dna methylation control of tissue polarity and cellular differentiation in the mammary epithelium

www.elsevier.com/locate/yexcr

Experimental Cell Research 298 (2004) 122–132

DNA methylation control of tissue polarity and cellular differentiation

in the mammary epithelium

Cedric Plachot and Sophie A. Lelievre*

Department of Basic Medical Sciences, Purdue University, West Lafayette, IN 47907-2026, USA

Received 28 February 2004, revised version received 14 April 2004

Available online 18 May 2004

Abstract

Alterations in gene expression accompany cell-type-specific differentiation. In complex systems where functional differentiation depends

on the organization of specific cell types into highly specialized structures (tissue morphogenesis), it is not known how epigenetic

mechanisms that control gene expression influence this stepwise differentiation process. We have investigated the effect of DNA methylation,

a major epigenetic pathway of gene silencing, on the regulation of mammary acinar differentiation. Our in vitro model of differentiation

encompasses human mammary epithelial cells that form polarized and hollow tissue structures (acini) when cultured in the presence of

basement membrane components. We found that acinar morphogenesis was accompanied with chromatin remodeling, as shown by

alterations in histone 4 acetylation, heterochromatin 1 protein, and histone 3 methylated on lysine 9, and with an increase in expression of

MeCP2, a mediator of DNA-methylation-induced gene silencing. DNA hypomethylation induced by treatment with 5-aza-2Vdeoxycytidineduring acinar differentiation essentially prevented the formation of apical tissue polarity. This treatment also induced the expression of CK19,

a marker of cells that are in a transitional differentiation stage. These results suggest that DNA methylation is a mechanism by which

mammary epithelial differentiation is coordinated both at the tissue and cellular levels.

D 2004 Elsevier Inc. All rights reserved.

Keywords: Nuclear organization; Three-dimensional culture; Tissue differentiation; Acinar morphogenesis; Chromatin structure; Tight junction; CK19

Introduction multicellular structures (tissue morphogenesis). In the mam-

The organization of the cell nucleus, and notably its

chromatin component, is considered to be critical for the

regulation of nuclear functions, including gene expression

[1–3], and for the control of cell and tissue differentiation

[4]. Differentiation is defined as the gain of a single or a

series of functions [5] characteristic of a specific cell or

tissue type. Cell differentiation can be categorized according

to the expression of markers characteristic of a recognizable

differentiation stage and/or cell type, while tissue differen-

tiation encompasses both the expression of specific diffe-

rentiation markers and the organization of cells into defined

0014-4827/$ - see front matter D 2004 Elsevier Inc. All rights reserved.

doi:10.1016/j.yexcr.2004.04.024

Abbreviations: 2-D, two-dimensional; 3-D, three-dimensional; 5Aza, 5-

aza-2Vdeoxycytidine; CK, cytokeratin; DAPI, 4V,6-diamidino-2-phenylin-

dole; ECM, extracellular matrix; EGF, epidermal growth factor; HP 1,

heterochromatin protein 1; MSP, methylation-specific PCR.

* Corresponding author. Department of Basic Medical Sciences,

Purdue University, 625 Harrison Street, LYNN, West Lafayette, IN

47907-2026. Fax: +1-765-494-0781.

E-mail address: [email protected] (S.A. Lelievre).

mary gland, the epithelial tissue displays phases of prolif-

eration and differentiation during adult life and as such it is

a flexible experimental model to investigate the relationship

between nuclear organization and tissue structure and func-

tion. The importance of nuclear organization in the diffe-

rentiation of adult mammary epithelial tissue was suggested

earlier by studies involving extracellular matrix (ECM)-

induced acinar morphogenesis of human mammary epithe-

lial cells in culture. In this model, mammary epithelial cells

are induced to differentiate into tissue-like glandular struc-

tures, called acini, in which cells display growth arrest and

express markers of the mammary cell type. The arrangement

into a single-layered epithelium, where mammary epithelial

cells are delineated by a basement membrane on one side

and a lumen on the opposite side, creates a baso-apical

polarity axis resulting in vectorial secretion of milk into the

lumen [6]. ECM-induced acinar morphogenesis was shown

to affect nuclear organization by influencing the distribution

of different nuclear proteins, including NuMA, Rb and

splicing factors. Interestingly, altering the chromatin struc-

C. Plachot, S.A. Lelievre / Experimental Cell Research 298 (2004) 122–132 123

ture in acinar cells by increasing the level of histone

acetylation upon inhibition of histone deacetylase with

trichostatin A induced proliferation. This suggested that

the organization of chromatin achieved upon acinar mor-

phogenesis was critical for the maintenance of this differ-

entiation stage [7]. Earlier reports using a murine model

showed the presence of an ECM-responsive element within

the promoter of the mammary-specific h-casein gene [8],

and ECM-directed inhibition of TGFh transcription [9],

which indicated that the communication between the ECM

and the cell nucleus ultimately controlled gene transcription

during acinar morphogenesis. A link between the ECM,

chromatin structure, and gene expression was demonstrated

recently by showing that trichostatin-A-induced alteration of

chromatin structure was sufficient to inhibit the expression

of the milk precursor h-casein in mammary epithelial cells

cultured in the presence of ECM [10]. However, despite

these scattered studies, the role of chromatin structure in

acinar differentiation remains mostly unexplored.

To further unravel the relationship between chromatin

structure and mammary acinar differentiation, it is impor-

tant to investigate the role of the pathways involved in the

control of chromatin remodeling and gene expression

during the morphogenesis process. In this study, we

focused our attention on DNA methylation, a major

epigenetic mechanism of regulation of gene expression

that has gained increasing importance in differentiation

processes [11]. DNA methylation occurs principally at

the 5V position of cytosines, most often when a cytosine

is followed by a guanosine (CpG dinucleotide). CpG

methylation within gene promoters can act directly by

affecting the binding of certain transcription factors to

DNA or by recruiting methyl binding proteins (e.g.,

MeCP2, MBD2) that in turn attach to chromatin modifier

complexes including histone deacetylases, causing deace-

tylation of adjacent histones and subsequent chromatin

condensation and gene silencing [12]. DNA methylation

is involved in major physiological processes including X-

chromosome inactivation, genetic imprinting and tissue-

specific gene expression [13]. Measurement of the level of

DNA methylation within promoters of markers of differ-

entiation and/or cell treatment with the DNA hypomethy-

lating agent 5-aza-2Vdeoxycytidine (5Aza) have revealed

that DNA methylation participates in the differentiation of

myocytes [14], trophoblasts [15], adipocytes [16], osteo-

blasts [17], spermatocytes [18], neuroepithelial cells [19],

and keratinocytes [20]. It was recently reported that the

expression of the methyl-binding protein MeCP2 is in-

creased during human fetal mammary gland development

[21], suggesting that DNA methylation may play a role in

mammary gland differentiation.

To decipher the role of DNA methylation in mammary

acinar differentiation, we have used the in vitro model of

differentiation described above, in which non-neoplastic

human mammary epithelial cells are induced to form acini

when cultured in the presence of exogenous ECM-enriched

in laminin [6]. We show that preventing DNA methylation

during the differentiation process inhibits the completion of

acinar morphogenesis, as shown by the absence of apical

polarity, and pushes a fraction of the cell population into a

state of differentiation characterized by the expression of

cytokeratin (CK) 19, a putative marker of pluripotence.

Materials and methods

Cell culture

Non-neoplastic human mammary HMT-3522 epithelial

cells, previously established from adult tissue (S1 passage 55

to 60) [22] were propagated as monolayers on plastic surface

(2-D culture), in 75-cm2 Falcon flasks (BD Biosciences,

Bedford, MA), at 37jC in 5% CO2, in chemically defined

H14 medium consisting of DMEM:F12 medium (GIBCO

BRL, St. Louis, MO), containing 250 ng/ml insulin (Boeh-

ringer Mannheim, Indianapolis, IN), 10 Ag/ml transferrin

(Sigma, St Louis, MO), 2.6 ng/ml sodium selenite (BD

Biosciences), 10�10 M estradiol (Sigma), 1.4 AM hydrocor-

tisone (BD Biosciences), 5 Ag/ml prolactin (Sigma), and 10

ng/ml epidermal growth factor (EGF; BD Biosciences). H14

medium was routinely changed every 2–3 days. S1 cells

were induced to recapitulate the formation of polarized

glandular structures (acini) when cultured in the presence

of exogenous ECM-enriched in laminin (Matrigelk, BD

Biosciences), a technique called three-dimensional (3-D)

culture [6]. Briefly, S1 cells (35,000 cells/cm2) were plated

on 41 Al/cm2 matrigel-coated surfaces and cultured in H14

medium containing 5% matrigel. Following 8 days of 3-D

culture, cells were induced to exit the cell cycle upon

incubation in H14 medium without EGF for 48 h. Acinar

morphogenesis, characterized by the formation of a single

layer of cells surrounding a lumen and delineated by an

endogenous basement membrane, was routinely observed by

days 9–10. Usually, at the end of the differentiation process,

well-formed acini represent 80–90% of the total acini

population. For treatment of S1 cells cultured under 3-D

conditions with 5Aza (Sigma), H14 medium containing

different concentrations of this drug was changed every

2 days for 10 days, starting the day of plating.

Preparation of genomic DNA

Cells cultured under 2-D monolayer conditions were

harvested using a cell scraper and acini were isolated from

3-D cultures by dispase treatment (5000 units per 100 ml

caseinolytic activity, BD Biosciences) [7]. Cells and acini

were then collected by centrifugation, washed once with

PBS, and incubated in digestion buffer (100 mM NaCl, 10

mM Tris–HCl, 25 mM EDTA, 0.5% SDS, 0.1 mg/ml

proteinase K) at 55jC for 12–16 h. Digests were deprotei-

nized by two steps of phenol/chloroform/isoamyl alcohol

[23], recovered by ethanol precipitation, dried, and resus-

C. Plachot, S.A. Lelievre / Experimental Cell Research 298 (2004) 122–132124

pended in water. DNA concentration was read using a Bio-

Rad spectrophotometer (Bio-Rad, Hercules, CA) at an

optical density (OD) wavelength of 260 nm, with an OD

of 1 equal to 50 Ag/ml of DNA.

Restriction endonuclease digestion

Restriction enzymes (MspI and HpaII; Roche, Indian-

apolis, IN) were used to determine the global methylation

pattern of genomic DNA. MspI cleaves at the restriction site

CC*GG regardless of the methylation status of the internal

cytosine (C*), whereas HpaII only cleaves when the internal

C-residue (C*) is nonmethylated [24]. Briefly, genomic

DNA was digested at 37jC for 1 h with either HpaII or

MspI (5 units/Ag of DNA) according to recommendations of

the manufacturer. Samples were analyzed by electrophoresis

in 1% agarose gel and visualized with ethidium bromide

(1.5 Ag/ml). Uncut DNA was used as control for DNA

integrity.

Methylation-specific PCR (MSP)

Two micrograms of genomic DNA were treated with

sodium bisulfite as described previously [25], and analyzed

by MSP using primer sets that covered CG dinucleotides

specific for p16 [25] and RARh2 [26] genes. For the p16

gene, primers specific for methylated DNA [sense primer: 5V-TTA-TTA-GAG-GGT-GGG-GCG-GAT-CGC-3V; antisense

primer: 5V-GAC-CCC-GAA-CCG-CGA-CCG-TAA-3V], andprimers specific for unmethylated DNA [sense primer: 5V-TTA-TTA-GAG-GGT-GGG-GTG-GAT-TGT-3V; antisense

primer: 5V-CAA-CCC-CAA-ACC-ACA-ACC-ATA-A-3V]yield a 150-bp product and a 151-bp product, respectively.

For the RARh2 gene, primers specific for methylated DNA

[sense primer: 5V-GGT-TAG-TAG-TTC-GGG-TAG-GGT-TTA-TC-3V; antisense primer: 5V-CCG-AAT-CCT-ACC-

CCG-ACG-3V], and primers specific for unmethylated DNA

[sense primer: 5V-TTA-GTA-GTT-TGG-GTA-GGG-TTT-ATT-3V; antisense primer 5V-CCA-AAT-CCT-ACC-CCA-ACA-3V] both yield a 234-bp product. The PCR conditions

were as follows: 1 cycle at 95jC for 5 min; 35 cycles at 95jCfor 30 s, 30 s at the desired annealing temperature (65jC and

55jC for methylated and unmethylated primers for p16,

respectively; 64jC and 55jC for methylated and unmethy-

lated primers for RARh2, respectively), 72jC for 30 s; and 1

cycle at 72jC for 4 min. The PCR samples were resolved by

electrophoresis in 2% agarose gel and visualized with ethi-

dium bromide.

Chromatin fractionation

Cells cultured under 2-D monolayer conditions were

harvested using a cell scraper and acini were isolated

from 3-D cultures by dispase treatment as described above

(see ‘‘Preparation of genomic DNA’’). Following incuba-

tion in hypotonic buffer (5 mM HEPES pH 7.4, 0.5 mM

EGTA, 1 mM MgCl2, 125 mM sucrose, 0.5 mM NaHCO3,

10 Ag/ml aprotinin, 1 mM PMSF, 250 AM NaF) for 30 min,

cells were lysed using a Kontes 2 ml Dounce homogenizer.

Cell nuclei were collected by centrifugation, washed twice

in buffer X (10 mM HEPES pH 7.9, 10 mM KCl, 1.5 mM

MgCl2, 340 mM sucrose, 10% glycerol, 1 mM DTT, 10 Ag/ml aprotinin, 1 mM pefabloc, 250 AM NaF, 0.1% Triton X-

100), and lysed 30 min in buffer Y (3 mM EDTA, 0.2 mM

EGTA, 1 mM DTT, 10 Ag/ml aprotinin, 1 mM pefabloc,

250 AM NaF). After centrifugation pellets were digested

with 1 unit of micrococcal nuclease (Sigma) for 5 min at

37jC. Proteins released upon chromatin digestion (super-

natant) were submitted to sodium dodecyl sulfate poly-

acrylamide gel electrophoresis (SDS-PAGE) and Western

blot analysis.

Preparation of whole cell protein extracts and Western blot

analysis

Cells were harvested from 2-D monolayer and 3-D

cultures as described above (see ‘‘Preparation of genomic

DNA’’) and whole cell extracts were prepared in Laemmli

buffer (2% SDS in phosphate buffered saline) including 10

Ag/ml aprotinin, 1 mM pefabloc, 250 AM NaF as described

previously [7]. For Western blot analysis, protein concen-

trations were determined using the Bio-Rad protein assay.

Equal amounts of proteins were separated and immuno-

blotted as described [7] with antibodies directed against

acetylated histone H4 (Upstate, Lake Placid, NY), hetero-

chromatin protein 1 (HP 1) g (Chemicon, Temecula, CA),

histone H2B (Santa Cruz Biotechnology, Santa Cruz, CA),

MeCP2 (ABR, Golden, CO), lamin A/C (Santa Cruz

Biotechnology), lamin B (Calbiochem-Novabiochem, Cam-

bridge, MA), MCM3 (a kind gift from Dr. Stillman, Cold

Spring Harbor Laboratories), and cytokeratin (CK) 18

(clone DC10, Oncogene Research Products, San Diego,

CA). Histone H4 acetylated, HP 1g and MeCP2 protein

levels were quantified using Scion NIH image software and

normalized to their respective loading control. There were

at least three independent experiments for each analysis.

Indirect immunofluorescence and apoptosis labeling

For immunostaining, cells were cultured in four-well

chamber slides (Nalge Nunc International, Naperville, IL).

Antibodies against mucin-1 (clone DF3, DAKO, Carpinte-

ria, CA), CK19 (clone A53-B/A2, Santa Cruz Biotechnol-

ogy), and vimentin (clone VIM 3B4, Chemicon) were used

on cells fixed in 2% paraformaldehyde. Antibodies against

a6-integrin (clone NKI-GoH3, Chemicon), h-catenin (clone

14, BD Biosciences), ZO-1 (Zymed, San Francisco, CA),

Ki67 (clone Ki67, DAKO), and antibody against branched

methylated histone H3-K9 (kindly provided by Dr Jenu-

wein, Research Institute of Molecular Pathology, Vienna,

Austria), were used on cells incubated in situ in permeabi-

lization buffer (0.5% Triton X-100, 100 mM NaCl, 300 mM


sucrose, 10 mM pipes, pH 6.8, 5 mM MgCl2, 1 mM

pefabloc, 10 Ag/ml aprotinin, 250 AM NaF), before fixation

in 2% paraformaldehyde. Immunostaining was performed as

described previously [7]. A minimum of 50–100 acini was

scored from three distinct experiments for immunostaining

involving mucin-1, ZO-1, and CK19. Cell apoptosis was

assessed using an in situ cell death detection kit (Roche),

which labels 3VOH DNA ends with FITC or Texas Red,

according to the manufacturer’s recommendations. A min-

imum of 500 cells was scored for each experiment, and

apoptotic cells were expressed as the number of cells

displaying a fluorescent and condensed nucleus over the

total number of cells scored. Two independent experiments

were run in duplicate for each condition. For all stainings,

DNAwas counterstained with 4V,6-diamidino-2-phenylindole

(DAPI) or 25 AM DRAQ5k (Alexis, San Diego, CA) and

samples were mounted in ProLong antifade solution (Mo-

lecular Probes, Eugene, OR).

Statistical analysis

Data are presented as means F SEM. The paired t test

was used to determine the probability (P value) that the

sample means are equal using Prism 3.0 software. A P <

0.05 was considered to be significant.

Fig. 1. Chromatin is remodeled during mammary acinar morphogenesis.

Western blot analysis of acetylated histone H4 (A), and heterochromatin

protein 1 (HP 1) g (B), and immunostaining for branched methylated

histone H3-K9 (C) in 2-D monolayer culture of S1 cells (S1 2-D) and acini

(S1 3-D) formed in 3-D culture. Histone H2B was used as loading control

in panels A and B. In panel C, confocal images represent single optical

sections. Arrows indicate large methylated H3-K9 aggregates. Nuclei are

counterstained with DRAQ5k. Size bar = 10 Am.

Results

Chromatin remodeling and increased MeCP2 expression

accompany mammary acinar morphogenesis

We have previously shown that the level of acetylation of

histone H4 measured in mammary acinar cells is critical for

the maintenance of this differentiation stage [7]. This

suggests that the chromatin structure present in acinar cells

is a critical component of the differentiation process. To

assess whether chromatin structure is a function of acinar

morphogenesis, the extent of open state chromatin (euchro-

matin) was measured by Western blot analysis of acetylated

histone H4 in breast epithelial S1 cells cultured under 2-D

monolayer conditions that do not permit acinar morphogen-

esis, and S1 cells induced to form acini under 3-D culture

conditions. In cells in 2-D monolayer culture, the level of

acetylated histone H4 was 60% higher (P < 0.05) compared

to cells organized into acini (Fig. 1A). Further investigation

of chromatin structure demonstrated that the decrease of

histone H4 acetylated in acinar cells was accompanied by a

change in markers of the repressed or silent state of

chromatin compared to cells in 2-D monolayer culture.

There was a 60% increase in the expression of heterochro-

matin protein HP 1g in acinar cells (P < 0.05; Fig. 1B).

Moreover, branched methylated histone H3-K9, which has

been proposed to be involved in higher-order organization

of silent chromatin and the binding of HP 1 to chromatin

[27,28], formed multiple small foci throughout the nuclei of

acinar cells, while it was faint and homogenous, and

sometimes formed one to two large aggregates in cells

cultured under 2-D monolayer conditions (Fig. 1C). Thus,

acinar morphogenesis is accompanied by changes in the

expression of chromatin markers as well as the redistribu-

tion of chromatin markers.

DNA methylation targets chromatin remodeling to spe-

cific DNA sequences by inducing local chromatin compac-

tion, which in turn leads to gene silencing. Notably, the

presence of DNA methylation has been associated with

local histone H4 deacetylation and methylation of histone

3 on lysine 9 (H3-K9) [29]. In addition, increased expres-

sion of MeCP2, a mediator of methylation-induced chro-


matin remodeling, has been observed during neuronal

differentiation [30], and during human fetal mammary gland

development [21]. To investigate the involvement of DNA-

methylation-related factors in acinar differentiation, expres-

sion levels of MeCP2 were compared between cells in 2-D

monolayer culture and acini (3-D culture). MeCP2 protein

level was 90% higher (P < 0.01) in acini compared to 2-D

monolayers (Fig. 2A). To verify that the high level of

MeCP2 expression observed in acini corresponded to an

accumulation of MeCP2 in the chromatin compartment,

chromatin fractions were prepared from cells in 2-D mono-

layer culture and acini. MCM3 was used as a marker of the

chromatin fraction and nuclear matrix protein lamin B was

used as a marker of the nonsoluble nuclear structure, to

assess the purity of the fractionation. Equal loading of

protein samples was confirmed by Ponceau red staining

(not shown). MeCP2 was strongly associated with chroma-

tin in acini, while it was almost not detectable in chromatin

fractions obtained from 2-D monolayers (Fig. 2B).

DNA hypomethylation prevents the establishment of apical

polarity

To investigate which steps of acinar differentiation are

influenced by DNA methylation, S1 cells were treated

during 10 days, in 3-D culture, with the hypomethylating

Fig. 2. Mammary acinar morphogenesis is accompanied by a dramatic

increase inMeCP2 expression.Western blot analysis of totalMeCP2 (A), and

MeCP2 present in chromatin fraction (B) in 2-Dmonolayer culture of S1 cells

(S1 2-D) and acini (S1 3-D) formed in 3-D culture. Lamin B was used as

loading control in panel A. In panel B, MCM3 and lamin B were used as

positive and negative markers of the soluble chromatin compartment,

respectively.

agent 5Aza. 5Aza is a deoxycytidine analog that incorpo-

rates into the DNA during replication and covalently traps

DNA methyltransferase, thereby depleting the cells of

enzyme activity and resulting in DNA hypomethylation

[31]. We assessed which concentration of 5Aza induced

minimum toxicity to the cells. A concentration of 0.1 AMgave a very low cytotoxic effect as shown by the apoptotic

index (5.5F 0.5% in cells treated with 0.1 AM 5Aza vs. 3F0.3% in nontreated cells), while further increase of 5Aza

concentration to 20 AM induced noticeable apoptosis (8.1 F0.5% for 0.5 AM and 15.2 F 0.8% for 20 AM 5Aza, Fig.

3A). In light of these results, a concentration of 0.1 AM was

used for the rest of the study. To verify whether 0.1 AM5Aza treatment was sufficient to induce DNA hypomethy-

lation, we used a test based on the differential response of

methylated cytosines to cleavage by methylation-sensitive

and -insensitive restriction enzymes. Digestion of genomic

DNA with HpaII, a restriction enzyme that cleaves at

nonmethylated sites only (see ‘‘Materials and methods’’),

gave a more extended degradation pattern in 5Aza-treated

cells compared to nontreated cells, indicating that genomic

DNA was indeed hypomethylated upon 5Aza treatment

(Fig. 3B). In addition, successful hypomethylation was

confirmed by methylation-specific PCR with RARh2 and

p16 genes, which have been shown to be regulated by DNA

methylation [25,26,32,33]. RARh2 and p16 genes were

methylated on the CpG containing-sequences assessed in

our control cells. They became partially hypomethylated in

cells treated with 0.1 AM of 5Aza (Fig. 3C).

Treatment with 0.1 AM of 5Aza was used to hypome-

thylate the DNA of S1 cells during acinar differentiation and

the resulting phenotype was analyzed. Mammary acinar

differentiation corresponds to a specific cascade of events

characterized by a progressive exit of the cell cycle, the

deposition of an endogenous basement membrane at the

periphery of the developing acinus, and the segregation of

different groups of proteins along the cell membrane to form

a polarity axis [34]. This tissue polarity results in the

formation of two distinct compartments, the basal and apical

poles that include the cell membrane in contact with the

basement membrane and lateral cell–cell contacts located

below tight junctions (baso-lateral polarity), and lateral

cell–cell contacts located at or above tight junctions and

the cell membrane in contact with the lumen (apical polar-

ity). Such a compartmentalization is critical for the organi-

zation and function of the acinus. The organization of

proteins at the basal cell membrane is established upon

contact with basement membrane molecules. Basal polarity

controls the survival of cells within the epithelial tissue

[35,36]. Completion of tissue polarity depends on the

establishment of tight junctions at the latero-apical cell

membranes [37,38]. Tight junctions are multiprotein com-

plexes that create selective barriers to water and solute flux

across the tissue and prevent the diffusion of membrane

components between the basal pole and the apical pole,

hence maintaining proper vectorial tissue function (e.g.,

Fig. 3. Cytotoxicity and demethylation induced by 5Aza treatment in S1

cells cultured under 3-D conditions. S1 cells were cultured in the absence or

the presence of different concentrations of 5Aza for 10 days under 3-D

conditions. (A) Dose–response effect of increasing 5Aza concentrations on

the viability of S1 cells. Cell viability was assessed by counting the number

of apoptotic cells within the population of acini. Results are expressed as

means F SE for two different experiments run in duplicate for each group.

(B) Effect of a 10-day treatment with 0.1 AM of 5Aza on the methylation

status of genomic DNA. The differential response to methylation-sensitive

restriction enzymes (MspI and HpaII) was used to determine the global

methylation pattern of genomic DNA in control (S1 3-D) and 5Aza-treated

cells (S1 3-D + 5Aza) cultured under 3-D conditions. Uncut genomic DNA

served as a control for DNA integrity. (C) Effect of a 10-day treatment with

0.1 AM of 5Aza on the methylation status of p16 and RARh2 genes.

Genomic DNAwas modified by sodium bisulfite in samples obtained from

control acini (S1 3-D) and cells treated with 5Aza (S1 3-D + 5Aza) before

proceeding with methylation specific PCR using methylated (M) and

unmethylated (U) primers for p16 and RARh2 genes. kbp = kilo base pairs;

Mk = marker of DNA sizes.


milk secretion into ducts or selective secretion and absorp-

tion of ions and metabolites) [39]. Typically, at day 3 of 3-D

culture, a high portion of the S1 cell population is in the cell

cycle as shown by the expression of Ki67, a marker of cell

cycle activity, whereas after days 5–6 of culture, the

fraction of Ki67-positive cells decreases to reach less than

5% of the cell population upon completion of acinar

morphogenesis at 10 days of 3-D culture (Fig. 4A) [7].

The treatment with 5Aza did not alter acinar-morphogen-

esis-induced exit of the cell cycle (Fig. 4A). However,

5Aza treatment prevented the complete formation of the

polarity axis as shown by immunostaining for basal,

lateral, and apical polarity markers. Nontreated cells were

used as control and displayed the typical localization of

a6-integrin, an ECM receptor, at the basal cell membrane,

and h-catenin at lateral cell–cell junctions. 5Aza treatment

did not affect the localization of these markers, suggesting

that baso-lateral polarity could still be established (Fig.

4B). In contrast, immunostaining of apical polarity markers

revealed a dramatic alteration of the organization of the

apical pole of acini upon 5Aza treatment. Mucin-like

glycoprotein mucin-1 is a milk precursor commonly used

as a functional marker of mammary acinar differentiation

[6,40]; it is typically located to the latero-apical side of

cells in the properly polarized acinus. Mucin-1 was relo-

cated to the basal compartment of 5Aza-treated cells,

hence displaying an inverted polarity and suggesting that

appropriate segregation between basal and apical compart-

ments had failed (Fig. 4C). In nontreated S1 cells, altered

polarity for mucin-1 accounted for 17 F 7% of acini while

it rose to 67 F 8% of acini in 5Aza-treated cells (Fig. 4C).

Tight junctions are critical for baso-apical segregation of

proteins and the function of the apical compartment [39].

Therefore, to test whether tight junctions were altered upon

5Aza treatment, we immunostained nontreated and 5Aza-

treated cells for ZO-1, a protein that plays a critical role in

the structural integrity of tight junctions [41,42]. ZO-1

formed the usual condensed foci at the apical side of cells

in the majority of control acini, while it was more diffused

in the apical compartment and often relocated to lateral

cell–cell junctions in 5Aza-treated cells (Fig. 4D). In

nontreated cells, the altered distribution of ZO-1 accounted

for 23 F 4% of acini, while it rose to 45 F 2% of acini in

5Aza-treated cells (Fig. 4D). 5Aza treatment did not

induce remarkable alterations in the microtubule and

microfilament networks, both known to participate in

polarity [38,43], indicating that flagrant disruption of these

cytoskeletal elements was not a likely cause of the lack of

apical polarity (not shown). Thus, hypomethylating the

DNA by 5Aza treatment affects preferentially the stages of

acinar differentiation that correspond to the formation of

the apical pole of the polarity axis, while growth arrest and

the formation of baso-lateral polarity are not influenced.

Induction of DNA hypomethylation during acinar

differentiation is accompanied by expression of CK19

The alteration of tissue polarity is commonly considered

as a sign of lack of tissue differentiation [7,44]. To inves-

tigate whether the loss of tissue differentiation induced by

DNA hypomethylation was accompanied by changes in the

state of epithelial differentiation of mammary cells, we

Fig. 4. 5Aza-induced DNA hypomethylation prevents the establishment of apical polarity in mammary epithelial S1 cells cultured under 3-D conditions. S1 cells

were cultured in the absence or the presence of 5Aza for 10 days under 3-D conditions. (A) Exit from the cell cycle was assessed by immunostaining for Ki67

(red), a marker of cell cycle activity. Immunostaining is shown during the proliferation phase of control cells (S1 3-D day 3), upon acinar differentiation of control

cells at day 10 of control cells (S1 3-D day 10), and in S1 cells treated with 0.1 AM of 5Aza up to day 10 (S1 3-D + 5Aza day 10). (B) The establishment of baso-

lateral polarity was investigated by assessing the localization of a6-integrin (green), and h-catenin [red; staining is typically lateral (see arrows) and also

accumulates towards the lumen] in nontreated cells (S1 3-D) and cells treated with 5Aza (S1 3-D + 5Aza). (C–D) Establishment of apical polarity was

investigated by assessing the localization of mucin 1 (green) (C) and the localization of ZO�1 (green) (D) in nontreated cells (S1 3-D) and cells treated with

5Aza (S1 3-D + 5Aza). Histograms represent the percentage of acini with altered polarity for mucin 1 and ZO-1 in nontreated cells and cells treated with

5Aza (with means F SE for three experiments in each group; * = P < 0.05; ** = P < 0.01). Nuclei are counterstained with DAPI (blue). Size bar = 10 Am.


studied the expression of cytokeratin (CK) 19 in nontreated

and 5Aza-treated S1 cells cultured under 3-D conditions.

CK19 is an intermediate filament protein restricted to

luminal mammary epithelial cells that are lined against the

lumen of the ductal-alveolar system [45,46]. In other

epithelial tissues, like the pancreas or the epidermis, the

expression of CK19 is characteristic of de-differentiation or

a state of flexible differentiation [47,48]. Interestingly, in the

mammary gland, only a subset of luminal cells express

CK19. Luminal mammary cells expressing CK19 are con-

sidered to be less differentiated compared to other luminal

mammary cells because they lack expression of certain

mammary differentiation markers [46]. CK19 was not

detectable by Western blot analysis in both nontreated and

5Aza-treated S1 cells (not shown). However, upon 5Aza

treatment, CK19 staining was consistently observed in the

cytoplasm of one or two cells in 12 F 1.5% of acini (Fig.

5A), while no cells positive for CK19 staining could be

detected within control acini. To further characterize the loss

of epithelial differentiation induced by 5Aza treatment, we

asked whether it could be correlated with epithelial–mes-

enchymal transition, which is accompanied by the loss of

epithelial markers and the strong expression of mesenchy-

mal markers. CK18 is another cytokeratin usually expressed

in luminal mammary epithelial cells. In contrast to CK19,

its expression does not seem to vary with the stage of

differentiation of luminal adult cells. CK18 was unchanged

in terms of localization and expression in 5Aza-treated cells

compared to untreated cells (Fig. 5B). The mesenchymal

marker vimentin is usually faintly present against the basal

Fig. 5. 5Aza treatment induces the expression of CK19. S1 cells were cultured in the absence (S1-3D) or the presence of 5Aza (S1-3D + 5Aza) for 10 days under

3-D conditions. (A) Immunostaining for CK19 (green). The histogram represents the percentage of acini with CK19 positive cells in nontreated cells and cells

treated with 5Aza (with meansF SE for three experiments in each group; *** = P < 0.001). (B) Western blot and immunostaining for CK18 (green). Expression

of lamins A and C is used as a loading control. (C) Immunostaining for vimentin (green). Nuclei are counterstained with DAPI (blue). Size bar = 10 Am.


side of S1 cells, probably due to their involvement in the

secretion and organization of basement membrane compo-

nents [49]. There was no dramatic alteration of vimentin

between nontreated and 5Aza-treated cells, as seen by

immunostaining. Taken together, these data suggest that

the alteration of the state of epithelial differentiation asso-

ciated with 5Aza-induced DNA hypomethylation of mam-

mary cells is restricted to alterations within the luminal

stage of differentiation.

Discussion

Our results show that mammary acinar differentiation of

adult epithelial cells is accompanied by chromatin remod-

eling. 5Aza-induced DNA hypomethylation of mammary

epithelial cells during acinar differentiation specifically

prevents the establishment of apical polarity. Moreover,

hypomethylation of DNA is accompanied by the expres-

sion of CK19, a marker of a less differentiated status of

luminal cells, in a small fraction of the cell population.

Thus, DNA methylation, a major nuclear pathway in-

volved in the regulation of chromatin structure and subse-

quently gene expression, seems to influence very specific

stages of mammary epithelial differentiation.

We have previously demonstrated that the nuclear orga-

nization of cells that form polarized glandular structures or

acini is critical for the maintenance of the advanced stage of

differentiation of the mammary epithelium [7]. Notably,

altering chromatin structure by enhancing the acetylation

of histones using trichostatin A, and thus promoting an open

chromatin structure, induced acinar cells to re-enter the cell

cycle, de-differentiate, and proliferate. Here, by comparing

2-D monolayer culture and acini culture of mammary epi-

thelial cells, we show that tissue morphogenesis leading to

acini formation is accompanied by substantial chromatin

remodeling that seems to promote a repressed state of

chromatin, as shown by decreased levels of histone 4

acetylated and increased levels of HP 1. These modifications

may mostly influence gene expression in a negative fashion

and restrict the protein profile to a tissue-specific phenotype.

This hypothesis is supported by the fact that MeCP2, one of


the major effectors of DNA-methylation-mediated gene

silencing, is highly expressed in acinar cells compared to

cells organized as 2-D monolayers.

Hypomethylating agent 5Aza was useful to establish that

DNA methylation is a critical mechanism for the control of

differentiation in several cell types. These studies dealt with

the differentiation of spermatocytes [18], adipocytes [16],

trophoblasts [15], and germ cells [50]. Our study reports the

involvement of DNA methylation in the control of the

differentiation of an exocrine type of tissue for which the

specific arrangement of cells into glandular structures is a

key element of functional differentiation. Interestingly,

5Aza treatment prevents the establishment of apical polarity

during the acinar differentiation process. Alterations in the

formation of the apical compartment are illustrated by the

inappropriate localization of mucin 1 and ZO-1. The disor-

ganization of these two markers may be linked. ZO-1 is a

critical organizer of tight junctions and consequently of the

apical compartment [41,42]; thus, an initial alteration of

ZO-1 organization is likely to have repercussions on other

polarity markers including mucin 1. The restriction of the

effect of 5Aza treatment to the apical compartment of acini

suggests that, in mammary epithelial cells, DNA methyla-

tion may regulate a set of genes that control the establish-

ment of apical polarity. Polarized sorting of plasma

membrane proteins and the formation of specific protein

complexes that define the baso-lateral and apical plasma

membranes are critical for the biogenesis and maintenance

of polarity. However, the signals and machinery involved in

polarized sorting and the mechanisms by which specific

protein complexes control polarization are far less under-

stood for apical polarity than for baso-lateral polarity [43].

Thus, many of the players involved in apical polarity remain

to be deciphered, especially for exocrine tissues like the

mammary gland. 5Aza treatment of 3-D culture of human

mammary epithelial cells may represent a useful tool to

further decipher the genetic determinism of apical polarity

in mammary tissue. Indeed, identifying the genes involved

in the control of apical polarity and/or the mechanisms

associated with their regulation would be of great interest to

advance the understanding of the mechanisms involved in

polarity-related disorders, including cancer [51].

5Aza treatment results also in the emergence of a

subpopulation of cells that express CK19, a marker of

epithelial cells that possess reduced characteristics of dif-

ferentiation [46–48]. Because the small percentage of acini

displaying altered apical polarity in control cultures non-

treated with 5Aza did not show any CK19 staining, the

expression of CK19 in 5Aza-treated cells is likely a conse-

quence of DNA hypomethylation rather than a consequence

of the lack of apical polarity. CK19 expression may be

regulated either directly by methylation or indirectly by the

action of other genes regulated by methylation. Our prelim-

inary investigation of CpG sites within the CK19 gene

sequence using the CpG island searcher website (http://

www.uscnorris.com/cpgislands/cpg.cgi) suggests the pres-

ence of a cluster of CpG sites that might be assimilated to a

CpG island in a region of the CK19 gene that overlaps the

starting sequence of the transcription site. Further investi-

gation will be required to test whether these sites can be

methylated. Although the exact significance of CK19 ex-

pression is still debated, there is growing evidence to

suggest that it is associated with stem or progenitor proper-

ties of epithelial cells found in adult tissues. CK19 distri-

bution coincides with the location of progenitor cells (basal

layer) in the epidermis, while it is not seen in layers

containing differentiated cells [47]. Similarly, in the ductal

pancreatic tree, cells expressing CK19 are considered as

potential precursors of both the endocrine and exocrine

compartments [48]. In the mammary gland, CK19 expres-

sion was recently demonstrated to pertain to luminal epi-

thelial cells that retain progenitor properties [46].

Interestingly, the limitation of CK19 expression to a small

fraction of the cell population in our culture system is

considerably similar to the distribution observed in breast

tissue biopsies [45,46]. The reason why there is only

sporadic expression of CK19 within the population of acini

remains to be determined. Although we have not shown that

the cells that express CK19 upon 5Aza treatment are

potential progenitors, our observation suggests that DNA

methylation might be one of the nuclear mechanisms

determining mammary cell precursor properties.

Taken together, our results indicate that DNA methyla-

tion may act at least on two critical aspects of mammary

epithelial differentiation, including tissue polarity and also

potentially progenitor capabilities. Alterations in apical

polarity and CK19 expression have been frequently ob-

served in breast cancer [52–55]. However, the mechanisms

involved are still unknown. DNA methylation might be a

promising path to explore with regards to the mechanisms

involved in the alteration of either of these differentiation

aspects in pathological situations.

Acknowledgments

We thank Eli Asem and Gurushankar Chandramouly for

critical reading of the manuscript, Bruce Stillman for the

MCM3 antibody, Thomas Jenuwein for the antibody against

branched methylated histone H3-K9, and Amelie Rodrigue

for technical assistance. This work was supported by grants

from the Walther Cancer Institute (WCI-110-114 to S.A.L.)

and the Jim and Diane Robbers Foundation at the Purdue

Cancer Center (S.A.L.), and a Fellowship from the Joyce

Fox Jordan Cancer Research Program at the Purdue Cancer

Center (C.P.).

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