Cell Reports Article Global Changes in the Mammary Epigenome Are Induced by Hormonal Cues and Coordinated by Ezh2 Bhupinder Pal, 1,3 Toula Bouras, 1,3,8 Wei Shi, 2,5,8 Franc ¸ ois Vaillant, 1,3 Julie M. Sheridan, 1,3 Naiyang Fu, 1,3 Kelsey Breslin, 1 Kun Jiang, 1 Matthew E. Ritchie, 2,3 Matthew Young, 2 Geoffrey J. Lindeman, 1,4,7,8 Gordon K. Smyth, 2,6,8 and Jane E. Visvader 1,3, * 1 ACRF Stem Cells and Cancer Division 2 Bioinformatics Division The Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, VIC 3052, Australia 3 Department of Medical Biology 4 Department of Medicine 5 Department of Computing and Information Systems 6 Department of Mathematics and Statistics The University of Melbourne, Parkville, VIC 3050, Australia 7 Department of Medical Oncology, The Royal Melbourne Hospital, Grattan Street, Parkville, VIC 3050, Australia 8 These authors contributed equally to this work *Correspondence: [email protected]http://dx.doi.org/10.1016/j.celrep.2012.12.020 SUMMARY The mammary epithelium is a dynamic, highly hormone-responsive tissue. To explore chromatin modifications underlying its lineage specification and hormone responsiveness, we determined genome- wide histone methylation profiles of mammary epithe- lial subpopulations in different states. The marked differences in H3K27 trimethylation between subpop- ulations in the adult gland suggest that epithelial cell-fate decisions are orchestrated by polycomb- complex-mediated repression. Remarkably, the mam- mary epigenome underwent highly specific changes in different hormonal contexts, with a profound change being observed in the global H3K27me3 map of luminal cells during pregnancy. We therefore exam- ined the role of the key H3K27 methyltransferase Ezh2 in mammary physiology. Its expression and phosphorylation coincided with H3K27me3 modifica- tions and peaked during pregnancy, driven in part by progesterone. Targeted deletion of Ezh2 impaired alveologenesis during pregnancy, preventing lacta- tion, and drastically reduced stem/progenitor cell numbers. Taken together, these findings reveal that Ezh2 couples hormonal stimuli to epigenetic changes that underpin progenitor activity, lineage specificity, and alveolar expansion in the mammary gland. INTRODUCTION The mammary gland, which comprises a branching ductal epi- thelial network embedded in an adipose-rich stromal matrix, is remarkably adaptive to physiological requirements and undergoes dramatic morphological changes during puberty and pregnancy. At birth, it manifests as a rudimentary branched structure, but ductal elongation and branching commence with puberty and pregnancy provokes the rapid expansion of alveolar units that differentiate into milk-secretory cells prior to parturi- tion. The steroid hormones estrogen and progesterone exert pivotal roles during mammary development via their cognate receptors, the estrogen receptor (ER) and progesterone receptor (PR) (Brisken and O’Malley, 2010). ER is essential for ductal morphogenesis in puberty (Mallepell et al., 2006; Mueller et al., 2002), whereas PR governs ductal side-branching and alveolar development during pregnancy (Brisken et al., 1998; Lydon et al., 1995; Mulac-Jericevic et al., 2003). The mammary epithelium can be divided into two primary line- ages: the myoepithelial lineage constitutes the outer layer of cells that contact the basement membrane, whereas the luminal lineage comprises both ductal and alveolar cells. Adult stem cells prospectively isolated from the mouse mammary gland display the requisite stem cell properties of multilineage differen- tiation and self-renewal (Shackleton et al., 2006; Stingl et al., 2006). A recent study has added a new layer of complexity to the prevailing model of the mammary epithelial hierarchy through the identification of unipotent cells that contribute to homeo- stasis of the gland (Van Keymeulen et al., 2011). It is not yet clear whether these correspond to stem or progenitor cells and what their relationship is to the prospectively isolated epithelial sub- sets. In the mouse, mammary stem cells (MaSCs) have been shown to be highly responsive to steroid hormones (Asselin- Labat et al., 2010; Joshi et al., 2010), while progesterone augmented the number of human bipotent stem-like cells in cellular assays (Graham et al., 2009). MaSCs lie at the apex of the hierarchy and give rise to progenitors and mature cells through progressive restriction. Two distinct types of luminal progenitor cells have been isolated from the mouse mammary Cell Reports 3, 411–426, February 21, 2013 ª2013 The Authors 411
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Cell Reports
Article
Global Changes in the Mammary EpigenomeAre Induced by Hormonal Cuesand Coordinated by Ezh2Bhupinder Pal,1,3 Toula Bouras,1,3,8 Wei Shi,2,5,8 Francois Vaillant,1,3 Julie M. Sheridan,1,3 Naiyang Fu,1,3 Kelsey Breslin,1
Kun Jiang,1 Matthew E. Ritchie,2,3 Matthew Young,2 Geoffrey J. Lindeman,1,4,7,8 Gordon K. Smyth,2,6,8
and Jane E. Visvader1,3,*1ACRF Stem Cells and Cancer Division2Bioinformatics Division
The Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, VIC 3052, Australia3Department of Medical Biology4Department of Medicine5Department of Computing and Information Systems6Department of Mathematics and Statistics
The University of Melbourne, Parkville, VIC 3050, Australia7Department of Medical Oncology, The Royal Melbourne Hospital, Grattan Street, Parkville, VIC 3050, Australia8These authors contributed equally to this work*Correspondence: [email protected]
http://dx.doi.org/10.1016/j.celrep.2012.12.020
SUMMARY
The mammary epithelium is a dynamic, highlyhormone-responsive tissue. To explore chromatinmodifications underlying its lineage specification andhormone responsiveness, we determined genome-wide histonemethylation profiles ofmammary epithe-lial subpopulations in different states. The markeddifferences in H3K27 trimethylation between subpop-ulations in the adult gland suggest that epithelialcell-fate decisions are orchestrated by polycomb-complex-mediated repression.Remarkably, themam-maryepigenomeunderwenthighly specificchanges indifferent hormonal contexts, with a profound changebeing observed in the global H3K27me3 map ofluminal cells during pregnancy. We therefore exam-ined the role of the key H3K27 methyltransferaseEzh2 in mammary physiology. Its expression andphosphorylation coincided with H3K27me3 modifica-tions and peaked during pregnancy, driven in partby progesterone. Targeted deletion of Ezh2 impairedalveologenesis during pregnancy, preventing lacta-tion, and drastically reduced stem/progenitor cellnumbers. Taken together, these findings reveal thatEzh2 couples hormonal stimuli to epigenetic changesthat underpin progenitor activity, lineage specificity,and alveolar expansion in the mammary gland.
INTRODUCTION
The mammary gland, which comprises a branching ductal epi-
thelial network embedded in an adipose-rich stromal matrix,
C
is remarkably adaptive to physiological requirements and
undergoes dramatic morphological changes during puberty
and pregnancy. At birth, it manifests as a rudimentary branched
structure, but ductal elongation and branching commence with
puberty and pregnancy provokes the rapid expansion of alveolar
units that differentiate into milk-secretory cells prior to parturi-
tion. The steroid hormones estrogen and progesterone exert
pivotal roles during mammary development via their cognate
receptors, the estrogen receptor (ER) and progesterone receptor
(PR) (Brisken and O’Malley, 2010). ER is essential for ductal
morphogenesis in puberty (Mallepell et al., 2006; Mueller et al.,
2002), whereas PR governs ductal side-branching and alveolar
development during pregnancy (Brisken et al., 1998; Lydon
et al., 1995; Mulac-Jericevic et al., 2003).
The mammary epithelium can be divided into two primary line-
ages: themyoepithelial lineage constitutes the outer layer of cells
that contact the basement membrane, whereas the luminal
lineage comprises both ductal and alveolar cells. Adult stem
cells prospectively isolated from the mouse mammary gland
display the requisite stem cell properties of multilineage differen-
tiation and self-renewal (Shackleton et al., 2006; Stingl et al.,
2006). A recent study has added a new layer of complexity to
the prevailingmodel of themammary epithelial hierarchy through
the identification of unipotent cells that contribute to homeo-
stasis of the gland (Van Keymeulen et al., 2011). It is not yet clear
whether these correspond to stem or progenitor cells and what
their relationship is to the prospectively isolated epithelial sub-
sets. In the mouse, mammary stem cells (MaSCs) have been
shown to be highly responsive to steroid hormones (Asselin-
Labat et al., 2010; Joshi et al., 2010), while progesterone
augmented the number of human bipotent stem-like cells in
cellular assays (Graham et al., 2009). MaSCs lie at the apex of
the hierarchy and give rise to progenitors and mature cells
through progressive restriction. Two distinct types of luminal
progenitor cells have been isolated from the mouse mammary
ell Reports 3, 411–426, February 21, 2013 ª2013 The Authors 411
Figure 1. Histone Methylation Profiles of Mammary Epithelial Subpopulations in the ‘‘Steady State’’ and Their Correlation with Gene
Expression Changes
(A) Genome-wide heat map showing the pattern of H3K4me3, H3K9me2, and H3K27me3marks in MaSC-enriched cells from 5 kb upstream to 5 kb downstream
of the TSS of each gene. Rows correspond to genes clustered by coverage pattern. All 26,310 genes in themm9 genome are shown. The first three columns show
(legend continued on next page)
412 Cell Reports 3, 411–426, February 21, 2013 ª2013 The Authors
gland, but committed basal progenitor cells remain elusive (As-
selin-Labat et al., 2007, 2011; Sleeman et al., 2007).
Although a number of transcription factors and pathways have
been implicated in controlling specific steps along the mammary
differentiation hierarchy (reviewed in Visvader, 2009), the role of
the epigenome in regulating cell-fate decisions and differentia-
tion within this epithelial compartment remains unclear. In other
systems, there is substantial evidence that histone methylation
governs lineage-specific developmental programs and that its
deregulation leads to oncogenesis (Bracken and Helin, 2009;
Sauvageau and Sauvageau, 2010). It is thought that histone
modifications establish discrete domains of active and inactive
chromatin to effect gene expression. Histone lysine methylation
can serve as either an active or repressive mark: trithorax-medi-
ated methylation of lysine 4 on histone H3 within nucleosomes is
associated with activated gene expression, while methylation of
lysine 27 by polycomb group (PcG) proteins is linked with gene
repression and chromatin condensation (Margueron and Rein-
berg, 2011). Ezh2, a member of the PcG family, is a histone
methyltransferase that forms the catalytic component of the
polycomb repressive complex PRC2. This complex silences
lineage specification genes to regulate the maintenance and
differentiation of embryonic and adult stem cells (reviewed in
Margueron and Reinberg, 2011). In embryonic stem cells, where
genome-wide histone methylation patterns have been exten-
sively studied, key developmental genes often exhibit both
repressive H3K27me3 marks and activating H3K4me3 marks
(Bernstein et al., 2006). This bivalent modification has been
proposed tomaintain these genes ‘‘poised’’ for subsequent acti-
vation or repression upon lineage specification. In vivo mapping
studies indicate that PcG-dependent H3K27me3 selectively
marks genes in the epidermal lineages and controls gene ex-
pression changes during the differentiation of skin stem cells
(Lien et al., 2011).
In this report, we examine the contribution of epigenetic mech-
anisms to regulation of the lineage hierarchy in the steady-state
mammary gland and in response to different hormonal milieu.
We determined genome-wide histone methylation profiles of
the MaSC-enriched, luminal progenitor, and mature luminal
subsets. Correlating the global H3K4me3 and H3K27me3 modi-
fication maps with gene expression signatures indicated that
the epigenome has an important role in directing cell-fate
changes from the basal to luminal cell lineage. Moreover, the
mammary epigenome was found to be highly sensitive to dif-
ferent hormonal environments. H3K27 trimethylation of chro-
matin emerged as a key mediator of gene expression changes
during pregnancy, concomitant with high levels of Ezh2, appar-
coverage depth on a linear color scale, with the x axis showing distance from the T
In this case, the x axis shows the scale from 4 (nonexpressed) to 12 (maximum
ordered by expression level: the fifth gene group shows little histone marking o
H3K4me3 and increasing H3K27me3 levels. Genes are sorted by expression wit
(B) Segmented bar graphs showing genome-wide percentage of genes with histon
marks in the TSS region are shown, but virtually identical data were obtained for
(C) Heatmaps of gene expression and histone modification changes as cells res
mature luminal (ML) cells. Columns give log2-fold changes for differential gene e
H3K27me3 marking across the broad gene, respectively, for the 200 most differ
See also Figure S1 and Table S1.
C
ently activated by phosphorylation. Targeted deletion of Ezh2 in
the mammary epithelium dramatically reduced both ductal and
alveolar morphogenesis. The expression of Ezh2 and its phos-
phorylation appear to be coordinated through progesterone,
a key pregnancy hormone. Thus, hormonally driven expansion
of the alveolar compartment is programmed, at least in part,
by Ezh2-mediated changes in chromatin modification. Given
the critical importance of both progesterone and EZH2 to breast
cancer, these data implicate progesterone-induced global
changes in chromatin structure in the genesis of this disease.
RESULTS
Histone Methylation Landscapes of Mammary EpithelialSubpopulations in the ‘‘Steady State’’To explore the relevance of histone modification to the regula-
tion of gene expression along the mammary differentiation hier-
expressed in the steady-state gland but downregulated during
Luminal Progenitor Genes
es are directly correlated with H3K4me3 changes (top panels) and inversely
elial subset 10 kb ± of the TSS region of three MaSC/basal-specific and three
le 0–10. Bar plots show ChIP qRT-PCR data for three independent biological
ell Reports 3, 411–426, February 21, 2013 ª2013 The Authors 415
Log
fold
cha
nge
MaSC/ basal Lum
Expression log fold change
OVX vs control pregnant vs virgin
H3K
4me3
H3K
27m
e3
3
10
-2
0 4-6
-4 0 2 4-2 6
-4 0 2 4-2 6
MaSC/ basal Lum
32
0
-2-3
0 5-5
-5
Expression log fold change
1
-1
-4-8 -2 2
2
-1
-3-4-4
3
10
-2
2
-1
-3-4
0 4-6 -4-8 -2 2
32
0
-2-3
0 5-5
1
-1
-4
32
0
-2-3
1
-1
-4
32
0
-2-3
1
-1
-4
32
0
-2-3
1
-1
-4
0 5
0 5-5
32
0
-2-3
1
-1
-4
FoxA1
Wnt7b
Hey1
Fol
d ch
ange
vs
Inpu
t
1.51.0
0.50
3.0
2.5
2.0
1.5
1.0
0.5
0
3.02.5
2.0
0.4
0
1.8
1.2
0.8
virgin
12.5 dP
virgin
12.5 dP
Wap
Elf5
Csn2
Fol
d ch
ange
vs
Inpu
t F
old
chan
ge v
s In
put
Fol
d ch
ange
vs
Inpu
t
virgin 12.5 dP
virgin 12.5 dP
virgin 12.5 dP
virgin
12.5 dP
1.2
0.6
1.2
0.4
0.8
1.2
0.4
0.8
1.6
0
virgin
12.5
0
0
BA
DC
virgin
12.5
virgin
12.5
virgin
12.5
virgin
12.5
virgin
12.5
Fol
d ch
ange
vs
Inpu
tF
old
chan
ge v
s In
put
virgin 12.5 dP
virgin 12.5 dP
virgin 12.5 dP
Figure 3. The Mammary Epithelial Epigenome Is Influenced by Hormonal Status
(A and B) Scatter plots of expression versus epigenetic log2-fold changes in the mammary epithelial subsets of (A) ovariectomized mice and (B) pregnant mice.
Increased H3K27me3 marking strongly mediates decreased expression in luminal cells from 12.5 day pregnant glands (p < 10�6). Other correlations were also
significant (p < 0.05), except H3K27me3 modifications in the MaSC subset at midpregnancy.
(C) Derepression of milk genes and Elf-5 in the luminal subset of pregnant glands at 12.5 days. The left panel shows read coverage of H3K27me3 marks around
the TSS of each gene. The right panel shows ChIP-qRT-PCR confirmation (n = 3; error bars show SEM).
(D) Repression of luminal commitment genes (Wnt7b, Foxa1, and Hey1) in the luminal subset during pregnancy correlates with increased H3K27me3 marks. The
left panel shows read coverage for H3K4me3 (red) and H3K27me3 (blue) around the TSS of each gene. The right panel shows ChIP-qRT-PCR confirmation (n = 3;
error bars show SEM).
See also Figure S2, and Tables S1 and S2.
416 Cell Reports 3, 411–426, February 21, 2013 ª2013 The Authors
pregnancy, such as wnt7b, and the luminal commitment genes
Foxa1 (Bernardo et al., 2010) and Hey1 (Bouras et al., 2008)
had abundant H3K27me3 modifications at 12.5 days of
pregnancy, as validated by ChIP-qRT-PCR (Figure 3D). These
changes likely reflect a shift in gene expression within the
emerging alveolar cells toward the highly specialized function
of milk production. In general, genes repressed in pregnancy
with a concomitant increase in H3K27me3 marking were en-
riched for the mammary morphogenesis and developmental
gene categories. Genes upregulated during pregnancy and
with reduced H3K27me3 modifications showed enrichment for
lipid biosynthesis and lipid catabolism (Table S2), commensu-
rate with the changed mammary function during pregnancy.
Although H3K27me3 was not consistently correlated with ex-
pression changes in the MaSC/basal subset of pregnant mice, it
nevertheless appeared to play an important role for specific
genes. In particular, it was associated with derepression of a
number of genes that are normally expressed only in the luminal
lineage. This observation is of particular interest given the
dramatic expansion of theMaSC pool and its altered gene signa-
ture during pregnancy (Asselin-Labat et al., 2010). Quantitative
RT-PCR confirmed expression of the milk protein genes Wap
and Csn2 and the luminal progenitor transcription factor Elf-5,
all of which are normally restricted to luminal subpopulations
(Lim et al., 2010; Figure S2B). Compatible with their derepres-
sion, each of these genes showed diminished H3K27me3 marks
at their TSS during pregnancy, as confirmed by ChIP-qRT-PCR
analysis (Figure S2C). Thus, lineage-priming may occur in the
expanded stem cell population during pregnancy prior to com-
mitment along the alveolar lineage. Intriguingly, expression of
the basal-specific gene Lgr5 was extinguished in the MaSC
pool during pregnancy, accompanied by augmented H3K27 tri-
methylation (Figures S2B and S2C).
At a more global level, the total number of genes within the
luminal subset with significant (FDR< 0.05) H3K27me3modifica-
tions relative to input increased in pregnancy but decreased in
ovariectomized mice significantly (Figure S2D). In summary,
striking epigenetic changes occurred within a specific cellular
subset during pregnancy and were selectively observed for
H3K27me3 but not H3K4me3 or H3K9Ac modifications (data
not shown), which showed small changes.
Dynamic Expression of the Polycomb Group RepressorEzh2 in the Mammary GlandIn view of the marked hormone-induced changes in the global
H3K27me3 profile during pregnancy, we examined the ex-
pression of Ezh2 during mammary ontogeny. Ezh2 is the core
enzymatic subunit of PRC2 that catalyzes K27 trimethylation
on H3 (Margueron and Reinberg, 2011) and has emerged as
an important prognostic marker in breast cancer. Western blot
analysis showed that Ezh2 expression was low in virgin glands
and peaked during early to midpregnancy before declining in
late pregnancy (Figure 4A). Interestingly, the profile of total
H3K27me3-modified protein closely mirrored that of Ezh2 (Fig-
ure 4A). Immunohistochemical staining confirmed the ex-
pression of Ezh2 during mammary morphogenesis and further
revealed that it was abundant in the terminal end buds (TEBs)
of the developing pubertal gland, with lower levels visible in the
C
nuclei of myoepithelial and luminal cells of mature ducts (Fig-
ure 4B). Ezh2 staining was most intense in the ducts and alveoli
during pregnancy (Figure 4B) and declined to low levels in
lactating and involuting glands (Figure 4B).
Ezh2 Deficiency Delays Mammary Morphogenesisduring PubertyTo investigate the physiological role of Ezh2 in the mammary
gland, we conditionally targeted the Ezh2 locus using cre recom-
binase driven by the mouse mammary tumor virus (MMTV)
CD29hiCD24+ cells frommammary glands of 8- to 9-week-old mice were
injected into the cleared mammary fat pads of 3-week-old nonobese dia-
betic-severe combined immunodeficient female recipients. Data are
pooled from three independent experiments collected 8 weeks post-
transplantation. The repopulation frequency was calculated using limiting
dilution analysis as described (Hu and Smyth, 2009).aShown as number of outgrowths per number of injected cleared
mammary fat pads.
mammary glands manifests in puberty, because this stage
requires large numbers of progenitor cells to orchestrate ductal
growth (Asselin-Labat et al., 2007).
Loss of Ezh2 Profoundly Affects the Expression of CellCycle and Epidermal GenesTo identify potential downstream effectors of Ezh2, the genome-
wide transcriptional profiles of the MaSC/basal and luminal pop-
ulations in their steady state were determined following ex vivo
cre-mediated excision of Ezh2. Similar to freshly sorted cells
(Figure 4G), the clonogenic capacity of these Ezh2-deficient
MaSC/basal and luminal populations was dramatically reduced
compared to control cultures (Figure S4A). Gene ontology anal-
ysis of the top 500 differentially expressed genes revealed a sig-
nificant association with cell cycle, DNA replication, and DNA
Figure 4. Ezh2 Is Required for Normal Mammary Gland Development
(A) Western blot analysis of mammary gland lysates for expression of Ezh2 and H3
provided the controls.
(B) Immunohistochemical staining of mammary gland tissue sections from FVB
(6 weeks), mature ducts in 8-week-old mice; alveoli in early pregnancy (6.5 d
bars, 50 mm.
(C) Immunohistochemical staining of TEBs for Ezh2 expression in MMTV–cre; Ez
(D) Whole-mounts of mammary glands from 7-week-old virgin MMTV–cre; Ezh2f/f
lymph node in the inguinal gland is marked by a white arrow. Scale bars, 2.0 mm
(E) Extent of fat pad filling was estimated in virgin mice at 6, 7, and 8 weeks of a
(F) Immunohistochemical staining of terminal end buds for BrdU incorporation, s
glands compared to those from Ezh2f/+ and MMTV–cre; Ezh2f/+ mice. Scale bar
(G) Colony-forming capacity of sorted MaSC-enriched (CD29hiCD24+; labeled C
fibroblast feeders from 8-week-old MMTV–cre; Ezh2f/f mice compared to Ezh2f
CD61+ luminal progenitors that could be isolated from the smaller targeted gland
quantitation of the colony forming capacity of the CD29loCD24+ and CD29hiCD2
represent mean ± SD of three independent experiments, with eight replicates fo
See also Figure S3.
C
repair (Figure S4B). Of the top ranked genes, three potent cell
cycle inhibitors were derepressed: Cdkn1c (p57), Cdkn2a
(Ink4a/Arf), and Cdkn1a (p21). Consistent with Arf being an
important target of Ezh2, Arf transcript levels were considerably
lower in the luminal population from pregnant glands than in
other subsets and Arfwas derepressed in Ezh2-deficient luminal
cells at midpregnancy (Figure S4C). Gene expression changes in
Ezh2-deficient cells were inversely correlated with changes in
pregnancy (Asselin-Labat et al., 2010), with 67% (52 of 78) of
differentially expressed genes in the MaSC/basal subset and
91% (146 of 160) in the luminal subset showing changes in oppo-
site directions.
Interestingly, one of the top derepressed gene sets in the
MaSC/basal population, other than those related to cell cycle
regulation, was keratinocyte differentiation (Figure S4B). The
expression of genes within the epidermal differentiation complex
on mouse chromosome 3, previously shown to be a target of
Ezh2 repression in skin (Ezhkova et al., 2009), was activated in
the MaSC-enriched subset from Ezh2-deficient glands. This is
consistent with previous reports of misexpression of nonlineage
genes associated with Ezh2 deletion (see Discussion). To inves-
tigate a potential relationship between the gene expression sig-
natures of Ezh2-deficient cells and metaplastic breast cancers,
the molecular profiles of the different breast cancer subtypes
were interrogated with the Ezh2-deficient MaSC/basal cell
signature. Intriguingly, this signature was found to bemost highly
represented in the claudin-low subgroup based on signature ex-
pression scores (Figure S4D). The claudin-low subtype exhibits
metaplastic features, expresses lower levels of Ezh2 than the
other subtypes, and can even display epidermal traits (Keller
et al., 2012), suggesting that they have undergone metaplasia
as a result of aberrant differentiation.
Ezh2 Deficiency Leads to Reduced AlveolarDevelopment and Failure of LactationWe next examined the effect of Ezh2 deficiency on pregnancy
and lactation. Although alveoli formed in MMTV-cre;Ezh2f/f
mammary glands during pregnancy (Figure 5A), they were fewer
and more disorganized than those in Ezh2f/+, Ezh2f/f, or MMTV-
cre mammary glands (Figure 5B; data not shown). This pheno-
type appeared most obvious from midpregnancy (n = 9), where
K27me3 protein during development. Ezh2 cKO tissue (12.5 dP) and antitubulin
/N females for Ezh2 expression, representing TEBs that characterize puberty
P) and midpregnancy (12.5 dP); lactation (1 dL); and involution (4 dI). Scale
h2f/f mice compared to littermate Ezh2f/+ glands. Scale bars, 50 mm.
mice compared to glands from Ezh2f/+ and MMTV-cre; Ezh2f/+ littermates. The
.
ge using ImageJ software, with four to nine mice for each time point.
howing reduced numbers of proliferating cells in MMTV–cre; Ezh2f/f mammary
s, 50 mm. Isotype control antibody panels are shown in insets.
D29hi) and luminal cells (CD29loCD24+; labeled CD29lo) grown on irradiated/+ littermates. The same results were obtained for 6-week-old mice. The few
s also had reduced clonogenic activity (data not shown). Histogram showing
4+ subpopulations (200 and 300 cells were plated per well, respectively). Data
r each. *t test p < 0.0001 compared to Ezh2f/+ controls for both subsets.
ell Reports 3, 411–426, February 21, 2013 ª2013 The Authors 419
A
B
16.5
dP
12.5
dP
MMTV-cre; Ezh2f/f
16.5
dP
Ezh2f/+
E
MMTV-cre; Ezh2f/fMMTV-cre
C
D
Tubulin
Ezh2
H3K27me3
MMTV-cre;Ezh2f/f Ezh2f/+
Cdk
n1c
Fol
d ch
ange
vs
Inpu
t
0
1.5
1.0
0.5
2.0
MMTV-cre; Ezh2f/f
Ezh2f/+
Fol
d ch
ange
vs
Inpu
tC
amk2
n1
0
3.5
2.5
1.5
0.5
MMTV-cre; Ezh2f/f
Ezh2f/+ Fol
d ch
ange
vs
Inpu
tW
nt7b
0
2.5
1.5
1.0
0.5
2.0
MMTV-cre; Ezh2f/f
Ezh2f/+
Cdk
n2a
Fol
d ch
ange
vs
Inpu
t
MMTV-cre; Ezh2f/f
Ezh2f/+0
4
3
2
1
Ezh2f/+
12.5
dP
MMTV-cre; Ezh2f/fMMTV-cre; Ezh2f/+ Ezh2f/+
Figure 5. Ezh2 Deficiency Leads to Abnormal Alveolar Development
(A) Whole-mounts of mammary glands from MMTV–cre; Ezh2f/f mice (right panel) compared to those from Ezh2f/+ and MMTV–cre; Ezh2f/+ mice at day 12.5 of
pregnancy show retardation of ductal growth. Mice were mated at 7 weeks of age. No gross abnormalities in the alveolar units were evident in early pregnancy
(6.5 dP; data not shown). Scale bars, 4.0 mm.
(B) H&E sections of mammary glands from MMTV–cre;Ezh2f/f, Ezh2f/+ littermates, and MMTV-cre mice at days 12.5 and 16.5 of pregnancy. Scale bars, 50 mm.
(C) Western blot analysis of mammary glands from MMTV-cre; Ezh2f/f and Ezh2f/+ littermate control mice for expression levels of Ezh2, H3K27me3 protein, and
tubulin.
(D) Derepression of cell cycle genes in pregnant glands lacking Ezh2. ChIP-qRT-PCR for H3K27me3 marks across Cdkn2a/Arf, Cdkn1c (p57), Wnt7b, and
Camk2n1 in the expanding luminal population from day 12.5 pregnant mice. Histograms show the mean of two independent samples with at least two technical
replicates for each.
(E) Immunostaining for milk protein of MMTV-cre; Ezh2f/f tissue sections (right panel) compared to an Ezh2f/+ littermate control at 16.5 days of pregnancy (left
panel). Scale bars, 100 mm.
See also Figures S3 and S4.
ductal elongation of the mammary tree remained stunted in 50%
of Ezh2-deficient glands (Figure 5A). Heterozygotes seemed to
have an intermediate phenotype, with less dense but apparently
normal alveoli (Figures 5A and S3F). Notably, progenitor cell
activity was severely compromised in all three epithelial subsets
ajver, S.D., and Kleer, C.G. (2005). The Polycomb group protein EZH2 impairs
DNA repair in breast epithelial cells. Neoplasia 7, 1011–1019.
Supplemental Information
EXTENDED EXPERIMENTAL PROCEDURES
Mouse StrainsAll animal experiments were conducted using mice bred at and maintained in our animal facility according to institutional and the
Melbourne Health Research Directorate Animal Ethics Committee guidelines. Ezh2 mice were on a pure FVB/N or mixed C57Bl/6
and FVB/N background; the same results were obtained for each. Adult female mice were subjected to timed pregnancies, scored
by the presence of vaginal plugs and confirmed by examination of embryos on collection of mammary glands. Mice were genotyped
using the primers listed below.
Mammary Cell Preparation and Cell SortingAntibodies against mouse antigens were purchased from BD PharMingen (San Diego, CA) unless otherwise specified, and included
CD24-PE, biotinylated CD31, CD45 and Ter119, CD29–FITC (Chemicon, Temecula, CA), CD61-APC and streptavidin–APC–Cy7,
CD14-biotin (eBioscience, San Diego, CA). For the luminal progenitor fractionation experiments, CD49b-FITC, Sca1-APC, CD45-
PECy-7 and CD31-PECy-7 were from eBioscience, and CD29-APC-Cy7 from Biolegend (San Diego, CA). Single cell suspensions
were sorted on a FACSAria or FACSDiva (BD PharMingen).
Histology and Whole MountingFor histological examination of mouse mammary glands, tissues were fixed in 4% paraformaldehyde overnight and embedded in
paraffin. Sections (5 mm) were prepared and stained with hematoxylin and eosin (H&E). For whole-mount analysis, mammary glands
were harvested and fixed in Carnoy’s solution (six parts 100% ethanol, three parts chloroform, one part glacial acetic acid) and
stained with hematoxylin.
Bromodeoxyuridine ImmunodetectionMicewere injected with BrdUCell Labeling Reagent (0.5mg/10 g body weight, AmershamBiosciences) 1 hr prior to tissue collection.
Tissueswere fixed in 4%paraformaldehyde and embedded in paraffin. For immunohistochemical detection of BrdU-labeled cells, rat
anti-BrdU (Becton Dickinson) and biotinylated rabbit anti-rat IgG antibody (Dako) were used, followed by HRP-conjugated strepta-
vidin (Dako, LSAB2).
Cell Culture and Retroviral-Mediated InfectionFor primary cell culture, freshly sorted mammary epithelial cells were plated on irradiated fibroblast feeders on collagen-coated
6-well plates and infected as described (Bouras et al., 2008). Colony assays in 2D have been described (Shackleton et al., 2006).
Primary cells following transduction with cre-MIG (MSCV-cre-IRES-GFP) or empty retrovirus were manually counted after sorting
for GFP and replated at 200 or 300 cells per well in a 24-well plate containing a feeder layer of irradiated fibroblasts. After 7 days,
cultures were fixed with ice-cold acetone/methanol and stained with Giemsa for enumeration.
For siRNA-mediated knockdown of PR in T-47D cells, the following siGENOME SMARTpools (Dharmacon) were used: Ezh2 (hu)
M-004218-03-0005, RISC-free D-001220-01 and progesterone receptor (hu) M-003433-01-0005. Transfections were performed as
described by the manufacturer (Dharmacon) using Dharmafect-1 and cells harvested for analysis after 60 hr.
ImmunohistochemistryParaffin-embedded sections (5 mm) were dewaxed in xylene and rehydrated through an alcohol series, blocked with 3% hydrogen
peroxide, and subjected to antigen retrieval by boiling in 10 mM citrate buffer pH 6.0 for 30 s at pressure using a DAKO pressure
cooker. The mouse-on-mouse (MOM) kit (Vector) was used for mousemonoclonal antibodies. Immunostaining with other antibodies
was performed using the streptavidin-biotin peroxidase detection system as per the manufacturer’s instructions (ABC reagent,
Vector Laboratories). 3,3-diaminobenzidine was used as substrate (DAKO). In all cases, an isotype-matched control IgG was
used as a negative control. The following antibodies were used: anti-SMA (Sigma), anti-milk (Accurate Chemical and Scientific),
Genome-wide heatmaps of methylation mark coverage were drawn using Repitools (Statham et al., 2010). Density plots were
drawn using in-house scripts written in R (http://www.r-project.org). Coverage graphs were generated for genes of interest using
the Integrated Genome Browser (Nicol et al., 2009).
ChIP-seq Statistical AnalysisFragment counts were formed for each gene. Fragments were counted if the center of the fragment was contained in the TSS or
broad region for that gene. Statistical analysis of the count data was performed using edgeR package (Robinson et al., 2010) of
the Bioconductor software project (Gentleman et al., 2004). The Benjamini-Hochberg method was used to control the FDR. Genes
were called as significantly enriched for each histone mark using the normalizeChIPtoInput function, which normalizes the ChIP
counts to input and evaluates enrichment using a negative binomial statistical model. Log2-fold-changes in histone mark coverage
between cell populations or between conditions were computed using the predFC function. This function adjusted the ChIP counts
for input by fitting a negative binomial log-linear generalized linear model (McCarthy et al., 2012), using a prior count of 0.5 per sample
to avoid unreliably large log-fold-changes that might otherwise arise from zero or small counts. The negative binomial dispersion was
set to 0.01 for all calculations, allowing for a degree of biological variation typical of mouse experiments (McCarthy et al., 2012). Gene
Ontology analysis used the DAVID tool (Huang da et al., 2009). Gene set enrichment used the mean-rank gene set enrichment test
(Michaud et al., 2008).
RNA Extraction and Quantitative RT-PCR AnalysisTotal RNA was isolated from primary mammary epithelial subpopulations with the RNeasy Micro kit (QIAGEN). Reverse transcription
by using oligo(dT) primer and Moloney murine leukemia virus reverse transcriptase (Invitrogen) was according to the manufacturer’s
protocol. Quantitative RT-PCR was carried out by using a Rotorgene RG-6000 (Corbett Research) and SensiMix (dT) DNA kit
(Quantace) under the following conditions: 10 min at 95�C followed by 35 cycles consisting of 15 s at 95�C, 20 s at 62�C, and20 s at 72�C. Gene expression was determined with the Rotor-Gene software (version 1.7). The primer sequences are listed below.
Microarray AnalysisMicroarray expression data for steady-state epithelial cell subsets, pregnant and ovariectomized mice were analyzed as described
previously (Asselin-Labat et al., 2010; Lim et al., 2010). For Ezh2-deficient profiles, cell subsets were sorted from three independent
mouse pools, and up to 250 ng of RNA per sample was labeled, amplified and hybridized to Illumina Mouse-WG6 V2 Expression
BeadChips according to Illumina standard protocols at the Australian Genome Research Facility (Melbourne, Australia). Summary
probe profiles were exported from GenomeStudio and was analyzed using the limma software package (Smyth, 2004). Expression
values were normexp background adjusted and quantile normalized using control probes (Shi et al., 2010). The data have been
uploaded into GSE38203. Probes were filtered if not expressed (detection p-value > 0.05 across all arrays) or poorly annotated
(Barbosa-Morais et al., 2010). Differential expression between between Ezh2-deficient and control cells was assessed using linear
models and empirical Bayes moderated t-statistics (Smyth, 2004). Reliability was improved using array quality weights (Ritchie et al.,
2006), and each mouse pool was treated as a random effect to allow for dependence between samples from the same pool
(Smyth et al., 2005). Gene ontology enrichment for biological process (BP) terms was carried out on the top 500 genes from each
contrast using the GOstats package (Falcon and Gentleman, 2007). Focused gene set testing using Ezh2 signatures obtained
from Kamminga et al. (2006) (34 genes matched by gene symbols) and Ezhkova et al. (2009) (65 genes matched by gene symbols)
were performed using the roast method (Wu et al., 2010). Microarray probes werematched to ChIP-seq profiles by gene symbol. The
probe with highest average expression data was chosen to represent each gene.
Genes with FDR < 0.05 and at least 50% fold-change were selected as Ezh2-deficient signature genes and compared with the
human breast cancer data set from Herschkowitz et al. (2007). Where multiple probes were present for the same gene on a given
platform, the probe with the highest average expression level was kept for further analysis. Genes were matched between platforms
usingGene symbols and a signature score calculated for each sample as in Lim et al. (2010). TheEzh2-deficient signature geneswere
also compared across tumor subtypes using roast gene set tests (Wu et al., 2010) with Ezh2–deficient log-fold changes as gene
weights.
Oligonucleotides Used in the StudyGenotyping oligonucleotides (50 to 30)
Ezh2
Fwd 1: TTATTCATAGAGCCACCTGG
Fwd 2: ACGAAACAGCTCCAGATTCAGGG
Rev: CTGCTCTGAATGGCAACTCC
ChIP-q-RT-PCR oligonucleotides
c-Kit
Fwd: TCAGGGGTGCCACGATCCGT
Rev: TAGTCGGGATTGCCGGGCGA
S2 Cell Reports 3, 411–426, February 21, 2013 ª2013 The Authors
Cell Reports 3, 411–426, February 21, 2013 ª2013 The Authors S3
Arf
Fwd: CGCAGGTTCTTGGTCACTGTGAGG
Rev: TGCCCATCATCATCACCTGGTCC
Rankl
Fwd: TGTACTTTCGAGCGCAGATG
Rev: CCACAATGTGTTGCAGTTCC
Rank
Fwd: ACACCCTGCCTCCTGGGCTT
Rev: AAGCCTGGGCCTCCTTGGGT
PR
Fwd: GCTTGCATGATCTTGTGAAACAGC
Rev: GGAAATTCCACAGCCAGTGTCC
Ezh2
Fwd: GCAATTTAGAAAACGGAAATGC
Rev: GTACAAAACACTTTGCAGCTGG
18S rRNA
Fwd: GTAACCCGTTGAACCCCATT
Rev: CCATCCAATCGGTAGTAGCG
SUPPLEMENTAL REFERENCES
Barbosa-Morais, N.L., Dunning, M.J., Samarajiwa, S.A., Darot, J.F., Ritchie, M.E., Lynch, A.G., and Tavare, S. (2010). A re-annotation pipeline for Illumina
BeadArrays: improving the interpretation of gene expression data. Nucleic Acids Res. 38, e17.
Falcon, S., and Gentleman, R. (2007). Using GOstats to test gene lists for GO term association. Bioinformatics 23, 257–258.
Gentleman, R.C., Carey, V.J., Bates, D.M., Bolstad, B., Dettling, M., Dudoit, S., Ellis, B., Gautier, L., Ge, Y., Gentry, J., et al. (2004). Bioconductor: open software
development for computational biology and bioinformatics. Genome Biol. 5, R80.
Herschkowitz, J.I., Simin, K., Weigman, V.J., Mikaelian, I., Usary, J., Hu, Z., Rasmussen, K.E., Jones, L.P., Assefnia, S., Chandrasekharan, S., et al. (2007).
Identification of conserved gene expression features between murine mammary carcinoma models and human breast tumors. Genome Biol. 8, R76.
Huang da, W., Sherman, B.T., and Lempicki, R.A. (2009). Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat.
Protoc. 4, 44–57.
Kamminga, L.M., Bystrykh, L.V., de Boer, A., Houwer, S., Douma, J., Weersing, E., Dontje, B., and de Haan, G. (2006). The Polycomb group gene Ezh2 prevents
McCarthy, D.J., Chen, Y., and Smyth, G.K. (2012). Differential expression analysis of multifactor RNA-Seq experiments with respect to biological variation.
Nucleic Acids Res. 40, 4288–4297.
Michaud, J., Simpson, K.M., Escher, R., Buchet-Poyau, K., Beissbarth, T., Carmichael, C., Ritchie, M.E., Schutz, F., Cannon, P., Liu, M., et al. (2008). Integrative
analysis of RUNX1 downstream pathways and target genes. BMC Genomics 9, 363.
Ritchie, M.E., Diyagama, D., Neilson, J., van Laar, R., Dobrovic, A., Holloway, A., and Smyth, G.K. (2006). Empirical array quality weights in the analysis of
microarray data. BMC Bioinformatics 7, 261.
Robinson, M.D., McCarthy, D.J., and Smyth, G.K. (2010). edgeR: a Bioconductor package for differential expression analysis of digital gene expression data.
Bioinformatics 26, 139–140.
Shi, W., Oshlack, A., and Smyth, G.K. (2010). Optimizing the noise versus bias trade-off for Illumina whole genome expression BeadChips. Nucleic Acids Res. 38,
e204.
Smyth, G.K. (2004). Linear models and empirical bayes methods for assessing differential expression in microarray experiments. Stat. Appl. Genet. Mol. Biol. 3,
Article 3.
Smyth, G.K., Michaud, J., and Scott, H.S. (2005). Use of within-array replicate spots for assessing differential expression in microarray experiments.
Bioinformatics 21, 2067–2075.
Vikstrom, I., Carotta, S., Luthje, K., Peperzak, V., Jost, P.J., Glaser, S., Busslinger, M., Bouillet, P., Strasser, A., Nutt, S.L., and Tarlinton, D.M. (2010). Mcl-1 is
essential for germinal center formation and B cell memory. Science 330, 1095–1099.
Wu, D., Lim, E., Vaillant, F., Asselin-Labat, M.L., Visvader, J.E., and Smyth, G.K. (2010). ROAST: rotation gene set tests for complex microarray experiments.
Bioinformatics 26, 2176–2182.
S4 Cell Reports 3, 411–426, February 21, 2013 ª2013 The Authors
Figure S1. Genome-Wide Heatmaps of Methylation Profiles for Luminal Progenitor and Mature Luminal Subpopulations in the ‘‘Steady
State’’, Related to Figures 1 and 2
(A) Heat maps for luminal progenitor (left) and mature luminal cells (right) show distribution of H3K4me3, H3K9me2, and H3K27me3 marks ± 5 kb of the TSS of
each gene. Genes are clustered into groups according to their histonemethylation profiles and ordered within groups by expression level. Far right column shows
log2-expression. Other columns show percentage coverage.
(B) Density plots of average histone methylation coverage stratified by gene expression. The x-axes show distance from TSS in base pairs. The y-axis shows
average number of covering reads at each base pair. For each cell subset, genes are stratified into four equal-size groups by expression level, and the average
coverage is shown for each group. Lines are red to green from high to low expression. Plots are shown for each epigenetic mark in each epithelial cell subset.
H3K4me3 marks peak sharply around the TSS and directly correlate with gene expression, whereas H3K27me3 shows a broad pattern and reverse association
with expression.
Cell Reports 3, 411–426, February 21, 2013 ª2013 The Authors S5
Wap
Elf5 Csn2
Lgr5
Fol
d ch
ange
vs
Inpu
t
virgin 12.5 dP
virgin 12.5dp
virgin 12.5 dP
virgin 12.5 dP
virgin
12.5 dP
1.0
0.5
0.75
1.2
0.4
0.8
1.6
2.0
0.5
1.0
2.5
1.5virgin
12.5 dP
virgin
12.5 dP
virgin
12.5 dP
Fol
d ch
ange
vs
Inpu
t F
old
chan
ge v
s In
put
0.25
Fol
d ch
ange
vs
Inpu
t
1.0
0.5
0.75
0
0.25
0
0
0
Csn2Elf5
MaSC Luminal MaSC Luminal
Wap
MaSC Luminal
Lgr5
MaSC Luminal
0.35
0.05
0.15
0.45
0.25
0.014
0.002
0.006
0.018
0.010
0.35X101.0X102
0.05
0.25
0.2
0.4
0.6
0.8
0.15
00 0 0
A
B
8 wk virgin 12.5 dP
Exp
ress
ion
rela
tive
to 1
8S r
RN
A
control
Ovx
Ovx
Ccnd2
Cdkn2a
Ovx
control Ovx
4
2
3
1
0
1.6
0.8
1.2
0.4
0
control
control
TSS
TSS
Fol
d ch
ange
vs
Inpu
t F
old
chan
ge v
s In
put
C
OVXvirgin 12.5 dP1.0
0.8
0.6
0.4
0.2
0
MaSC/basal Luminal
1.0
0.8
0.6
0.4
0.2
0
OVXvirgin 12.5 dP
H3K4me3 and H3K27me3H3K27me3 onlyH3K4me3 onlyNo H3K4me3/H3K27me3
D
Figure S2. Genome-Wide Histone Modification Changes in Mammary Epithelial Cells from Pregnant and Ovariectomized Mice, Related to
Figure 3
(A) Read coverage maps and corresponding ChIP-qRT-PCR for H3K27me3 marks on Ccnd2 and Cdkn2a (Ink4a/arf) in the MaSC/basal population. n = 3
independent biological samples for each, error bars represent SEM.
(B) Pregnancy induces derepression of luminal genes in the MaSC-enriched subset invoking ‘‘lineage-priming’’. Quantitative RT-PCR was performed to
determine the levels of b-casein (Csn2), Wap, Elf-5, and Lgr5 mRNA in the MaSC/basal and luminal subsets isolated from virgin (8 weeks old) and 12.5 day
(C) Read coverage maps and corresponding ChIP-qRT-PCR for H3K27me3 marks on three luminal genes (Elf5, Csn2, Wap) and the MaSC/basal-specific
gene Lgr5 in the MaSC/basal population. Data are shown for virgin and 12.5 day pregnant (12.5 dP) mice. Coverage graphs show fragments per million on the
scale 0–10. ChIP-qRT-PCR was performed on three independent biological samples; error bars represent SEM.
(D) Genome-wide proportions of genes with histone methylation marks in pregnant or ovariectomized mice. Segmented bar graphs show the percentages of all
genes significantly marked (FDR < 0.05) with H3K4me3 and/or H3K27me3 for MaSC/basal or luminal subsets from virgin, ovariectomized, or pregnant mice.
S6 Cell Reports 3, 411–426, February 21, 2013 ª2013 The Authors
A
E
C
K18
Ezh2f/+ MMTV-cre; Ezh2f/f
p63
D
MM
TV
-cre
; Ezh
2f/f
6 wk 8 wk
8 wk
Ezh
2f/+
lactation (8 d)
F
Brd
U p
ositi
ve c
ells
per
TE
B
4
0
14
12
8
10
6
2
16Ezh2f/+
MMTV-cre; Ezh2f/f
GMMTV-cre; Ezh2f/f
2 dL
MMTV-cre
Ezh
2f/+
12 wk 6 wk
MM
TV
-cre
; Ezh
2f/f
B
MM
TV
-cre
; Ezh
2f/+
16.5 dP12.5 dP
hCD
4
FSC-A FSC-A
FSC-A FSC-A
Lum MaSC/basal
MM
TV
-cre
; Mcl
-1/h
CD
4 (f
/+)
Mcl
-1/h
CD
4 (f
/+) 1.74% 0.20%
83.5% 99.0%
hCD
496.9% 99.3%
12.7% 0.91%
Figure S3. Delayed Mammary Morphogenesis in Ezh2-Deficient Mice, Related to Figures 4 and 5
(A) Efficient MMTV-cre-mediated deletion in luminal and basal mammary epithelial cells based on hCD4 reporter mice. FloxedMcl-1-hCD4 reporter mice in which
human CD4 surface expression is activated upon cre-mediated excision of Mcl-1, thus serving as a reporter of Mcl-1 deletion (Vikstrom et al., 2010). Mcl-1 is
expressed throughout luminal and myoepithelial cells in the mammary gland (unpublished data). MMTV-cre induced effective deletion of the floxed Mcl-1-hCD4
reporter in the luminal (83%) andMaSC/basal (99%) subsets, respectively. MMTV-cre mediated deletion in GTRosa26 reporter mice confirmed activity of MMTV
in mammary epithelial cells but not stroma (data not shown).
(B) Whole-mounted mammary glands from virgin MMTV–cre; Ezh2f/f mice compared to Ezh2f/+ littermate controls. Stunted development was evident in
Ezh2-deficient glands at 6 weeks but generally not at 12 weeks of age. Scale bars, 2.0 mm.
(C) Cell fate appears unchanged in Ezh2-deficient mammary glands. Immunostaining for lineage markers in mammary gland tissue sections from virgin 8-week
old MMTV–cre; Ezh2f/f mice compared to Ezh2f/+ controls. Scale bars, 50 mm.
(D) Npt2b immunostaining of sections fromMMTV–cre; Ezh2f/f glands at 6 and 8weeks of age did not reveal premature alveolar differentiation in the ducts or TEBs
(top panels). A small minority of cells (<2%) were positive for Npt2B staining (middle right panel). Immunostaining of mammary gland sections from Ezh2f/+ mice
for Npt2b at 8 days of lactation (bottom left panel) serves as a positive control. Red = Npt2b, Green = E-cadherin, Blue = DAPI. Scale bars, 50 mm.
(E) Histogram showing the number of BrdU-positive cells per TEB in pubertal mammary glands from MMTV–cre; Ezh2f/f mice compared to Ezh2f/+ mice (n = 3
mice; error bars represent SEM).
(F) H&E sections of mammary glands from MMTV–cre; Ezh2f/+ mice at days 12.5 and 16.5 of pregnancy. Scale bars: 50 mm.
(G) H&E sections of mammary glands at day two of lactation from MMTV–cre; Ezh2f/f and MMTV–cre mice. Scale bars: 100 mm.
Cell Reports 3, 411–426, February 21, 2013 ª2013 The Authors S7
cell cyclecell cycle processmitotic cell cyclecell cycle phasecell divisionM phase of mitotic cell cyclenuclear divisionmitosisorganelle fi ssionM phasecellular component organizationpolyol metabolic processresponse to stimulusresponse to DNA damage stimuluscellular response to stimulusDNA-dependent DNA replicationDNA replicationDNA metabolic processDNA replication initiationmyotube differentiationkeratinizationcellular response to stressorganelle organizationinositol metabolic process
or empty control retrovirus in 2D cultures and harvested after 72 hr. PCR analysis of genomic DNA confirmed excision. The top and bottom bands represent the
wild-type and floxed alleles respectively. Ex vivo cre-mediated excision of Ezh2 in the MaSC/basal (and luminal subsets) severely impaired clonogenic capacity.
Shown here are data for the MaSC/basal (CD29hi) population.
(B) Microarray analysis of the MaSC/basal (CD29hi) and luminal (CD29lo) subsets from control (undeleted) and Ezh2-deficient cells following ex vivo excision
revealed significant enrichment of gene signatures related to cell cycle regulation. Comparative analyses of the Ezh2 signature genes fromKamminga et al. (2006)
and Ezhkova et al. (2009) with our gene expression profiles showed that the signatures were significantly differentially expressed in the basal and luminal subsets:
1) for upregulated genes in the MaSC/basal and luminal subsets, p = 0.0009 and 0.0003 respectively (Kamminga et al., 2006) and 2) for up or downregulated
genes in the MaSC/basal and luminal subsets, p = 0.0009 and 0.0099 respectively.
(C) Quantitative RT-PCR analysis of Arf expression in cellular subsets from virgin versus pregnant glands (left panel). qRT-PCR showed that Arf is derepressed in
Ezh2-deficient luminal cells isolated from 12.5 day pregnant mice (right panel) (n = 3 independent experiments; error bars represent SEM).
(D) Ezh2 transcriptional signature by breast cancer tumor subtype. Box plots show the aggregate gene expression score in each tumor for genes associated
with Ezh2-deficiency in the MaSC/basal subset. The Ezh2-deficient expression score is highest in the claudin-low subtype and lowest in the basal and luminal
B subtypes. Gene set testing (Wu et al., 2010) confirms that these comparisons are statistically significant (p = 0.0002 for claudin-low versus basal subtypes and
p = 0.0027 for claudin-low versus luminal B).
S8 Cell Reports 3, 411–426, February 21, 2013 ª2013 The Authors
A
MM
TV
-cre
; Ezh
2f/f
CD14+ CD14–CD29hi
Col
onie
s pe
r 10
0 ce
lls
10
0
40
30
20
CD14+
CD29lo
CD24+
CD29hi
CD24+
Ezh2f/+
MMTV-cre; Ezh2f/f
B
C
MMTV-cre; Ezh2f/f
CD
24
CD29
CD
24
CD14
CD
24
CD29
CD
24
MMTV-cre; Ezh2f/f
CD14
CD
24
CD29
CD
24
CD14
CD
24
CD29
CD
24
CD14
virg
inpr
egna
nt
CD14_
CD29lo
CD24+
Ezh
2f/+
Ezh2f/+
Ezh2f/+
H3K27Me3
Tubulin
4 w
k
8 w
k6.
5 d
12.5
d16
.5 d
6 w
k
Ezh2
f/+
Virgin Pregnancy
cKO
12.5 dP
D
8 w
k
6 w
k
Ezh2f/+
8 w
k8
wk
H3K27me3
Tubulin
Virgin
MMTV-cre;Ezh2f/f
Figure S5. Ezh2 Deficiency Dramatically Reduces Progenitor Activity among the Epithelial Subsets during Pregnancy, Related to Figure 6
(A) CD24/CD29 and CD24/CD14 flow cytometric plots of lineage-negative mammary epithelial cells (CD45�CD31�Ter119�) from MMTV–cre; Ezh2f/f mice and
Ezh2+/f littermate controls in either the virgin or pregnant (day 12.5) states. CD14 was used to subdivide the luminal population since CD61 is rapidly down-
regulated during pregnancy. In virgin glands, CD14 expression enriches for luminal progenitor activity whereas the CD14� subset is enriched for mature luminal
cells (Asselin-Labat et al., 2011). In pregnancy, the spectrum of CD14 expression is broader, with progenitor activity detectable in all subsets, thus suggesting
a continuum of alveolar and ductal progenitor cells. Ezh2-deficiency leads to diminution of all progenitor activity.
(B) Colony forming capacity of freshly sorted CD29hiCD24+MaSC/basal (denoted CD29hi), CD29loCD24+CD14+ (denoted CD14+), and CD29loCD24+CD14�
(denoted CD14�) luminal subfractions from mid-pregnant MMTV–cre; Ezh2f/f mice or Ezh2f/+ mice, grown on irradiated fibroblast feeders.
(C) Histogram showing the colony forming capacity of each subpopulation. Data represent the mean of two independent biological experiments with eight
replicates for each.
(D) Global diminution of H3K27me3 protein in Ezh2-deficient mammary epithelial cells: Western blot analyses of total H3K27me3 protein and tubulin in lysates
from Ezh2-deficient (MMTV-cre; Ezh2f/f) versus littermate control mammary glands (Ezh2f/+) (upper panel). Loss of trimethylated H3K27 protein in Ezh2-deficient
glands at 12.5 days pregnancy (lower panel, right lane).
Cell Reports 3, 411–426, February 21, 2013 ª2013 The Authors S9
FSC-H
FS
C-W
SSC-HS
SC
-WFSC-A
PI
FSC-A
Line
age
Sca
1-A
PC
CD49b-FITC
CD
24-P
E
CD29-APC-Cy7
Sca
1-A
PC
CD49b-FITCC
D24
-PE
CD29-APC-Cy7
Oil Pg
Mat Lum Lum Prog (PR+)
Lum Prog (PR–)
Figure S6. Isolation of Hormone Receptor-Positive and Negative Luminal Progenitor Populations from Young Adult Mammary Glands,
Related to Figure 6
CD24/CD29 and CD49b/Sca-1 flow cytometric plots of lineage-negative mammary epithelial cells (CD45�CD31�) from 8 week-old mice. CD49b/Sca-1 flow
cytometric plots are shown for lineage-negative CD29loCD24+ cells derived from mice treated with progesterone or vehicle (oil) for 48 hr. The three luminal
subsets used for qRT-PCR analysis are depicted: mature luminal, PR+, and PR� luminal progenitor cells.
S10 Cell Reports 3, 411–426, February 21, 2013 ª2013 The Authors