3454
Research Article
Polycomb repressive complex 2 impedes intestinal cell
terminal differentiation
Yannick D. Benoit1,2, Manon B. Lepage1, Taoufik Khalfaoui1, Éric Tremblay1, Nuria Basora1, Julie C. Carrier2,
Lorraine J. Gudas3 and Jean-François Beaulieu1,*
1
CIHR Team on the Digestive Epithelium, Département d’Anatomie et Biologie Cellulaire, Faculté de Médecine et des Sciences de la Santé,
Université de Sherbrooke, Sherbrooke, QC J1H 5N4, Canada
2
CIHR Team on the Digestive Epithelium, Département de Médecine, Faculté de Médecine et des Sciences de la Santé, Université de Sherbrooke,
Sherbrooke, QC J1H 5N4, Canada
3
Department of Pharmacology, Weill Cornell Medical College, New York, NY 10065, USA
*Author for correspondence ([email protected])
Journal of Cell Science
Accepted 15 February 2012
Journal of Cell Science 125, 3454–3463
ß 2012. Published by The Company of Biologists Ltd
doi: 10.1242/jcs.102061
Summary
The crypt–villus axis constitutes the functional unit of the small intestine, where mature absorptive cells are confined to the villi, and
stem cells and transit amplifying and differentiating cells are restricted to the crypts. The polycomb group (PcG) proteins repress
differentiation and promote self-renewal in embryonic stem cells. PcGs prevent transcriptional activity by catalysing epigenetic
modifications, such as the covalent addition of methyl groups on histone tails, through the action of the polycomb repressive complex 2
(PRC2). Although a role for PcGs in the preservation of stemness characteristics is now well established, recent evidence suggests that
they may also be involved in the regulation of differentiation. Using intestinal epithelial cell models that recapitulate the enterocytic
differentiation programme, we generated a RNAi-mediated stable knockdown of SUZ12, which constitutes a cornerstone for PRC2
assembly and functionality, in order to analyse intestinal cell proliferation and differentiation. Expression of SUZ12 was also
investigated in human intestinal tissues, revealing the presence of SUZ12 in most proliferative epithelial cells of the crypt and an
increase in its expression in colorectal cancers. Moreover, PRC2 disruption led to a significant precocious expression of a number of
terminal differentiation markers in intestinal cell models. Taken together, our data identified a mechanism whereby PcG proteins
participate in the repression of the enterocytic differentiation program, and suggest that a similar mechanism exists in situ to slow down
terminal differentiation in the transit amplifying cell population.
Key words: Polycomb, SUZ12, Differentiation, Intestine, Crypt
Introduction
The epithelium of the small intestine is in continuous and rapid
renewal. The dynamics of this system involves cell generation
and migration from the stem cell population, located in the lower
part of the crypt, to extrusion of senescent cells at the tip of the
villus. The renewal kinetics of the intestinal epithelium has been
characterized in great detail over the last three decades (Cheng
and Leblond, 1974; Bjerknes and Cheng, 2005; Barker et al.,
2008; Scoville et al., 2008; Potten et al., 2009; Quante and Wang,
2009; Li and Clevers, 2010; Shaker and Rubin, 2010). These
studies have established that the crypt contains two physically
distinct proliferative compartments: the first, located in the lower
third of the crypt containing the slow growing stem cells is called
‘the stem cell zone’ and the second, located in the middle of the
crypt containing the fast growing and differentiating transit cells
is called ‘the transit amplifying (TA) zone’. Movement from the
stem cell zone to the TA zone triggers the irreversible process of
intestinal cell determination towards either secretory (mucous,
enteroendocrine and Paneth cells) or absorptive (absorptive cells)
lineages. Except for Paneth cell precursors that down-migrate and
complete their differentiation at the crypt base, terminal
differentiation occurs in the upper third of the crypt so that all
cells reaching the villi are fully functional. More recent studies
have yielded a wealth of complementary information involving
signalling pathways such as Wnt, Bmp and Notch in intestinal
stem cell regulation and lineage specification as well as evidence
for the co-existence of two types of stem cells, the crypt base
columnar cells and the label-retaining cells found at position ,4.
Taken together, these observations indicate that the intestinal
crypt is a highly hierarchized structure.
In agreement with The Unitarian Theory (Cheng and Leblond,
1974), all cell lineages within the intestinal epithelium are
derived from a common stem cell precursor (Barker and Clevers,
2010). Specification for each lineage begins at a common origin
just above the stem cell zone (approximately cell position 6 and
above) (Bjerknes and Cheng, 2005) and involves Notch
signalling (Yang et al., 2001). It is well documented that the
secretory population represents less that 10% of intestinal
epithelial cells, the rest being absorptive cells. Absorptive cells
thus need to divide several times more than secretory cells before
undergoing terminal differentiation (Bjerknes and Cheng, 2005)
in order to explain why absorptive cells are in the majority on the
villi.
Minimal absorptive cell differentiation in the TA zone has
been well documented in rodents with a variety of intestinal cell
markers such as sucrase-isomaltase (SI) and apolipoproteins
Journal of Cell Science
PRC2 impedes terminal differentiation
which are not detected in the crypt epithelium (Trier and Madara,
1981; Demmer et al., 1986; Traber, 1999; Sauvaget et al., 2002),
suggesting that absorptive cell differentiation is not induced in the
TA zone. However, in man, some absorptive cell differentiation
markers have been identified in the epithelium of the crypt. These
include dipeptidylpeptidase IV (DPPIV) and aminopeptidase N
(Gorvel et al., 1991; Quaroni et al., 1992) but also immature
forms of other markers (Beaulieu et al., 1989; Beaulieu et al.,
1990; Gorvel et al., 1991; Hoffman and Chang, 1993; Giannoni
et al., 1995; Basque et al., 1998) including SI, one of the best
characterized intestinal cell markers. The latter data are
consistent with the fact that all cells in the TA zone express
the transcription factors CDX2, HNF1a and HNF4a known to
promote expression of differentiation markers such as SI (Traber,
1999; Mitchelmore et al., 2000; Boudreau et al., 2002b; Escaffit
et al., 2005a; Olsen et al., 2005; Fang et al., 2006; Bosse et al.,
2007; Boyd et al., 2009; Benoit et al., 2010; Boyd et al., 2010).
These data suggest that in the TA zone, repressive mechanisms
are responsible for the full suppression of terminal differentiation
in rodents. In man, the low levels of SI precursor in the human
TA zone suggests an apparent leakiness in the mechanism(s) that
repress terminal differentiation. Because of the existence of this
leakiness, human intestinal cells represent an interesting system
to investigate the mechanisms that repress differentiation in
proliferating epithelial cells.
Mechanisms responsible for the transient repression of
absorptive cell differentiation in the TA zone have not yet been
investigated. Because the main pro-differentiation transcription
factors are expressed in these cells, one may consider various
mechanisms known to negatively modulate their transcriptional
activity such as post-translational modifications (Houde et al.,
2001; Rings et al., 2001), expression of transcriptional repressors
(Boudreau et al., 2002a) and/or epigenetic mechanisms such as
histone modifications (Tou et al., 2004; Mariadason, 2008; Verzi
et al., 2010). Another possibility not yet investigated could be
epigenetic control of gene expression via polycomb group (PcG)
proteins. Indeed, PcGs have been extensively characterized for
their ability to repress differentiation and promote self-renewal in
embryonic stem cells via histone methylation (Boyer et al., 2006;
Lee et al., 2006; Margueron and Reinberg, 2011), a step catalysed
by the polycomb repressive complex 2 (PRC2) (Sparmann and
van Lohuizen, 2006; Margueron and Reinberg, 2011). The PRC2
core component SUZ12 is essential for the assembly, stability
and functionality of PRC2 (Margueron and Reinberg, 2011).
While a role for PcGs in the preservation of stemness
characteristics of embryonic stem cells is now well established,
recent evidence has suggested that PcGs may have a more direct
role in mediating cell differentiation (Caretti et al., 2004;
Ezhkova et al., 2009) and cancer progression (Martinez-Garcia
and Licht, 2010; Mills, 2010; Margueron and Reinberg, 2011).
In the present study, we have investigated the potential
implication of PcG-mediated epigenetic regulation in human
intestinal cell differentiation. Our data show that inactivation of
PRC2 leads to significant upregulation of specific terminal
differentiation markers including SI in the two well characterized
intestinal cell models Caco-2/15 and human intestinal epithelial
crypt (HIEC) cells. In this context, predominant distribution of
SUZ12 in the TA zone in the normal intestine and its detection in
adenocarcinoma cells in primary tumours suggests that PcGs also
exert repressive activity in situ on intestinal cell terminal
differentiation and in colorectal tumour cells.
3455
Results
PRC2 core member SUZ12 expression decreases as
differentiation proceeds in human intestinal cells
The Caco-2/15 cell line has been widely exploited for its unique
ability to spontaneously undertake a differentiation program upon
reaching confluence, which is reflected by robust expression of
specific biomarkers (Beaulieu and Quaroni, 1991; Sambuy et al.,
2005) such as SI that increases with confluence (Fig. 1A). The
analysis of expression of the major polycomb factors constituting
the PRC2 complex in Caco-2/15 cells revealed that transcripts
encoding the SET-domain-containing histone methyltransferase
EZH2, as well as its natural partner embryonic ectodermal
development (EED) PcG protein, were constitutively expressed
in Caco-2/15 cells (data not shown). Since previous findings have
described that SUZ12 is essential for EZH2 and EED
stabilization (Cao and Zhang, 2004), we used the expression of
SUZ12 at the protein level as a marker for functional PRC2
complexes in Caco-2/15 cell cultures at various stages of
confluence. Western blot analyses revealed that SUZ12 was
expressed in Caco-2/15 cells and that protein levels significantly
decreased between 0 days post-confluence (PC) and 30 days PC,
which inversely correlated with the appearance of SI (Fig. 1A).
Disruption of PRC2 induces the enterocytic differentiation
program of Caco-2/15 cells
To study the role of PRC2 in intestinal differentiation, RNAimediated stable knockdown of SUZ12 was generated in Caco-2/15
cells. These cultures showed significantly reduced protein levels of
SUZ12 at all stages examined as compared to shCNS cultures
(Fig. 1B). Using an antibody directed against trimethylated
lysine27 of histone 3 (H3K27me3), western blot analyses on
shCNS and shSUZ12 protein lysates confirmed that the
knockdown of SUZ12 was accompanied by a loss of PRC2
methyltransferase activity (Fig. 1B).
The impact of the loss of SUZ12 on differentiation determined by
measuring SI protein levels revealed a strikingly early appearance
and higher levels of SI expression in confluent SUZ12 knockdown
cell cultures compared to controls (Fig. 1C,D). Since regulation of
SI is mainly mediated by transcription factors HNF1a and CDX2,
we examined the expression of these factors. Interestingly, while
HNF1a rose gradually in Caco-2/15 cells between 0 and 8 days PC,
no significant difference was noted between control and shSUZ12
cells (Fig. 1E). Expression of other biomarkers associated with cell
polarization such as villin, E-cadherin and Li-cadherin were not
modulated by SUZ12 ablation (Fig. 1F) suggesting that PRC2
activity specifically targets terminal differentiation markers. To
evaluate the functional relevance of the upregulated expression of SI
in Caco-2/15 cells upon SUZ12 knockdown, SI mRNA levels were
determined in control and shSUZ12 cells at 0, 4 and 8 days of
postconfluence and compared to those of Caco-2/15 cells
maintained at confluence up to 30 days. As shown in Fig. 2, SI
levels in shSUZ12 cells at 4 and 8 days were comparable to those
observed in wild-type Caco-2 cells maintained at confluence for 12
and 30 days, respectively. However, the amounts of SI transcript
never exceeded 25% of those found in the purified intestinal
epithelium (Fig. 2).
Distribution pattern of the PRC2 core factor SUZ12 in the
human intestinal tract
The aforementioned results suggested that PRC2 activity
participates in the repression of SI expression. To further explore
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Journal of Cell Science 125 (14)
Fig. 1. Impact of SUZ12 knockdown on the Caco-2/
15 differentiation program. (A) Western blot analysis
demonstrates the expression profile of SI and SUZ12
proteins in 0-, 8-, 15- and 30-day post-confluent Caco2/15 cultures. Relative optical density values of
immunodetected SI and SUZ12 at each confluence
stage in Caco-2/15 as compared to the actin loading
control are shown on the right (n53, *P,0.001). (B) A
representative western blot for the assessment of
SUZ12 protein expression levels and the PRC2associated epigenetic marker H3K27me3, in Caco-2/15
stable SUZ12 knockdown (shSUZ12) and control
(shLUC) cell populations at 0, 4 and 8 days of
postconfluence. Actin was used as the loading control.
(C) Representative western blot of SI expression as a
function of confluence (+0, +4 and +8 days) in control
(shLUC) and SUZ12 knockdown (shSUZ12) Caco-2/15
cultures. (D) Amounts of SI at each confluence stage in
control (shLUC) and SUZ12 knockdown (shSUZ12)
Caco-2/15 cultures relative to the loading control actin
(n53, ***P,0.0001). (E,F) Relative amounts of (E)
HNF1a protein in Caco-2/15 at 0, 4 and 8 days
postconfluence and (F) CDX2, E-cadherin and villin in
Caco-2/15 cultures at 8 days post-confluence. Values
were obtained by optical density measurements of
western blot analyses, relative to the loading control
actin, in control (shLUC) and SUZ12 knockdown
(shSUZ12) stable SUZ12 knockdown (shSUZ12; n53).
this hypothesis, the expression and distribution pattern of SUZ12
was studied by indirect immunofluorescence on cryosections of the
normal human adult small intestine. As previously documented in
detail in the human, SI is mainly expressed by the mature absorptive
cells located in the terminal differentiation compartment and on the
villus but also as weaker expression under a precursor form in the
proliferative cells of the crypt (Beaulieu et al., 1989; Beaulieu et al.,
1990). Interestingly, as determined in matched serial adjacent
sections (Fig. 3A–D), specific nuclear staining for SUZ12 was
observed in the epithelial cells of the crypt that were negative for the
mature form of SI (Fig. 3A,B) but stained for the detection of the SI
precursor (Fig. 3C,D). SUZ12 was absent from the Paneth cells at
the crypt bottom and villus enterocytes, stained with anti-group IIA
phospholipase A2 (PLA2G2A) antibody and anti-mature SI
antibody, respectively (Fig. 3B). Further analyses on serial
sections confirmed the mutually exclusive patterns of expression
of SUZ12 and both mature SI and PLA2G2A, suggesting the
presence of active PRC2 complex in the TA cells of the middle crypt
(Fig. 3E). Even though SUZ12 is predominantly expressed in the
TA zone, the consistent expression of an immature form of SI, even
detected at low levels, suggests that the repressive mechanism(s)
counteracting the promoting effects of CDX2 and HNF1a on SI
expression is leaky.
PRC2 impedes functional enterocytic differentiation in
human normal crypt cells
The observation that SUZ12 expression was associated with the
proliferative and poorly differentiated epithelial cell population in
Fig. 2. SI transcript levels following SUZ12 knockdown in Caco-2 cells
relative to their fully differentiated counterparts and pure intestinal
epithelium. Relative amounts of SI transcript were determined by
quantitative PCR in control (shLUC, black columns) and SUZ12 knockdown
(shSUZ12, white columns) Caco-2/15 cells at 0, 4 and 8 days postconfluence
compared to wild-type Caco-2/15 cells at subconfluence (SC), confluence
(C), 3, 12 and 30 days of post confluence (curve). Amounts were determined
relative to the average of five purified intestinal epithelial fractions. In all
cases, RPLP0 amplification was used as a control reference gene.
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PRC2 impedes terminal differentiation
3457
Fig. 3. Distribution pattern of the PRC2 core factor SUZ12 in human small intestinal mucosa. (A–D) Indirect immunofluorescence detection of SUZ12 in
the normal human adult small intestine revealed restricted expression in the undifferentiated cells of the crypts. SUZ12 was not detected in differentiated
cells of the upper crypt and villus regions [identified with the mature form of SI in B (red staining)] or in Paneth cells (identified with PLA2, shown in green in B
and D and brackets and asterisks in A and C). Note that in the human, cells in the transit amplifying zone express a precursor form of SI (D). Evans blue was used
as a histological counterstain (A,C) and nuclei were stained with DAPI (blue) in the corresponding serial sections (B,D). (E) Higher magnification of a
representative crypt region sequentially stained for SI, PLA2 and SUZ12, as described above. Images were digitally merged and deconvoluted. Differentiated
populations in the crypt bottom (Paneth cells; yellow) and the villus (SI positive; red) are indicated by arrowheads and the SUZ12-positive zone (green) in the
middle of the crypt is delimited by brackets. The weak expression of SI in the luminal domain of absorptive cells in the middle portion of the crypt is visible.
Nuclei were stained with DAPI (blue). Scale bars: (A–D) 50 mm; width of section in E is 80 mm.
situ prompted the use of HIEC cells to further study the role of
PRC2 on the enterocytic differentiation program. The HIEC model
had been previously shown to display many characteristics
associated with undifferentiated intestinal crypt cells (Perreault
and Beaulieu, 1996; Pageot et al., 2000; Tremblay et al., 2006;
Beaulieu and Ménard, 2012). Ectopic expression of transcription
factors such as HNF1a and CDX2 in HIEC induced initiation of an
intestinal cell differentiation program (Benoit et al., 2010). It is
noteworthy that under these conditions, expression of cell
polarization markers such as E-cadherin, Li-cadherin and various
tight junction components was triggered while terminal
differentiation remained limited as evaluated by expression of
low levels of SI mRNA transcript. Taking together, these data
indicating that HIECHNF1a/CDX2 are committed but still poorly
differentiated suggest that they may share characteristics with the
TA cells (Benoit et al., 2010). Consistent with this, we observed
significant endogenous expression of the SUZ12 transcript in wildtype HIEC cells which was greatly increased following ectopic
expression of HNF1a and CDX2 (Fig. 4A). Adult small intestine
total epithelium fractions were used as a reference point for SUZ12
expression. Hence, we hypothesized that the increased SUZ12
expression in committed HIEC could be related to the weak net
effect of HNF1a and CDX2 on HIEC cell differentiation and that
knocking down this PRC2 component would release repression of
completion of the differentiation program.
We confirmed that the SUZ12 protein was endogenously
expressed in HIEC cells and that it could be efficiently knocked
down by shSUZ12 (Fig. 4B). The abolition of SUZ12 expression
was accompanied by the loss of an H3K27me3 signal, confirming
the loss of PRC2 methylation activity (Fig. 4B). As polycomb
group protein actions are associated with repressed differentiation
and maintained proliferative activity in other models (Margueron
and Reinberg, 2011), BrdU incorporation assays were performed
to determine if SUZ12 knockdown affected cell growth in these
normal cells. A significant decrease in BrdU incorporation was
observed in shSUZ12 cells when compared to shCNS cells
(Fig. 4C). Furthermore, loss of PRC2 activity, via SUZ12
knockdown, was not sufficient to trigger the expression of
HNF1a or CDX2 pro-enterocytic differentiation factors in HIEC
cells (Fig. 4D).
Consistent with the intestinal progenitor status of HIEC,
previous studies confirmed the absence, or trace expression of
digestive enzymes such as SI and DPPIV in wild-type cells
(Perreault and Beaulieu, 1996; Pageot et al., 2000) but forced
expression of HNF1a and CDX2 induced these enterocytic
differentiation markers (Benoit et al., 2010). The impact of
SUZ12 abolition on differentiation was thus studied in wild type
and HNF1a/CDX2 overexpressing HIEC. RT-PCR analyses
showed that abolition of SUZ12 in wild type HIEC cultures did
not significantly induce SI and DPPIV expression (Fig. 5A,B).
This result is in accordance with the above observations that
SUZ12 knockdown is not sufficient to induce the HNF1a or
CDX2 transcription (Fig. 1E,F9, Fig. 4D) necessary for SI and
DPPIV expression. In HIECCDX2/HNF1a cells, however, abolition
of SUZ12 resulted in a significant increase in SI and DPPIV
mRNA expression (Fig. 5A,B) as well as that of lactasephlorizine hydrolase (LPH) and intestinal alkaline phosphatase
(ALP), two other well-characterized enterocytic differentiation
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Journal of Cell Science 125 (14)
Fig. 4. Use of normal HIEC cells to study the regulatory mechanisms of
crypt cell proliferation and differentiation. (A) Quantitative PCR of SUZ12
expression in wild-type (wt) HIEC cells and HIEC cells committed toward
differentiation (CDX2/HNF1a-expressing cells). Total adult small intestinal
epithelial fractions were used as control. Results were normalized to the
RPLP0 housekeeping gene (n53, ***P, 0.0001). (B–D) RNAi-mediated
knockdown of SUZ12 in HIEC cells. (B) Representative western blots
showing the expression levels of SUZ12 protein in wild-type (wt), stable
control (shCNS), and knockdown (shSUZ12) HIEC cultures. The PRC2associated epigenetic marker H3K27me3 was assessed in each cell type.
Actin was used as loading control. (C) The percentage of BrdU-labelled cells
in the total cell populations of shCNS and shSUZ12 HIEC cultures (n54,
**P50.001). (D) RT-PCR illustrating the absence of HNF1a and CDX2
transcription factors in shCNS and shSUZ12 HIEC cultures. HNF1a- and
CDX2-expressing HIEC cells were used as positive controls. RPLP0 was used
as a control housekeeping gene.
markers (Fig. 5C). It is noteworthy that another wellcharacterized intestinal cell differentiation marker, fatty acid
binding protein (FABPi), was not significantly modulated in
response to SUZ12 depletion suggesting that not all terminal
differentiation-related genes are under the dependence of the
PRC2 complex. Furthermore, as observed in Caco-2/15 cells,
SUZ12 knockdown in HIECHNF1a/CDX2 cells failed to alter
expression of polarization markers such as Li-cadherin (Fig. 5D)
and E-cadherin (not shown) but resulted in a significant decrease
in the expression of CD44 and EpCAM (Fig. 5D), two markers
recently associated with colorectal cancer stem cells and
invasiveness (Kuhn et al., 2007; Lugli et al., 2010; Hou et al.,
2011). Taken together, these results show that abolition of PRC2
epigenetic regulation via SUZ12 knockdown promotes the
expression of a number, but not all terminal differentiation
markers in normal intestinal cells.
Suz12 expression pattern in human colorectal cancers
Recent studies reported a putative role for the polycomb group
proteins in solid tumour progression, including colorectal cancers
(Fluge et al., 2009; Du et al., 2010; Mills, 2010; Margueron and
Reinberg, 2011). SUZ12 expressing human colorectal cancers
were first analysed by quantitative PCR (qPCR) in patientmatched pairs of colon resection margins and corresponding
primary carcinoma samples. On average with 44 cases tested, a
significantly higher expression of SUZ12 was observed in the
tumour group (Fig. 6A, column A). Analysing each pair
Fig. 5. Expression of intestinal cell markers in HIEC crypt cells following
SUZ12 knockdown. Transcript expression levels of enterocytic-specific
differentiation markers (A) SI (n53, ***P,0.0001) and (B) DPPIV (n55,
**P50.0056) were measured by qRT-PCR, in control (shCNS) and SUZ12
knockdown (shSUZ12) cultures of wild-type (wt) and ‘committed’ (HNF1a/
CDX2) HIEC cells. (C,D) Expression of the differentiation-associated genes
lactase-phlorizine hydrolase (LPH; n53, **P50.0018), intestinal alkaline
phosphatase (ALP; n53, *P50.0345) and intestinal fatty acid binding protein
(FABPi) (n53, aP50.052), the cell–cell junction marker Li-cadherin (n53,
non-significant), and the stem-cell-associated markers CD44 and EpCAM
(n53, *P50.0127, **P50.0099) were also evaluated by qPCR in shCNS and
shSUZ12 committed HIEC cells. In all cases, RPLP0 amplification was used
as a control reference gene.
individually revealed that 12 of the cases exhibited a 26 or
higher increased expression of SUZ12 in the tumour sample
compared to normal tissue. However, higher levels of SUZ12
expression was not correlated with tumour stage as shown in
Fig. 6A (r250.038; P50.75, not significant) nor with tumour
grade (r250.074; P50.65, not significant). In order to determine
the SUZ12 distribution pattern in such a pathological context,
indirect immunofluorescence experiments were performed on 16
different human colonic adenocarcinoma cases. E-cadherin coimmunostaining was carried out to distinguish the epithelial
organized regions from stromal cells. Of the cases studied, all
well-differentiated adenocarcinomas displayed limited SUZ12
positive staining (three cases; not shown), while the abundance of
SUZ12-positive nuclei in epithelial structures varied from ‘weak’
to ‘strong’ in the moderately differentiated samples (10 cases) as
shown in two representative samples (Fig. 6B,C). Although
tested in a limited number of samples (three cases), relative
abundant SUZ12-positive nuclei were observed in poorly
differentiated tumours (not shown).
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PRC2 impedes terminal differentiation
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Fig. 6. SUZ12 expression pattern in human colorectal
cancers. (A) Quantitative PCR data of SUZ12 expression in
tumours relative to their corresponding resection margins used as
reference (R) for each of the 44 patient-matched tissue pairs
tested at stages 0 (adenomas; n53), I (n55), II (n59), III (n519)
and IV (n58). The average change of relative SUZ12 transcript
expression for the 44 cancer pairs (A) was found to be
statistically significant (P50.0007, paired t-test).
(B,C) Immunodetection of SUZ12 in representative moderately
differentiated human colon carcinoma sections showing variable
staining intensity (weak, 1; moderate, 2; strong, 3). Epithelial
structures were counterstained using anti-E-cadherin and nuclei
were stained with DAPI. Scale bar: 50 mm.
Discussion
A tightly coordinated balance between proliferation and
differentiation is essential for the intestinal epithelium to
preserve its constant renewing capacity. The crypt–villus axis
of the small intestine confines the proliferative/stem cell
population from the differentiated absorptive cells in two
physically distinct regions, the crypt and the villus. As cells
move out of the stem cell region they become committed to either
an absorptive lineage or a secretory lineage. Bjerknes and Cheng
provided experimental evidence that absorptive progenitors
divide approximately four times before terminal differentiation
while secretory progenitors divide only once or twice (Bjerknes
and Cheng, 2005) explaining why absorptive cells are so strongly
in the majority on the villi. While polycomb group proteins have
been extensively described as regulators of pluripotency and selfrenewal in human and murine stem cells (Boyer et al., 2006; Lee
et al., 2006; Margueron and Reinberg, 2011), little is known
about the participation of such epigenetic transcriptional
regulatory mechanisms in committed cells outside the stem cell
niche. Our findings showing that abolition of PRC2 activity in
two experimental cell models triggers acceleration of absorptive
cell terminal differentiation markers such as SI suggests that
epigenetic regulation by PRC2 is a novel mechanism involved in
regulating the balance between intestinal absorptive cell
amplification and differentiation, summarized in Fig. 7. Based
on this model, we propose that PRC2 activity allows TA cells to
maintain their proliferation potential while delaying acquisition
of a number of terminal differentiation characteristics. Moreover,
we describe the distribution pattern of SUZ12 in human
colorectal cancer and found a significant increase of this
marker of PRC2 activity in 27% of the tumours, suggesting
that PcG might play a role in cancer progression in at least a
subset of patients.
PRC2 core members, EZH2, EED1/2 and SUZ12, were found
to be expressed in the human intestinal epithelial models Caco-2/
15 and HIEC, and in order to study PRC2 involvement in the
differentiation of these two models, we disrupted this complex by
RNAi-based knockdown of SUZ12. Previous studies have shown
the critical importance of SUZ12 in PRC2 assembly and
subsequent EZH histone methyltransferase activity (Cao and
Zhang, 2004). EZH1 has previously been shown to have the
capacity to compensate for the loss of EZH2 among PRC2.
Fig. 7. Proposed model for the regulation of enterocytic differentiation in
the human intestinal crypt. Intestinal stem cells are located in the same
region as Paneth cells. Stem cells lack the pro-differentiation factors CDX2
and HNF1a and express basal levels of the components responsible for PRC2
histone methyltransferase activity (PRC2 HMTase). Exit from the stem cell
niche and entry into the transit amplifying zone coincides with induction of
CDX2 and HNF1a, which enhances expression of the PRC2 HMTase activity
machinery. Only basal levels of enterocyte differentiation markers such as SI
are detected in these proliferating cells. The loss of PRC2 HMTase activity in
cells reaching the terminal differentiation compartment triggers a reduction of
cell proliferation and allows for full enterocytic differentiation. Thus,
although transcription factors CDX2 and HNF1a are responsible for the
ultimate expression of the enterocyte differentiation markers such SI, their
expression is repressed by PcGs in the expanding cell population of the transit
amplifying zone.
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Moreover, four distinct EED isoforms, which can be
differentially used by the cell in specific physiological/
pathological contexts, were also identified (Margueron and
Reinberg, 2011). In order to limit any compensation
mechanisms by known isoforms of PRC2 core proteins, we
opted for SUZ12 knockdown as a way to silence PcG-mediated
histone methyltransferase activity. H3K27 tri-methylation, a
hallmark of PRC2 activity (Sparmann and van Lohuizen, 2006;
Martinez-Garcia and Licht, 2010; Margueron and Reinberg,
2011), was shown to be efficiently downregulated following
SUZ12 knockdown in both Caco-2/15 and HIEC cells.
SI expression has been extensively characterized as a major
enterocyte terminal differentiation marker (Beaulieu and
Quaroni, 1991; Boudreau et al., 2002b; Benoit et al., 2010).
Significant SI induction was found to occur earlier when PRC2
was silenced in the human intestinal differentiation model
Caco-2/15. We and others have previously demonstrated the
importance of CDX2 and HNF1a transcription factors in SI
induction (Boudreau et al., 2002b; Benoit et al., 2010; Boyd et al.,
2010). No significant changes in either CDX2 or HNF1a
expression were observed in PRC2 invalidated cells ruling out
a differential effect of these factors on precocious induction of SI
expression.
In the intact small intestine, SUZ12 was found to be
predominantly expressed in the cells of the TA zone, being
below detection levels in villus and Paneth cells, both terminally
differentiated. Interestingly, the pro-differentiation transcription
factors HNF1a and CDX2 are expressed by virtually all intestinal
committed epithelial cells in the normal small intestine, including
those of the TA zone (Traber, 1999; Mitchelmore et al., 2000;
Boudreau et al., 2002b; Escaffit et al., 2005a; Olsen et al., 2005;
Fang et al., 2006; Bosse et al., 2007; Benoit et al., 2010). These
observations suggest that epigenetic PRC2 activity may
participate in the mechanism responsible for counteracting the
pro-differentiation influence of CDX2 and HNF1a in the TA
cells before they reach the terminal differentiation compartment.
Abolition of PRC2 activity results in significant enhancement of
SI expression in Caco-2/15 cells but one may argue that this
result may not necessary reflect the normal situation considering
the cancerous nature of Caco-2/15 cells. Therefore, this
hypothesis was further investigated using the human normal
intestinal epithelial crypt HIEC cells (Perreault and Beaulieu,
1996). Previous studies on HIEC cells showed that they
expressed intestinal epithelial crypt cell markers such as
epithelial keratins, junctional proteins, a trace of DPPIV, MIM1/39, integrin a6b4, a8b1 and a9b1 (Perreault and Beaulieu,
1996; Desloges et al., 1998; Basora et al., 1999; Pageot et al.,
2000; Benoit et al., 2009; Beaulieu and Ménard, 2012) but no Ecadherin, CDX2, HNF1a or SI (Escaffit et al., 2005a; Escaffit
et al., 2005b; Escaffit et al., 2006; Benoit et al., 2010). Forced
expression of CDX2 and HNF1a in HIEC resulted in the
expression of polarization/junctional markers such as E- and Licadherin, a decrease in the expression of stem cell related
markers such as DCAMKL1 and Mushashi-1 and the induction of
SI and DPPIV expression (Benoit et al., 2010). Considering that
these phenotypic changes resulting from the expression of CDX2
and HNF1a expression were reminiscent of what occurs in
position 6–7 and above in the normal crypt (Escaffit et al., 2005a;
Escaffit et al., 2005b; Escaffit et al., 2006; Benoit et al., 2010),
including expression of detectable levels of the two terminal
intestinal cell differentiation markers SI and DPPIV (Beaulieu
et al., 1989; Quaroni et al., 1992), we hypothesized that
HIECCDX2/HNF-1a cells may mimic the in vivo situation in the
TA zone. If right, abolition of the PRC2 repressive influence
should result in enhancement of terminal differentiation features
such as SI and DPPIV expression and remain without significant
effect on polarization markers such as E-cadherin. As expected,
knock down of SUZ12 in HIECCDX2/HNF-1a cells caused a
significant increase in the differentiation markers SI and DPPIV
as well as lactase-phlorizin hydrolase and intestinal alkaline
phosphatase, two other digestive brush border enzymes
associated with the terminal differentiated phenotype
(Mitchelmore et al., 2000) and did not affect E- and Licadherins as observed in Caco-2/15 cells. This phenomenon
diverges from studies performed in embryonic stem cells where
PRC2 activity can regulate E-cadherin repression (Herranz et al.,
2008). The data presented herein on committed cells points out a
distinct role for PRC2 activity in cells located outside of the stem
cell compartments, restraining their terminal differentiation. It
has to be pointed out however that not all terminal differentiation
markers have their expression controlled by PRC2 activity as
illustrated with intestinal fatty acid binding protein, another wellcharacterized intestinal terminal differentiation marker (Levy
et al., 2009).
Interestingly, PRC2 inactivation in HIEC caused a decreased
expression of the epithelial cell adhesion molecule EpCAM and
the cell-surface antigen CD44. Both proteins have been reported to
be expressed in the proliferative cells of normal crypts (MerlosSuárez et al., 2011). EpCAM expression has recently been shown
to be under PcG control in human embryonic stem cells, being
specifically expressed by undifferentiated embryonic stem cells
and repressed upon differentiation (Lu et al., 2010). Moreover, the
transcriptionally active EpCAM gene was associated with high
H3K27 trimethylation at the promoter region. On the other hand,
CD44 expression has been observed in colon tumour-initiating
cells and was associated with an aggressive phenotype (MerlosSuárez et al., 2011). Remarkably, both EpCAM and CD44 were
identified, along with claudin-7 and tetraspanin CO-029, as
members of a complex which promotes colorectal cancer
progression and apoptosis resistance (Lugli et al., 2010).
Many recent studies have suggested a participation of PcGs in
the progression of human cancers, including colon (Fluge et al.,
2009; Du et al., 2010; Mills, 2010; Margueron and Reinberg,
2011). On this basis, and considering the association between
PRC2 and the proliferative region of the crypt–villus axis, we
investigated SUZ12 expression and its distribution pattern in
human colon adenocarcinomas. SUZ12 expression was found to
be increased in a significant portion of the colorectal cancer pairs
tested but there was no correlation with either the stage or grade
of the tumours and the nuclear staining was found to be relatively
heterogeneous in the primary tumours. These results suggest that
overexpression of SUZ12 may define a subset of colon cancers.
Taken together, these data establish a new molecular
mechanism limiting terminal differentiation in the intestinal
cell proliferative compartment. Indeed, besides the previously
reported inhibitory mechanisms of intestinal cell differentiation
(Houde et al., 2001; Rings et al., 2001; Boudreau et al., 2002a;
Tou et al., 2004; Mariadason, 2008; Verzi et al., 2010) detailed
above, our studies using the two distinct intestinal cells models
Caco-2/15 and HIECCDX2/HNF-1a showed that the activity of the
PRC2 component of PcG specifically repressed the expression of
a number of terminal differentiation markers. In situ, the
PRC2 impedes terminal differentiation
cornerstone PRC2 component SUZ12 was identified in a
significant proportion of colon tumours and found
predominantly in the TA zone in the normal small intestine
suggesting that PcG proteins may also participate in the
repressive mechanism of terminal differentiation in vivo.
Materials and Methods
Tissues
Tissues from the normal adult proximal ileum were obtained from Quebec
Transplant (Quebec, Canada) in accordance with protocols approved by the local
Institutional Human Subject Review Board for the use of human material. Forty
four patient-matched pairs of colon resection margins and primary tumour samples
were obtained from the Cooperative Human Tissue Network (n515), funded by
the National Cancer Institute, and from patients undergoing surgical resection at
the Centre Hospitalier Universitaire de Sherbrooke (n529). Patients did not
receive neoadjuvant chemotherapy or radiotherapy. Tissues were obtained after
patient’s written informed consent, according to a protocol approved by the
Institutional Human Subject Review Board. In all cases, clinical and pathological
information were obtained from medical records. Patient’s cancers were
histologically classified and graded according to overall TNM staging criteria
(based on tumour, lymph node and metastatic status). Tissue arrays of colonic
adenocarcinomas, including pathology diagnosis and tumour grade, were
purchased at US Biomax Inc (Rockville, MD).
Journal of Cell Science
Cell culture and establishment of SUZ12 knockdown HIEC and Caco-2/15
cells
Human intestinal crypt HIEC cells have been characterized elsewhere (Perreault
and Beaulieu, 1996; Pageot et al., 2000) and extensively used as a human intestinal
crypt cell model (Beaulieu and Ménard, 2012). CDX2/HNF1a inducible HIEC
cells were used as a normal human differentiation model and were cultured as
recently described (Benoit et al., 2010). Caco-2/15 cells were used as a wellcharacterized human intestinal differentiation model. Their characterization and
culture conditions have been previously described (Beaulieu and Quaroni, 1991;
Vachon and Beaulieu, 1992). Stable shRNA SUZ12 knockdown (shSUZ12) and
control non-silencing (shCNS) HIEC and Caco-2/15 cell populations were
established by lentiviral infection which allowed for an optimal rate of gene
transfer and long-term transgene expression. The pLKO.1-puro vector containing
SUZ12-silencing (shSUZ12) and luciferase-silencing (shCNS) short hairpin RNA
were purchased from Sigma-Aldrich (MISSION shRNA). The five pLKO.1/
shSUZ12 and pLKO.1/shCNS vectors were used to produce virus in 293T cells as
previously described (Benoit et al., 2009). Sub-confluent HIEC and Caco-2/15
cells were infected with viral suspensions and selected for 10 days with 10 mg/ml
puromycin to generate stable cell populations. Four of shSUZ12 were found to be
efficient in blocking SUZ12 expression (not shown).
Indirect immunofluorescence staining
The preparation and optimum cutting temperature (OCT; Canemco-Marivac,
Lakefield, QC) embedding of specimens, cryosectioning as well as staining
procedures were performed as previously described (Escaffit et al., 2005b; Benoit
et al., 2010). Paraffin-embedded tissue arrays were rehydrated, prior to
immunostaining, as previously described. The rabbit monoclonal antibody
targeting the human Suppressor of Zeste 12 (SUZ12; D39F6, Cell Signaling)
was used at a 1:2000 dilution while the mouse monoclonal antibodies targeting the
mature and immature forms of human sucrase-isomaltase (HSI-5 and HSI-13,
respectively) were produced and characterized as described previously (Beaulieu
and Quaroni, 1991) and used at a 1:500 dilution. Rabbit polyclonal antibody raised
against the Paneth cell specific PLA2G2A was used at a 1:5000 dilution (H-74,
Santa Cruz Biotech). Mouse monoclonal antibody used for E-cadherin detection
was used at a 1:5000 dilution (BD Biosciences). Secondary antibodies were FITCconjugated anti-mouse and anti-rabbit (Chemicon). Nuclei were stained with
DAPI.
Western blot analysis
Proteins were extracted, separated by SDS-PAGE and transferred onto
nitrocellulose membranes as previously described (Perreault and Beaulieu, 1996;
Escaffit et al., 2005a). Membranes were blocked in PBS containing 5% skim milk
and 0.1% Tween 20 (Bio-Rad). The rabbit monoclonal anti-SUZ12 (D39F6, Cell
Signaling, 1:1000), rabbit polyclonal anti-H3K27me3 (07-449, Millipore, 1:5000),
goat polyclonal anti-HNF1a (C-19, Santa Cruz Biotech, 1:500), mouse
monoclonal anti-sucrase-isomaltase (HSI-14, 1:10) (Beaulieu and Quaroni,
1991), anti-Cdx2 (88, Biogenex, 1:500), anti-E-cadherin (36, BD Biosciences,
1:2000), anti-Li-cadherin (C-17, Santa Cruz Biotech, 1:500) and anti-actin (C4,
1:75,000, Chemicon) were incubated with membranes for 12 hours at 4 ˚C in
blocking solution. After washing in PBS, membranes were incubated with
horseradish peroxidase-conjugated secondary antibodies (anti-mouse, anti-rabbit,
Amersham) and developed using the Immobilon Western kit (Millipore).
3461
RNA extraction, RT-PCR and quantitative PCR
Total RNA was extracted using TriPure isolation reagent (Roche). mRNA was
reverse-transcribed using the reverse transcriptase Omniscript (Qiagen). PCR assays
were performed as described previously (Dydensborg et al., 2006; Benoit et al.,
2009). Human SI, CDX2, HNF1a, Li-cadherin, DPPIV, B2M and RPLP0 were
evaluated by qPCR as previously described (Escaffit et al., 2005a; Dydensborg et al.,
2006; Escaffit et al., 2006; Benoit et al., 2010). For EZH2 detection we used the
forward 59-TTTTTGCCAAGAGAGCCATC-39 and the reverse 59-AAGGCAGCTGTTTCAGAGGA-39 primers while EED (variants 1 and 2) was detected
using the forward 59-CGATGGTTAGGCGATTTGAT-39 and the reverse 59CCAAATGTCACACTGGCTGT-39 primers. For human SUZ12 detection, the
forward 59-TGAAACTCTGGAATCTCCATGTC-39 and the reverse 59TGCATGCTGACTAGATGAAGC-39 primers were used. For lactase-phlorizine
hydrolase detection we used the forward 59-GCCACAGGCTTTTCAGAGAG-39
and the reverse 59-GAACTGCACCTCCTCCTGTC-39 primers, intestinal fatty acid
binding protein (FABP2) was detected using the forward 59-AACTGAACTCAGGGGGACCT-39 and the reverse 59-CCTTTTGGCTTCTACTCCTTCA-39
primers, intestinal alkaline phosphatase (ALPI) was detected using the forward 59TCCCCTCAGGTTGTTCTCTG-39 and the reverse 59-CCAAGTCCTTGGGTCTACCA-39 primers, CD44 was detected using the forward 59-TAAGGACACCCCAAATTCCA-39 and the reverse 59-CCACATTCTGCAGGTTCCTT-39 primers,
and EpCAM was detected using the forward 59-CACAACGCGTTATCAACTGG39 and the reverse 59-CCAGCTTTTAGACCCTGCAT-39 primers. Due to technical
limitations that prevented a PCR efficiency of between 90% and 110%, LPH PCR
amplification products were migrated on an agarose gel and the optical density of the
bands were quantified using Scion Image software (NIH).
BrdU incorporation assays
BrdU incorporation experiments in cells were performed in accordance with the In
Situ Cell Proliferation Kit FLUOSH protocol (Roche) as described previously
(Benoit et al., 2009).
Statistical analysis
Data were expressed as means 6 s.e.m. Each experiment was repeated at least
three times and representative results were shown. All values were subjected to the
two-tailed Student’s t-test. P,0.05 was considered significant.
Acknowledgements
The authors thank Cory Glowinski (Bitplane) for assistance with the
generation of the digital images created using Imaris software, Timo
J. Nevalainen (Turku University, Finland) for the gift of the antiphospholipase A2, Gérald Bernatchez and Aline Simoneau for
technical help in the preparation of the colon cancer specimens and
qRT-PCR, respectively, and Elizabeth Herring and Alison Urvalek
for reviewing the manuscript. We also acknowledge the excellent
collaboration of Québec Transplant in obtaining fresh specimens of
normal adult intestine and of the Cooperative Human Tissue
Network, funded by the National Cancer Institute, in obtaining
human colon cancer samples. Thanks to Pascale Roy for assistance
with preparation for the immunofluorescence analysis and to
Nathalie Carrier for statistical analysis.
Funding
This work was supported by the Canadian Institutes of Health [grant
number MOP-123415 to J.F.B.]; and the National Institutes of Health
[grant number RO1 CA0043796-22 to L.J.G.]. J.F.B. is the recipient
of the Canada Research Chair in Intestinal Physiopathology. J.F.B.
and J.C. are member of the Fonds de la Recherche en Santé du
Québec-funded Centre de Recherche Clinique Étienne Le Bel of the
Centre Hospitalier Universitaire de Sherbrooke. Deposited in PMC
for release after 12 months.
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