MYC-Induced Epigenetic Activation of GATA4 in Lung Adenocarcinoma

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Author Manuscript Published OnlineFirst on December 13, 2012; DOI: 10.1158/1541-7786.MCR-12-0414-T Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

MYC-induced epigenetic activation of GATA4 in lung adenocarcinoma

Authors: Inês C. Castro1, Achim Breiling2, Katharina Luetkenhaus1, Fatih Ceteci3, Simone Hausmann4, Sebastian Kress1, Frank Lyko2, Thomas Rudel1, Ulf R. Rapp5.

Inês C. Castro, Achim Breiling and Katharina Luetkenhaus contributed equally to this work.

Authors´ affiliations: 1Biocenter, Department of Microbiology, University of Würzburg, Würzburg, Germany; 2Division of Epigenetics, DKFZ-ZMBH Alliance, German Cancer Research Center, Heidelberg, Germany; 3Beatson Institute for Cancer Research, Glasgow, United Kingdom; 4Department of General Surgery, University Hospital rechts der Isar, Technical University of Munich, Munich, Germany; 5Max Planck Institute for Heart and Lung Research, Bad Nauheim, Germany

Running title: Epigenetic switch induced by MYC in lung adenocarcinoma

Keywords: GATA4, MYC, Lung adenocarcinoma, Metastasis, Epigenetics.

Corresponding Author: Ulf R. Rapp, Division Molecular Mechanisms in Lung Cancer, Max Planck Institute for Heart and Lung Research, Parkstraße 1, 61231 Bad Nauheim; Phone: Tel.: 0049-6032705420; Fax: 0049-6032705471; E-mail: [email protected]

Conflict of Interest: No potential conflicts of interest

Financial Support: Deutsche-Krebshilfe Foundation (grant-109081) and German Center for Lung Research (DZL)

Words number: 4896

Figures number: 6

Downloaded from mcr.aacrjournals.org on September 25, 2021. © 2012 American Association for Cancer Research. Author Manuscript Published OnlineFirst on December 13, 2012; DOI: 10.1158/1541-7786.MCR-12-0414-T Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Abstract

Human lung cancer is a disease with high incidence and accounts for most cancer related deaths

in both men and women. Metastasis is a common event in Non-Small Cell Lung Cancer

(NSCLC), diminishing the survival chance of the patients with this type of tumor. It has been shown that MYC is involved in the development of metastasis from NSCLC, but the mechanism underlying this switch remained to be identified. Here we focus on GATA4 as a MYC target in the development of metastasis with origin in lung adenocarcinoma, the most common type of

NSCLC. Epigenetic alterations at the GATA4 promoter level were observed after MYC expression in lung adenocarcinoma in vivo and in vitro. Such alterations include site-specific demethylation that accompanies the displacement of the MYC-associated zinc finger protein

(MAZ) from the GATA4 promoter, which leads to GATA4 expression. Histone modification analysis of the GATA4 promoter revealed a switch from repressive histone marks to active histone marks after MYC binding, which corresponds to active GATA4 expression. Our results thus identify a novel epigenetic mechanism by which MYC activates GATA4 leading to metastasis in lung adenocarcinoma suggesting novel potential targets for the development of anti-metastatic therapy.

Introduction

Non-Small-Cell lung cancer (NSCLC) is the most frequent type of lung cancer with high

metastatic potential and a low cure rate. Cancer has been classified as a genetic disease ever

since the discovery of retroviral oncogenes and the finding of frequent mutations in their cellular

counterparts in human tumors. Growing evidence now suggests that epigenetic alterations are at

least as common as mutational events in the development of cancer (1,2). Tumor-specific

promoter hypermethylation is well-documented (1). Epigenetic silencing is also known to be a

frequent event in NSCLC, i.e. of p16, H-cadherin, death-associated-protein (DAP) kinase1

(DAPK1), 14-3-3 sigma and the candidate tumor suppressor gene RASSF1A (3). However,

comparatively little is known about the role of promoter hypomethylation in gene activation in

cancer, especially in NSCLC.

We previously identified MYC as a metastasis inducer in a mouse model for NSCLC and in the human NSCLC cell line A549. Based on our observations in this mouse model, we suggested a

lineage switch mechanism to be involved in progression to metastasis and identified GATA4 as a

MYC-target in this context (4). Our earlier findings of RAF/MYC cooperation in the murine

hematopoietic system revealed that MYC affects tumor cell reprogramming (5). Furthermore,

amplifications and rearrangements of MYC genes were found in a fraction of human NSCLC (6).

The GATA family of transcription factors consists of six members, GATA1-6. These proteins

have two highly conserved zinc-finger domains and recognize a consensus DNA binding motif

of (A/T)/GATA/(A/G) (7). They are involved in the regulation of several biological processes

including organogenesis, differentiation, proliferation and apoptosis (7). While the GATA1-3

proteins are important for the development of the nervous system and hematopoietic cell

lineages, GATA4-6 are involved in organogenesis, especially of the heart, ovary and extraembryonic tissues. In the murine lung, GATA6 drives the expression of the thyroid transcription factor TTF-1, which is an upstream effector necessary for surfactant protein C (Sp-

C) expression (8). Furthermore, GATA6-Wnt signaling was shown to be important for epithelial stem cell development and airway regeneration (9). In contrast to GATA6, expression of

GATA4 was detected neither in adult lung nor in lung adenomas induced by oncogenic C-RAF.

(4). GATA4 is, however, involved in the maintenance of the intestine of the adult mouse.

Here we show that MYC induces epigenetic changes in the GATA4 promotor leading to its expression. Furthermore, MYC-dependent GATA4 upregulation was accompanied by site- specific demethylation and the acquisition of active histone modification marks in its promoter.

Our results thus suggest a further auto-regulation mechanism of GATA4 upon MYC expression.

Materials and Methods

Molecular Cloning

The coding sequence of GATA4 was subcloned into the EcoRI site of the retroviral vector pEGZ/GFP/Neo by using the oligonucleotides 5'-CCGGAATTCCGGATGTATCAGAGCTT

GGCCATGG-3' and 5'-CCGGAATTCCGGTTACGCAGTGATTATGTCCCC-3'. The resulting plasmid was confirmed by sequencing.

Cell Culture and Cell Lines

The A549 and Phoenix amphotrophic cell lines were obtained from the ATCC, where they are regularly verified by genotypic and phenotypic tests. All cell lines were cultured in DMEM medium supplemented with 10% FCS and 1% Pen/Strp. Cells were maintained in a humidified incubator at 37°C with 5% CO2 and harvested using 0.15% Trypsin-EDTA. For viral transfection, the MYC-ERTM virus encoding a fusion of the murine oestrogen receptor and human c-MYC, GATA4 shRNA and MAZ shRNA-producing virus were independently produced in the Phoenix amphotrophic packaging cell line. 10μg of pBpuro c-MYC-ERTM (10), pLKO.1-puro-shGATA4 (MISSION® shRNA Plasmid DNA from Sigma Aldrich; Clone

ID:NM_002052.2-1592s1c1) and pLKO.1-puro-shMAZ (MISSION® shRNA Plasmid DNA from Sigma Aldrich; Clone ID:NM_002383.1-761s1c1) were individually transfected into

Phoenix amphotrophic cells using Lipofectamine (Invitrogen) according to manufacturer’s instructions. After 24 hours the medium was changed. The viruses were produced overnight, and supernatant was then harvested and used for infection of A549 or A549 J5-1 (4) cells in the presence of 8μg/ml of polybrene. 24 hours after infection, infected cells were selected with 5

250μg/ml of puromycin. For viral infection of A549 cells with pEZG-GATA4, the procedure described above was used. After infection, cells were selected with 500μg/ml of zeocin. 1 week after selection, pools of 10 cells were seeded in a 96 wells plate and for each pool, GATA4 mRNA levels were measured. The pool number 11 showed the highest level of GATA4 mRNA and was used for further experiments. If mentioned, TSA (Sigma-Aldrich) was added to the culture medium at a concentration of 50ng/ml.

Soft Agar Assay

For the soft agar assay, the bottom agar was prepared using an autoclaved stock of 5% Sea

Plaque Agarose, which was microwaved and mixed with DMEM medium to a final concentration of 0.5% Agarose. 5ml of 0.5% Agarose were poured in a 60mm dish. For the top agar the 5% stock was diluted to 0.6% Agarose with DMEM medium and stored in a waterbath at 40°C. Dilutions of the cells were prepared in 1ml of medium (10000 cells per 60mm dish) and then mixed 1:1 with the 0.6% Agarose. The mixture was poured on top of the solidified bottom agar. After solidification of the top agar, the dishes were incubated in a wet chamber at 37°C for

21-28 days, when the colonies number was counted.

Immunohistochemistry

Immunohistochemistry was performed on paraffin sections. After deparaffinization and rehydration, sections were boiled in 10mM citrate buffer for 10-20min for antigen retrieval. To quench the endogenous peroxidase activity sections were incubated with methanol or PBS containing 1-3% H2O2. Non-specific antibody binding was prevented by incubation with 5% of

serum with 0.2% Trition X-100 in PBS for 1 hour at room temperature. After blocking, sections

were incubated with the primary antibody (GATA4, sc-1237, Santa Cruz (1:200); TTF1, M3575,

Dako (1:200); Pro Sp-C, gift from Jeffrey A. Whitsett (1:5000) over night at 4°C. After washing, sections were incubated with the corresponding biotinylated secondary antibodies (Dako) at

1:200-600 for 1 hour at room temperature. For staining ABC reagent was applied (Vactastain

Elite ABX Kit, Vector Labs) and colour was developed with diaminobenzidine (DAB). For counterstaining haematoxylin was used. After dehydration the stainings were mounted with enthellan. For immunofluorescence staining, the following secondary antibodies (all obtained from Jackson ImmunoResearch Laboratories, at 1:200 dilutions) were used: donkey anti-goat

Cy5 and donkey anti-rabbit Cy3.

Immunocytochemistry

Immunocytochemistry was performed on cells grown on coverslips. After fixation using 4%

PFA-PBS for 15min at room temperature, cells were washed with PBS. To prevent unspecific antibody binding, cells were blocked with 4% serum, 0.2% Triton X-100 in PBS for 1 hour at room temperature. The primary antibody was diluted in 4% serum in PBS and applied over night at 4°C (GATA4, sc-1237, Santa Cruz (1:250); GATA6, sc-9055, Santa Cruz (1:100)). After washing with PBS the cells were incubated with the secondary antibody for 2 hours at room temperature. The antibody was diluted in 4% serum in PBS (anti-goat-Alexa 647, 1:200; anti- rabbit-Cy3, 1:200). Before mounting with mowiol, cells were counterstained using DAPI.

Luciferase Reporter Assay

A 442 bp fragment upstream of the transcription start site of Gata4 was amplified from genomic

DNA from HeLa cells using the following primers: 5´- AAAAAACTCGAGG

GAACTAGCATCCAGCC-3´ and 5´-AAAAAAAAGCTTGCTGCA GCGGCGACGAA-3´. 7

The fragment was subcloned into XhoI and HindIII sites of the pGL3 Luciferase Reporter- Basic

Vector (Promega) by enzymatic reaction. A549 and A549 J5-1 cells were transfected independently with the pGL3 vector containing the GATA4 promoter and with the pGL3 control vector containing luciferase under control of a SV40 promoter. Caco-2 cells were transfected with the same vectors and used as a positive control, as they normally express GATA4 (11). To measure Luciferase activity, ONE-Glo Luciferase Assay System was used and performed in 96 well plates according to the recommendations in the manual. The lumimometer was pre-heated to 37°C and the luminescence was measured with an integration time of 100 ms.

Chromatin-Immunoprecipitation

Crosslinked chromatin from A549 control cells or A549 J5-1 cells was prepared and immunoprecipitated as described previously (12). ChIP-grade antibodies specific for

H3K27me3 (07-449), H3K4me2 (07-030) and H3K4me3 (07-473) were purchased from

Upstate. ChIP-grade antibodies specific for H3K9me2 (ab1220) and H3K9me3 (ab8898) were obtained from abcam. Other antibodies employed were against MAZ (abcam ab85725), EZH2

(Cell Signalling AC22), DNMT1 (active motive 39204), DNMT3a (abcam ab13888), DNMT3b

(activ motive 39207), POLII (4H8, abcam ab5408), GATA4 (Santa Cruz sc-1237) and GATA6

(Santa Cruz sc-9055). The chicken-v-MYC antibody was a gift from Klaus Bister.

Immunoprecipitates were finally dissolved in 30 µl of TE buffer (10mM Tris-HCL pH 8, 1mM

EDTA). 1µl was analysed by real time PCR using a primer pair specific for the GATA4 promoter region (CHIPGATA4_up 5’-CGGAGACCCCAGAGCCTG-3’; CHIPGATA4_lo 5’-

CTCTCTACCTCCAGACAAGC-3’) in 10 µl PCR reactions, using the Absolute QPCR SYBR

Green Mix (Themo Scientific) and a Roche LightCycler 480. PCR conditions: 1 cycle: 95°C x

15min; 42 cycles: 95°C x 15sec / 60°C x 40sec, read; melting curve 65°C - 95°C, read every

1°C. Cycle threshold numbers for each amplification were measured with the LightCycler 480 software and enrichments were calculated as percentage of the input.

DNA-isolation from paraffin sections

After GATA4 staining, sections were air-dried and GATA4-positive and –negative tumor regions were scratched from the slide using a scalpel. Material was collected in 30μl of lysis buffer (10mM TrisHCl, 1mM EDTA, 1% w/v Tween 20, 100μg/ml proteinaseK) and incubated overnight at 37°C. For inactivation of proteinaseK samples were heated at 95°C for 20min.

Bisulfite sequencing

For next generation bisulfite sequencing DNA from cultured cells was prepared using the

DNeasy Blood & Tissue Kit (Quiagen) according to manufacturer´s specifications. DNA from paraffin section was prepared as described above. Ca. 250ng up to 1μg of DNA was bisulfite treated with the EpiTect Bisulfite Kit (Qiagen) according to manufacturer’s instructions.

Deaminated DNA was amplified by PCR using the primer mentioned above, but with the 454- adapter sequence added and an identifier 4-base code for each tissue sample (here referred to as

XXXX): 454-COGATA4-prom_up 5’-

GCCTCCCTCGCGCCATCAGXXXXTAATAAAGTTGATTTTGGGTATTATAG-3’

454-COGATA4-1_lo 5’-

GCCTTGCCAGCCCGCTCAGXXXXCCCTACCTACTAAACCTAAAAATTC-3’. PCR conditions were as follows: 95°C for 3min followed by 40 cycles at 95°C for 30sec, annealing temperature for 40sec and 72°C for 45sec, followed by a 3min incubation at 72°C. PCR products

were subsequently purified using the QIAquick gel extraction Kit (Qiagen). For sequencing,

equimolar amounts of all amplicons were combined in a single tube Roche 454 FLX Standard sequencing was provided by the Core Genomics and Proteomics Core Facility of the DKFZ.

Real-Time PCR

RNA was isolated from cultured cells using TriFast reagent (Peqlab). cDNA-synthesis was performed using 1μg of RNA per reaction with first strand cDNA synthesis kit (Fermentas). qPCR was performed in a 20μl reaction using Finnzymes SYBER Green Master Mix (NEB) and

Rox as a reference dye. The amplification efficiency was raised to the power of the threshold

cycle (Ct-value) to calculate the relative amount of the transcript. This gives the number of

cycles necessary for the product to be detectable. The resulting value was normalized to the level

of the housekeeping gene. Assays were performed in triplicates in a StepOnePlus detection

system (Applied Biosystems). The primer sequences used were for Chicken-v-MYC 5´-

CGGCCTCTACCTGCACGACC-3´ and 5´-GACCAGCGGACTGTGGTGGG-3´, for hsGATA4

5´-CTACATGGCCGACGTGGGAG-3´and 5´- CTCGCCTCCAGAGTGGGGTG-3´, for

hsGATA6 5´-TCCATGGGGTGCCTCGACCA-3´ and 5´-CCCTGAGGTGGTCGCTTGTG, for

hsmucin2 5´-CGACTAACAACTTCGCCTCCG-3´and 5´-CGCGGGAGTAGACTTTGGTG-3´,

for hsMYC 5´-GCCCCTGGTGCTCCATGA-3´and 5´-

CAACATCGATTTCTTCCTCATCTTCT-3´ and for β-actin 5´-

GTCGTACCACAGGCATTGTGATGG-3´ and 5´-GCAATGCCTGGGTACATGGTGG-3´.

Ethics Statment

All animal studies were approved by the Bavarian State authorities for animal experimentation.

Statistical Analysis

Results are expressed as the mean ± s.e.m.. One-way analysis of variance was used to determine the differences among several groups, followed by Student-Newman-Keul's test using GraphPad

InStat software. Student’s t-test was used for two group comparisons. For all tests, statistical significance was considered to be at p<0.005.

Results

GATA4 expression is accompanied by GATA4 promoter demethylation in c-MYC/KRas-

mutant typeII-pneumocytes

Previously we observed a GATA6 to GATA4 switch in Sp-C-c-MYC and Sp-C-CRAF- BxB/Sp-

C-c-MYC lung tumors and liver metastasis (4). In this work we observed low or absent expression of pro Sp-C in GATA4-positive lung tumor regions from mice with the same

genotype (Fig. 1A), which suggests that GATA4-positive tumor cells might become independent

of any oncogenes under Sp-C promoter, including c-MYC, by an epigenetic mechanism. We have therefore surmised an epigenetic mechanism for the underlying mechanism of GATA4- induction in lung tumor.

We analyzed a 389 bp long CpG-rich region, which contains the promoter of the GATA4 gene

(Fig. S1A) by next generation bisulfite sequencing, using genomic DNA from GATA4-positive and –negative regions of a Sp-C-c-MYC/KRas-mutant lung tumor and a GATA4-positive liver metastasis (Fig. S1B). We did not observe global methylation changes for the examined region, but significantly reduced methylation levels for the CpG-sites 13-22 (with the exception of CpGs

15, 19 and 20 - see Table S1 for statistical tests) in the GATA4-positive tumor and metastasis material (Fig. 1B). Thus, this region showed clear hypomethylation in GATA4-expressing tissues.

MYC is necessary to induce the expression of GATA4 and its target gene mucin2 in human lung adenocarcinoma cells

If MYC expression is a prerequisite for induction of GATA4 expression in lung adenocarcinoma, the introduction of MYC into a GATA4-negative human lung adenocarcinoma cell line should lead to the expression of GATA4 and its target gene mucin2 (13). To test this possibility, we performed qPCR and immunofluorescence staining on the chicken-v-MYC expressing A549 J5-

1 cells (4). As shown in Fig. 2A, the mRNA expression of GATA4 and its target gene mucin2 was significantly upregulated in MYC-expressing A549 cells.

Moreover, using an inducible cell line carrying a fusion of MYC with the Estrogen Receptor (ER) which is dependent on tamoxifen presence to be located to the nucleus, we could show GATA4 and mucin2 mRNA upregulation after 3 weeks of MYC induction, although MYC induction occurs as early as 1 week after tamoxifen addition (Fig. 2B). This suggests that epigenetic changes at the GATA4 promoter take effect at this time point.

In contrast to our in vivo data GATA6 was also significantly upregulated in the A549 cells upon

MYC-expression (Fig. 2A). This suggests that other factors might superimpose positive regulation. Furthermore, we performed immunofluorescence staining for GATA4 and GATA6 and were able to detect both in the A549 J5-1 cells (Fig. 2C).

GATA4 is necessary for MYC induced anchorage independent growth in lung adenocarcinoma cells

The ability of cancer cells to form colonies without anchorage support is a key aspect of the tumor phenotype, particularly with respect to metastatic potential (14). A549 J5-1 cells showed

an increased ability to form colonies in soft agar in comparison to the parental cell line (Figs. 3A

and 3B). The inducible cell line A549 MYC-ER showed the same increased ability in the

presence of tamoxifen (Figs. 3A and 3B). The knockdown of GATA4 in A549 J5-1 (Fig. 3C) restored the behavior of wild-type A549 cells regarding anchorage-independent growth ability

(Figs. 3D and 3E). These data suggest that GATA4 might be necessary for the transformation phenotype of MYC-expressing A549 cells. Based on these data, a newly prepared A549 cell line overexpressing GATA4 (Figs. 3F and 3G) was tested for the same feature, which showed that

GATA4 alone is able to mimic MYC induced ability to grow anchorage independently in A549 cells (Figs. 3H and 3I), strengthening the evidence that GATA4 is necessary for MYC induced metastasis. While growing in adherent culture, the A549 GATA4-11 cells did not show a difference in proliferation when compared to A549 cells (data not shown).

MYC is able to induce GATA4 promoter demethylation in human lung adenocarcinoma cells

To test whether ectopic MYC expression is sufficient to induce demethylation of the GATA4 promoter in a human lung adenocarcinoma cell line, we performed next generation bisulfite sequencing of the same region analyzed above in A549 and A549 J5-1 cells. Also, in A549 cells, the GATA4 promoter region was strongly methylated, which did not change significantly upon

MYC expression (72% and 71%, respectively). Nevertheless, as shown in Figs. 4A and 4B we observed a significant reduction in methylation at CpG sites 19-22 (with the exception of site 21

- see Table S2 for statistical tests) in these cell lines. These results thus confirm the data obtained in mouse lung tumors and strongly suggest that ectopic MYC expression is sufficient to induce local demethylation of the GATA4 promoter.

Epigenetic signature of the GATA4 promoter

To complement our analysis of epigenetic modifications in the GATA4 promoter, we mapped

histone modifications by chromatin immunoprecipitation (ChIP) in the same region. Chromatin

was prepared from A549 control and A549 J5-1 cells and precipitated with antibodies against

histone H3 dimethylated at lysine 9 (H3K9me2), histone H3 trimethylated at lysine 9

(H3K9me3), histone H3 dimethylated at lysine 4 (H3K4me2), histone H3 trimethylated at lysine

4 (H3K4me3) and histone H3 trimethylated at lysine 27 (H3K27me3). Immunoprecipitated DNA

was analysed by qPCR using a primer pair specific for the GATA4 promoter region. This PCR

fragment of 372 bp spans a region starting with CpG 8 and extending about 200 bp into the

GATA4 gene body. In agreement with the strong methylation of the promoter in A549 control

cells, we found mainly repressive histone marks in the GATA4 promoter region (H3K9me3 and

H3K27me3), most prominently H3K27me3, which would indicate repression by Polycomb

Group (PcG) proteins (Fig. 5A). In contrast, in the MYC expressing line J5-1, the levels of

repressive marks were low, whereas both active H3K4 marks were enriched, indicating ongoing

transcription at the GATA4 promoter. These results suggest a significant change in the epigenetic

makeup of the GATA4 promoter upon MYC expression, which is characterized by local DNA

hypomethylation and a switch from repressive to active histone marks.

Protein occupancy in the GATA4 promoter region

In order to identify proteins interacting with the GATA4 promoter in A549 control and A549 J5-1

cells, we analyzed a fragment of the GATA4 promoter region corresponding to the segment

amplified by the bisulfite primers with the MatInspector software (15). This revealed the

presence of a MYC-related E-box, the potential binding site for the MYC-associated zinc finger

protein MAZ, which is usually shared by the SP1 transcription factor (Fig. S1C). In addition we

identified a Y-box and consensus sequences for the general transcription factor TFIID between

CpGs 15 and 22. To validate these potential binding sites and to gain insight into the protein

presence at the GATA4 promoter in control and MYC expressing cells, we performed ChIP using

antibodies against chicken-v-MYC (chk-MYC), GATA4, GATA6, MAZ, the three major DNA methyltransferases (DNMT1, DNMT3a and DNMT3b), the histone H3 lysine 27 methyltransferase Enhancer of Zeste 2 (EZH2) and the large subunit of the DNA-polymerase II

(POL II) in chromatin prepared from A549 control cells and the J5-1 cell line.

Immunoprecipitated DNA was analysed by qPCR using the same primer pair as for the analysis of histone modifications. In the repressed case (A549 control), we found strong binding of MAZ and EZH2 in the GATA4 promoter, which was paralleled by the presence of DNMT1 (Fig. 5B).

In A549 J5-1, MAZ and EZH2 binding was greatly reduced, whereas chk-MYC (the protein expressed in this cell line) and GATA4 itself were found enriched on the GATA4 promoter.

While DNMT3a and DNMT3b were detectable only at background levels, DNMT1 showed a significant interaction with the GATA4 promoter region (Fig. 5B). DNMT1 levels did not differ significantly between the two cell lines, which is in line with the observation that the overall methylation of the region did not change in MYC expressing cells. GATA6, as GATA4 antagonist, could not be found interacting either in control or in J5-1 cells. In addition to the observed activation of the GATA4 promoter in J5-1 cells, we found increased interaction of POL

II with the promoter (Fig. 5B), which is again consistent with increased transcriptional activity.

MAZ plays a role in GATA4 induction by MYC

MAZ has been described as a recruiting factor of DNA methyltransferases (DNMTs) and histone deacetylases (HDACs) (16). As we found MAZ to be displaced from the GATA4 promoter after

MYC expression in A549 cells, we predicted that the knockdown of MAZ in A549 cells could also lead to GATA4 upregulation. MAZ absence might result in the blocking of DNMTs and/or

HDACs recruitment to GATA4 promoter leading to GATA4 transcription activation and mimicking the transformation phenotype induced by GATA4. Therefore we used a shRNA- mediated knockdown of MAZ in A549 cells and which were afterwards seeded in soft agar to test if the lack of MAZ in A549 is able to induce anchorage-independent growth. Depletion of

MAZ was sufficient to induce anchorage independent growth in A549 cells in a significant manner (Figs. 5C and 5D), suggesting that the knockdown of MAZ might be sufficient for the phenotype transformation of A549 cells. Moreover, as postulated above, MAZ-depleted A549 cells also showed an upregulation of GATA4 and its target mucin2 (Fig. 5E).

In conclusion, our findings suggest a scenario where GATA4 is usually kept repressed by MAZ,

DNMT1 and PcG containing complexes. Upon MYC expression, MYC interacts with its binding site (E-box) near the GATA4 promoter, displacing MAZ and EZH2. This leads to specific DNA- hypomethylation of the region containing the MAZ-binding site and the E-box due to the absence of DNMT1 recruitment. The recruitment of GATA4 to its own promoter suggests some further auto-regulation of this transcription factor (Fig. 6).

Discussion

A mutually exclusive expression of GATA4/GATA6 triggered by MYC was previously reported

both in lung tumors and corresponding metastasis from animals expressing c-MYC in type II

cells under the control of the Sp-C promoter, which suggests that this lineage switch occurring in the primary tumor is important for the development of c-MYC-induced metastasis (4).

In this work we stained lung tumor tissues from animals with the same genotype and observed that GATA4 positive regions show low pro Sp-C expression. This can represent loss of differentiation or reprogramming and suggests that these regions are presumably poor for any transgene expression that is under Sp-C promoter, like c-MYC (Fig. 1A). These assumptions led us to the investigation of the epigenetic mechanism behind MYC-induced GATA4 expression suggested in this work.

MYC drives GATA4 expression in human lung adenocarcinoma cells

The upregulation of GATA4 and its functional target mucin2 was also detected in MYC expressing A549 cells (Figs. 2A and 2B) and occurred 3 weeks after ectopic MYC expression

(Fig. 2 B), suggesting that a multi-step mechanism might be involved in the changes induced by this oncogene. Accompanying GATA4 upregulation, MYC expressing A549 cells showed a metastatic behaviour (14), in particular increased ability to grow anchorage independently in soft agar, which was reverted after GATA4 knock-down (Figs. 3A, 3D, 3E, 3H and 3I). These data indicate the direct involvement of GATA4 in MYC induced metastasis. In fact, GATA4 is a lineage selector gene, and its upregulation suggests dedifferentiation and loss of organ identity, which is in line with the theory that the metastatic process is a recapitulation of ontogeny (5). 18

GATA4 promotes the expression of the anti-apoptotic factor Bcl2 and cyclin D2 (17), increases

proliferation and migration capacities in colorectal cancer (18) and its altered expression is

correlated with a broad range of tumors emerging from gastrointestinal tract, lungs, ovaries,

brain and with poor prognosis in ovarian granulose cell tumors (19).

MYC induces GATA4 promoter demethylation

Growing evidence now suggests that epigenetic alterations at the promoter level are at least as common as mutational events in the development of cancer (1,2). Tumor-specific promoter hypermethylation is well documented in NSCLC (1), but comparatively little is known about gene activation by promoter hypomethylation. In the present work, we show that GATA4 upregulation in lung adenocarcinoma is accompanied by site-specific demethylation of the CpG islands 13-22 of its own promoter upon MYC-expression in vitro, in primary tumors and in

corresponding liver metastasis (Figs. 1B, 4A and 4B). Interestingly, this region contains an E-

BOX, a potential binding site for MYC (20,21), which was previously described as a key

regulatory element of GATA4 expression in vitro (22) and in vivo (23). A Y-BOX sequence is located directly downstream. The Y-BOX binding protein 1 regulates the expression of many important genes (24) and is involved in the development of metastasis in patients with gastric and breast cancer (25). This protein may bind to the hypomethylated region of the GATA4

promoter and thus regulate its expression, or even act as a partner in metastasis development.

Although controversial, hypermethylation of GATA4 in lung cancer has been reported (18,26). A

study from Guo et al. on the methylation of GATA genes in lung cancer used several lung cancer

cell lines revealing GATA4 silencing by hypermethylation in most of the tested cells, including

A549 (26). Curiously, the only cells extracted from metastatic tissue - H157 - were unmethylated

in the GATA4 promoter region analyze (26). Montavon et al. showed that the GATA4 promoter was kept unmethylated in all tissues collected from patients with a rapidly invasive ovarian cancer (High-Grade Serous Ovarian Cancer), while tumors from patients with less invasive ovarian cancers, showed hypermethylation of the GATA4 promoter (27). This supports our observations that hypomethylation-dependent upregulation of GATA4 is involved in metastasis development. Since MYC preferentially binds to promoter regions and CpG-rich regions (21), our data highlights this oncogene as a key-player in epigenetic modifications occurring at

GATA4 promoter level.

MYC induces the enrichment of active histone marks at the GATA4 promoter

DNA methylation acts in cooperation with histone tail modifications (28). In the present work,

corresponding to the strong methylation of the GATA4 promoter of the A549 control, mainly

repressive histone marks H3K9me3 (1), and most prominently trimethylation of lysine 27 of

histone H3 (29) were found in control cells (Fig. 5A). The enrichment of H3K27me3 indicates

repression by the Polycomb repressive complex 2 (PRC2) (30) which methylates H3K27 via its

catalytic subunit – Enhancer of Zeste 2 (EZH2) (29,30) -, which was indeed found to be enriched

at the GATA4 promoter of A549 wild-type cells, in contrast to the low binding of EZH2 detected at the GATA4 promoter of MYC-expressing A549 cells. This supports our finding that GATA4 expression is repressed in A549 cells, and that this event is accompanied by an enrichment of the methylated target of PRC2, H3K27.

In contrast to the enrichment of repressive histone marks at the GATA4 promoter of wild-type

A549 cells, in the MYC expressing line J5-1, the levels of repressive marks are low, while both active H3K4 marks (1) are enriched, indicating ongoing transcription at the GATA4 promoter

(Fig. 5A). The recruitment of PolII - the key rate-limiting step in gene activation (31) - co-occurs with the enrichment of the active H3K4 marks (32) and was observed in MYC-expressing A549 cells. Altogether, these data demonstrate significant changes in the epigenetic landscape of the

GATA4 promoter upon MYC expression leading to the transcriptional activation of GATA4.

MYC induces changes in protein occupancy at GATA4 promoter

Broad changes in protein occupancy at the GATA4 promoter of A549 cells upon MYC expression were also observed. Among other proteins tested, GATA4 markedly bound to its own promoter upon MYC expression. On the other hand, MAZ (16) and the already mentioned EZH2 protein were displaced from the GATA4 promoter in MYC-expressing A549 cells (Fig. 5B).

Judging from the specific and very restricted hypomethylation observed in MYC positive tumors or MYC expressing A549, most likely also DNMT1 is displaced from or looses access to the region encompassing CpGs 13-22. Nevertheless the resolution of the ChIP-method employed

(ca. 400 bp) is not suitable to resolve such restricted changes in DNMT1 levels. MAZ displacement from this region nevertheless suggests that it has a role as DNMT1 recruiter, which may be a key event for GATA4 repression in wild-type A549 cells. MAZ displacement might prevent maintenance methylation due to the absence of DNMT1-recruitment and therefore provoke very site-specific hypomethylation at the GATA4 promoter. Although the knock-down of MAZ in A549 cells did not lead to a very pronounced GATA4 upregulation, it led to a dramatic increase in mucin2 mRNA levels and in the ability of these cells to form colonies anchorage-independently. These data suggest that moderate changes in GATA4 levels can result in strong upstream effects and highlight MAZ as a key-player in MYC-induced metastasis via

GATA4 activation (Figs. 5C, 5D and 5E). Indeed, a previous study from Bartoszewski et al

showed that the repression of the gene coding for the cystic fibrosis trans-membrane

conductance regulator (CFTR) is maintained during endoplasmatic reticulum stress by

recruitment of methyl binding proteins and/or HDACs. The repressed state of CFTR depends on

the binding of MAZ to a hypermethylated region of the promoter, demonstrating methylation-

specific repression by MAZ in this case as well (33).

Our findings suggest a scenario where GATA4 is usually kept repressed by MAZ, DNMT1, and

PcG containing complexes. Upon MYC expression, chk-MYC interacts with its binding site (E-

box) near the GATA4 promoter, which leads to MAZ and EZH2 displacement and therefore

MAZ-recruitment of DNMT1 to the MAZ-binding site and the E-box is blocked. Demethylation

of these regions occurs as a consequence of replication proceeding in the absence of maintenance

methylation, provided by DNMT1 (34), leading to subsequent GATA4 activation. Activated

transcription of GATA4 might induce its auto-regulation by interacting with its own promoter

(Fig. 6). We propose a novel molecular mechanism for GATA4 activation in lung

adenocarcinoma, which is dependent on epigenetic changes on its own promoter and leads to

metastatic conversion. Moreover, we were able to identify GATA4 as a MYC target in malignant lung adenocarcinoma, and highlighted GATA4 as a potential target for anti-metastatic therapy.

Acknowledgments

We are grateful to Jeffrey A. Whitsett for pro Sp-C antibody, Roland Halter for Sp-C-c-MYC mice, Klaus Bister for chicken-v-MYC antibody and Daniel J. Murphy for the MYC-ER construct. We also thank Tanja Musch and Francesca Tuorto for supporting bisulfite sequencing.

Grant Support

Inês Castro was supported by the grants GRK1411/1, BMBF–01GU0709 and FORPROTECT and Katharina Luetkenhaus by the grant GRK1411/1.

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Figure Legends

Fig. 1- Ectopic expression of GATA4 in Sp-C-c-MYC/Ras-mutant lung adenocarcinoma is accompanied by site-specific GATA4 promoter demethylation.

Fig. 1A - Staining of mouse lung tumor sections for Sp-C and Gata4 shows mutually exclusive expression patterns between them. Lung tumor serial sections were stained for Sp-C and Gata4

(left and middle panel). Gata4 high/Sp-C negative- and Gata4 low/Sp-C positive tumor regions were encircled with red and green dashed lines, respectively. The most right panel shows double immunofluorescence staining of a mouse lung tumor section for Sp-C and Gata4. Note that the majority of Gata4 positive tumor cells (red) are negative for Sp-C (green). Dapi (blue) shows nuclei. Scale bar = 50 µm.

Fig. 1B – Next generation bisulfite sequencing of the CpG-rich region near the GATA4 promoter

(for exact primer locations see Fig. S1A). DNA was isolated from lung tumor regions stained negatively and positively for GATA4 and from GATA4-positive liver metastasis, respectively.

Sequencing results are shown as heatmaps in which each row represents one sequence read.

Individual red boxes indicate methylated and green boxes indicate unmethylated CpG dinucleotides. Sequencing gaps are shown in white. CpGs showing the highest degree of change between DNA from GATA4-positive and –negative tumor tissue are boxed. The overall methylation of the region is 76% for both GATA4-positive and –negative lung tumors and 73% for the liver metastasis. A significant decrease in methylation of the CpGs 13-22 (boxed, 66% in

GATA4 negative tissues) was observed in GATA4 positive tissue (44% in GATA4 positive

tissues). For statistical tests see Table S1. The DNA used for the next generation sequencing was isolated from GATA4-stained paraffin-sections as illustrated in Fig. S1B.

Fig. 2 - MYC-expression in A549 cells leads to upregulation of GATA4 and its target mucin2.

Fig. 2A- qPCR analysis of A549 cells infected with chicken-v-MYC expressing J5 virus shows highly significant expression of exogenous MYC. The expression of MYC in the human lung adenocarcinoma cell line A549 leads to a highly significant upregulation of GATA4 and its target gene mucin2 on mRNA level. In parallel, GATA6 was also significantly upregulated.

Experiments were performed on the basis of three independent RNA-isolations.

Fig. 2B - mRNA levels of human MYC, GATA4 and mucin2 in induced and not induced A549

MYC-ER cells over time. GATA4 and its target mucin2 are upregulated after 3 weeks of MYC induction in A549 MYC-ER cells, although MYC upregulation is detected as early as 1 week after induction.

Fig. 2C - Consistent with the RNA-expression, immunocytochemistry demonstrates an induction of GATA4 expression on the protein level in the A549 J5-1 cells. Green (Cy3) colored cells are

GATA4 positive; red (Alexa 647) colored cells are GATA6 positive; blue (DAPI). Scale bars =

50 µm.

All error bars represent the SD of the mean. Statistical differences between groups are indicated.

Fig. 3 - GATA4 is necessary for anchorage independent growth induced by MYC

Fig. 3A - Anchorage independent growth of A549 MYC-ER cells in the presence of OHT.

Colonies formed by A549 and A549 EV (Empty Vector) in the presence of OHT (controls), and

A549 J5-1 and A549 MYC-ER in the presence of OHT, after 3 weeks in soft agar are shown.

Fig. 3B - Quantification of colonies formed by the different cells lines (conditions as indicated).

A549 MYC-ER cells show, in the presence of OHT, a comparable ability to form colonies as

A549 J-51 cells, indicating the induction of anchorage independent growth by MYC.

Fig. 3C - mRNA levels of human GATA4 in A549, A549 J5-1 and A549 J5-1 shGATA4 26.

The sh-RNA mediated knock-down of GATA4 in A549 J5-1 cells restored the low values of

GATA4 mRNA levels of A549 wild-type cells.

Fig. 3D - Anchorage independent growth of A549 J5-1 cells after knockdown of GATA4.

Quantification of colonies formed by the 3 different cell lines indicated. The soft agar assay with the A549 J5-1 cells shows a highly significant reduction in the number of colonies after infection with the shRNA26-expressing virus.

Fig. 3E - Colonies formed by A549, A549 J5-1 and A549 J-51 shGATA4 26 cells after 3 weeks in soft agar are shown.

Fig. 3F - A549 cells infected with the pEGZ/GATA4 vector show GATA4 expression. mRNA levels of GATA4 and EGFP in A549 cells infected with the plasmid pEGZ/GATA4 are shown.

Pools of 10 cells were seeded in a 96 well plate after infection and selected with zeocin. The best

candidate was pool number 11, showing the highest level of GATA4 mRNA levels and used for

further experiments.

Fig. 3G - Immunocytochemistry for human GATA4 in A549 and A549 GATA4–11 cells.

GATA4 and GFP expression was detected in the nucleus of A549 GATA4–11 cells. Red (Cy5)

colored cells are GATA4 positive; Green cells are GFP positive; Scale bars = 50 µm.

Fig. 3H - Growth in soft agar resulted in an increase of number and size of colonies in the case of A549 GATA 4–11 cells compared to the parental cell line A549.

Fig. 3I - Quantification of the colonies formed by A549 and A549 GATA4-11, showed a significant increase of the colonies formed in GATA4 expressing cells.

All error bars represent the SD of the mean. Statistical differences between groups are indicated. ns: not significant

Fig. 4 - Site-specific demethylation of CpG dinucleotides in the promoter region of GATA4

upon MYC expression in A549 cells.

Next generation bisulfite sequencing of the GATA4 promoter in A549 (A) and A549 J5-1 (B)

cells (for exact primer locations see Fig. S1A). Sequencing results are shown as heatmaps in

which each row represents one sequence read. Individual red boxes indicate methylated and

green boxes indicate unmethylated CpG dinucleotides. Sequencing gaps are shown in white.

There is no significant change in the overall methylation-status of the region (72% in A549 and

71% in A549 J5-1), but significant demethylation of the CpGs 19-22 (right box) was observed

(49% in A549 versus 37% in A549 J5-1). For statistical tests see Table S2.

Fig. 5 - MYC and GATA4 bind to the GATA4 promoter upon expression of exogenous

MYC in the A549 cells, while MAZ is displaced

Fig. 5A - Chromatin immunoprecipitation (ChIP) analysis of the GATA4 promoter region in control (A549) and MYC expressing cells (A549 J5-1). ChIP was repeated at least three times with chromatin from biological replicates using antisera specific for H3K9me2, H3K9me3,

H3K4me2, H3K4me3 and H3K27me3. Immunoprecipitated DNA was analyzed by real time

PCR and a primer specific for the GATA4 promoter region. Enrichments are shown as percentage of the total input. Error bars show the SD of the mean.

Fig. 5B - ChIP assay as in (A) monitoring occupancy of the GATA4-promoter by candidate proteins chicken-v-MYC (chk-MYC), GATA4, GATA6, MAZ, the three major DNA methyltransferases (DNMT1, DNMT3a and DNMT3b), Enhancer of Zeste 2 (EZH2) and the large subunit of the DNA-polymerase II (POL II). Binding of chicken-v-MYC and GATA4 on the GATA4 promoter was significantly higher in the MYC expressing A549 J5-1 cells than in the parental cell line. Error bars show the SD of the mean.

Fig. 5C - Anchorage independent growth of A549 cells after knockdown of MAZ. MAZ knockdown in A549 cells lead to GATA4 activation and increased anchorage independent growth.

Pictures from the colonies formed by A549 and A549 shMAZ 345 cells after 3 weeks in soft

agar are shown.

Fig. 5D - Quantification of the colonies formed by A549 cells after infection with shRNA-

producing virus 354. The soft agar assay plate with the A549 shMAZ 345 cells showed a highly

significant increase in the number of colonies formed, when compared with the parental cell line,

A549.

Fig. 5E - GATA4 and mucin2 mRNA levels were measured in A549 and A549 shMAZ 345

cells. Results showed a significant increase in both GATA4 and mucin2 mRNA levels in MAZ-

depleted cells, compared with the parental cell line, A549.

Error bars show the SD of the mean. Statistical differences between groups as indicated.

Fig. 6 - Locked Switch Model of GATA4 expression induced by MYC.

Fig. 6A - Under physiological conditions MAZ binds to the GATA4 promoter. MAZ recruits

DNMT1, which in turn methylates the CpG-sites.

Fig. 6B - When MYC expression rises above a given threshold, MYC binds to the GATA4 promoter displacing MAZ. As a consequence DNMT1 is not recruited to this DNA-region any more.

Fig. 6C - After replication CpG-sites remain unmethylated due to the absence of DNMT1.

Fig. 6D - Activated transcription of GATA4 might induce its auto-regulation by interacting with its own promoter.

A #22620 (Sp-C-c-MYC/Kras*)

GATA4 Sp-C Sp-C / GATA4 red= areas of higggh GATA4 staining red= areas of higggh GATA4 staining. green= areas of low GATA4 staining green= areas of low GATA4 staining.

B #16740 (Sp-C-c-MYC/Kras*) #16740 (Sp-C-c-MYC) #22620 (Sp-C-c-MYC/Kras*) GATA4 neg. lung tumor GATA4 pos. lung tumor GATA4 pos. liver metastasis

159131619222630331591316192226303315 913161922263033 66% 44% 44%

Figure 1

A p<0.001 p<0.01 p<0.01 p<0.01 0.6 1.0 0.025 0.005 *** ** ** **

0.8 0.020 0.004 0.4 0.6 0.015 0.003 YC mRNA levels YC mRNA TA4 mRNA mRNA TA4 levels TA6 mRNA mRNA TA6 levels ucin 2 mRNA levels mRNA ucin 2 A A M M A A 00010.010 m 004.4 m 0.002 0.2 0.2 0.005 0.001 relative hs relative GG relative hs G 0.0 relative hs G 0.0 0.000 0.000 A549 A549 J5-1 A549 A549 J5-1 A549 A549 J5-1 A549 A549 J5-1

B A549 MYC-ER C 0.10 A49A549 MY C-ER + OHT p<0.01 A549 A549 J5-1 p<0.01 ** ** 0.08 p<0.05 0.06 * p<0.001 0.04 *** GATA4

ive hs MYC mRNA MYC mRNA levels hs ive 0.02 t t

rela 0.00 Week 1 Week 2 Week 3 Week 4

0.0020 p<0.01 **

0.0015 merge

p<0.01 4 mRNA 4 mRNA levels

A A 0.0010 **

ns ns 0.0005

relative hs GAT 0.0000 Week 1 Week 2 Week 3 Week 4 GATA6

0.005 p<0.01 levels ** 0.004

0.003 p<0.05 *

0.002 merge

0.001 ns ns

relative hs mucin2 mRNA 0.000 Week 1 Week 2 Week 3 Week 4

Figure 2

A549 A549 EV + OHT A549 J5-1 A549 MYCER + OHT B C D p<0.0001 ns *** p<0.0001 p<0.05 p<0.0001 60 *** * 0.08 100 *** m2 m2 evels l p<0.001 l p<0.01 m m m *** ** m 80 0.06 40 60 0.04 40 20 0.02 20 Number of colonies/78,5 colonies/78,5 of Number Number of colonies/78,5 colonies/78,5 of Number 0 hs GATA4 mRNA relative 0000.00 0

E F 0.004 p<0.0001 *** levels A A 0.003

0.002

0.001 A549 A549 J5-1 A549 J5-1 shGATA4 26

relative hs GATA4 mRN relative 0.000

G H I GFP GATA4 25 P<0.0001 *** 2

m m 20

15 A549 A549 10

0 -11 4 - 11 Number of colonies/78,5 m colonies/78,5 of Number 4 4 A A A549 GATA A549 GAT Figure 3

A B A549 A549 J5-1

1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 59% 49% 60% 37%

Figure 4

A B 14 4 0.77 A549 A549 12 2 A549 J5-1 0.66 A549 J5-1

10 0 0.55 t t 8 8 004.4

6 6 0.33 % inpu % input 4 4 0.22

0.1 2 2

0 0 0.00

C D E p<0.0001 p<0.05 40 p<0.0001 0.0005 0.003 *** *** * 0.0004 30 0.002 549 s/78,5 mm2 s/78,5 mRNA levels mRNA mRNA levels mRNA e e A A 000003.0003 20 0.0002 0.001 10 0.0001 Number of coloni of Number relative hs mucin2 relative hs mucin2 0 relative hs GATA4 0.0000 0.000 hMAZ 345 hMAZ s s A549

Figure 5

AB Methylation

GATA4 gene

GATA4 promoter

MAZ DNMT1 DNMT1

MAZ MYC CpG CpG CpG CpG CpG CpG CpG CpG CpG CpG CpG CpG CpG CpG 13 14 15 16 17 18 19 13 14 15 16 17 18 19

Replication

MYC CpG CpG CpG CpG CpG CpG CpG CpG CpG CpG CpG CpG CpG CpG 13 14 15 16 17 18 19 13 14 15 16 17 18 19 GATA 4

GATA 4 GATA 4

Figure 6

MYC-induced epigenetic activation of GATA4 in lung adenocarcinoma

Ines C Castro, Achim Breiling, Katharina Luetkenhaus, et al.

Mol Cancer Res Published OnlineFirst December 13, 2012.

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