Aberrant de novo methylation of the p16INK4ACpG
island is initiated post gene silencing in association
with chromatin remodelling and mimics
Rebecca A. Hinshelwood1, John R. Melki2, Lily I. Huschtscha3, Cheryl Paul1, Jenny Z. Song1,
Clare Stirzaker1, Roger R. Reddel3,4and Susan J. Clark1,?
1Cancer Program, Garvan Institute of Medical Research, 384 Victoria St, Darlinghurst, NSW 2010, Australia,2Human
Genetic Signatures, PO Box 184, North Ryde, NSW 1670, Australia,3Children’s Medical Research Institute, 214
Hawkesbury Rd, Westmead, NSW 2145, Australia and4University of Sydney, NSW 2006, Australia
Received February 26, 2009; Revised April 21, 2009; Accepted May 21, 2009
Changes in the epigenetic landscape are widespread in neoplasia, with de novo methylation and histone
repressive marks commonly enriched in CpG island associated promoter regions. DNA hypermethylation
and histone repression correlate with gene silencing, however, the dynamics of this process are still largely
unclear. The tumour suppressor gene p16INK4Ais inactivated in association with CpG island methylation
during neoplastic progression in a variety of cancers, including breast cancer. Here, we investigated the tem-
poral progression of DNA methylation and histone remodelling in the p16INK4ACpG island in primary human
mammary epithelial cell (HMEC) strains during selection, as a model for early breast cancer. Silencing of
p16INK4Ahas been previously shown to be necessary before HMECs can escape from selection. Here, we
demonstrate that gene silencing occurs prior to de novo methylation and histone remodelling. An increase
in DNA methylation was associated with a rapid loss of both histone H3K27 trimethylation and H3K9 acety-
lation and a gradual gain of H3K9 dimethylation. Interestingly, we found that regional-specific ‘seeding’
methylation occurs early after post-selection and that the de novo methylation pattern observed in HMECs
correlates with the apparent footprint of nucleosomes across the p16INK4ACpG island. Our results demon-
strate for the first time that p16INK4Agene silencing is a precursor to epigenetic suppression and that sub-
sequent de novo methylation initially occurs in nucleosome-free regions across the p16INK4ACpG island
and this is associated with a dynamic change in histone modifications.
Widespread changes in genomic DNA methylation patterns
occur during the transition from a normal cell to a cancer
cell, and this is associated with chromatin remodelling and
modified gene expression. Chromatin consists of nucleosomes,
each containing 147 bp of DNA wrapped around an octamer of
core histone proteins, which are separated from each other by
?50 bp of linker DNA (1). Nucleosome occupancy along
DNA promoters plays an important role in transcriptional
regulation (2), where both sliding and loss of nucleosomes
affect the accessibility of the DNA to transcription factors
(reviewed in 3). Malignant cells are characterized by a
global reduction in genomic DNA methylation and a localized
increase in methylation of CpG island-associated promoter
regions (reviewed in 4–7). CpG islands have a frequency of
CpG dinucleotides approximately five times greater than the
genome, and commonly span the promoter region of ubiqui-
tously expressed ‘housekeeping’ genes, and the 50or 30
regions of many tissue-specific genes (8,9). Typically the
CpG islands of transcriptionally active genes in normal cells
are unmethylated and associated with permissive histone
marks, whereas the CpG islands of transcriptionally inactive
genes in cancer cells are densely methylated and associated
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Human Molecular Genetics, 2009, Vol. 18, No. 16
Advance Access published on May 28, 2009
by guest on December 21, 2015
with repressive histone marks. However, the dynamics of
aberrant de novo methylation in CpG island-associated promo-
ters that mediate the transition from the unmethylated, active
state to the densely methylated, inactive state remain largely
unknown (10,11). It is not easy to address this question in
tumour tissue because DNA hypermethylation is often an
early event and therefore once the tumour is large enough to
detect the aberrant methylation process has already occurred
and the hypermethylated genes are already silenced.
In this study, we investigated the temporal change in epige-
netic modifications in the CpG island of the p16INK4Agene
(also known as CDKN2A and MTS-1). The p16INK4A
protein binds and inhibits the activities of cyclin-dependent
kinases CDK4 and CDK6 resulting in the hypophosphorylated
form of pRb which acts as an inhibitor of cell-cycle pro-
gression (reviewed in 12). Many human tumour cell lines
and primary tumours have lost expression of wild-type
p16INK4Aand this has led to the suggestion that the
p16INK4Agene encodes a tumour suppressor (13). Indeed, the
p16INK4ACpG island promoter is often hypermethylated in
many tumours including breast tumours, and this appears to
occur early in the oncogenic pathway (14–16). We and
others have shown that loss of p16INK4Aexpression in
human mammary epithelial cells (HMECs) is necessary for
in vitro lifespan extension (17), and this also correlates with
hypermethylation of the CpG island promoter (18–23).
HMECs, when cultured in serum-free medium, exhibit two
phases of growth (24,25). The first growth phase lasts for
several population doublings (PDs), after which growth tem-
porarily ceases (termed selection or M0). Within 2–4 weeks,
colonies of small cells with a basal mammary epithelial phe-
notype (26,27) appear with enhanced growth capacity and
these colonies continue to proliferate for another 20–40 PDs
before entering a second growth plateau resembling cell
crisis, termed agonescence (28). HMECs isolated during the
first growth phase are termed pre-selection cells, and those
isolated during the second growth phase are termed either
post-selection or variant HMECs (vHMECs) (19,22,24). Post-
selection HMECs have been shown to share many character-
istics of pre-malignant breast-cancer cells, including both
genetic and epigenetic lesions (21,23,29,30), and therefore
provide an ideal primary cell model to study early epigenetic
changes in malignancy.
To investigate the temporal relationship between gene silen-
cing, DNA methylation and chromatin remodelling, we ana-
lysed in detail de novo methylation of the p16INK4ACpG
island in post-selection HMECs that were not expressing
p16INK4A. Using this primary tissue model, we first found
that hypermethylation of the p16INK4ACpG island occurs
only after the gene is silenced, and secondly we demonstrated
that a low level of de novo methylation is associated with a
dynamic remodelling of associated chromatin, as early as the
first passage following selection. Lastly, we demonstrated
that individual post-selection strains share a common pattern
of regional-specific initial ‘seeding’ methylation within the
p16INK4ACpG island. Using a high-resolution foot-printing
technique, known as methylase-based single-promoter analy-
sis assay (MSPA) (31), which exploits the fact that nucleo-
somes and binding factors restrict M. Sss I CpG methylase
from methylating the DNA (32), we found that the ‘seeding’
methylation ‘hot spots’ correlated with the position of nucleo-
somes in the post-selection HMECs. Our results demonstrate
for the first time that p16INK4Agene silencing is a precursor
to epigenetic changes in post-selection HMECs and that
subsequent de novo methylation occurs primarily in nucleo-
some-free regions across the p16INK4ACpG island and then
progressively spreads to adjacent regions with proliferation.
p16INK4Ais inactivated and heterogeneously methylated
in post-selection HMECs
To study the underlying epigenetic changes that occur during
p16INK4Asilencing in HMECs that have undergone selection,
we first used quantitative reverse transcription-PCR (qRT–
PCR) to show that p16INK4AmRNA expression is inactivated,
or down regulated, in the individual post-selection HMECs
strains, Bre-12 and Bre-40 (30), and Bre-56, Bre-60 and
Bre-80, compared with their isogenic pre-selection HMECs
(Fig. 1A). We also confirmed that a loss of p16INK4AmRNA
expression correlates with a loss in p16INK4A
(Fig. 1B), as previously shown in Bre-40, Bre-60, Bre-70
and Bre-80 post-selection cells (19). Using direct PCR bisul-
phite sequencing, we reported that p16INK4Asilencing in
Bre-40, Bre-60, Bre-70 and Bre-80 post-selection HMECs cor-
relates with extensive DNA hypermethylation of the CpG
island-associated p16INK4Apromoter (19). To better under-
stand the dynamics and initial events leading to CpG island
methylation, we have analysed the DNA methylation profiles
in more detail by bisulphite clonal sequencing across three
neighbouring regions of the p16INK4ACpG island spanning
1035 bp across the start of transcription and first exon (Sup-
plementary Material, Fig. S1). The post-selection HMECs
from each strain comprised 10–40 independent colonies that
escaped selection and these colonies were pooled prior to
analysis to gain a comprehensive view of the DNA methyl-
ation patterns. Figure 2 summarizes the bisulphite methylation
sequencing data from Bre-38 and Bre-40 pre- and post-
selection cells across 71 CpG sites (numbered from –19
CpG to þ52 CpG relative to the start of transcription). The
actively p16INK4Aexpressing pre-selection cells (Bre-38 and
Bre-40) were essentially unmethylated from CpG sites –19
to þ38, even though there was evidence of low-level sporadic
CpG methylation noted in Bre-40 (Fig. 2A and C). In the pre-
selection cells, the level of methylation was further enriched
downstream from the start of transcription at CpG sites þ40
to þ52 in both Bre-38 and Bre-40 (Fig. 2A and C) correlating
to the boundary region of the p16INK4ACpG island (Sup-
plementary Material, Fig. S1). In contrast, in the correspond-
ing Bre-38 and Bre-40 post-selection HMECs [passage 11
(p11) and 10 (p10), respectively] there was extensive DNA
methylation across the three regions analysed (Fig. 2B and
D) and this occurred in nearly all molecules, suggesting that
DNA methylation of p16INK4Ais bi-allelic.
Even though the DNA methylation was generally extensive,
there was also considerable heterogeneity noted, with some
molecules having no DNA methylation or methylation at
only a few of the CpG sites, despite p16INK4Aexpression
Human Molecular Genetics, 2009, Vol. 18, No. 163099
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being suppressed in these cells. The DNA methylation was
also not uniformly distributed between the CpG sites. Blocks
of adjacent CpG sites appeared to be more resistant to methyl-
ation, whereas other CpG sites appeared to be predominately
methylated in ‘hot spots’ (Fig. 2B and D). Four such ‘hot
spots’ were found to occur in similar locations in Bre-38
and Bre-40 post-selection HMECs across regions I and II.
To ascertain whether the same CpG sites were also ‘hot
spots’ for the de novo methylation in different post-selection
HMEC strains, we further analysed region II in other post-
selection HMEC strains, Bre-60 (p7) and Bre-70 (p9)
(Fig. 3). Focal ‘hot spots’ of methylation of adjacent CpG
sites were also observed for these post-selection HMECs,
even though the location of the specific sites were slightly
different from strain to strain.
p16INK4Asilencing in post-selection HMECs occurs prior
to DNA hypermethylation
We previously showed that p16INK4Ais inactivated and
methylated in post-selection HMECs (19), but what was not
clear at the time was if DNA methylation was causing
p16INK4Asilencing or if p16INK4Asilencing was promoting
DNA methylation. The fact that the methylation pattern in
all the different post-selection HMEC strains was hetero-
geneous, and a number of molecules remained unmethylated
or had only minimal methylation in the early passages after
selection (Figs 2B,D and Fig. 3), led us to ask if p16INK4A
silencing preceded DNA methylation. To address this ques-
tion, we used laser capture microscopy to isolate individual
post-selection HMECs that had just emerged from selection
and stained negative for p16INK4Aexpression (Fig. 4A–C).
At this early time point, each post-selection colony had only
expanded to approximately 30 cells or 4–5 PDs. We per-
formed bisulphite sequencing on pools of 20 individually
laser-captured cells that were negative for p16INK4Aexpression
from a colony at the time of selection (Fig. 4B), and compared
the methylation profile to 20 pooled individually laser-
captured pre-selection or senescent cells, from the same
dish, that were positive for p16INK4Aexpression (Fig. 4A,
Supplementary Material, Fig. S2). Figure 4D summarizes the
methylation results and shows that there was little or no
methylation evident in either the p16INK4Asilent HMECs at
the time of selection, or the surrounding p16INK4Aexpressing
pre-selection cells (Fig. 4D). The silencing of the p16INK4A
gene therefore appears to occur independently and prior to
subsequent de novo methylation.
p16INK4Ahypermethylation originates from focal CpG
sites and progressively spreads with proliferation
We performed a detailed analysis on post-selection cells
derived from a single HMEC colony to ascertain if the hetero-
geneity in DNA methylation observed in post-selection
HMECs was due to the fact that we had pooled individual
colonies prior to passaging or if the heterogeneity reflected
the inherent stochastic nature of de novo and or maintenance
methylation. We isolated HMECs from a single Bre-80
colony at selection and cultured the cells for an additional
39 passages and analysed methylation at passages 1, 4, 10,
23, 26 and 39. Figure 5 shows the progressive expansion of
DNA methylation we observed across region II of the
p16INK4ACpG island. In the pre-selection Bre-80 cells,
region II was essentially unmethylated with only a few
single methylated CpG sites; however, by passage 1 after
selection, ‘hot spots’ of methylation were already observed
in some of the molecules. With increasing passage number,
the density of methylation also progressively increased, and
by passage 39 all molecules were hypermethylated in nearly
50% of CpG sites, with two discrete ‘hot spots’ flanking a
region of CpG sites that was more resistant to methylation.
Indeed, the overall methylation pattern generated from the
single Bre-80 clone was remarkably similar to the patterns
observed from the pooled post-selection colonies in each of
different HMEC strains (for example Bre-60 and Bre-70)
(Fig. 5B), suggesting a directive process.
Nucleosome occupancy of the p16INK4ACpG island mimics
the post-selection HMEC methylation profile
The regularity of methylation ‘hot spots’ that we observed
across the p16INK4ACpG island, in each of the different
Figure 1. Suppression of p16INK4Aexpression in post-selection HMECs. (A) mRNA levels of p16INK4Ain Bre-12, Bre-40, Bre-56, Bre-60 and Bre-80 post-
selection HMECs were determined by quantitative RT–PCR. After normalizing expression to 18S rRNA, the fold change in expression levels was made relative
to pre-selection HMECs. All donor post-selection strains show reduced levels of p16INK4AmRNA. (B) Western blot showing p16INK4Aprotein expression in
Bre-56 pre-selection cells, and p16INK4Asilencing in Bre-56 and Bre-80 post-selection cells. Actin levels were used as a loading control.
3100Human Molecular Genetics, 2009, Vol. 18, No. 16
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post-selection HMEC strains, prompted us to address if this sig-
used the new high-resolution foot-printing technique, known as
MSPA (31), to determine the position of the nucleosomes
across the three neighbouring regions of the p16INK4ACpG
island promoter. The advantage of this method is that it provides
an in vitro map of regions that are protected from the DNA
methyltransferase enzyme. Nuclei were prepared from Bre-38
pre-selection HMECs and analysed for accessibility to M.Sss I
CpG methylase. M.Sss I CpG methylase treatment of control
naked genomic DNA was also performed as a control for the
extent of enzyme activity. Figure 6A confirms that p16INK4A
was essentially unmethylated in Bre-38 pre-selection HMECs
and after M.Sss I treatment the control genomic DNA became
extensively methylated (Fig. 6B). In contrast, when the Bre-38
pre-selection nuclei were treated with M.Sss I, the p16INK4A
DNA molecules were methylated in three distinct patterns
(Fig. 6C). Class I clones, exhibited two focal regions of methyl-
ation flanking a central region of protection from M.Sss I treat-
ment, whereas class II and III clones appeared to be protected
from methylation at one or other end of the p16INK4Aamplicons
examined. When the methylation pattern of the clones were ana-
of ?150 bpinlengthareshowntobemoreprotectedfromM.Sss
suggesting the loss of a nucleosome in the actively expressing
Bre-38 pre-selection HMECs (Fig. 6D). Interestingly, the
regions that were more susceptible to in vitro M. Sss I
Figure 2. p16INK4Asilencing in post-selection cells is accompanied by DNA hypermethylation. Bisulphite methylation clonal sequencing analysis was performed
across three neighbouring regions of the p16INK4ACpG island associated promoter region. Amplicon locations: region I, region II and region III. The start of
transcription, as indicated by Genbank accession number p16INK4A(AB060808), is indicated by a black arrow. CpG sites are numbered relative to the start of
transcription. (A) Bre-38 pre-selection cells [passage 5 (p5)]. (B) Bre-38 post-selection cells (p11). (C) Bre-40 pre-selection cells (p5). (D) Bre-40 post-selection
cells (p10). White circle, unmethylated CpG site; black circle, methylated CpG site. Red bars indicate focal ‘hot spots’ of DNA methylation in post-selection
Human Molecular Genetics, 2009, Vol. 18, No. 16 3101
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signature pattern of methylation observed in vivo in the Bre-38
Figure 6E. Conversely, the regions that were protected from
M.Sss I methylation in vitro (Fig. 6D) correspond to the
regions protected in vivo in the Bre-38 post-selection HMECs
(Fig. 6E). In both cases, the region spanning the start of tran-
scription was more susceptible to methylation, but in contrast
to the in vitro M.Sss I methylation data, the in vivo methylation
profile observed in the Bre-38 post-selection HMECs where
ent with a gain of a nucleosome near the start of transcription
(Fig. 6D,E). Supplementary Material, Figure S3 shows a com-
parative analysis of in vitro MSPA pre-selection Bre-38 data
terns using lowess curves that were generated and smoothing
applied over 10 data points. A clear correlation can be observed
between the apparent nucleosome footprint generated using the
MSPA technique and the DNA methylation wave signature
pattern observed in the post-selection HMECs. However, after
the start of transcription, the phasing of the methylation foot-
print, reflecting nucleosome positioning, appeared to be more
compact in the post-selection cells, which is consistent with
the inactive state of p16INK4Ain these cells and de novo methyl-
ation in the nucleosome-free linker regions.
breast-cancer cells, we performed MSPA on MDAMB453 cells,
a breast-cancer cell line in which p16INK4Ais also expressed and
the p16INK4ACpG island are essentially unmethylated in the
expressing MDAMB453 cells
Fig. S4A). Secondly, when MDAMB453 nuclei were treated
with M.Sss I, we also observed distinct focal regions of in vitro
methylation in similar focal regions to the pre-selection Bre-38
M.Sss I treated nuclei (Supplementary Material, Fig. S4B).
Thirdly, the M.Sss I methylation clonal pattern found in the
MDAMB453 cells also mimics the in vivo wave pattern of
methylation observed for the post-selection cells Bre-38 (Sup-
plementary Material, Fig. S4C). The combined data supports
the hypothesis that the nucleosome footprint dictates the de
novo methylation signature pattern across the p16INK4ACpG
island in both normal breast cells and breast-cancer cells.
p16INK4Aepigenetic silencing in post-selection HMECs
is associated with dynamic chromatin remodelling
To determine the relationship between p16INK4Asilencing in
post-selection HMECs, DNA methylation and chromatin remo-
delling, we performed chromatin immunoprecipitation (ChIP)
assays with acetylated H3K9 (H3K9Ac), dimethylated H3K9
(H3K9Me2) and trimethylated H3K27 (H3K27Me3) antibodies
to compare histone modifications in pre- and post-selection
cells. p16INK4A-associated chromatin isolated from actively
expressing pre-selection HMECs was enriched for active
selection cells at the first passage after selection which remained
deacetylated with further passaging (Fig. 7A). In contrast,
p16INK4Aassociated chromatin from pre-selection HMECs was
depleted in the repressive H3K9Me2 mark, but after selection
slowly accumulated histone methylation marks over several
Figure 3. DNAhypermethylationofp16INK4Aoccursatfocal‘hotspot’regions.
the p16INK4ACpG island-associated promoter, revealing that there is a distinct
scription. (A) Bre-38 post-selection cells (p11). (B) Bre-40 post-selection cells
(p10). (C) Bre-60 post-selection cells (p7). (D) Bre-70 post-selection cells
(p9). White circle, unmethylated CpG site; black circle, methylated CpG site.
Red bars indicate focal ‘hot spots’ of DNA methylation.
3102Human Molecular Genetics, 2009, Vol. 18, No. 16
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in pre-selection cells, and this was rapidly depleted in post-
selection HMECs at the first passage after selection (Fig. 7C).
Similar results were observed using different HMEC strains
(Supplementary Material, Fig. S5). Together these data show
that p16INK4Asilencing at selection promotes a progressive
increase in de novo CpG methylation in post-selection HMECs
(Fig. 7D) and a parallel deacetylation of H3K9 and loss of tri-
sequently and gradually increases with DNA methylation
prehensive analysis of the temporal steps involved in p16INK4A
silencing, epigenetic reprogramming and nucleosome position-
lated from disease-free breast tissue provide a uniquely
(reviewed in33).Post-selectionorvariantHMECsexhibit many
preneoplastic characteristics, including transcriptional silencing
of the p16INK4Atumour suppressor gene and overexpression
of cyclo-oxygenase 2 (Cox-2) (18–23) as well as epigenetic
of p16INK4Aalso occurs commonly in pre-malignant lesions and
rare foci of morphologically normal epithelial cells exhibiting
precursors, which can promote malignancy with additional epi-
genetic and or genetic changes (29).
One of the biggest challenges in dissecting the processes
involved in epigenetic reprogramming in cancer is that the
changes occur early in oncogenesis and are consequently dif-
ficult to study in clinical samples. We therefore used HMECs
grown in serum-free conditions to study the early epigenetic
changes that are associated with p16INK4Asilencing. We and
others have previously shown that p16INK4Asilencing in post-
selection cells that arise under these culture conditions is
associated with DNA hypermethylation of the CpG island pro-
moter and this occurs just as the first post-selection cells
emerge from selection (18–23). We now demonstrate that
DNA hypermethylation of p16INK4ACpG island occurs only
after the gene is silent and that the initial ‘seeds’ of de novo
methylation accumulate in the accessible regions of the
chromosome and this is associated with a dynamic remodel-
ling of the chromatin.
Over the years, there has been constant debate over the
mechanism of epigenetic silencing of tumour suppressor
genes in cancer (11,34,35). Central to this debate is the
Figure 4. p16INK4silencing in HMECs occurs prior to DNA hypermethylation. Laser capture dissection techniques were used to isolate single Bre-70 pre- and
post-selection cells that were positive or negative for p16INK4Aexpression. (A) Bre-70 pre-selection cells expressing p16INK4A(positive staining) and small post-
selection cells silenced for p16INK4A. (B) Selection of p16INK4Asilenced cells for dissection. (C) Cells after dissection, showing specific isolation of p16INK4A
silenced cells. (D) Bisulphite methylation clonal sequencing analysis of p16INK4Afrom single Bre-70 post-selection (p16INK4Asilenced) and pre-selection cells
(p16INK4Aexpressed) isolated by laser capture microscopy. Newly emerging post-selection HMECs that were negative for p16INK4Adid not exhibit methylation.
CpG sites are numbered relative to the start of transcription. White circle, unmethylated CpG site; black circle, methylated CpG site.
Human Molecular Genetics, 2009, Vol. 18, No. 163103
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question of whether DNA methylation and histone modifi-
cations are a consequence or cause of gene silencing? A
favoured dogma is that DNA methylation causes gene silen-
cing and was demonstrated in early in vitro studies (36–38).
However, more recently the hypothesis that DNA methylation
is a consequence of gene silencing is gaining support. We have
previously reported that transcriptional silencing of a geneti-
cally manipulated GSTP1 gene in LNCaP cells precedes
DNA methylation (39), and the low level ‘seeding’ methyl-
ation observed in normal cells promotes H3K9 deacetylation
and H3K9 methylation. In contrast, Bachman et al. 2003
(35) reported that silencing of p16INK4Aand H3K9 methylation
preceded DNA methylation also using an experimentally con-
trived situation. The advantage of the HMEC system is that it
allows us to address the interplay between gene inactivation
and epigenetic changes at the earliest time points after
p16INK4Asilencing in an in vivo setting. Using laser capture
dissection of single cells clearly demonstrated that de novo
methylation occurred post gene silencing, and analysis of a
single colony showed that methylation was progressive
rather than being a single aberrant event that encompasses
the entire island, supporting previous findings (10,19). Even
though the methylation of individual CpG sites was stochastic,
it was clear that there was an overall similar pattern between
different HMEC strains. Using the new single-molecule foot-
printing technique developed to visualize nucleosome occu-
pancy at high resolution (31), we were able to demonstrate
that the signature wave pattern of methylation observed in
the post-selection cells was similar to the observed nucleo-
some footprint across the p16INK4ACpG island. Our results
suggest that chromatin accessibility is dictating the initial
access to the DNA methyltransferase enzyme, thereby only
permitting aberrant de novo methylation to limited regions
within the p16INK4ACpG island which then spreads progress-
ively with PD. Our data also suggests that the nucleosomes are
quite mobile in the actively expressing cells, and not precisely
positioned along the p16INK4Apromoter. This is not unex-
pected, as it is well known that nucleosomes are not a
simple static unit; rather, they are a dynamic element that
can slide along the DNA, thus permitting access or constraints
to transcriptional machinery (3). Indeed, the transitory nature
of nucleosomes was emphasized in a recent MSPA analysis
of the GRP78 promoter during endoplasmic reticulum (ER)
stress (40). Similar to our finding, silent promoters have gen-
erally been shown to be enriched for nucleosomes relative to
their active counterparts (41), and nucleosome depletion at
CpG islands has been described for several epigenetically
regulated human gene promoters (31,42). For example, the
methylated and silent MLH1 promoter was found to be occu-
pied by three nucleosomes in RKO cells, which were evicted
upon demethylation and activation of the promoter by
The final implication of our work relates to chromatin remo-
selection cells was associated with the lack of a nucleosome at
the start of transcription and bivalent histones consisting of
the active H3K9Ac mark and the repressive polycomb
EZH2-associated H3K27Me3 mark. After p16INK4Ainacti-
vation, the nucleosomes are remodelled with the gain of a
nucleosome across the start of transcription, and a rapid deace-
tylation of H3K9 in concert with the rapid removal of the
H3K27Me3 polycomb mark. In contrast, dimethylation of
H3K9 accumulates more gradually in parallel with the accumu-
lation of DNA methylation, resulting in consolidation of
p16INK4Agene silencing. Our data supports recent findings
Figure 5. DNA hypermethylation of p16INK4Ain a single Bre-80 colony
increases and expands with successive passaging. (A) Bisulphite methylation
clonal sequencing analysis of p16INK4Ain a single Bre-80 colony at selection
(top panel). A single Bre-80 colony was cultured until the end of its lifespan,
and the DNA methylation status of p16INK4Adetermined at passage numbers 1,
4, 10, 23, 26 and 39 (relative to selection). Density of methylation of the CpG
sites increases with increasing passage number. CpG sites are numbered rela-
tive to the start of transcription. White circle, unmethylated CpG site; black
circle, methylated CpG site. Red bars indicate focal ‘hot spots’ of DNA
methylation. (B) Quantitation of bisulphite clonal sequencing data for
Bre-60 (p7), Bre-70 (p9) and Bre-80 (p23) post-selection cells, highlighting
similar methylation patterns.
3104 Human Molecular Genetics, 2009, Vol. 18, No. 16
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Figure 6. MSPA of p16INK4Ain Bre-38 pre-selection cells. Bisulphite methylation clonal sequencing analysis of p16INK4Ain M.Sss I treated Bre-38 pre-selection
cells was performed to ascertain whether nucleosomes influence p16INK4Amethylation patterns in post-selection HMECs. (A) Untreated Bre-38 pre-selection
cells. (B) M.Sss I treated control genomic DNA. (C) M.Sss I treated Bre-38 pre-selection nuclei. Methylation patterns were divided into three distinct
classes; class I (top), class II (middle) and class III (bottom). (D) Quantitation of in vitro Bre-38 M.Sss I MSPA clonal data. (E) Quantitation of in vivo
Bre-38 post-selection clonal methylation data from Fig. 2B. The methylation footprints at the single molecule level reveal ?150 bp protected regions consistent
with nucleosome units across each of the three regions. Proposed nucleosome units (?150 bp) are indicated by green ovals, while nucleosome devoid regions are
indicated by dotted white ovals. CpG sites are numbered relative to the start of transcription. White circle, unmethylated CpG site; black circle, methylated CpG
site. Red bars indicate focal ‘hot spots’ of DNA methylation in Bre-38 post-selection cells (as shown in Fig. 2B).
Human Molecular Genetics, 2009, Vol. 18, No. 163105
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that many genes including those involved in cell-fate determi-
nation, stem-cell renewal, cell growth and cell division are
marked by polycomb in the normal cells, but are susceptible
to aberrant DNA methylation in cancer cells (43,44). What
remains unclear is the mechanism responsible for rapid
change from the bivalent histone mode to one associated with
DNA methylation and histone deacetylation and methylation.
Our studies demonstrate that gene silencing in pre-malignancy
is key to tipping the balance from an epigenetically plastic con-
dition to one that becomes repressively locked.
MATERIALS AND METHODS
Cells and cell culture
Breast tissue removed from reduction mammoplasties was
obtained with institutional Ethics Committee approval and
informed donor consent. HMEC cultures were prepared from
normal breast tissue for donor strains Bre-12, Bre-38, Bre-40,
Bre-56, Bre-60, Bre-70 and Bre-80 as previously described
cultured according to the protocol described in (19,24).
Quantitative real-time reverse transcription PCR
RNA was extracted from Bre-12, Bre-40, Bre-56, Bre-60 and
Bre-80 pre- and post-selection HMECs using Trizol Reagent
protocol.cDNAwas reverse transcribedfrom1 mg of total RNA
using SuperScript III RNase H2Reverse Transcriptase (Invitro-
gen) according to the manufacturer’s protocol. The reaction was
primed with 150 ng of random primers (Boehringer-Mannheim,
Castle Hill, NSW, Australia). The reverse transcription reaction
was diluted 1:10 with sterile H2O before addition to the reverse
transcription PCR. p16INK4Aexpression primer sequences are
described in Supplementary Material, Table S1. p16INK4A
expression was quantitated using the 7900HT Applied Biosys-
tems Sequence Detection System as described (30).
selection (p12), and Bre-80 post-selection (p52) cells was per-
formed essentially as described in (45). p16INK4Awas detected
with an anti-p16 (Ab-1) mouse monoclonal antibody (DSC-
50.1/H4: Calbiochem/Merck KGaA, Darmstadt, Germany).
DNAwasisolatedfromlessthan1 ? 106pre-andpost-selection
cells using simple lysis buffer (2 mg tRNA, 280 ng/ml
proteinase K, 1% SDS) (46), or from 1–3 ? 106cells using
either Puregene DNA isolation kit (Gentra Systems, Inc., Min-
neapolis, USA) according to the manufacturer’s instructions,
Figure 7. Interplay between accumulation of repressive chromatin and de novo CpG methylation associated with p16INK4Aepigenetic silencing. Chromatin from
pre- and post-selection HMECs was immunoprecipitated with (A) acetylated H3K9 (H3K9Ac), (B) dimethylated H3K9 (H3K9Me2) and (C) trimethylated
H3K27 (H3K27Me3) antibodies. The amount of immunoprecipitated DNA (relative binding) was quantified by real-time PCR and was calculated as a ratio
of immunoprecipitated DNA to the total amount of input. (D) Bisulphite clonal sequencing data for Bre-80 (as described in Fig. 5) was quantitated as
average CpG methylation at each passage. A rapid deacetylation of H3K9 was associated with a loss of H3K27me3 and gain of DNA methylation followed
by a slower gain of H3K9 methylation.
3106Human Molecular Genetics, 2009, Vol. 18, No. 16
by guest on December 21, 2015
or with Trizol Reagent with the following modifications to the
manufacturer’s protocol: 300 ml of 100% ethanol and 20 mg
of tRNA was added to the reserved organic phase, the
samples inverted and incubated at room temperature for
3 min. Samples were centrifuged at 12 000 g for 15 min at
48C. The supernatant was carefully removed, the DNA pellet
resuspended in 18 ml of simple lysis buffer and the sample
incubated at 558C overnight prior to bisulphite conversion.
Laser capture microdissection
HMECs were stained for p16INK4Aexpression with a Envision
DAKO kit as previously described (19). Single pre-selection
Bre-70 cells that stained p16INK4Apositive and post-selection
Bre-70 cells that were p16INK4Anegative, were isolated using
the PALM Robot Microbeam laser microdissection system
(P.A.L.M GmbH, Bernried, Germany) (47). Twenty cells for
each cell type were captured in the tube cap in duplicate for
buffer (100 mM Tris–HCl pH 8.0, 3% SDS, 50 mM EDTA,
200 mg/ml Proteinase K).
placedin 18 ml lysis
DNA methylation studies
Bisulphite genomic sequencing was used to analyse the
methylation status of three neighbouring regions of p16INK4A
in pre- and post-selection HMECs (Supplementary Material,
Fig. S1). The bisulphite reaction was carried out on extracted
Figure 8. Summary of epigenetic silencing of p16INK4Ain post-selection HMECs. In pre-selection HMECs, the p16INK4Atranscription start site (TSS) is devoid
of a nucleosome. The CpG island-associated promoter region is marked by DNA methylation ‘seeds’, there is active gene transcription and the chromatin is in a
bivalent state as it is marked by both active (H3K9Ac) and repressive (H3K27Me3) histone modifications. As a consequence of stochastic gene silencing,
p16INK4Aundergoes epigenetic deregulation in post-selection HMECs through a DNA methylation-associated mechanism. In early post-selection cells, there
is nucleosome gain across the TSS, which is accompanied by loss of H3K27Me3, deacetylation of H3K9, and evidence of DNA methylation expansion
from ‘seeds’ within nucleosome linker regions. In late passage post-selection HMECs, there is consolidation of gene silencing through an enrichment of
H3K9Me and extensive spreading and accumulation of DNA methylation.
Human Molecular Genetics, 2009, Vol. 18, No. 163107
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DNA for 16 h at 558C on up to 2 mg of digested DNA, under
conditions described previously (48,49). Laser-captured cells
were incubated in 18 ml of DNA lysis buffer for 30 mins at
378C prior to bisulphite treatment for 4 h, as described (49).
After bisulphite conversion, the DNA was ethanol precipi-
tated, dried, resuspended in 10–50 ml H2O and stored at
2208C. Triplicate PCR amplifications were performed for
p16INK4Aand pooled. The primer sequences and location of
the p16INK4Aamplicons in relation to the CpG island and
start of transcription are summarized in Supplementary
Material, Table S1 and Supplementary Material, Figure S1.
The methylation status of p16INK4Awas determined by bisul-
phite clonal sequencing of the pooled PCR products, as
described (50) to ensure representative clonal analysis.
Example DNA sequence traces are shown in Supplementary
Material, Fig. S6.
Methylase-based single-promoter analysis assays
Nucleosome positioning assays were performed by a high-
resolution, MSPA, essentially as described in (31,42) and
with the following modifications. Actively growing Bre-38
pre-selection HMECs, or MDAMB453 breast cancer cells,
were trypsinized and washed twice with cold PBS. Cells
were resuspended in 1 ml of cold RSB buffer (10 mM Tris–
HCl; pH 7.4, 10 mM NaCl and 3 mM MgCl2) per 10 ? 106
cells and incubated on ice for 10 min. One microlitre of
10% NP-40 detergent per 10 ? 106cells was added and the
cells were homogenized at 48C with the tight pestle in a
glass dounce homogenizer for at least 15 strokes. Nuclei
were washed with 1 ml of cold RSB buffer and resuspended
in 74.25 ml of 1? M. Sss I bufferþsucrose per 1 ? 106
nuclei. 1 ? 106nuclei were treated with 60 units of M. Sss I
(New England BioLabs, Inc., Beverly, MA, USA) in a final
volume of 150 ml for 15 min at 378C. M. Sss I treated purified
control gDNA (6 mg) was used as a positive control. Reactions
were stopped by the addition of an equal volume of stop sol-
ution (20 mM Tris–HCl; pH 7.9, 600 mM NaCl, 1% SDS and
10 mM EDTA) and proteinase K treatment for 48 h at 378C.
DNA was purified by phenol chloroform extraction, ethanol
precipitated and resuspended in water. One microgram of
sheared M. Sss I treated nuclei or control gDNA was bisulphite
converted for 16 h at 558C, under conditions previously
described (48). After bisulphite conversion, the DNA was
ethanol precipitated, dried, resuspended in 50 ml water and
stored at 2208C. Bisulphite genomic sequencing was used
to analyse the methylation status of individual molecules
modified by M. Sss I across the three neighbouring regions
of the p16INK4ACpG island associated promoter region
described in Supplementary Material, Figure S1. Lowess
curves were generated and smoothing applied over 10 data
points in order to highlight similarities between the methyl-
ation patterns seen for the in vitro MSPA data with the in
vivo Bre-38 post-selection methylation data.
Chromatin immunoprecipitation assays
Chromatin immunoprecipitation assays were carried out
according to themanufacturer’s
Millipore, Temecula, CA, USA) using pooled Bre-60 and
Bre-70 pre-selection cedllsd, Bre-80 post-selection ceslls,
Bre-12 pre- and post-selection cells, Bre-40 pre- and post-
selection cells and Bre-38 pre- and post-selection cells, essen-
tially as described (30). The eluted complexes were immuno-
precipitated with antibodies specific for acetylated H3K9
(H3K9Me2, Upstate/Millipore) and trimethylated H3K27
measured by quantitative real-time PCR as described (30).
Chromatin immunoprecipitation amplification primers are
described in Supplementary Material, Table S1.
and DNAyield was
Supplementary Material is available at HMG online.
We would like to thank Marcel Coolen for critical reading of
the manuscript and help with figures, Christopher Molloy and
Jane Noble for experimental assistance and Professors Thea
Tlsty and Peter Jones for technical advice.
Conflict of Interest statement. None declared.
This work is supported by Cancer Institute NSW (CI NSW),
National Breast Cancer Foundation (NBCF) and National
Health and Medical Research Council (NH&MRC) project
grants; Dora Lush Biomedical Postgraduate Scholarship
from the NH&MRC, CI NSW Research Scholar Award and
NBCF Excellence Award (R.A.H.); and NH&MRC fellow-
ships (S.J.C. and R.R.R.).
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