2013). lncRNAs mediate chromatin-based gene regulation

through interactionswith histonemodifiers, chromatin remodeling

complexes, transcriptional regulators, or theDNAmethylationma-

chinery. Like other epigenetic modifications, DNA methylation is

dynamic and reversible. DNAdemethylation occurs through either

inhibition of the maintenance methyltransferase DNMT1 or by

active enzymatic reactions, leading to loss of methylation in

response to developmental or environmental signals (Bergman

andCedar,2013;WuandZhang,2011).ActiveDNAdemethylation

involvesoxidation ofmethyl groups via the TET family ofmethylcy-

tosine dioxygenases (Guo et al., 2011; He et al., 2011; Kriaucionis

andHeintz, 2009; Tahiliani et al., 2009). The DNA repairmachinery

playsan importantpart inDNAdemethylation, involvingactivation-

induced cytidine deaminase (AICDA) and thymine-DNA glycosy-

lase (TDG) (Bhutani et al., 2010; Cortazar et al., 2011; Kangaspe-

ska et al., 2008; Metivier et al., 2008; Rai et al., 2008). An adaptor

protein that tethers the nucleotide excision repair (NER) and

base excision repair (BER) machineries to sites of DNA demethy-

lation is the stress response protein GADD45A (growth arrest and

DNA-damage-inducible, alpha). GADD45A interacts with compo-

nents of DNA repair complexes, including XPG and TDG, thereby

recruiting DNA repair complexes to specific sites and resulting in

the replacement of methylated cytosines by unmethylated cyto-

sines (Barreto et al., 2007;Cortellino et al., 2011; Niehrs and Scha-

fer, 2012; Schmitz et al., 2009). While GADD45A binds to

promoters undergoing demethylation (Barreto et al., 2007; Cortel-

lino et al., 2011; Schafer et al., 2013; Schmitz et al., 2009), it re-

mains unresolved how specific promoters are targeted.

Epigenetic silencing of tumor suppressor genes is a well-

known phenomenon in tumorigenesis, but themolecular mecha-

nisms underlying transcriptional repression are poorly under-

stood. We have studied epigenetic regulation of the tumor

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Figure 1. TARID Is an Intergenic Long Noncod-

ing RNA

(A) DNA methylation analysis of TCF21 in tumor

samples. Heatmap of DNA methylation for CGI1,

CGI2, and CGI3 in amplicons (a–d) analyzed by

MassARRAY. Each square represents a single CpG or

a group of two or three CpG units analyzed, each row

representing a tissue sample.Methylation frequencies

extend from yellow (0%) to blue (100%). Samples

include tumor (T) or normal (N) tissue DNAs from pa-

tients with non-small cell lung cancer (NSCLC), head

and neck squamous cell carcinomas (HNSCC), and

ovarian cancers (OVC). A scheme representing the

TCF21 locus including the transcription start site

(arrow), coding exons (black boxes), 50 and 30 un-

translated regions (white boxes), and CpG islands 1–3

(green bars) is displayed above. See also Figure S1.

(B) Diagram showing the relative promoter activity of

TCF21 fragments cloned in sense (S) and antisense

(AS) orientation into the pGL4 reporter plasmid. pGL4-

SV40 (SV40) was used as positive control. The relative

luciferase activities (FL/RL) over vector control (pGL4)

are displayed. Data are represented as mean ± SD of

six independent experiments (n = 6). Constructs

yielding significantly increased promoter activity over

pGL4 (t test, p < 0.05) are marked (*).

(C) RT-PCR products from cDNAs generated using

oligo(dT) (right, lane i) or a TARID-specific primer (lane

ii). The genomic structure ofTARID splice variants (T1–

T5) is shown at the right. Primers used for RACE are

indicated by green and red arrows.

(D) Northern blot of RNA from primary skin fibroblasts

using 10 mg of total RNA or 500 ng of poly(A)-enriched

RNA and a TARID-specific RNA probe.

Molecular Cell

LncRNA Demethylates the Tumor Suppressor TCF21

suppressor, transcription factor 21 (TCF21, also known as

CAPSULIN/POD1/EPICARDIN), a tissue-specific, basic helix-

loop-helix transcription factor regulating themetastasis suppres-

sor KISS1 (Arab et al., 2011). TCF21 is essential for embryonic

development (Cui et al., 2004; Lu et al., 2002; Quaggin et al.,

1999) and shows frequent aberrations in human cancers (Arab

et al., 2011; Smith et al., 2006; Tessema et al., 2008).TCF21 is en-

coded by three exons, each associated with a CpG island (CGI).

In cancer cells the promoter CGI is hypermethylated, leading to

epigenetic silencing of TCF21 (Arab et al., 2011; Richards et al.,

2011; Tessema et al., 2008). Here we provide evidence that the

third CGI of TCF21 harbors a promoter that directs transcription

of an unknown lncRNA in antisense orientation to TCF21. This

lncRNA, termed TARID, activates TCF21 transcription by

inducing TET protein-dependent DNA demethylation. We show

that TARID binds to the TCF21 promoter and recruits GADD45A

and TDG to direct base excision repair for demethylation. Our

work reveals a function of lncRNAs in epigenetic regulation to

serve as an address label for gene-specific DNA demethylation.

RESULTS

Concordant Promoter Methylation and Expressionof TCF21 and TARID

TCF21 is encoded by three exons, all being associated with a

CpG island, termed CGI1, CGI2, and CGI3 (Figure 1A). Aberrant

methylation of the CGI1 correlates with epigenetic silencing of

Mo

TCF21 in multiple human malignancies (Arab et al., 2011; Smith

et al., 2006). Analysis of DNAmethylation revealed that CGI1 and

CGI3, but not CGI2, were hypermethylated in samples from pa-

tients with non-small cell lung cancer, head and neck squamous

cell carcinomas, and ovarian cancers, suggesting a regulatory

mechanism that coordinates methylation and transcription of

CGI1 and CGI3 (Figures 1A and S1 available online). To examine

whether CGI3 might represent an alternative transcription start

site (TSS) of TCF21, we performed luciferase reporter assays

with constructs that cover CGI3 sequences in sense and anti-

sense orientation. A 554 bp fragment comprising the TCF21 frag-

ment in antisense orientation (F3) directed luciferase activity,

delineating the minimal active promoter and demonstrating

that CGI3 harbors a promoter that directs the synthesis of anti-

sense transcripts (Figure 1B). RT-PCR revealed that the TCF21

antisense transcripts are differentially spliced, yielding five

PCR products (T1–T5) comprising the TSS, the first exon 1 and

the last exon, but differing in the region that overlaps the

TCF21 promoter (Figure 1C). Exon-intron junctions in all splice

variants follow canonical splice-donor and splice-acceptor sites

(Table S1). Rapid amplification of cDNA ends (RACE) of these

transcripts, hereafter referred to as TARID (for TCF21 antisense

RNA inducing demethylation), identified the 50 end at

position +3,682 and the 30 end at position �4,399 relative to

the TCF21 transcription start site (Figure 1C). Accordingly, the

major transcript (T2) has a length of about 4.5 kb on northern

blots (Figure 1D). Consistent with a role as a nuclear lncRNA,

lecular Cell 55, 604–614, August 21, 2014 ª2014 Elsevier Inc. 605

TCF21TARID

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Figure 2. TARID Is Required for TCF21

Expression

(A) Relative levels of TARID and TCF21 mRNA in

HEK293 cells transfected with a BAC encoding

wild-type TCF21 (WT, HEK293TARIDWT) or amutant

BAC carrying a deletion of the TARID promoter

(MUT, HEK293TARIDMUT). Data are represented

as mean ± SD of three independent experiments

(n = 3).

(B) Relative levels of TARID and TCF21 mRNA

after locked nucleic acid (LNA)-mediated knock-

down of TARID in human primary skin fibroblasts

(48 hr). Scrambled control (Ctrl) LNA was used at

100 nM. RNA levels were normalized to HPRT1

and SDHA mRNAs. Data are represented as

mean ± SD of three independent experiments

(n = 3).

(C) Chromatin immunoprecipitation showing

RNAP II and p300 occupancy at the TCF21 locus

after knockdown of TARID in primary human skin

fibroblasts. Locations of amplicons (C1–C6) are

indicated in the scheme above. Values represent

the enrichment of bound protein fractions relative

to input. Data are represented as mean ± SD of

six independent experiments (n = 6). The asterisk

marks significant enrichment over control (t test,

p < 0.05).

(D) DNA methylation at the TCF21 promoter in

human primary skin fibroblasts after knockdown

of TARID with LNA (48 hr). The MassARRAY data

of amplicons comprising the TCF21 promoter is

shown below. Data are represented as mean ±

SD of three independent experiments (n = 3).

Molecular Cell

LncRNA Demethylates the Tumor Suppressor TCF21

TARID is localized preferentially in the nucleus and does not

encode an open reading frame longer than 85 amino acids

(Figures S2A and S2B). The coding potential was tested by Fick-

ett’s algorithm (Fickett, 1982), yielding test scores below values

commonly observed for coding transcripts (<0.95). Moreover,

computer-based prediction of the RNA structure suggested

that TARID exhibits a thermodynamically strong secondary

structure, a common feature of lncRNAs (Figures S2B and S2C).

In normal lung, oral, and ovarian epithelium, CGI1 and CGI3

are unmethylated and both TCF21 and TARID are expressed.

In corresponding tumor samples, CGI1 and CGI3 are methylated

and both TCF21 and TARID are silent. The correlation between

both TCF21 and TARID expression levels and methylation of

their corresponding promoter CGIs suggested that TARID acti-

vates TCF21 expression by decreasing promoter methylation

(Figures S3A and S3B).

TARID Activates TCF21 Transcription by InducingDemethylation of the TCF21 PromoterAs some lncRNAs alter epigenetic signatures through interac-

tions with chromatin remodeling enzymes (Gupta et al., 2010;

Li et al., 2013; Rinn and Chang, 2012; Zhao et al., 2008), we es-

tablished human embryonic kidney 293 cell lines (HEK293TARIDWT

and HEK293TARIDMUT) that harbor a bacterial artificial chromo-

some comprising the TCF21 locus or a mutated version lacking

the TARID promoter region (Figure S3C). In several subclones,

HEK293TARIDWT cells expressed both TCF21 and TARID,

whereas in HEK293TARIDMUT cells the level of both RNAs was

606 Molecular Cell 55, 604–614, August 21, 2014 ª2014 Elsevier Inc.

strongly reduced (Figure 2A). The positive correlation between

TARID and TCF21 mRNA suggested that expression of TCF21

is functionally linked to transcription of TARID (Figure S3D).

Indeed, knockdown of TARID in primary skin fibroblasts (ex-

pressing both TCF21 and TARID) led to decreased expression

of TCF21 (Figure 2B) and decreased occupancy of both

RNAP II and the histone acetyltransferase p300 at the TCF21

promoter (Figure 2C). In support of TARID being required for

maintaining the TCF21 promoter hypomethylated, knockdown

of TARID led to a 30% gain of DNA methylation around the

TSS of TCF21 (Figure 2D). To test whether elevated levels of

TARID would affect promoter methylation and TCF21 expres-

sion, we transfected all TARID splice variants (T1–T5) into three

cancer cell lines (H387, C8161, and A549) in which expression

of TCF21 is repressed by promoter hypermethylation. Expres-

sion of TCF21 was induced after transfection of transcripts T1

and T2, whereas transcripts T3, T4, and T5 did not activate

transcription. For comparison, TCF21 expression levels in

normal lung fibroblast, epithelial cells, and melanocytes are dis-

played in Figure 3A. Thus, sequences that promote transcrip-

tional activation of TCF21 are contained within exon 2 of TARID.

Transfection of TARID splice variant T2 resulted in a significant

increase of RNAP II, p300, and H3K4me3 at the TCF21 pro-

moter (Figure 3B). Moreover, ectopic TARID triggered demethy-

lation of the TCF21 promoter in all cancer cell lines assayed.

Demethylation was very specific, being restricted to a small re-

gion comprising the TSS, whereas no change was observed in

flanking sequences (Figure 3C). While TARID regulates TCF21,

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Figure 3. TARID Activates TCF21 by Pro-

moter Demethylation

(A) Left: relative levels of TCF21 expression in lung

fibroblast (LF), epithelial cells (EC), and melano-

cytes (ML). Middle: TCF21 expression in H387,

C8161, and A549 cancer cells transfected with

in vitro-synthesized TARID splice variants (T1–T5).

Right: scheme of TARID splice variants high-

lighting the minimal region required for TCF21

activation (shaded). RNA levels were normalized to

HPRT1 and SDHA mRNAs. Data are represented

as mean ± SD of three independent experiments

(n = 3).

(B) ChIP assaymonitoring the association of RNAP

II, p300, and H3K4me3 with different regions of

TCF21 in H387 cells transfectedwith TARID (splice

variant T2) or control RNA (Ctrl RNA). Values

represent enrichment of bound protein fraction

relative to input. The locations of amplicons (C1–

C6) are indicated in the scheme above. Actin B

(ACTB) was used as positive control. Data are

represented as mean ± SD of four independent

experiments (n = 4, *p < 0.05).

(C) DNA methylation levels around the TSS of

TCF21 in H387, C8161, and A549 cells transfected

with TARID (splice variant T2) or with control RNA

(Ctrl RNA). The position of CpG residues in

amplicon a (indicated by lollipops) is shown above.

Data are represented as mean ± SD of three in-

dependent experiments (n = 3).

Molecular Cell

LncRNA Demethylates the Tumor Suppressor TCF21

the reverse was not true, since no significant change in TARID

expression and promoter methylation was observed upon

TCF21 siRNA knockdown (data not shown).

TARID-Mediated Demethylation Requires GADD45Aand TDGPrevious results have established that GADD45A, a stress

response protein involved in growth control, genomic stability,

DNA repair, and apoptosis, influences epigenetic gene regula-

tion by promoting active DNA demethylation (Barreto et al.,

2007). As GADD45A binds RNA and shows characteristics of

a ribonucleoprotein particle (Sytnikova et al., 2011), we

reasoned that TARID might guide GADD45A to the TCF21 pro-

moter. In support of GADD45A being involved in activation of

TCF21, knockdown of GADD45A inhibited TARID-mediated de-

methylation of the TCF21 promoter and prevented TCF21

expression in H387, C8161, and A549 tumor cells (Figures 4A

and 4B). GADD45A has been shown to recruit the nucleotide

excision repair (NER) or deamination-base excision repair

(BER) machinery, which removes methylated cytosine residues

(Barreto et al., 2007; Cortellino et al., 2011; Schmitz et al.,

2009). To examine whether demethylation of TCF21 is medi-

ated by NER or BER, we monitored TARID-mediated TCF21

expression after knockdown of XPA, XPC, XPF and XPG

(essential components of the NER pathway), and TDG

Molecular Cell 55, 604–614

(thymine-DNA glycosylase), an essential

component of the BER pathway. While

expression of TCF21 was clearly

reduced upon TDG knockdown, knock-

down of XPA, XPC, XPF, and XPG did not affect TCF21 expres-

sion (Figure 4C). To approve that active demethylation is

brought about by GADD45A-dependent recruitment of the

BER machinery, we treated cells with low doses of the BER in-

hibitors CRT0044876, betulinic acid, and ABT888 and moni-

tored TCF21 expression. All three BER inhibitors prevented

TARID-dependent expression of TCF21, whereas GADD45A

was not affected (Figure 4D). Moreover, coimmunoprecipitation

experiments confirmed the association of TDG with GADD45A

in H387 cells (Figure 4E). In addition, TARID-mediated DNA de-

methylation and expression of TCF21 were inhibited upon

knockdown of TDG (Figure 4F), indicating that TDG cooperates

with TARID to activate TCF21 expression.

TARID Is Bound by GADD45A and Interacts with theTCF21 PromoterTo examine whether TARID associates with GADD45A, we

incubated bead-bound GADD45A with radiolabeled RNA

comprising different regions of TARID and monitored

GADD45A-associated RNA (Figures 5A and S4). These pull-

down assays revealed that GADD45A interacts with full-length

TARID (RNA1) and with a truncated version comprising exon 2,

the region that is complementary to the TSSofTCF21. Fragments

comprising the first exon of TARID, intronic sequences, and

fragments lacking the TSS overlapping region did not bind

, August 21, 2014 ª2014 Elsevier Inc. 607

A B

C D

E F

Figure 4. TARID-Mediated DNA Demethyla-

tion Requires GADD45A-TDG Base Excision

Repair

(A) Relative levels of TCF21 mRNA in H387, C8161,

and A549 cells transfected with TARID or control

RNA (Ctrl RNA) after siRNA-mediated knockdown

of GADD45A (siGA45A) or GADD45B (siGA45B).

RNA levels were normalized to HPRT1 and SDHA

mRNAs. Data are represented as mean ± SD of

three independent experiments (n = 3).

(B) H387 cells were transfected with siRNA against

GADD45A or a scrambled control siRNA (siCtrl) and

cotransfected with TARID or control RNA (Ctrl RNA).

DNA methylation was measured by MassARRAY.

Data are represented as mean ± SD of three inde-

pendent experiments (n = 3).

(C) Relative levels of TCF21 mRNA in H387 cells

transfectedwith TARID (48 hr) after siRNA-mediated

knockdown of NER genes (XPA, XPC, XPF, XPG) or

the BER gene TDG. RNA levels were normalized to

HPRT1 and SDHAmRNAs. Data are represented as

mean ± SDof three independent experiments (n = 3).

(D) Relative levels of TCF21 and GADD45A (GA45A)

mRNA in H387 cells transfected with TARID (splice

variant T2) for 48 hr, followed by treatment with BER

inhibitors CRT0044876 (CRT), betulinic acid (BA), or

ABT888 (ABT) for 48 hr. DMSO was used as a

solvent control and GADD45A expression as a DNA

damage response control. RNA levels were

normalized to HPRT1 and SDHA mRNAs. Data are

represented as mean ± SD of three independent

experiments (n = 3).

(E) FLAG-tagged GADD45A was immunoprecipi-

tated from lysates of transfected H387 cells, and

coprecipitated TDG was monitored on immunoblots

with antibodies against TDG and FLAG. The posi-

tions of molecular weight marker proteins (MW) are

indicated.

(F) Methylation analysis of the TCF21 promoter of H387 cells after treatment with siRNA against TDG (siTDG) or a scrambled control (siCtrl). Cells were

cotransfected with TARID or control RNA (Ctrl RNA). Methylation of CpGs 1–14 was measured by MassARRAY. Data are represented as mean ± SD of three

independent experiments (n = 3).

Molecular Cell

LncRNA Demethylates the Tumor Suppressor TCF21

(Figure 5A). As a control, all RNAs were bound with similar effi-

ciency to the nonspecific RNA binding protein PTB1 (polypyrimi-

dine tract binding protein-1). These results demonstrate that

GADD45A binds selectively to TARID sequences that overlap

the TCF21 promoter.

To monitor the interaction of TARID with GADD45A in vivo, we

immunoprecipitated GADD45A from HEK293 cells that harbor

the TCF21 BAC construct HEK293TARIDWT and measured

GADD45A-associated TARID RNA (Figure 5B). Consistent with

the in vitro pull-down assays, these RNA immunoprecipitation

(RIP) assays revealed specific binding of GADD45A to TARID,

but not to SDHA (succinate dehydrogenase subunit A) mRNA or

to 28S rRNA used as controls. Control assays with FLAG-tagged

PTB1showedassociation of all threeRNAs, validating the interac-

tion of TARID with GADD45A in vivo. Moreover, chromatin immu-

noprecipitation (ChIP) experiments showed a clear enrichment of

GADD45A at the TCF21 promoter in cells transfected with TARID,

but not with control RNA (Figure 5C), reinforcing that TARID re-

cruitsGADD45Ato theTCF21promoter tomediatedemethylation.

The observation that TARID is associated with chromatin and

interacts with GADD45A suggested that TARID might guide

608 Molecular Cell 55, 604–614, August 21, 2014 ª2014 Elsevier Inc.

GADD45A to the TCF21 promoter. To test whether TARID phys-

ically interacts with the TCF21 promoter, we transfected H378

cells with biotin-labeled TARID and captured TARID-associated

DNA with streptavidin-coated beads. TARID captured TCF21

sequences overlapping the 50 end of TCF21 (amplicons 3

and 4), whereas upstream or downstream regions (amplicons

1, 2, 5, 6, and 7) were not bound (Figure 5D). TCF21 DNA was

not captured by biotinylated RNA harboring TARID sequences

in the reverse orientation or by intronic RNA, emphasizing the

specificity of the binding assay (Figure 5D). These results reveal

that TARID physically interacts with both the TCF21 promoter

and GADD45A, implying an RNA-guided mechanism to recruit

GADD45A and TDG to facilitate DNA demethylation and activa-

tion of TCF21.

TARID-Mediated DNA Demethylation Requires TETProteinsOnemajor pathway of active DNA demethylation involves oxida-

tion of methyl groups via the TET family of methylcytosine diox-

ygenases (Guo et al., 2011; He et al., 2011; Kriaucionis and

Heintz, 2009; Tahiliani et al., 2009). To examine whether

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28S rRNATARID SDHA mRNA

Bou

nd R

NA

(% to

inpu

t)

Bou

nd R

NA

(% to

inpu

t)

PTB1 IgG

1

2

3

4

5

6

1 2 3 4 5 6

1

2

3

4

5

6

Inpu

t 5%

PTB

1

Figure 5. TARID Targets GADD45A to the

TCF21 Promoter

(A) Pull-down assay using immobilized FLAG-tag-

gedGADD45AorPTB1 incubatedwith radiolabeled

RNAs. Genomic regions encoding RNA fragments

1–6 relative to TCF21 is shown above. Bound RNA

was quantified by liquid scintillation counting (left)

followed by gel electrophoresis and autoradiog-

raphy (right). Seealso FigureS4. Theminimal region

in TARID that interacts with GADD45A is shaded.

Data are represented as mean ± SD of three inde-

pendent experiments (n = 3).

(B) RNA immunoprecipitation (RIP) of FLAG-tag-

ged GADD45A (top) and FLAG-tagged PTB1 (bot-

tom) transfected into HEK293TCF21WT cells. Levels

of TARID, SDHAmRNA, and 28S rRNA associated

with precipitated proteins were determined by

qRT-PCR and displayed as bound RNA relative to

input. Data are represented as mean ± SD of three

independent experiments (n = 3).

(C) Chromatin immunoprecipitation assays show-

ing the enrichment of GADD45A at TCF21 in H387

cells transfected with TARID or control RNA. Co-

precipitated DNA was analyzed by qPCR using

ampliconsC1–C5. Data are represented asmean±

SD of three independent experiments (n = 3, t test,

p < 0.05).

(D) DNA capture assay using H387 cells trans-

fected with biotin-labeled RNAs shown above.

Biotinylated RNA was captured with streptavidin-

coated beads, and associated TCF21 DNA

(amplicons 1–7) was analyzed by qPCR. Data are

represented as mean ± SD of three independent

experiments (n = 3, t test, p < 0.05).

Molecular Cell

LncRNA Demethylates the Tumor Suppressor TCF21

TARID-mediated demethylation of the TCF21 promoter is

brought about by TET proteins, we depleted H387 cells from

TET1, TET2, or TET3 by knocking down protein either alone or

in different combinations. Consistent with redundant functions

of individual TET proteins, depletion of all three TET proteins

was required to inhibit TARID-mediated demethylation and

TCF21 expression (Figures 6A and 6B). To monitor changes in

5-hydroxymethylcytosine (5hmC), we assayed 5hmC by quanti-

tative PCR (qPCR) following glucosyl-5-hydroxymethylcytosine-

sensitive MspI restriction digest (Kinney et al., 2011). We found

that hydroxymethylation of CpG#7 at the TCF21 promoter was

increased 5-fold in HEK293TARIDWT cells overexpressing TET1

(Figure S5). Significantly, ectopic TARID increased 5hmC

levels already 1–2 hr after TARID transfection (Figure 6C). In

support of TARID-induced demethylation involving hydroxyme-

thylation of CpG residues, 5hmC levels were 2-fold lower in

HEK293TARIDMUT compared to that in HEK293TARIDWT cells (Fig-

ure 6D, left). Moreover, 5hmC levels were reduced 3-fold after

knockdown of TARID in primary skin fibroblasts (Figure 6D,

right). These results indicate that TET protein(s)-mediated hy-

droxymethylation of CpG residues represents an intermediate

step of TARID-induced demethylation of the TCF21 promoter.

DISCUSSION

Two main mechanisms of DNA demethylation have been docu-

mented in mammals: global DNA demethylation and gene-spe-

Mo

cific DNA demethylation, the latter of which occurs at specific

promoter and enhancer sequences. One of the key questions

in gene-specific DNA demethylation is how locus specificity is

achieved. Because of their sequence specificity, noncoding

RNAs are excellent candidates for targeting protein complexes

to appropriate locations in the genome. In support of this view,

we show that lncRNAs can provide such specificity by binding

the adaptor protein GADD45A, which in turn recruits TDG and

TETs (seemodel Figure 7). TARID and GADD45Amediate deme-

thylation of TCF21 by utilizing BER and TDG, an essential

component of the BER pathway essential for DNA demethylation

(Cortazar et al., 2011; Pastor et al., 2013; Zhu et al., 2000). TDG

can efficiently excise the iterative oxidation products of 5mC and

5hmC, namely 5-formylcytosine (5fC), and 5-carboxylcytosine

(5caC), replacing 5-methyl cytosine with unmethylated cytosine

(He et al., 2011; Ito et al., 2010; Maiti and Drohat, 2011).

Demethylation of TCF21 is restricted to a few CpG dinucleo-

tides at the transcription start site, a characteristic feature of

gene-specific demethylation. Significantly, the part of TARID

that directs demethylation is associated with the TCF21 pro-

moter. The potential of RNA to bind to complementary DNA

sequences has led to the hypothesis that lncRNAs may play

important guiding roles in the establishment and transmission

of chromatin states. TARID may act as a scaffold to bring two

or more proteins into spatial proximity or may act as a guide to

recruit proteins, such as GADD45A to the TCF21 promoter.

This may occur through interactions of TARID with DNA or

lecular Cell 55, 604–614, August 21, 2014 ª2014 Elsevier Inc. 609

A

B

TCF2

1 m

RN

A le

vels

siC

trl

siTE

T1

Ectopic TARID

Moc

k

siTE

T2

siTE

T1/2

siTE

T1/3

siTE

T2/3

siTE

T1/2

/3

0.006

0.012

0.018

siTE

T3

0.20.4

0.6

0.81.0

TARID + siTET1TARID + siCtrlsiCtrl

TARID + siTET2TARID + siTET3D

NA

met

hyla

tion

siTET singly

siTET in combinations

TARID+ siTET1/3 TARID + siTET2/3TARID+ siTET1/2/3

Ctrl RNA + siCtrlTARID+ siTET1/2

CpG 1 2 3-4 5 7 8 9 10 11 14

CpG 1 2 3-4 5 7 8 9 10 11 14

0.2

0.4

0.6

0.8

1.0

DN

A m

ethy

latio

n

2

4

6

8

10

Rel

ativ

e 5h

mC

leve

l(M

spI r

esis

tanc

e)

Ectopic TARIDMoc

k

(0 h

r) 1 2 (hr)

Ctrl RNATARIDC

D

1

2

3

4

5

6

Rel

ativ

e 5h

mC

leve

l(M

spI r

esis

tanc

e)

HEK293

MUTWT

Skin fibroblasts

TARIDLNA

CtrlLNA

Figure 6. Demethylation by TARID Involves

TET Proteins and Hydroxymethylation of

CpG

(A) Relative levels of TCF21 mRNA in H387 cells

transfected with TARID (48 hr) after siRNA-medi-

ated knockdown of the indicated TET proteins.

RNA levels were normalized to HPRT1 and SDHA

mRNAs. Data are represented as mean ± SD of

three independent experiments (n = 3).

(B) Top: methylation of the TCF21 promoter after

treatment of H387 cells expressing ectopic TARID

with siRNAs against individual Tet proteins.

Bottom: methylation of the TCF21 promoter after

treatment of H387 cells with combinations of TET

siRNAs or a scrambled control (siCtrl) in the

presence of ectopic TARID. Methylation of CpGs

1–14 was measured by MassARRAY. Data are

represented as mean ± SD of three independent

experiments (n = 3).

(C) 5hmC levels at CpG#7 of the TCF21 promoter

measured by quantitative hydroxymethylcytosine

analysis. 5hmC levels at CpG#7 of the TCF21

promoter of H387 cells transfected with TARID or

with control RNA (Ctrl RNA) after 1 and 2 hr. Data

are represented as mean ± SD of three indepen-

dent experiments (n = 3).

(D) 5hmC levels at CpG#7 of the TCF21 promoter

measured by quantitative hydroxymethylcytosine

analysis. Left: 5hmC levels in HEK293TARIDWT (WT)

and HEK293TARIDMUT (MUT) cells. Right: 5hmC

levels in human primary skin fibroblasts after LNA-

mediated knockdown of TARID. Data are repre-

sented as mean ± SD of three independent

experiments (n = 3).

Molecular Cell

LncRNA Demethylates the Tumor Suppressor TCF21

with a promoter-bound protein. Interactions with DNA could be

brought about by direct physical interaction of RNA with duplex

DNA, forming an RNA:DNA triplex structure as reported for

pRNA, a noncoding RNA that is complementary to the rRNA

gene promoter (Schmitz et al., 2010). Alternatively, TARID could

associate with the TCF21 promoter by forming an R-loop, a

characteristic structure of CpG island promoters (Ginno et al.,

2012).

In accord with TARID being required to maintain the TCF21

promoter in an open, demethylated chromatin state, knockdown

of TARID leads to decreased 5hmC levels, promoter hyperme-

thylation, and gene silencing. Thus, TARID, GADD45A, TDG,

and TET proteins may function together as part of a surveillance

mechanism that protects TCF21 from hypermethylation and

transcriptional silencing. This is consistent with the need for

the continued presence of GADD45A for maintaining target

genes active (Barreto et al., 2007; Cortellino et al., 2011; Schafer

et al., 2013; Schmitz et al., 2009). It remains to be shown how

widespread the phenomenon of targeting of GADD45A-medi-

ated DNA demethylation by lncRNAs is and whether demethyla-

tion complexes other than GADD45A are also targeted by

lncRNAs. Given that loss of DNA methylation and inactivation

of tumor suppressor genes frequently occur in cancer cells,

610 Molecular Cell 55, 604–614, August 21, 2014 ª2014 Elsevier Inc.

RNA-guided DNA demethylation as described in this work may

not be limited to TARID-directed recruitment of GADD45A and

TDG/BER to the TCF21 promoter but may be relevant for under-

standing global epigenetic changes during tumorigenesis.

EXPERIMENTAL PROCEDURES

Patient Samples

Patient samples were obtained from the University Hospital Heidelberg (head

and neck tumors, n = 20; healthy oral epithelial tissue, n = 18), the Thoraxkli-

nik Heidelberg (lung adenocarcinoma, n = 18; lung squamous cell carcinoma,

n = 27; together with adjacent healthy tissue), and from the Northern Sweden

University Hospital (ovarian tumor, serous invasive tumors, n = 14; benign

and healthy controls, n = 18). Samples were collected with written consent

from the patients as approved by the ethical committees of the Medical Fac-

ulty of the University of Heidelberg and the Northern Sweden University

Hospital.

Cell Lines and Culture Conditions

HEK293 cells, head and neck cancer cells (HNO387), and human primary skin

fibroblasts (PromoCell) were cultured in Dulbecco’s modified Eagle’s medium

supplemented with 10% fetal calf serum, 5 mM L-glutamine, 100 U/ml peni-

cillin, and 100 mg/ml streptomycin. Epithelial cells and melanoytes (PromoCell)

were cultured in their specific mediums, melanocyte growth medium M2 and

epithelial growth medium, respectively (PromoCell). Melanoma (C8161) and

Figure 7. Model Depicting the Role of TARID in Regulation of TCF21

In cancer cells, the promoters of both TCF21 and TARID are frequently hypermethlyated, and both genes are silent. In normal cells, TARID lncRNA is expressed

and associates with the TCF21 promoter. TARID bindsGADD45A and recruits TDG together with TETs to induce promoter demethylation via base excision repair.

Demethylated TCF21 harbors RNAP II and active chromatinmarks H3K4me3, permitting TCF21 expression. Methylated and unmethylated CpGs are indicated by

black and white circles, respectively.

Molecular Cell

LncRNA Demethylates the Tumor Suppressor TCF21

lung cancer cells (A549) were cultured in RPMI 1640 medium. HEK293 cells

were transfected with either wild-type (RP11-706B15, carrying the TCF21

locus) or mutant (see below) BAC clone using the TransIT reagent (Mirus). Cells

were subjected to G418 selection (400 mg/ml; Sigma-Aldrich), and single cell-

derived clones were established by dilution seeding of G418-resistant col-

onies. DNAs and RNAs were isolated from cells or tissues using TRIzol (Life

Technologies).

Plasmids and BAC Constructs

RP11-706B15 (TARIDWT), a BAC clone carrying a 183 kb fragment including

the TCF21 locus, was received from BACPAC Resources Center (http://

bacpac.chori.org/). RP11-706B15-MUT-NEO (TARIDMUT-NEO) was gener-

ated by recombination, replacing the TARID promoter (GRCh/hg19, position

chr6: 134,213,747–134,214,300, termed F3) by the Neomycin resistance

(Neo) cassette. The Neo cassette was deleted by arabinose-induced flpe re-

combinase, yielding RP11-706B15-MUT (TARIDMUT) lacking the TARID pro-

moter. For the preparation of stable cell lines, all three constructs were

engineered to contain a Neo cassette 22 kb downstream of F3. The TARID

expression vector was generated by RT-PCR amplification of TARID cDNA,

using RNA from primary human skin fibroblasts and cloned into the Gateway

pT-REX-DEST30 vector (Life Technologies). 50 RACE and 30 RACE for TARID

were conducted as described (Frohman, 1993) using 5 mg of total RNA. The

genomic localization of the TARID isoforms (T1–T5) at chromosome 6mapped

between positions 134,213,940 and 134,205,890. Plasmids for luciferase re-

porter assay were generated by PCR amplification of DNA from primary hu-

man skin fibroblasts using primers with built in KpnI and XhoI restriction sites

allowing directional cloning into pGL4.10[luc2] (Promega). The genomic coor-

dinates of the luciferase reporter inserts are listed in Table S2.

Mo

Chromatin Immunoprecipitation and Coimmunoprecipitation

Assays

Cells were crosslinked with 1% formaldehyde at room temperature for 10 min;

quenched with 0.5 M glycine; lysed in 1% SDS, 10 mM EDTA, 50 mM Tris-HCl

(pH 8.1); and sonicated to yield 150–250 bp DNA fragments. Chromatin was

diluted 10-fold with IP buffer (16.7 mM Tris-HCl [pH 8.1], 167 mM NaCl,

1.2mMEDTA, 0.01%SDS, 1%Triton X-100), precleared for 1 hr at 4�Con pro-

tein A/G agarose in the presence of 20 mg/ml sonicated salmon sperm DNA,

and incubated overnight with the respective antibodies. Protein-DNA com-

plexes were captured on protein A/G agarose followed by two washes in

low salt buffer (150mMNaCl, 50mMTris-HCl [pH 8.0], 5 mMMgCl2, 1% Triton

X-100), high salt buffer containing 500 mM NaCl, and with LiCl buffer (250 mM

LiCl, 10 mM Tris-HCl [pH 8.0], 5 mM EDTA, 0.5% Na-deoxycholate, 0.5%

Triton X-100) and TE buffer. After elution and reversal of the crosslink, DNA

was extracted and amplified by qPCR. The amount of precipitated DNA was

normalized to input DNA and to immunoglobulin G (IgG)-bound DNA. For

coimmunoprecipitation assays, FLAG-tagged GADD45A or FLAG-tagged

PTB overexpressed in HNO387 cells were affinity purified by binding to M2

beads (Sigma-Aldrich). After elution with the FLAG peptide, coprecipitated

proteins were detected by immunoblotting.

RNA Immunoprecipitation

HEK293 cells expressing TARID were transfected with GADD45 expression

vector (pCS2+-FLAG-GADD45A), harvested after 48 hr, and lysed in IP buffer

containing 200 mM NaCl, 5 mM MgCl2, 10 mM HEPES (pH 7.0), 0.5% Non-

idet P-40, 1 mM dithiothreitol (DTT), 100 U/ml RNasin. After digestion with

DNase I (5 U/ml, 20 min) and centrifugation, the supernatant was cleared

by addition of 50 ml protein A beads (Sigma-Aldrich) and rotation for 1 hr

lecular Cell 55, 604–614, August 21, 2014 ª2014 Elsevier Inc. 611

Molecular Cell

LncRNA Demethylates the Tumor Suppressor TCF21

at 4�C. The cleared lysate was incubated with M2 beads (Sigma-Aldrich) for

4 hr at 4�C. After stringent washing, coprecipitated RNA was extracted with

TRIzol and analyzed by qRT-PCR. IgGs were used as a negative control.

DNA Capture Assay

HNO387 cells were transfected with 2 pmoles of biotin- and 4-thiouridine-

labeled TARID using the TransIT transfection reagent (Mirus). After 24 hr, cells

were UV irradiated at 2,500 kJ (365 nm, 40 s) and lysed in hypotonic buffer

(10 mM HEPES [pH 7.9], 10 mM KCl, 340 mM sucrose, 10% glycerol, 1 mM

DTT, 1.5 mM MgCl2, 100 U/ml RNase inhibitor). Isolated nuclei were lysed in

10 mM Tris-HCl (pH 7.4), 150 mM NaCl, 5 mM MgCl2, 0.5% NP-40, 1 mM

DTT, 100 U/ml RNasin. Chromatin was sheered by sonication, centrifuged

(12,000 3 g, 10 min, 4�C), and the supernatant was incubated for 1.5 hr at

room temperature with streptavidin-coated magnetic beads (Pierce, Thermo

Scientific) that were blocked for 3 hr with salmon spermDNA (5 mg/10 ml beads)

and BSA (20 mg/10 ml beads). After treatment with proteinase K (50 ng/10 ml

beads) and stringent washing, RNA was isolated and RNA-associated DNA

was quantified by qPCR.

Locked Nucleic Acids, siRNAs, PCR Primers, Drugs, and Antibodies

Custom designed locked nucleic acids (LNAs) against TARID (GGTGATGG

AAAAGTGGAG) and a scrambled LNA (GGTGTATTGAATGACGTG) were syn-

thesized by Exiqon and transfected by electroporation using Amaxa (Lonza).

siRNAs against GADD45A, GADD45B, XPA, XPC, XPG, XPF, TDG, TET1,

TET2, TET3, and control siRNAs were purchased from Life Technologies

and transfected with Dharmafect (Thermo Scientific). All knockdown experi-

ments showed at least 70% reduction in target gene expression. Primers

were synthesized by Sigma-Aldrich. Sequences of siRNAs and primers used

in this study are listed in Tables S3 and S4. CRT 0044876 and betulinic acid

were purchased from Sigma-Aldrich, ABT-888 from tebu-bio, all the drugs

were diluted in DMSO and added at 0.5 and 10 mM final concentration to the

cell culture media and incubated for 48 hr. The following antibodies were

used: RNA polymerase II (Ab5408, Abcam), p300 (sc-585, Santa Cruz),

TDG (c10996, AssayBioTech), H3K4me3 (pAb-003-050, Diagenode), and

GADD45A (sc-797 Santa Cruz).

RNA Pull-Down Assays and Northern Blot Analysis

FLAG-GADD45A and FLAG-PTB were overexpressed in HEK293TARIDWT cells

and immobilized on FLAG epitope (M2) beads. Beads (10 ml) containing 800 ng

of immobilized proteins were washedwith high salt buffer, equilibrated in bind-

ing buffer (50 mM Tris-HCl [pH 7.9], 10% glycerol, 100 mM KCl, 5 mM MgCl2,

10 mM b-mercaptoethanol, 0.1% NP-40), and incubated with 4 pmoles of32P-labeled RNA (5,000 cpm/ng). Equimolar amounts of RNA and bound pro-

teins were incubated for 1.5 hr at room temperature. After stringent washing,

bound RNA was extracted and subjected to gel electrophoresis and autoradi-

ography. For northern blot analysis, 500 ng of poly(A)-enriched RNA from pri-

mary skin fibroblasts were subjected to gel electrophoresis. After blotting, UV

crosslinking, and prehybridization, the membrane was incubated overnight at

68�C in hybridization buffer (63 SSC, 53 Denhardt’s solution, 0.5% SDS,

400 mg yeast tRNA) with a 463 nt 32P-labeled RNA probe that is complemen-

tary to the 30 end TARID. The genomic coordinates of the RNA northern blot

probe are chr6: 134,207,789–134,208,251 (Table S4). After stringent washing,

hybridization signals were detected by phosphorimaging.

Cell Fractionation

HEK293TARIDWT cells were harvested in cold PBS, washed twice, and incu-

bated in buffer A (HEPES-KOH 50 mM [pH 7.5], 10 mM KCl, 350 mM sucrose,

1 mM EDTA, 1 mM DTT, 0.1% Triton X-100) for 10 min on ice with occasional

shaking. The nuclei were collected by centrifugation (2,0003 g, 5 min), and the

supernatant was saved as the cytoplasmic fraction. The nuclei were washed

twice with buffer A without Triton X-100. RNA was purified using TRIzol and

checked by electrophoresis. Quantitative RT-PCR was used to measure the

enrichment of nuclear and cytoplasmic RNA.

Luciferase Reporter Assays

The Dual-Luciferase Reporter Assay System (Promega) was used to monitor

the promoter activity of the genomic inserts. Transfections were performed us-

612 Molecular Cell 55, 604–614, August 21, 2014 ª2014 Elsevier Inc.

ing TransIT reagent (MoBiTec). HEK293 cells were cotransfected with 50 ng of

the respective reporter constructs and 1 ng pRL-CMV in 384-well plates.

Firefly and Renilla luciferase activity were measured after 48 hr using Spectra

Max M5 plate reader (Molecular Device). The firefly luciferase signal was

normalized against the Renilla signal and against the empty vector pGL4.10

[luc2] (pGL4-basic) as control.

Quantitative DNA Methylation Analysis

DNA methylation was analyzed on bisulfite-converted genomic DNA (EZ DNA

Methylation Kit; Zymo Research) using the MassARRAY system (Sequenom)

as reported (Ehrich et al., 2005). DNA methylation levels were quantified from

mass spectra using Epityper software v.1.2 (Sequenom). The MassARRAY

primers for TCF21 locus are listed in Table S4.

Quantitative Hydroxymethylcytosine Analysis

The assay was performed as previously reported with minor modifications

(Kinney et al., 2011). Genomic DNA was purified with a commercial kit

(QIAGEN). A total of 500 ng of each DNA sample were resuspended in 50 ml

buffer (50 mM potassium acetate, 20 mM Tris-acetate, 10 mMmagnesium ac-

etate, 1 mM DTT [pH 7.9], and 100 mM uridine diphosphoglucose (NEB). DNA

was glucosylated with 4 units T4 phage beta glucosyltransferase (+ T4-BGT)

(NEB) or no enzyme (� T4-BGT). Samples were incubated at 37�C for 12 hr

and heat inactivated at 75�C for 10 min. One 1 unitMspI was added and incu-

bated for an additional 12 hr at 37�C, followed by heat inactivation at 75�C for

10min. DNAs were purified (QIAGEN kit), and the collected DNA was analyzed

by quantitative PCR. To normalize for differences ofMspI digestion efficiency,

the Cp values of the TCF21 promoter MspI site (CpG#7) was normalized to

qPCRs monitoring another MspI site located in CpG island 2. The ratio be-

tween (+T4-BGT) and (�T4-BGT) values was calculated, and the MspI resis-

tance is shown as 5-hydromethylcytosine relative level. A level of one is

considered background. In vitro methylation of TCF21 promoter plasmid

was carried as previously reported (Smith et al., 2006).

Quantitative RT-PCR

Relative expression was determined by qRT-PCR using the Universal Probe

Library System (Roche). cDNA was generated from 1 mg total RNA using oli-

go(dT) priming and Superscript II RT Kit (Invitrogen). Values were normalized

to the housekeeping genes HPRT1 and SDHA. Alternatively, gene-specific

primers were used (Table S3).

In Vitro Synthesis of TARID

DNA templates were generated by PCR using forward primers harboring the

T7 promoter. In vitro transcription assays contained 1 mg template DNA,

40 mM Tris-HCl (pH 7.9), 10 mM NaCl, 6 mM MgCl2, 10 mM DTT, 2 mM sper-

midine, 0.05% Tween-20, 0.5 mM each of ATP, GTP, CTP, and UTP, 0.5 U

RNase inhibitor, and 40 U T7 RNA polymerase (Promega). After incubation

for 16 hr at 37�C, RNA was purified and checked by gel electrophoresis. Biotin

and 4-thiouridine-labeled RNAs were synthesized using 1 mg template DNA in

40 mM Tris-HCl (pH 7.9), 10 mM NaCl, 6 mM MgCl2, 10 mM DTT, 2 mM sper-

midine, 0.05% Tween-20, 0.5 mM each of ATP, GTP, and CTP, 0.3 mM UTP,

0.05 mM biotin 16-UTP (Roche), and 0.05 mM 4-ThioUTP (Jena Bioscience),

0.5 U RNAsin, and 40 U T7 RNA polymerase (Promega). The genomic position

for RNA control intronic region (RNA6) used in RNApull-down experiments and

the capture assays is chr6: 134,211,494–134,212,061.

Bioinformatic and Statistical Analysis

Fickett’s test code algorithm was used to calculate Fickett’s TestCode scores

of TCF21 and TARID (Fickett, 1982). For prediction of secondary structure,

RNAz (http://rna.tbi.univie.ac.at/cgi-bin/RNAz.cgi) was used to calculate z

scores. To predict the transcription starting site in the TARID promoter, an on-

line tool was used (http://www.fruitfly.org/seq_tools/promoter.html). Fisher’s

exact test was used to test for differences in RNA expression or methylation

levels between two groups. The threshold for low and high methylation was

defined by the mean value plus two SD of the healthy tissues. The threshold

for low and high RNA expression was defined by values below and above

the mean of the cancerous tissues plus two SD. To test for the significance

of Spearman correlation coefficients, t distribution was calculated using Prism

Molecular Cell

LncRNA Demethylates the Tumor Suppressor TCF21

5 software (GraphPad). The statistical significance is considered to be signif-

icant if p < 0.05.

ACCESSION NUMBERS

The GenBank accession numbers for TARID T1–T5 reported in this paper are

KF484511, KF484512, KF484513, KF484514, and KF484515.

SUPPLEMENTAL INFORMATION

Supplemental Information includes five figures and four tables and can be

foundwith this article online at http://dx.doi.org/10.1016/j.molcel.2014.06.031.

ACKNOWLEDGMENTS

We thank Oliver Mucke and Jana Petersen for technical assistance. This work

was supported by funding from the Helmholtz Foundation, the German Con-

sortium for Cancer Research, and the National Institutes of Health, DE13123

to C.P. and by an ERC senior investigator grant N�249826-‘‘DNAdemethylase’’

to C.N. I.G.’s work has been supported by the DFG (GR475/22-1, SFB1036),

the excellence cluster CellNetworks, and the ERC (N�232645). Melanoma

cell line C8161 and pEF-FH-TET1 were kindly provided by Dr. Mary Hendrix

(Children’s Hospital of Chicago Research Center) and Dr. Anjana Rao (La Jolla

Institute for Allergy and Immunology), respectively.

Received: January 28, 2014

Revised: May 14, 2014

Accepted: June 24, 2014

Published: July 31, 2014

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