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Epigenetic modulation of the miR-200 family isassociated with transition to a breast cancer stem-cell-like state

Yat-Yuen Lim1,2,3, Josephine A. Wright1,2, Joanne L. Attema1,2, Philip A. Gregory1,2, Andrew G. Bert1,Eric Smith4, Daniel Thomas1, Angel F. Lopez1,2, Paul A. Drew5, Yeesim Khew-Goodall1,6 andGregory J. Goodall1,2,6,*1Division of Human Immunology, Centre for Cancer Biology, SA Pathology, Adelaide, SA 5000, Australia2Discipline of Medicine, The University of Adelaide, Adelaide, SA 5005, Australia3Institute of Biological Sciences, Faculty of Science, University of Malaya, Kuala Lumpur, Malaysia4Discipline of Surgery, The University of Adelaide, Adelaide, SA 5005, Australia5School of Nursing and Midwifery, Flinders University, Bedford Park, SA 5042, Australia6School of Molecular and Biomedical Science, The University of Adelaide, Adelaide, SA 5005, Australia

*Author for correspondence (greg.goodall@health.sa.gov.au)

Accepted 27 February 2013Journal of Cell Science 126, 2256–2266� 2013. Published by The Company of Biologists Ltddoi: 10.1242/jcs.122275

SummaryThe miR-200 family is a key regulator of the epithelial–mesenchymal transition, however, its role in controlling the transition between cancer stem-

cell-like and non-stem-cell-like phenotypes is not well understood. We utilized immortalized human mammary epithelial (HMLE) cells toinvestigate the regulation of the miR-200 family during their conversion to a stem-like phenotype. HMLE cells were found to be capable ofspontaneous conversion from a non-stem to a stem-like phenotype and this conversion was accompanied by the loss of miR-200 expression. Stem-

like cell fractions isolated from metastatic breast cancers also displayed loss of miR-200 indicating similar molecular changes may occur duringbreast cancer progression. The phenotypic change observed in HMLE cells was directly controlled by miR-200 because restoration of its expressiondecreased stem-like properties while promoting a transition to an epithelial phenotype. Investigation of the mechanisms controlling miR-200expression revealed both DNA methylation and histone modifications were significantly altered in the stem-like and non-stem phenotypes. In

particular, in the stem-like phenotype, the miR-200b-200a-429 cluster was silenced primarily through polycomb group-mediated histonemodifications whereas the miR-200c-141 cluster was repressed by DNA methylation. These results indicate that the miR-200 family plays a crucialrole in the transition between stem-like and non-stem phenotypes and that distinct epigenetic-based mechanisms regulate each miR-200 gene in this

process. Therapy targeted against miR-200 family members and epigenetic modifications might therefore be applicable to breast cancer.

Key words: Epithelial–mesenchymal transition, Breast cancer stem cells, miR-200, DNA methylation, Histone modifications, Gene regulation

IntroductionEpithelial-derived tumours contain a heterogeneous population of

cells which can be characterized by differences in histopathology

and functional properties including proliferative and apoptotic

responses to therapies and capacity for anchorage-independent

growth (Hanahan and Weinberg, 2011). Recent evidence supports

the existence of a cellular hierarchy within epithelial tumours. At the

apex of this hierarchy is a tumour-initiating cell (T-IC) or cancer

stem cell (CSC) population that can self-renew and differentiate to

progeny cells, thus resulting in the observed cellular and functional

heterogeneity within epithelial tumours (Polyak and Hahn, 2006;

Reya et al., 2001). CSCs have been prospectively isolated from a

variety of solid tumours, including breast (Al-Hajj et al., 2003;

Ginestier et al., 2007), brain (Singh et al., 2004), colorectal (Dalerba

et al., 2007; Ricci-Vitiani et al., 2007), head and neck (Prince et al.,

2007), pancreatic (Li et al., 2007), prostate (Patrawala et al., 2007;

Patrawala et al., 2006), melanoma (Schatton et al., 2008), and

bladder (Chan et al., 2009). The characterization of these rare

tumorigenic cells has the potential to provide important prognostic

and therapeutic value for epithelial cancers.

Breast cancer stem-like cells (bCSCs) enriched in the CD44hi/

CD242/low subpopulation are proposed to be largely responsible

for cancer progression and metastasis (Al-Hajj, 2007; Al-Hajj et

al., 2003). They possess stem-like properties including the ability

to self-renew and differentiate into CD442/low/CD24hi progeny,

show resistance to standard therapies and increase in numbers

after short courses of fractionated irradiation (Phillips et al.,

2006). At the molecular level, bCSC gene signatures are

associated with decreased overall patient survival, poor

metastasis-free survival and tumour recurrence (Liu et al.,

2007). Furthermore, bCSCs become more pronounced in

tumour tissue following endocrine therapy or chemotherapy,

consistent with their selective post-treatment survival (Creighton

et al., 2009). Therefore, an improved understanding of bCSCs

potential to drive breast cancer progression could inform

therapeutic targeting of breast cancer.

The epithelial–mesenchymal transition (EMT) is a crucial

embryonic developmental process that is characterized by the

losses of E-cadherin, cell polarity, cell–cell and cell–matrix

contact as well as gain in motility and fibroblast-like morphology

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(Bracken et al., 2009; Kalluri and Weinberg, 2009). Recently,EMT has been shown to confer cells with stem-like properties,

with migratory and invasive capabilities associated withmetastatic competence (Mani et al., 2008). Multipletranscription factors including ZEB1, TWIST, SNAI1, andFOXC1 have been shown to induce EMT (Polyak and

Weinberg, 2009; Thiery et al., 2009). An EMT core signaturederived from the changes in gene expression shared byupregulation of Goosecoid, SNAI1, TWIST and TGF-b1, and

by downregulation of E-cadherin was closely associated with theclaudin-low and metaplastic breast cancers subtypes (Taube etal., 2010). Importantly, these two breast cancers subtypes were

reported to have a significant similarity to a ‘tumorigenic’ genesignature defined using patient-derived CD44+/CD242 breasttumour-initiating stem-like cells (Hennessy et al., 2009), furthersupporting the notion that EMT associates with stem-like

properties. Cancerous cells that have acquired a moremalignant undifferentiated state with worse outcome usuallydisplay mesenchymal-like characteristics that are more metastatic

and likely to relapse (Thiery et al., 2009). Hence, these studiessuggest that the bCSCs and cells with stem-like properties mayalso be EMT-like.

MicroRNAs (miRNAs) have been shown to regulate geneexpression and can control crucial cellular pathways, includingstem cell identity and lineage commitment (Gangaraju and Lin,2009). The acquisition of stem-like and EMT-like properties

involves major changes in gene expression, including miRNAs.Different transcription factors and miRNAs can interact withinhighly interconnected protein–protein, protein–DNA, and

protein–non-coding RNA networks (Orkin and Hochedlinger,2011). These networks can be governed by a milieu of chromatin-remodelling and modifying complexes to regulate chromatin

organization and gene expression including DNA methylationand histone modifications (Prezioso and Orlando, 2011; Young,2011).

The miR-200 family consists of five members (miR-200a,miR-200b, miR-200c, miR-141 and miR-429) which areclustered and expressed as two separate polycistronic pri-miRNA transcripts with the miR-200b-200a-429 cluster at

chromosomal location 1p36 and miR-200c-141 cluster atchromosomal location 12p13. A double negative feedback loopbetween the ZEB transcription factors and expression of miR-200

family members, for example, regulates the induction of EMTand the reverse process, mesenchymal to epithelial transition(MET) (Bracken et al., 2008; Burk et al., 2008; Gregory et al.,

2008). While loss of miR-200 family gene expression has beenassociated with EMT, understanding of the molecular linksbetween the miR-200 family and the stem-like cellular stateremains incomplete. Here, we report the use of immortalized

human mammary epithelial cells (HMLE) to investigate thefunction and regulation of the miR-200 during their conversionfrom a non-stem to a stem-like phenotype and during EMT. We

found that HMLE cells spontaneously converted from a non-stemto a stem-like phenotype and this change was accompanied byloss of miR-200 expression. Restoration of miR-200 in stem-like

cells partially reprogrammed these cells back to the non-stemphenotype, which was accompanied by distinct phenotypic andmolecular changes. Investigation of the epigenetic-based

mechanism controlling miR-200 expression revealed bothhistone modifications and promoter DNA methylation weresignificantly altered in non-stem and stem-like phenotypes. These

results highlight the importance of epigenetic regulation in themaintenance of miR-200 family expression in epithelial cells, andtheir aberrant silencing in EMT and stem-like cells.

ResultsHMLE populations include CD44hi/CD24low cells thatspontaneously arise from CD44low/CD24hi cells

To investigate the role of miR-200 in regulating stem-likeproperties in breast epithelial cells, we studied the immortalized

human mammary epithelial cell line designated HMLE. An earlypassage of this cell line contains a small subpopulation ofCD44hi/CD24low stem-like cells which is distinct from theparental CD44low/CD24hi cells (Mani et al., 2008). The natural

existence of two definitive populations prompted us to usefluorescence activated cell sorting (FACS) to separate the twopopulations and assess the role of the miR-200 family in

regulating stem-like properties. Upon serial passaging of theparental cells we observed an increase in the proportion of cellsthat displayed a mesenchymal morphology, which suggested that

epithelial cells spontaneously converted to mesenchymal cellsduring culture (Fig. 1A). This correlated with an increasedproportion of the CD44hi/CD24low subpopulation (Fig. 1A). To

further explore the nature of these spontaneously derived CD44hi/CD24low/mesenchymal-like HMLE cells, we isolated these cellsby FACS.

To determine whether the increase in CD44hi/CD24low HMLEcells was due to different proliferation rates, we measured the cell

proliferation rate using the MTS cell proliferation assay. Wefound no significant difference in growth rate between theCD44hi/CD24low and CD44low/CD24hi sub-populations (Fig. 1B).

This result suggested that the increase in proportion of CD44hi/CD24low cells was due to direct conversion from CD44low/CD24hi cells. Next, we determined whether the CD44hi/CD24low

HMLE cells could revert back to being CD44low/CD24hi, sincenormal tissue stem cells are able to give rise to differentiatedprogeny. Interestingly, the CD44hi/CD24low cells retained theirCD44hi/CD24low profile and mesenchymal morphology even

after multiple passages (Fig. 1C). These results support thenotion that there is a spontaneous conversion of CD44low/CD24hi/epithelial cells to a stable CD44hi/CD24low/mesenchymal-like

state over time. Henceforth, we termed these de novo

spontaneously derived CD44hi/CD24low/mesenchymal stem-likeHMLE cells, ‘sl-HMLE’ and the CD44low/CD24hi/non-stem

epithelial cells, ‘nsl-HMLE’.

sl-HMLE cells exhibit stem-like and EMT-like properties

To further explore the stem-like nature of the sl-HMLE cells, weassessed their ability to form mammospheres in culture, whichrelies on the ability of cells to survive and grow in non-adherent

culture conditions (Dontu et al., 2003). These experimentsrevealed that the frequency of mammosphere formation from sl-HMLE cells was initially similar to that from nsl-HMLE cells

(Fig. 1D). However, second generation cultures from sl-HMLEcells produced significantly more mammospheres than secondgeneration cultures from nsl-HMLE cells, indicating sl-HMLE

cells possess increased self-renewal potential (Fig. 1D). Stem-likecells have been described as being undifferentiated cells that do notexpress, or have lower expression of mammary lineage epithelial

markers including the luminal epithelial cytokeratin 18 (CK18)and the basal/myoepithelial cytokeratin 14 (CK14) (Dontu et al.,2003; Ponti et al., 2005; Yu et al., 2007). We found that sl-HMLE

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cells and cells from second generation sl-HMLE mammospheres

had lower expression of CK18 and CK14, consistent with a less

differentiated, stem-like phenotype (Fig. 1E).

To determine if the sl-HMLE cells have an EMT-like gene

signature, as suggested by their elongated mesenchymal

morphology, we analysed the expression of E-cadherin, N-

cadherin, ZEB1, ZEB2 and Twist1 (Fig. 1F). Consistent with their

mesenchymal morphology, the sl-HMLE cells had low

expression of E-cadherin, and high expression of the

mesenchymal associated genes, N-cadherin, ZEB1, ZEB2 andTwist1 (Fig. 1F). Taken together these data indicate that sl-

HMLE cells exhibit stem-like and EMT-like properties in

comparison to the nsl-HMLE cells including a less

differentiated phenotype and increased self-renewal potential.

The miR-200 family coordinately regulates EMT and stem-like features of mammary epithelial cells

The miR-200 family is an important regulator of EMT and recent

studies have demonstrated they also play a role in inhibiting

stem-like properties (Iliopoulos et al., 2010; Shimono et al.,

2009). To assess whether the miR-200 family can coordinately

regulate the stem-like and/or EMT-like properties observed in the

sl-HMLE cells, we measured these miRNAs by qPCR. These

experiments revealed that miR-200a, miR-200b and miR-200c

were downregulated in the sl-HMLE cells and sl-HMLE-derivedmammospheres compared to HMLE and nsl-HMLE cells

(Fig. 2A). To assess whether the miR-200 levels were similarly

reduced in CD44hi/CD24low/2 prospective bCSCs, cells were

isolated from pleural or ascites effusion samples obtained from

Fig. 1. sl-HMLE cells display stem-like and EMT-like properties. (A) Changes in cell morphology and CD44 and CD24 cell surface markers over several

passages. In the flow cytometry two-dimensional plots, the y-axis shows CD44 and the x-axis shows CD24 fluorescence intensities. Scale bar: 50 mm. (B) Growth

curve of CD44low/CD24hi and CD44hi/CD24low HMLE cells measured by the MTS assay. Data are means 6 s.e.m., n53. (C) Contour plots from flow cytometry

for CD44 (y-axis) and CD24 (x-axis) of sorted CD44hi/CD24low HMLE cells after 4 weeks in culture. (D) Sphere-forming efficiency of first and second generation

mammospheres formed from sl-HMLE and nsl-HMLE cells. Only spheres of 150 mm diameter or more were quantified. Data are means 6 s.e.m.; n54;

**P,0.01. (E) Changes in two epithelial lineage markers, CK18 and CK14. mRNAs were measured by real-time PCR. Data are means 6 s.e.m.; n53; **P,0.01,

normalized to GAPDH. (F) Changes in expression of epithelial and mesenchymal markers as measured by real-time RT-PCR. Data are means 6 s.e.m.; n53;

*P,0.05, **P,0.01, normalized to GAPDH and shown relative to the level in HMLE cells (for Twist1 n52). sl-HMLE M2 represents sl-HMLE-derived second

generation mammospheres.

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breast cancer patients (supplementary material Table S1) and the

CD452 subpopulation further sorted for their CD44/CD24 status.

We found that the miR-200 family members were consistently

downregulated in the CD452/CD44hi/CD24low/2 breast cancer

cells (prospective bCSCs) compared to the CD452/CD44low/

CD24hi and CD452/CD44hi/CD24hi non-CSC subpopulations

(Fig. 2B). These findings together with the results obtained from

sl-HMLE cells and sl-HMLE-derived mammospheres suggest

that the miR-200 family members are involved in cellular

pathways that give rise to stem-like properties. To further

investigate this role we generated stable sl-HMLE cell lines that

overexpress the miR-200b-200a-429 cluster or the miR-200c-141

cluster using pMSCV-puro retroviral vectors (hereafter referred

to as sl-HMLE MSCV; sl-HMLE MSCV miR-200b-200a-429; sl-

HMLE MSCV miR-200c-141). Enforced expression of miR-200

caused downregulation of ZEB1, ZEB2 and Twist1 with

concomitant increase in E-cadherin mRNA and protein

(Fig. 3A,B) and rearrangement of the actin cytoskeleton from

stress fibre to cortical pattern as well as junctional localization of

E-cadherin and ZO-1 (Fig. 3C). Thus, enforced expression of

miR-200 caused the sl-HMLE cells to undergo mesenchymal to

epithelial transition (MET).

Next, we investigated whether restoration of miR-200 in the sl-

HMLE cells results in loss of stem cell markers and functional

properties. sl-HMLE cells were analysed for CD44 and CD24

marker expression using flow cytometry and in vitro sphere

assays were performed to assess stem cell-like function.

Expression of miR-200 caused a shift from a predominantly

CD44hi/CD24low to the CD44low/CD24hi phenotype (Fig. 4A)

and reduced their sphere-forming potential, especially in the

growth of second generation spheres (Fig. 4B,C). Expression of

miR-200 in the sl-HMLE cells also partially restored CK18 and

CK14 expression (Fig. 3A,C), consistent with these cells

undergoing differentiation. Expression of miR-200 family

members in the sl-HMLE cells had little effect on proliferation

rate (Fig. 4D). In summary, these results demonstrate that the

miR-200 family can coordinately control EMT and stem-like

properties of mammary epithelial cells.

The miR-200 family exhibit differential DNA methylation

states in non-stem and stem-like cells

The sustained reduction in miR-200 gene expression in sl-HMLE

cells suggested that epigenetic changes might be involved in the

initiation and/or maintenance of the stem-like sl-HMLE

subpopulation. We and others have reported that repressed

miR-200 genes acquire promoter CpG hypermethylation

(Gregory et al., 2011; Neves et al., 2010; Vrba et al., 2010;

Wiklund et al., 2011). To investigate the degree of DNA

methylation occurring at the miR-200b-200a-429 and miR-200c-

141 genes (Fig. 5A) in the different HMLE cell types compared

to other breast epithelial and mesenchymal cancer cell lines, we

examined CpG promoter methylation using bisulfite PCR melt

curve analysis (Smith et al., 2009). The promoters proximal to the

transcription start site (TSS) of the miR-200 genes in HMLE and

MDA-MB-468 cells were unmethylated which is consistent with

the expression of the genes in these cells (Fig. 5B). In the

mesenchymal MDA-MB-231 and Hs578T cell lines that no

longer express the miR-200 genes, CpG hypermethylation was

detected across the promoter. Intriguingly, the CpG methylation

profiles of the miR-200b-200a-429 gene in HMLE and sl-HMLE

cells were quite similar; the TSS and promoter region was mostly

unmethylated except for region B where sl-HMLE cells had a

slight increase in DNA methylation (Fig. 5B). In contrast, miR-

200c-141 gene silencing and CpG promoter and primary

transcript methylation were positively correlated in the two cell

types. The sl-HMLE cells had a high degree of methylation in the

miR-200c TSS compared to HMLE cells (Fig. 5B). Similar

differences in CpG methylation profiles of miR-200b-200a-429

and miR-200c-141 were also observed in HMLE cells treated

with TGFb1 to enrich for the CD44hi/CD24low mesenchymal

HMLE population (HMLE+TGFb1) (Fig. 5B).

Although informative, the bisulphite PCR melt curve analysis

is limited in scope, allowing for the analysis of only a small

number of CpG sites within a defined genomic region. To

increase the resolution of this analysis, we employed the Illumina

HumanMethylation450 BeadChip array. This approach enabled

us to determine the changes in DNA methylation across the entire

Fig. 2. Stem-like cells and bCSCs express low levels of the miR-

200s. (A) Comparison of miR-200a, miR-200b and miR-200c levels

in sl-HMLE cells compared to nsl-HMLE cells and in sl-HMLE-

derived second generation mammospheres (M2) compared with

HMLE cells as measured by TaqMan real-time PCR. Data are means

6 s.e.m.; n52; *P,0.05, normalised to snRNA U6. (B) Expression

of the miR-200 family members in sorted CD452/CD44hi/

CD24low/2 prospective bCSCs and CD452/CD44low/CD24hi or

CD452/CD44hi/CD24hi non-CSC cells. miRNAs were measured by

TaqMan real-time PCR. Data are representative results from three

patients, normalized to RNU6B or snRNA U6 and are shown

relative to CD44lowCD24hi cells. Error bars represent standard

deviation of triplicate PCR assays.

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miR-200b-200a-429 and miR-200c-141 chromosomal regions in

HMLE and HMLE+TGFb1 cells. The results from the array were

consistent with the melt curve analysis of the miR-200 promoter

regions. Comparison of beta-values of all probes near the

promoter of the miR-200c-141, but not miR-200b-200a-429,

revealed a progressive increase in the level of DNA methylation

during transition to a mesenchymal/stem-like state (Fig. 5C).

Moreover, we found the miR-200b-200a-429 gene body was

heavily methylated compared to the miR-200c-141 gene body

region (data not shown). This observation was in line with other

described polycomb group target genes that typically have a

hypermethylated gene body and unmethylated TSS regardless of

gene expression or silencing (Deaton and Bird, 2011).

The miR-200b-200a-429 and miR-200c-141 gene promoters

exhibit distinct histone modification profiles in non-stem

and stem-like cells

The reduced levels of promoter CpG methylation at the miR-200b-

200a-429 gene compared to the heavily methylated miR-200c-141

promoter prompted us to consider whether additional epigenetic

modifications took place at these promoters in the sl-HMLE cells.

Thus, we performed chromatin immunoprecipitation coupled to

quantitative PCR analysis (ChIP-qPCR) to detect specific histone

modifications near the TSS of the miR-200 genes. Early passage

parental HMLE (,97% CD44low/CD24hi) cells and sl-HMLE cells

were analysed for activating (H3K4me3, H3K9/14ac and

H3K27ac) and silencing histone modifications (H3K27me3,

H3K9me2, and H3K9me3). The unmodified histone H3 control

was included for normalization between cell types and we verified

the various ChIP assays by performing ChIP analysis of the

characterised promoters of the b-ACTIN, GAPDH, MYT1, PAX5

and MYOD genes, which served as positive and negative controls

to indicate the expected levels of enrichment of histone

modifications and chromatin modifying enzymes for the two cell

types (data not shown). Analysis of the miR-200 genes revealed

that H3K4me3 and H3K9/14ac occupied the miR-200b-200a-429

and miR-200c-141 gene promoters in HMLE cells that express

these miRNAs (Fig. 6A). A higher level of enrichment of the

activating histone modifications was detected for miR-200c-141

compared to the miR-200b-200a-429 promoter region, which

could reflect increased expression levels of miR-200c-141

compared to miR-200b-200a-429 in HMLE cells (Gregory et al.,

Fig. 3. Effects of stable overexpression

of the miR-200s in sl-HMLE cells.

(A) Expression of E-cadherin, ZEB1, ZEB2,

Twist1, CK18 and CK14 in the stable pMSCV-

puro sl-HMLE cell lines as measured by

qPCR, normalised to GAPDH. Data are

representative of two independent viral

transductions (means 6 s.d.) and are expressed

relative to sl-HMLE MSCV EV. Error bars

represent standard deviation of triplicate PCR

assays. (B) Western blots showing E-cadherin,

ZEB1 and ZEB2 proteins in the cell lines.

Alpha-tubulin was used as loading control.

(C) Phase-contrast images (first row) and

E-cadherin, ZO-1, F-actin, CK18 and CK14

immunofluorescence staining of sl-HMLE

stable cell lines transduced with EV or miR-

200b-200a-429 or miR-200c-141 clusters.

DAPI staining (blue) was used to detect nuclei

and is merged with the indicated specific

antibody staining (red) in their respective

panels. Scale bar: 50 mm.

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2008; Wiklund et al., 2011). Consistent with the miR-200

expression in these cells, the silencing histone modifications,

H3K27me3, H3K9me2 and H3K9me3, were not detected across

the promoter regions of the miRNAs (Fig. 6A). In contrast to

HMLE cells, the activating marks across both miR-200 promoters

were erased in sl-HMLE cells. However, whereas the activating

marks at the miR-200b-200a-429 TSS promoter were replaced by

the polycomb-mediated H3K27me3 mark, this particular silencing

histone modification was not detected at miR-200c-141 gene

(Fig. 6A). ChIP-qPCR analysis of HMLE+TGFb1 cells revealed

similar correlations between the activating and silencing histone

modifications and miR-200 gene expression (Fig. 6A).

Fig. 4. Stable overexpression of the miR-200s in sl-

HMLE cells results in a change from stem-like to non-

stem phenotype. (A) Flow cytometry analysis of CD44

and CD24 expression on sl-HMLE cells and stable

pMSCV-puro sl-HMLE cell lines. (B) Formation of first

generation and second generation mammospheres from

the stable pMSCV-puro sl-HMLE cell lines. Only spheres

§150 mm in diameter were quantified. The data are

means 6 s.e.m.; n56; *P,0.05, **P,0.01. (C) Phase-

contrast images of sl-HMLE cells and stable pMSCV-

puro sl-HMLE cells cultured in the sphere assay (first

generation). Scale bars: 100 mm. (D) Proliferation rates

of the stable pMSCV-puro sl-HMLE cell lines measured

using MTS assays. The data are means 6 s.e.m.; n52.

Fig. 5. Methylation profiles of the miR-200 gene promoters.

(A) Schematic showing the regions (labelled A,B,C) analysed by

PCR melt curve analysis. (B) PCR melt curve analysis of the CpG-

rich regions encompassing the miR-200b-200a-429 and miR-

200c-141 transcription start sites in high miR-200 (HMLE, MDA-

MB-468) and low miR-200 (sl-HMLE, HMLE+TGFb1, Hs578T,

MDA-MB-231) human cell lines. The breast cancer cell lines were

characterized for miR-200 expression by Gregory et al. (Gregory

et al., 2008). Methylase-treated DNA from healthy donor

lymphocytes (M-ref) were used as a methylation reference/

positive control. (C) CpG methylation analysis of the miR-200b-

200a-429 and miR-200c-141 loci in HMLE cells following 8, 24

and 46 days of TGFb1 treatment using the Illumina HM450K

methylation array.

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Furthermore, miR-200b-200a-429 but not miR-200c-141 gene

repression was linked with polycomb-mediated H3K27me3 gene

silencing. Taken together, these results suggest that dynamic

changes in chromatin states across the miR-200 genes between

non-stem and stem-like cells are mediated by distinct and

complementary epigenetic mechanisms – DNA methylation in

the case of miR-200c-141 and PcG-mediated silencing in the case

of miR-200b-200a-429.

Polycomb group proteins associate with miR-200b-200a-

429 but not miR-200c-141 in sl-HMLE and EMT-induced

HMLE cells

To confirm that polycomb repressive complex 2 (PRC2)

complexes are associated with the miR-200b-200a-429

promoter in sl-HMLE and HMLE+TGFb1 cells, ChIP analysis

was performed using antibodies to the PRC2 components EZH2

and SUZ12. This analysis revealed that EZH2 and SUZ12

occupied the TSS of miR-200b-200a-429 but not miR-200c-141

in the sl-HMLE and HMLE+TGFb1 cells (Fig. 6A). By contrast,

EZH2 and SUZ12 were not detected across these promoters in

HMLE cells consistent with miR-200 expression (Fig. 6A).

These results indicated that in sl-HMLE and HMLE+TGFb1

cells, miR-200b-200a-429 but not miR-200c-141 is occupied by

PRC2, leading to H3K27me3-mediated gene silencing.

Interestingly, the levels of EZH2 and SUZ12 were higher in sl-

HMLE compared with nsl-HMLE cells and were repressed after

expression of miR-200 in sl-HMLE cells (Fig. 6B,C). This is

consistent with reports indicating miR-200b can directly repress

SUZ12 (Iliopoulos et al., 2010) and suggest loss of miR-200 in sl-

HMLE may enhance PRC2 mediated repression of miR-200b-

200a-429.

To investigate whether PgC-mediated silencing of miR-200b-

200a-429 gene may occur in breast cancers, we examined breast

cancer cell lines. We found that the luminal/epithelial breast

cancer cell line, MDA-MB-468, had a similar histone

modification profile to HMLE cells, consistent with their

expression of the miR-200 genes (Fig. 7). In contrast, in the

basal/mesenchymal breast cancer cell line, MDA-MB-231, which

no longer expresses the miR-200 family members, the cells had

H3K27me3 marks and EZH2 and SUZ12 association indicative

of polycomb repression at the miR-200b-200a-429 but not the

miR-200c-141 gene (Fig. 7). Similarly, H3K27me3 was found at

Fig. 6. Histone modification profiling of HMLE, sl-HMLE and

HMLE+TGFb1 cells. (A) Association of histone modifications with

the promoters of the miR-200b-200a-429 and miR-200c-141 genes in

HMLE, sl-HMLE and HMLE+TGFb1 cells. The x-axis shows the

distance from the transcription start site (marked with vertical dashed

line) and the y-axis shows the log2 enrichment values over input,

normalized to H3. Error bars represent standard deviation from

duplicate PCR assays. (B) Expression of SUZ12 and EZH2 in sl-

HMLE and nsl-HMLE. Data are means 6 s.e.m.; n52; *P,0.05 and

normalized to GAPDH. (C) Measurement of SUZ12 and EZH2 in the

stable pMSCV-puro sl-HMLE cell lines as measured by qPCR,

normalised to GAPDH. Data are representative of two independent

viral transductions (means 6 s.d.) and are expressed relative to sl-

HMLE MSCV EV. Error bars represent standard deviation of

triplicate PCR assays.

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the miR-200b-200a-429 promoter but not the miR-200c-141promoter in Hs578T cells (data not shown). Collectively, these

results indicate that repression of miR-200b-200a-429 and miR-200c-141 genes is mediated by distinct cell context-dependentepigenetic mechanisms involving specific and dual combinations

of histone and DNA methyltransferases.

DiscussionThe miR-200 family is a well-established mediator of EMT, buttheir ability to regulate transition between non-stem and stem-like states in a model system has not been well characterised.

Here, we show that the miR-200 family can coordinately regulateepithelial cell plasticity and stem-like properties of immortalizedhuman epithelial breast cells (HMLE). During the conversion to a

stem-like state, the miR-200 family genes are epigeneticallysilenced by distinct de novo processes involving DNA

methylation (in the case of miR-200c-141) and polycombprotein-associated repression (in the case of miR-200b-200a-429). These findings suggest genetic and epigenetic regulation ofmiR-200 plays an important role in plasticity between non-stem

and stem-like phenotypes.

In this study, we identified a spontaneously generatedsubpopulation of CD44hi/CD24low/2 stem-like cells derived

from HMLE which we termed sl-HMLE. The sl-HMLE cellsappeared to fit the dynamic EMT-CSC interconversion modelmore than the conventional stem cell/CSC model (Gupta et al.,

2009; Shackleton, 2010). This is an attractive model whichpermits the study of pathways that contribute to the gain in stem-like properties including miRNA and epigenetic changes. The sl-HMLE cells described here, although similar to EMT-induced

HMLE cells (Mani et al., 2008), have several differences: (a) thesmall subpopulation of CD44hi/CD24low fraction increased overtime in culture, (b) sl-HMLE cells are CK14-low, CK18-low, and

do not revert back to the HMLE or nsl-HMLE phenotype. Whilethis work was being finalised, Chaffer et al. (Chaffer et al., 2011)reported similar findings in transformed HMEC cultures. They

observed that the floating transformed HMECs have enrichedCD44hi/CD24low stem-like cells that do not revert back to theparental bulk population which is mainly CD44low/CD24hi.

Furthermore, these cells gave rise to ductal structures thatstained for both CK14 and pan-cytokeratin when injectedorthotopically into the humanized mammary fat pad of nudemice. Thus sl-HMLE behave as stem-like cells, which as an in

vitro model system, can be utilized to characterize the molecularand cellular mechanisms regulating conversion between non-stem and stem-like states.

The observation that HMLE cells undergo spontaneous andprogressive conversion into a stem-like/mesenchymal counterpartin culture led to the hypothesis that molecular-based switches

might be regulating this process. We recently showed thatMDCK cells are able to switch and maintain their cell phenotype(epithelial or mesenchymal) in culture due to an autocrine TGF-b/ZEB/miR-200 signalling network (Gregory et al., 2011).

Although the balance of miR-200 and ZEB levels via miR-200/ZEB feedback loop (Bracken et al., 2008; Burk et al., 2008) wascrucial for the MDCK cells to initiate a mesenchymal phenotype,

it required secretion of autocrine TGFb to drive stable ZEBexpression in order to maintain the mesenchymal phenotype inculture after removal of exogenous TGFb1 treatment (Gregory

et al., 2011). Similarly, sl-HMLE cells have increased expressionof genes in the TGFb, IL-6 and HAS-CD44 pathways. Thesepathways could potentially be involved in the conversion of

HMLE cells to de novo sl-HMLE cells in culture and alsomaintaining the stem-like/EMT-like phenotype of sl-HMLE cellsvia autocrine and paracrine signalling. Recent studies support theproposal that autocrine and paracrine signalling induce and

maintain embryonic stem (ES) cells (Berge et al., 2011) andstem-like/mesenchymal state in the breast (Scheel et al., 2011).

By stable re-expression of each miR-200 genomic cluster in

sl-HMLE, we demonstrate here that the miR-200 familyfunctionally represses stem-like properties. In addition we findthat bCSCs isolated from metastatic breast cancer patients, like

the sl-HMLE cells, have decreased levels of the miR-200 family.These results are consistent with other studies that show thatmiR-200 family members regulate stemness in normal mammary

Fig. 7. Histone modification profiling of MDA-MB-468 and MDA-MB-

231 cells. Association of histone modifications with the promoters of miR-

200b-200a-429 and miR-200c-141 clusters in MDA-MB-468 and MDA-MB-

231 cells. The x-axis shows the distance from the transcription start site

(marked with vertical dashed line) and the y-axis shows the log2 enrichment

values over input, normalized to H3. Error bars represent standard deviation

from duplicate PCR assays.

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stem cells (Shimono et al., 2009), bCSCs (Iliopoulos et al., 2010;Iliopoulos et al., 2009; Shimono et al., 2009), pancreatic and

colorectal CSCs (Wellner et al., 2009) and with the reversion ofEMT observed when miR-200 is expressed in the MDA-MB-231breast cancer cell line (Park et al., 2008; Burk et al., 2008).Furthermore, we showed that the expression of miR-200 family

members are dynamically associated with and at least partiallymaintained by epigenetic changes. Importantly, we found that thesilencing mechanism of the two clusters of miR-200 family

members in sl-HMLE and HMLE+TGFb1 cells were controlledby distinct mechanisms. The miR-200b-200a-429 cluster wasoccupied by the PcG proteins while miR-200c-141 was silenced

by DNA methylation. Intriguingly, this contrasts findings inMDCK cells induced to undergo EMT where DNA methylationoccurred at both miR-200 cluster promoters (Davalos et al., 2011;Gregory et al., 2011). During the preparation of this study for

publication, other groups demonstrated that combinatorialhistone and DNA modifications regulate the miR-200 familymembers (Au et al., 2012; Cao et al., 2011; Davalos et al., 2011;

Vrba et al., 2011). Cao et al. and Davalos et al. showed that PcGproteins can regulate both the miR-200b-200a-429 and miR200c-141 genes (Cao et al., 2011; Davalos et al., 2011). However, data

obtained from the sl-HMLE and HMLE+TGFb1 cells isconsistent with the findings from Vrba et al. and Au et al. thatPcG mediates the silencing of the miR-200b-200a-429 and not

the miR-200c-141 gene (Vrba et al., 2011; Au et al., 2012). Thisis supported by our findings in the basal breast cancer cell lines,MDA-MB-231 and Hs578T indicating that H3K27me3 occupiesthe miR-200b-200a-429 cluster and not miR-200c-141 cluster.

Together, these studies suggest that different cell lines mayacquire distinct silencing mechanisms to regulate miR-200expression. It will be interesting to determine whether distinct

epigenetic mechanisms regulate the miR-200 genes in othercontexts where plasticity between non-stem and stem-like statesoccurs.

PcG proteins are critical regulators of gene silencing importantfor tissue development, stem cell function, and X chromosomeinactivation (Prezioso and Orlando, 2011). While the cause forpolycomb-mediated silencing on the miR-200b-200a-429 but not

miR-200c-141 promoter remains unclear, it is consistent with theobservation that the miR-200b-200a-429 promoter shows higherGC content (.60%) compared to the miR-200c-141 promoter.

GC-rich promoters are often targeted by PcG and are commonlyprotected from CpG methylation (Deaton and Bird, 2011).However, in different types of cancers including breast and

prostate, PcG proteins such as EZH2 and SUZ12 are usuallyfound to be elevated (Iliopoulos et al., 2010; Kleer et al., 2003).This may cause aberrant PcG-mediated silencing of non-PcG

targets of genes that might contribute to aggressive poorlydifferentiated cancer. Interestingly, miR-200b was shown toregulate SUZ12 post-transcriptionally in bCSCs (Iliopoulos et al.,2010). This would result in another double-negative feedback

loop that adds a further layer of regulation on the miR-200b-200a-429 cluster in addition to the miR-200-ZEB1/2 feedbackloop (Bracken et al., 2008). Future experiments are needed to

determine if both PRC2 and ZEB1/2 act separately orsynergistically as a complex in this setting.

In summary, we have established that the miR-200 gene family

plays a functional role in regulating stem-like properties observedin the HMLE cell line model. Moreover, we found that thetranscriptional activation and repression of miR-200 family

members is maintained by dynamic and distinct epigeneticmechanisms. These findings suggest targeting miR-200

transcripts as a therapeutic means to restore its expression inbCSCs may be of benefit in the treatment of breast cancer.

Materials and MethodsCell culture

HMLE cells and their derivatives were cultured in HuMEC ready medium (Gibco),and other human breast cancer cell lines were cultured as previously described(Bracken et al., 2008). Cell proliferation was measured using the MTS assay usingthe CellTiter 96 AQueous Non-radioactive Cell Proliferation Assay (Promega). sl-HMLE MSCV EV, sl-HMLE MSCV miR-200b-200a-429 and sl-HMLE MSCVmiR-200c-141 stable cell lines were generated by transducing sl-HMLE withpMSCV-GFP retroviral vectors expressing the primary transcript of miR-200b-200a-429 and miR-200c-141 followed by selection with 1 mg/ml puromycin.Sphere assay was performed as described in (Dontu et al., 2003) with minormodifications. Cells were grown in DMEM:F12 (1:1), supplemented with 2% B27(Invitrogen), 20 ng/ml EGF and 20 ng/ml bFGF (basic fibroblast growth factor;R&D), 4 mg/ml heparin (Sigma), 5 mg/ml insulin, 0.5 mg/ml hydrocortisone.Single cell suspensions were cultured for 10–14 days at a different specifieddensity in 6- or 24-well ultra low attachment plates (Corning, Co-Star) orPolyhema-coated plates (Sigma). Spheres with diameter §150 mm were usuallycounted at day 10–12 to determine the sphere-forming efficiency (SFE). Here, SFEis presented as the number of spheres formed in day 10–14 per number of singlecells seeded (spheres/number of cells seeded). Spheres were harvested using70 mm cell strainers (BD Biosciences) and then dissociated to single cells withtrypsin for subsequent passages. Only 20,000 or 10,000 dissociated cells wereseeded in subsequent passages. To induce EMT in HMLE cells, the HMLE cellswere cultured as described in Mani et al. (Mani et al., 2008) in DMEM:F12 media(1:1) supplemented with 10 mg/ml insulin, 20 ng/ml EGF, 0.5 mg/mlhydrocortisone, and 5% fetal calf serum (FCS) and treated with 2.5 ng/ml ofTGFb1 (R&D).

Isolation of cells from human breast cancer pleural and ascites samples

The research using human samples was performed according to the Declaration ofHelsinki. Breast cancer pleural or ascites effusion samples were obtained withinformed consent from the Royal Adelaide Hospital (RAH Hospital EthicsCommittee Protocol No. 081013). Access to patient tumour samples was approvedby the appropriate institutional human ethics review boards. Patient details can beobtained at supplementary material Table S1. Patient samples were processedimmediately upon received from thoracentesis or paracentesis. Cells were pelletedby centrifuging pleural or ascites fluid mix with acid citrate dextrose (ACD) (5%final concentration). The pellet was washed twice with 16 phosphate-bufferedsaline (PBS) with 5% ACD and 0.4% human serum albumin (HSA), thenincubated in red blood cell lysis buffer (0.15 M ammonium chloride, 10 mMpotassium bicarbonate, 0.1 mM EDTA) for 3 min at room temperature. The cellswere then washed twice with 16 PBS with 5% ACD and 0.4% HSA beforesuspension in HuMEC ready medium + 20% FCS. After Ficoll density gradientcentrifugation, cells at the interface layer were collected and washed once with 16PBS with 5% ACD and 0.4% HSA. Cells were counted using white cell fluid dyeexclusion. Cells were then cryopreserved at no more than 16108 cells per vial in8:1:1 ratio of HuMEC ready medium:10% FCS:10% DMSO.

Cell staining and flow cytometry

Cells were stained at a concentration of 16105 cells per 50 ml or 16106 cells per100 ml of 16 PBS with 2% FCS (staining medium). Antibodies were added atappropriate dilution determined from titration experiments. Antibodies includedCD45-PeCy5 (1:10 vol:vol; 2652U; Beckman Coulter), CD24-PE (1:5 vol:vol;555428; BD Biosciences) or biotin (0.5:100 vol:vol; 13-0247; eBiosciences), andCD44-FITC (1:5 or 10 vol:vol; 555478; BD Biosciences) or APC (559942; BDBiosciences). Cells were stained for 25 min on ice in the dark and washed with 3–4 ml of staining media each. When biotinylated primary antibodies were used,cells were further stained with streptavidin conjugated fluorophores Pe-Cy7(0.3125:100 vol:vol; 25-4317; eBiosciences) and washed. Cells were analysedusing a FACS Aria cell sorter (BD Biosciences) or FC500 Terpsichore (BeckmanCoulter) and sorted using a FACSAria cell sorter. Side scatter and forward scatterprofiles were used to eliminate debris and cell doublets. Contaminating humanCD45 leucocytes cells were eliminated by CD45+ cell exclusion. In cell-sortingexperiments, sorted cell populations final purities ranged from 70 to 95%. In somecases, to prevent cell–cell aggregation in single cells suspension, DNase (type IV)(61362; Sigma) was added to the cells in suspension at 50 U/ml. FCS Express 4software (De Novo) was used to re-analyze the flow cytometry data.

Isolation of RNA and real-time PCR

Total RNA was extracted from cell lines, and real-time PCR was performed byusing primers as previously described (Gregory et al., 2008) and as shown in

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supplementary material Table S2. When small numbers of cells (#16105) wereobtained especially through cell sorting, RNeasy Plus mini kit (Qiagen) was usedto isolate for RNA. MicroRNA PCRs were performed using TaqMan microRNAassays (Applied Biosystems, Foster City, CA). Real-time PCR data for mRNA andmicroRNA were expressed relative to glyceraldehyde 3-phosphate dehydrogenase(GAPDH) or snRNA U6/RNU6B, respectively.

Western blotting

Western blotting was performed as previously described (Gregory et al., 2008).The following primary antibodies were used: ZEB1 (1:200 vol:vol, E20; SantaCruz), ZEB2 (1:5000 vol:vol; Christoffersen et al., 2007), E-cadherin (1:1000vol:vol, 610182; BD Transduction Laboratories), and tubulin (1:5000 vol:vol,ab7291; Abcam). Membranes were exposed using enhanced chemiluminescence(GE Healthcare) and imaged using the LAS4000 Luminescent Image Analyzer(Fujifilm).

Immunofluorescence

Cells were seeded on poly-L-lysine and mouse rat tail collagen type I-coated eight-well chamber slides (Nunc) and stained using anti-E-cadherin (1:500 vol:vol; asmentioned earlier in the text), ZO-1 (1:500 vol:vol; 61-7300 Zymed), CK18 (1:500vol:vol; ab49824 Abcam), CK14 (1:50 vol:vol; ab9220 Abcam) or F-actin aspreviously described (Gregory et al., 2008). Nuclei were visualized by co-stainingwith DAPI. Cells were examined using an Olympus IX81 microscope, and pictureswere taken using a Hamamatsu Orca camera. Images were analysed with OlympusCell software. All matched samples were photographed (control and test) usingidentical exposure times.

DNA methylation analysis

Genomic DNA was isolated from cells using either DNeasy Blood and Tissue kit(Qiagen), Trizol, or phenol chloroform ethanol precipitation method (Invitrogen).DNA (0.5–2 mg) was bisulphite modified with the EZ DNA Methylation-Gold Kitaccording to the manufacturer’s protocol (Zymo Research). For melt curveanalysis, bisulphite-modified DNA was PCR amplified using primers andconditions that did not discriminate between methylated and unmethylated DNAand did not amplify unmodified DNA (supplementary material Table S3) (Smithet al., 2009). Bisulphite-modified DNA from HMLE (unmethylated reference),M.SssI CpG Methylase treated normal donor lymphocytes (methylated reference),and unmodified DNA from normal donor lymphocytes (negative control) wasincluded in each PCR. The PCR and melt curve was performed using a Rotor-GeneQ (Qiagen) with a 95 C̊ activation step for 15 min; 95 C̊ for 30 s, 55 C̊ for 45 s for45 cycles; and a final extension step of 72 C̊ for 4 min. The melt of the PCRproduct was performed from 60 to 90 C̊, rising in 0.1 C̊ increments, waiting for30 s at the first step and for 2 s at each step thereafter, and acquiring fluorescenceat each temperature increment. The raw melt data were normalized using the HRMAnalysis module of the Rotor-Gene 6000 Series Software (Qiagen) as describedpreviously (Smith et al., 2009).

Bisulphite-modified DNA was used for hybridization on InfiniumHumanMethylation 450 BeadChip, following the Illumina Infinium HDMethylation protocol, and the BeadChip was scanned using an Illumina HiScanSQ scanner (Illumina, San Diego, CA, USA). The methylation score for each CpGwas represented as a b-value according to the fluorescent intensity ratio. b-valuesmay take any value between 0 (non-methylated) and 1 (completely methylated).

ChIP-qPCR assays

ChIP assays using 16106 cells per reaction were performed as below. Antibodiesincluded anti-histone H3 (ab1791; Abcam), and anti-trimethyl histone H3K4(ab8580; Abcam), anti-acetyl histone H3K9/14 (06-599; Millipore), anti-acetylhistone H3K27 (07-449; Millipore), anti-trimethyl histone H3K9 (ab8898;Abcam), anti-dimethyl histone H3K9 (ab1220; Abcam), anti-SUZ12 (3737; CellSignaling), and anti-EZH2 (pAb-039-050; Diagenode). In brief, 66106 cells werecrosslinked in 1% formaldehyde (final concentration) for 10 min at roomtemperature with gentle rocking or inversion every 2–3 min. Cells were pelletedand washed in ice-cold 16 HBSS (Gibco) containing protease inhibitor mixture(PIC) (Roche). The cells was lysed in 300 ml of lysis buffer (10 mM Tris pH 7.5/1 mM EDTA/1% SDS) containing PIC and incubated on ice for 10 min. Afterlysis, 900 ml of 16 HBSS containing PIC was added and then aliquoted 200 mleach into six individual tubes. Each 200 ml aliquot was sonicated by using abioruptorH sonicator (Diagenode), which was empirically determined to give riseto genomic fragments ranging from 200 to 800 bp. The soluble chromatin wascollected by 4 C̊ ultracentrifugation (13,000 rpm for 5 min) and pooled into a new15 ml falcon tube. The supernatant was diluted twofold with 26 RIPA buffer(10 mM Tris-HCl pH 7.5; 1 mM EDTA; 1% Triton X-100; 0.1% SDS; 0.1%sodium deoxycholate; 100 mM NaCl; PIC), 1/10 volume (40 ml) input wasremoved, and 400 ml of soluble chromatin was distributed to new Eppendorf tubes.Each respective antibody was added at appropriate amount as tested in titrationexperiments using control promoters. Immunoprecipitations were performed for2 h at 4 C̊ with rotation, and antibody:protein:DNA complexes were then collected

with 30 ml of protein A and/or G Dynabeads (Invitrogen) for 2 h of rotation. Thebeads were washed three times using 200 ml of RIPA buffer and once with TEbuffer, then incubated with 200 ml of fresh elution buffer with Proteinase K for 2 hin a thermomixer (1300 rpm, 68 C̊) to reverse the protein:DNA cross-links. Afterincubation, eluates were collected into new Eppendorf tubes. Genomic DNA wasrecovered by using phenol chloroform extraction and ethanol precipitation. Pelletswere washed in 70% ethanol, briefly air-dried, and resuspended in T10E (10 mMTris pH 7.5; 0.1 mM EDTA) buffer. Quantitation of ChIP DNA (relativeenrichment) using the Rotor-Gene 6000 (Corbett Life Science) was carried outin triplicate qRT-PCRs with gene specific primers to assess antibody enrichment(supplementary material Table S4). Enrichment of histone modifications atgenomic regions were expressed as the percentage input normalized to respectivecell type H3 levels to account for differences in H3 levels in different celltypes. [Percentage input was calculated using the formula % (ChIP/Input)52[Ct(ChIP)2Ct(Input)6Input dilution factor]6100%, to account for chromatinsample preparation differences].

Author contributionsY.-Y.L., J.A.W., J.J.A., P.A.G., D.T., A.F.L., P.A.D., Y.K.-G. andG.J.G. conceived and designed experiments. Y.-Y.L., J.A.W., J.J.A.,A.G.B. and E.S. performed experiments and analysed the data.Y.-Y.L., J.A.W., J.J.A., P.A.G., Y.K.-G. and G.J.G. wrote themanuscript.

FundingThis work was supported by a fellowship from the National CancerFoundation Australia [grant number ECF-09-08 to P.A.G.]; ascholarship from the University of Malaya Skim LatihanAkademik IPTA program [to Y.-Y.L.]; and grants from theNational Health and Medical Research Council [grant numbers626918 and 1008440 to G.J.G. and Y.K.-G.]; and the Cancer CouncilSouth Australia [grant number 626956 to G.J.G., Y.K.-G. and P.A.G.and 1005078 to P.A.D.].

Supplementary material available online at

http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.122275/-/DC1

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