Báo cáo khoa học: The Promyelocytic Leukemia Zinc Finger (PLZF ) gene is a novel transcriptional target of the CCAAT-DisplacementProtein (CUX1) repressor ppt

The Promyelocytic Leukemia Zinc Finger (PLZF ) gene is a
novel transcriptional target of the CCAAT-Displacement-
Protein (CUX1) repressor
Isabelle Fre
´
chette, Mathieu Darsigny, Karine Brochu-Gaudreau, Christine Jones and Franc¸ois
Boudreau
De
´
partement d’Anatomie et Biologie Cellulaire, Faculte
´
de Me
´
decine et des Sciences de la Sante
´
, Universite
´
de Sherbrooke, Que
´
bec,
Canada
Introduction
The establishment of the functional adult intestine is
the result of several steps, beginning with gross mor-
phogenesis of the digestive tract and followed by cyto-
differentiation of the epithelium and the induction of
intestine-specific genes. An exchange of signals between
the endoderm and mesoderm leads to successive transi-
tions that result in the specialization of the intestinal
tube into the small intestine, cecum and colon along
the proximal–caudal axis [1]. The coordination of gene

transcription is crucial in the orchestration of position-
dependent intestinal epithelial development, differentia-
tion and homeostasis. An interesting hypothesis is that
intestinal disorders, such as colorectal cancer (CRC),
could evolve from the subtle accumulation of genetic
alterations in the transcriptome that leads to a diver-
gence of these cells from their original identities [2].
Colorectal adenomas and carcinomas are often associ-
ated with phenotypic and genetic characteristics of the
Keywords
colorectal cancer; CUX1; intestinal
epithelium; PLZF; transcriptional repression
Correspondence
F. Boudreau, De
´
partement d’Anatomie et
Biologie Cellulaire, Faculte
´
de Me
´
decine
et des Sciences de la Sante
´
, 3001 12e ave
Nord, Sherbrooke, Que
´
bec J1H 5N4,
Canada
Fax: 001 819 564 5320
Tel: 001 819 820 6876

E-mail:
(Received 2 June 2010, revised 11 August
2010, accepted 16 August 2010)
doi:10.1111/j.1742-4658.2010.07813.x
The CCAAT-Displacement-Protein (CUX1) can transcriptionally repress
sucrase–isomaltase gene expression, a specific product of enterocytes that
becomes re-expressed during human colonic polyposis. Little is known of
the gene repertoire that is directly affected by CUX1 in the intestinal
epithelial context. This article identifies the Promyelocytic Leukemia Zinc
Finger (PLZF) gene as a transcriptional target for the CUX1 repressor.
CUX1 interacts in vivo with multiple DNA-binding sites in the 5¢-UTR and
promoter of the PLZF gene in colorectal cancer cells, a region that is func-
tionally targeted by CUX1 in cotransfection assays. PLZF was found to be
induced in colorectal cancer cell lines, correlating with a low detectable
level of CUX1, a pattern that was reversed in normal human colonocytes.
Reduction of p200CUX1 expression by RNAi in the Caco-2 ⁄ 15 cell line
increased PLZF gene transcript expression. Because of the implication of
Plzf in the regulation of stem cell maintenance, as well as Wnt and Ras sig-
naling, in other systems, our observations suggest that the novel genetic
relationship between CUX1 and PLZF could be of relevance to human
diseases, such as leukemia, and open up a new field of investigation for the
implication of these regulators during intestinal polyposis and cancer.
Abbreviations
BTB ⁄ POZ, bric-a
`
-brac, tramtrack, brad complex ⁄ poxvirus zinc finger; ChIP, chromatin immunoprecipitation; CR, cut repeat; CRC, colorectal
cancer; CRESIP, colonic repression of the sucrase–isomaltase gene; CUX1, CCAAT-Displacement-Protein; EMSA, electrophoretic mobility
shift assay; HD, homeodomain; PLZF, Promyelocytic Leukemia Zinc Finger; RARa, retinoic acid receptor a; shRNA, short hairpin RNA;
SI, sucrase–isomaltase.
FEBS Journal 277 (2010) 4241–4253 ª 2010 The Authors Journal compilation ª 2010 FEBS 4241

normal adult small intestinal epithelium [3–6]. During
embryonic life, the colon passes through a transient
developmental stage that reproduces the small intesti-
nal phenotype with the expression of small intestinal-
specific genes, such as sucrase–isomaltase (SI) [7]. It
has been suggested that the mechanisms involved in
fetal colon development are recapitulated during colo-
nic neoplasia in adults. An example is the re-expression
of the SI gene in the majority of colonic adenomatous
polyps and adenocarcinomas [3,4,8]. Therefore, it is
clear that the identification of the molecular regulators
responsible for intestinal epithelial establishment,
determination and maturation is crucial to the
provision of a better understanding of the intestinal
homeostasis that is challenged during intestinal tumori-
genesis.
In the past, we have identified a novel regulatory
element for colonic repression of the sucrase–isomal-
tase gene (CRESIP) [9]. The CCAAT-Displacement-
Protein (CUX1) interacts with CRESIP and represses
SI gene transcription based on transfection and Cux1
deletion studies. These findings suggest that CUX1
could represent an important regulator of colonic epi-
thelium homeostasis, but there is limited knowledge
about the nature of the molecular pathways susceptible
to direct influence by its transcriptional activity.
The Cux1 gene encodes for a transcriptional repres-
sor that belongs to the homeodomain protein family
and contains four evolutionarily conserved DNA-bind-
ing domains, three cut repeats (CR1, CR2 and CR3)

and a cut homeodomain (HD) [10]. CUX1 contains
two repressive regions (R1 and R2) at the carboxy ter-
minus that account for its repressive activity [11]. Sev-
eral Cux1 isoforms are produced from the proteolytic
processing of p200Cux1 (e.g. p110Cux1) as well as
alternative splicing (e.g. p75Cux1) [12]. Many in vitro
gene targets of CUX1 are implicated in the control of
the cell cycle [10,13,14]. Genetic deletion of Cux1 in
mice has confirmed its crucial role during development.
Cux1 hypomorphic mice are smaller than wild-type
mice [15,16] and display organ-specific phenotypes,
including growth retardation, delayed differentiation of
lung epithelia, curly whiskers and altered hair follicle
morphogenesis [17]. These mutant mice also show a
deficiency in the hematopoiesis system with decreases
in lymphocyte T- and B-cell populations, as well as an
excessive production of myeloid cells in the liver, bone
marrow and peripheral blood, a feature that is com-
monly observed during the progression of human
myeloid leukemia [16].
In the search for novel transcriptional targets of
CUX1, we investigated the colonic changes in the gene
repertoire of Cux1 mutant mice by gene expression
analysis. This approach identified the Promyelocytic
Leukemia Zinc Finger (PLZF) gene as a putative gene
target of CUX1. PLZF was originally identified as a
t(11;17) reciprocal chromosomal translocation with the
retinoic acid receptor a (RARa) gene in individuals
with acute promyelocytic leukemia [18]. PLZF is also
expressed in immature hematopoietic cells and is

downregulated after differentiation [19]. PLZF is a
transcription factor composed of a repression domain
BTB ⁄ POZ (bric-a
`
-brac, tramtrack, brad complex ⁄ pox-
virus zinc finger) and nine Kru
¨
ppel zinc fingers [20].
This repression domain allows PLZF to recruit core-
pressors such as SMRT (silencing mediator of retinoid
and thyroid hormone receptor), N-CoR (nuclear recep-
tor corepressor), mSin3a (murine Swi-independent 3a)
and HDAC (histone deacetylase) to exert its transcrip-
tional action [21]. Recent genetic experimental
approaches have shown a function for Plzf in the
maintenance of spermatogonial stem cells [22,23], as
well as axial skeletal patterning with alterations in the
expression of Hox genes [24]. Other genetic evidence
has identified the Caenorhabditis elegans PLZF ortho-
log eor-1 as a positive regulator of the Ras and Wnt
pathways [25]. Interestingly, the deregulation of these
pathways is a common step in the molecular cascade
that promotes CRC [26].
In this article, we provide evidence that PLZF is a
direct target gene of CUX1. Expression analyses show
a reciprocal expression pattern of CUX1 and PLZF
among CRC cell lines and normal isolated colono-
cytes. Because of the well-documented role of PLZF in
acute promyelocytic leukemia [27], as well as its impli-
cation in the regulation of several pathways well docu-

mented to be upregulated in CRC, we suggest that the
modulation of PLZF via CUX1 action may be func-
tionally relevant to cancer pathology.
Results and Discussion
CUX1 physically interacts with the PLZF gene
both in vitro and in vivo
CUX1 is a crucial molecular contributor to SI gene
transcriptional repression in colonocytes during murine
post-natal development [9]. Although SI gene re-expres-
sion is commonly observed in human colonic polyposis
[4,8,28], the transcriptional effect of CUX1 on cancer-
related genes has not been explored in the intestinal
context. We thus decided to perform a gene expression
analysis with colon isolated from newborn Cux1 mutant
and control mice in order to identify novel CUX1 gene
targets. A nonstatistical analysis of gene transcript vari-
ations among pooled mutant versus pooled control
CUX1 represses PLZF transcriptional activity I. Fre
´
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4242 FEBS Journal 277 (2010) 4241–4253 ª 2010 The Authors Journal compilation ª 2010 FEBS
samples predicted that the Plzf gene would be one of
the most induced genes in the Cux1 mutant pooled sam-
ples (Table S1). A quantitative RT-PCR analysis
revealed that Plzf mRNA expression was induced in
some, but not all, Cux1 mutant versus control colonic
samples. However, this observation was not significant
overall (data not illustrated). The variability of Plzf
induction of expression among different Cux1 mutant
individuals could be explained by putative compensa-

tory mechanisms, as well as the possibility that the
Cux1 mutation results in a hypomorphic allele because
of the production of an N-terminal, 160-kDa truncated
form of Cux1 in mutant mice, as already observed for
other CUX1 gene targets [9,16].
To further determine whether this prediction could
be of importance in human genetic regulation, we
promptly investigated the presence of potential interac-
tion sites for the transcription factor CUX1 in the
PLZF gene. The human sequence of the PLZF gene
has been characterized previously and the transcrip-
tional start position has been well identified [29].
A computer analysis of the human PLZF gene using
matinspector matrix software [30] predicted 15 poten-
tial binding sites for the transcription factor CUX1
within 6.6 kb of the 5¢-UTR and flanking regions of
the gene (Fig. 1). Noteworthy, the 5¢-UTR genomic
region contained a high-density cluster of 12 predicted
binding sites for CUX1 (Fig. 1). We then evaluated the
capacity of CUX1 to interact with different potential
binding sites of the genomic 5¢-UTR and promoter
regions of the PLZF gene. Electrophoretic mobility
shift assay (EMSA) was performed using double-
stranded
32
P-labelled probes that corresponded to the
15 predicted CUX1 interacting sites included within
the 5¢-UTR and the promoter region of PLZF.
Nuclear extracts isolated from HEK293T cells, trans-
fected or not with an expression vector for CUX1,

were used for the assays. Overexpression of CUX1
protein in HEK293T cells led to the formation of sev-
eral retarded complexes that harbored similar patterns
with the binding sites 1, 2, 4, 5, 6, 7, 10, 12, 13 and 15,
as opposed to HEK293T crude nuclear extracts
(Fig. 2A). The pattern of retarded complexes between
HEK293T crude and CUX1-enriched nuclear extracts
was similar for sites 8, 9, 11 and 14, suggesting that
these sites could be of better affinity for CUX1 recog-
nition in vitro (Fig. 2A). The binding pattern for site 3
was somewhat different from that of the other sites,
suggesting that the presence of other putative elements
could be interfering with CUX1 interaction with this
region (Fig. 2A). In order to better characterize the
nature of the different patterns for the CUX1-retarded
complexes, some sites representative for these different
patterns were analyzed further. The introduction of
mutations in the CUX1 consensus binding site resulted
in a loss of CUX1-formed complexes, as observed for
sites 8, 10, 12, 13, 14 and 15 and, to a lesser extent,
for site 3 (Fig. 2B). The addition of an excess amount
of nonlabelled oligonucleotide for these specific sites
competed for the formation of CUX1-related complex,
whereas corresponding mutated oligonucleotides vali-
dated for the absence of CUX1 interaction did not
compete, as illustrated for sites 8, 10, 12, 13, 14 and 15
in Fig. 2C. The addition of affinity-purified polyclonal
CUX1 antibodies to the binding reaction for each of
these positive interacting sites resulted in a supershift
Fig. 1. Identification of 15 putative CUX1

binding sites in 6.6 kb of the PLZF gene. (A)
Schematic representation of the PLZF gene
with its predicted binding sites for the tran-
scription factor CUX1. (B) Nucleotide
sequence of the 15 potential binding sites
for CUX1 in the PLZF gene. Base pairs in
capital letters denote the CUX1 core
sequence and italics show high conservation
content [30].
I. Fre
´
chette et al. CUX1 represses PLZF transcriptional activity
FEBS Journal 277 (2010) 4241–4253 ª 2010 The Authors Journal compilation ª 2010 FEBS 4243
of the four major specific CUX1-containing complexes
relative to IgG control lanes, as observed for sites 5, 7,
8, 10 and 14 (Fig. 2D). These observations suggested
that these sites can be recognized by multiple CUX1
cleaved forms specifically produced from the peG-
FP ⁄ CUX1 expression vector, as observed in transfect-
ed HEK293T cells (Fig. 2A, right panel). However, the
exact contribution of these cleaved forms could not be
distinguished in these conditions.
To further verify whether the in vitro interaction of
CUX1 with the PLZF gene could be reflected in the cel-
lular context in vivo, chromatin immunoprecipitation
A
B
CD
Fig. 2. The eGFP ⁄ CUX1 fusion protein interacts with different 5¢-UTR and promoter binding sites of the PLZF gene in vitro. (A) Nuclear pro-
tein extracts from transfected HEK293T cells (empty vector, lanes 2; eGFP ⁄ CUX1, lanes 3) were used for EMSA with labelled oligonucleo-

tides. A condition with no protein included within the binding reaction was also tested for each probe (lanes 1). The retarded complexes are
indicated by arrows. The right panel is a Western blot analysis representing the different isoforms produced from p200CUX1 in HEK293T
cells transfected with the peGFP-CUX1 expression vector. (B) EMSA was carried out as described in (A), except that the consensus site for
CUX1 for each oligonucleotide was mutated. (C) Competition of site 3, 8, 10, 12, 13, 14 and 15 complexes were obtained with the addition
of a 10-fold molar excess of nonlabelled wild-type sites or CUX1 mutated site oligonucleotides. (D) Supershift experiments of the interacting
sites 3, 5, 7, 8, 10 and 14 were performed by including a specific CUX1 antibody or rabbit IgG in the binding reactions.
CUX1 represses PLZF transcriptional activity I. Fre
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4244 FEBS Journal 277 (2010) 4241–4253 ª 2010 The Authors Journal compilation ª 2010 FEBS
(ChIP) experiments were performed. The human Caco-
2 ⁄ 15 cell line was used as the p200CUX1 protein was
shown to be expressed in these cultured cells [9]. Sub-
confluent Caco-2 ⁄ 15 cell chromatin was cross-linked
and immunoprecipitated with the CUX1 antibody. PCR
amplifications of different PLZF regions were per-
formed on the immunoprecipitated purified chromatin
(Fig. 3B). CUX1 consistently interacted with a specific
region of the genomic 5¢-UTR that contained the
in vitro validated CUX1 binding sites 5–7 (Chip2,
Fig. 3A, B) and a specific region of the promoter that
contained the validated binding site 15 (Chip3, Fig. 3A,
B). Because of the proximity of sites 13, 14 and 15, it
remains probable that these additional sites also contrib-
ute to the enrichment of the promoter region as revealed
by this assay. As a negative control, a portion of the
PLZF gene for which CUX1 was not predicted to inter-
act (Chip1) was not immunoprecipitated with the CUX1
antibody as determined by PCR (Fig. 3B, C). Overall,
these data confirmed that CUX1 can interact strongly

with the genomic 5¢-UTR and promoter regions of the
PLZF gene both in vitro and in vivo.
CUX1 represses the transcriptional activity of the
genomic 5¢-UTR and promoter regions of the
human PLZF gene
Because the genomic 5¢-UTR may be of importance in
gene transcriptional regulation [31–34], we decided to
test whether this region was functionally responsive to
CUX1 transcriptional action. A 3-kb 5¢-UTR that con-
tained eight interacting CUX1 binding sites was PCR
amplified and subcloned into a luciferase reporter vec-
tor. To measure the transcriptional effect of CUX1 on
the 5¢-UTR genomic region, murine (CMV-Cux1) and
human (eGFP ⁄ CUX1) expression vectors were
cotransfected with the pGL3basic ⁄ PLZF 5¢-UTR
reporter construct in HEK293T cells. The addition of
CUX1 expression vectors to the cotransfection assay
resulted in a two-fold reduction in 5¢-UTR ⁄ luciferase
activity (Fig. 4A). A western analysis confirmed that
both the murine Cux1 and human CUX1 proteins
were synthesized when cotransfected in HEK293T
cells, with the production of multiple isoforms of the
proteins, as reported previously (Fig. 4B) [35]. The
murine Cux1 protein was shorter than the human
CUX1 protein because of the use of an N-terminally
truncated mouse Cux1 construct [36]. The effect of
CUX1 was next verified on a 776-bp portion of the
PLZF gene promoter that contained the initiation start
site for transcription (Fig. 4C). The addition of
increasing amounts of CUX1 expression vector to the

cotransfection assay resulted in a two-fold maximal
reduction of the PLZF promoter activity in HEK293T
cells (Fig. 4D). Individual abrogation of CUX1 inter-
acting sites 13 and 15 of the PLZF promoter did not
influence significantly the repressive activity of CUX1,
in contrast with site 14, for which deletion resulted in
a reduction of CUX1-dependent repression of the
PLZF promoter (Fig. 4D, E). Simultaneous mutations
of CUX1 interacting sites 13, 14 and 15 completely
abolished the repressive effect of CUX1 on the PLZF
promoter (Fig. 4D, E). Taken together, these data
demonstrate that CUX1 has the ability to occupy and
repress PLZF gene transcriptional activity. The loca-
tion of several CUX1 elements within the 5¢-UTR was
surprising because this region typically contains post-
transcriptional elements controlling RNA stability or
translation. Only a few transcriptional response ele-
ments have been identified in 5¢-UTR genomic regions
[31–34]. Interestingly, a recent report identified a single
RHOX5 repressor element within the 5¢-UTR region
of the netrin-1 receptor gene Unc5c that accounted for
repression of the gene within Sertoli cells [34]. As we
identified multiple CUX1 elements within the 5¢-UTR
of the PLZF gene, we speculate that CUX1 could
interfere with PLZF
gene transcription by forming a
physical barrier in the PLZF genomic region that cor-
responds to the 5¢-UTR, causing a limitation of PLZF
transcriptional initiation and gene processing via the
transcriptional basal machinery.

PLZF exhibits a reciprocal expression pattern to
CUX1 in human colonocytes and CRC cell lines
The expression of CUX1 is altered in breast cancer
[37] and uterine leiomyoma [38,39]. The profile of
CUX1 expression in colon cancer has not been
reported. We thus aimed to compare the level of
CUX1 protein expression among human CRC cell
lines in comparison with other cell lines of cancer ori-
gin. p200CUX1 was weaker in CRC cell lines
(Colo205, T84, Caco-2 ⁄ 15 and DLD-1 cells) when
compared with colonocytes isolated from human
fetuses that were used as normal controls (Fig. 5A).
This reduction was not observed in the liver cancer cell
line HepG2, the pancreatic cancer cell line MIA PaCa-
2 and the transformed kidney cell line HEK293T
(Fig. 5A). This indicates that the p200CUX1 protein
reduction observed in the CRC context is most likely
not a general phenomenon of cancer. We then asked
whether the profile of PLZF expression was interre-
lated with CUX1 expression in these cell lines. PLZF
protein was detected in every CRC cell line, but was
not detectable in isolated normal colonocytes
(Fig. 5A), a pattern that was confirmed at the gene
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FEBS Journal 277 (2010) 4241–4253 ª 2010 The Authors Journal compilation ª 2010 FEBS 4245
transcript level (Fig. 5B). However, we cannot exclude
the possibility that a minor subpopulation of epithelial
crypt cells, for example stem cells, could support

PLZF expression. Although PLZF expression was
weakly detected in HepG2 and MIA PaCa-2 cancer
cell lines that harbored high levels of p200CUX1
A
B
C
Fig. 3. CUX1 interacts with the PLZF gene in vivo. (A) Consensus CUX1 binding sites of the PLZF sequence are indicated in brackets. Bases
in capital letters denote the CUX1 core sequence and italics show high conservation content in comparison with the consensus sequence.
Primer sets used for the ChIP experiments are indicated by bold arrows (Chip2 ⁄ 3up and Chip2 ⁄ 3dw). For clarity, primer sets Chip1up and
Chip1dw are not represented in the figure. Primer Chip1up started at position +3165 bp on the PLZF sequence and primer Chip1dw at posi-
tion +3304 bp. (B) ChIP assays were performed with subconfluent Caco-2 ⁄ 15 cell chromatin. Chromatin was immunoprecipitated with the
CUX1 antibody or with rabbit IgG as a negative control. Purified immunoprecipitated chromatin was subjected to PCR amplification of three
independent regions of the PLZF gene (1–3); 10% of the chromatin extract (Input) was also amplified by PCR to determine the amount of
DNA prior to immunoprecipitation. A representative result of three to four independent experiments is illustrated. (C) Chromatin immunopre-
cipitated in (B) was analysed by quantitative real-time PCR with oligonucleotides amplifying three independent regions of the PLZF gene
(1–3). Results are expressed as the fold increase over normal rabbit IgG normalized to input.
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4246 FEBS Journal 277 (2010) 4241–4253 ª 2010 The Authors Journal compilation ª 2010 FEBS
AB
C
E
D
Fig. 4. CUX1 functionally influences the PLZF 5¢-UTR and promoter genomic regions in transient reporter assays. (A) HEK293T cells were
transiently cotransfected with 0 or 0.4 lg of the CMV-Cux1 (murine) or peGFP-CUX1 (human) expression vector and 0.2 lg of the pGL3ba-
sic ⁄ PLZF 5¢-UTR luciferase reporter construct. The pcDNA3.1 plasmid was used as an empty control vector to calibrate for the addition of
the expression vectors. Cells were harvested after transfection and analyzed for luciferase activity. Data were normalized with Renilla values.
Results obtained in triplicate were reported as a percentage of the controls (means ± SD) and are representative of three independent
experiments. (B) A western blot with CUX1 and actin antibodies was performed on pooled lysates used for the luciferase detection in (A).

The molecular masses for each Cux1 and CUX1 protein isoform are indicated. (C) Schematic representation of the wild-type, single, double
and triple mutants of the 776-bp region of the PLZF promoter. The bold crosses represent the site(s) mutated in each construct. (D)
HEK293T cells were transfected with increasing amounts of the peGFP-CUX1 (0, 5 and 12 ng) expression vector and 0.2 lg of the different
pGL3basic ⁄ PLZF 776-bp promoter luciferase constructs illustrated in (C). The pcDNA3.1 plasmid was used as an empty control vector to
calibrate for the addition of the expression vector. Cells were harvested after transfection and analyzed for luciferase activity. Data were
normalized with Renilla values. Results obtained in triplicate were reported as a percentage of the controls (means ± SD) and are representa-
tive of three independent experiments. (E) The percentage of repression activity of CUX1 on both wild-type and mutant pGL3basic ⁄ PLZF
776-bp promoter luciferase constructs was monitored as described in (A). ***P < 0.001.
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expression, this correlation was not maintained in the
HEK293T cell line (Fig. 5A). Thus, PLZF-positive
expression correlated well with the relatively low level
of CUX1 repressor detection in colonic cancer cells,
but other regulatory mechanisms are likely to be
involved in the context of transformed human cells.
We then tested whether PLZF expression could be
responsive to CUX1 variations in colon cancer cells.
The Caco-2 ⁄ 15 cell line was used to generate stable
populations of cells expressing a short hairpin RNA
(shRNA) against CUX1. The efficiency of CUX1
mRNA reduction was more than 70% (P < 0.0001),
as assessed by quantitative RT-PCR (Fig. 5C),
and correlated with an efficient loss of CUX1 pro-
tein abundance, as determined by western blotting
(Fig. 5D). Coincidently, PLZF mRNA expression was
induced more than 2.8-fold in shCUX1 Caco-2 ⁄ 15 cell
populations as opposed to shCUX1 mutated controls

(P < 0.05) (Fig. 5E).
CUX1 and PLZF as a novel molecular link in
human diseases
Human CUX1 is located on chromosome 7q22, a
region that is often deleted in 7q-related acute myeloid
leukemia and myeloid dysplasia [40–44]. These studies
suggest that CUX1 is a tumor suppressor and that its
loss is a significant event in the generation or progres-
sion (or both) of myeloid disorders. The demonstration
that PLZF is a direct target for CUX1 repressive
action represents an interesting avenue for the specific
investigation of this molecular interaction during leu-
kemia progression. However, the PLZF ortholog was
demonstrated to promote the expression of Ras and
AB
C
E
D
Fig. 5. CUX1 and PLZF expression in human colon cancer cell lines. (A) Western blot analysis was performed with CUX1, PLZF and actin
antibodies on total protein extracts from various human cancer cell lines [colorectal cancer cell lines, liver cancer cell line (HepG2), pancreas
cancer cell line (MIA PaCa-2) and the transformed kidney cell line (HEK293T)] and from epithelial cells isolated from human fetal colon. (B)
Total RNA was isolated from Colo205, Caco-2 ⁄ 15, DLD-1 and normal colonocytes derived from human fetus and subjected to quantitative
real-time PCR. PLZF expression was quantified and normalized using Tata-box binding protein (TBP) reference gene. (C) Total RNA was iso-
lated from stable Caco-2 ⁄ 15 cell populations that contained an integrated shRNA against the CUX1 mRNA or a mutated shRNA as a control.
Real-time PCR was performed and CUX1 expression was normalized using human b2-microglobulin (b2-mic) reference gene. (D) Western
blot experiment using CUX1 and actin antibodies was performed to monitor CUX1 expression in the Caco-2 ⁄ 15 cell populations described in
(C). (E) Total RNA isolated as described in (C) was used to monitor PLZF mRNA expression. *P < 0.05.
CUX1 represses PLZF transcriptional activity I. Fre
´
chette et al.

4248 FEBS Journal 277 (2010) 4241–4253 ª 2010 The Authors Journal compilation ª 2010 FEBS
Wnt responsive genes in Caenorhabditis elegans [25].
Sustained activation of Wnt and Ras signaling is very
frequent in human CRC [26,45] and results in intesti-
nal polyposis and adenocarcinoma in transgenic mice
[46,47]. Taken together, our findings demonstrate that
CUX1 is a negative regulator of the PLZF gene.
Future investigations into the molecular status of
CUX1 in human CRC, as well as the comprehensive
role of PLZF in intestinal homeostasis, represent
future challenges that will enable us to expand our
knowledge about the mechanisms related to intestinal
disease.
Experimental procedures
Animals
The generation of Cux1 mutant mice has been described
elsewhere [16]. C57BL ⁄ 6J mice heterozygous for the tar-
geted allele were subsequently bred with normal CD1 mice.
Homozygous Cux1 mice were identified by PCR, and their
identity was confirmed on the basis of their typically small
size and curly hair. Mice were treated in accordance with a
protocol approved by the Institutional Animal Research
Review Committee.
Isolation of epithelial cells from mouse and
human intestine
Intestinal epithelial cells were isolated from the intestinal
epithelium of mouse youngsters and human fetuses as
described previously [48]. Human intestine from fetuses
ranging from 17 to 20 weeks of age (post-fertilization) were
obtained after legal abortion. The project was in accor-

dance with a protocol approved by the Institutional
Human Research Review Committee for the use of human
material. Briefly, the intestine was separated in sections
and the colon was opened longitudinally and rinsed with
cold NaCl ⁄ P
i
. The colon sections were further cut into
5-mm pieces and incubated in 5 mL of cold MatriSperse
(Becton-Dickinson Canada, Oakville, ON, Canada) in
15-mL tubes at 4 °C for 18–24 h. The epithelial layer was
dissociated by gentle manual shaking. The epithelial sus-
pension was collected, centrifuged, washed with cold
NaCl ⁄ P
i
and processed for RNA and protein isolation as
described previously [48].
Cell culture and shRNA knockdown
The HepG2 human liver carcinoma cell line, MIA PaCa-2
human pancreatic carcinoma cell line and the human ade-
nocarcinoma colorectal cell lines DLD1, T84 and Colo205
were all obtained from the American Type Culture Collec-
tion (ATCC, Rockville, MD, USA). The Caco-2 ⁄ 15 cell
line [49] was kindly provided by Dr J. F. Beaulieu. DLD1
cells were cultured in RPMI1640 medium and T84 cells in
Ham’s:DMEM. Caco-2 ⁄ 15, Colo205, HepG2, MIA PaCa-2
and HEK293T cells were cultured in DMEM. All culture
media were supplemented with 10% fetal bovine serum
(ICN Biomedicals, Aurora, OH, USA), 2 mm glutamine
(Gibco, Burlington, ON, USA), 0.01 m Hepes (Gibco) and
100 lgÆmL

)1
penicillin ⁄ streptomycin (Gibco). Fetal bovine
serum was heat inactivated as recommended for DLD1 cell
culture (ATCC). The cell lines were maintained at 37 °Cin
a humidified atmosphere with 5% CO
2
. An shRNA cloned
in pSuper.retro.puro (Oligoengine, Seattle, WA, USA) that
targeted a conserved sequence in rat, mouse and human
CUX1 was kindly provided by Dr Julian Downward [50].
Two bases (in capitals) were further mutated (5¢-aagaaga
acaGAccagaggattt-3¢) to be used as a control. The Caco-
2 ⁄ 15 cell line was infected with the retroviral shRNA con-
structs and selected with 5 lgÆmL
)1
of puromycine for
1 week (Sigma Chemical Co., St Louis, MO, USA).
Microarray analysis
Total RNA was isolated from the colon of newborn Cux1
hypomorphic mutant and control mice as described previ-
ously [9]. Briefly, 5 lg of total RNA from three indepen-
dent individuals of both normal and mutant mice were
pooled and submitted to the Penn Microarray Facility
(University of Pennsylvania, Philadelphia, PA, USA) for
target preparation and hybridization to murine MG_U74Av2
GeneChips (Affymetrix, Santa Clara, CA, USA), followed
by microarray analysis as described elsewhere [51]. Micro-
array Analysis Suite 5.0 (MAS, Affymetrix) was used to
quantify microarray signals [51].
EMSA

EMSAs were performed essentially as described previously
[9,52] with some modifications. The reactions were per-
formed in a volume of 24 lL of binding buffer D (10 mm
Hepes, pH 7.9, 10% glycerol, 0.1 mm EDTA and 0.25 mm
phenylmethanesulfonyl fluoride) containing 5 lg of nuclear
protein extracts from HEK293T cells, transfected or not with
the peGFP-CUX1 expression vector, 50 mm KCl, 50 ng of
poly(dI–dC) and 25 000 cpm of
32
P-labeled DNA probes for
10 min. For the supershift analysis, 200 ng of CUX1
M-222X antibody or rabbit IgG (Santa Cruz Biotechnology,
Santa Cruz, CA, USA) were added and the binding reactions
were pursued for 10 min at room temperature. Competition
assays were performed with the addition of an excess
(10-fold) of nonlabelled double-stranded oligonucleotides.
Retarded complexes were then separated on a 5% polyacryl-
amide gel at 4 °C for 4 h, dried for 1 h at 80 °C and exposed
overnight on a Molecular Imager FX screen (Biorad, Missis-
sauga, ON, Canada). The running buffer used was a Tris–
glycine 0.5· buffer (0.2 m glycine, 0.025 m Tris and 1 mm
I. Fre
´
chette et al. CUX1 represses PLZF transcriptional activity
FEBS Journal 277 (2010) 4241–4253 ª 2010 The Authors Journal compilation ª 2010 FEBS 4249
EDTA). The DNA probes consisted of double-stranded oli-
gonucleotides of 15 potential CUX1 binding sites within the
5¢-UTR and 776-bp promoter regions of the human PLZF
gene (Fig. 1) (matinspector software tool; -
omatix.de) [30]. Mutated oligonucleotides for CUX1 inter-

acting sites were designed as follows (upper strand, mutation
in bold): site 3, 5¢-gctgtaggggactcgatttaactcgagtctctctcca-3¢;
site 8, 5¢-gcgccaagcttctcgtcttttggagcttccctccct-3¢; site 10, 5¢-tcg
cttcgacatcactgccccgcggacct-3¢; site 12, 5¢-tgggccctcgagtccttat-
caaac-3¢; site 13, 5¢-ggccagcttcgctattcctctgtc-3¢; site 14, 5¢-ac-
cctcctgttgtccttcgtgagctctgaaag-3¢; site 15, 5¢-tctgatgttttcga
gtcctacagt-3¢.
Genomic DNA amplification, reporter plasmid
constructs and mutagenesis
The 3-kb 5¢-UTR region of the human PLZF gene was
amplified by PCR from purified genomic DNA isolated from
the human normal intestinal epithelial cell line HIEC [53]
and Herculase DNA polymerase (Stratagene Cloning Sys-
tems, La Jolla, CA, USA). The 776-bp promoter region was
amplified with iProof DNA polymerase (Biorad) from the
vector pGL3basic already containing a 2-kb region of the
human PLZF gene. The primer sequences used for the 3-kb
5¢-UTR were 3¢HPLZF (5¢-gaggggaagaagcaaaagaga-3¢) and
(+)2878 HPLZF (5¢-gatccggaggctttgtacc-3¢). The primer
sequences for the 776-bp region were pPLZF742SmaIA
(5¢-tcactacccgggaagcccttgcttccttcatc-3¢) and pPLZF742SmaIB
(5¢-agtgatcccgggagataaagcagcagcagctg-3¢). The 3-kb 5¢-UTR ⁄
PLZF amplified fragment was subcloned into the pBluescript
KS(–) vector (EcoRV). The integrity of the subcloned PCR
products was confirmed by sequence analysis. The 3-kb
5¢-UTR ⁄ PLZF fragment was then released from pBluescript
KS(–) with BamHI and KpnI (Roche Diagnostics, Laval, QC,
Canada) and subcloned into the KpnIandBglII restriction
sites of the pGL3basic luciferase reporter plasmid. The
776-bp PLZF promoter fragment was released with SmaI

(Roche Diagnostics) restriction enzyme and subcloned into
the SmaI restriction site of pGL3basic. Mutagenesis of the
CUX1 sites 13, 14 and 15 of the 776-bp PLZF promoter was
performed by overlap extension with mutated oligonucleo-
tides. The integrity of the mutated promoter was confirmed in
each case by sequence analysis.
Transient transfections and luciferase assays
HEK293T cells were seeded in 24-well plates for 24 h.
Transfections were performed with Lipofectamine 2000
(Invitrogen, Burlington, ON, Canada) according to the
manufacturer’s recommendations. Cells at 90% confluence
were cotransfected with 200 ng of luciferase reporter con-
struct (pGL3basic ⁄ PLZF 3-kb 5¢-UTR, pGL3basic ⁄ PLZF
776-bp or pGL3basic ⁄ PLZF 776-bp mut13, pGL3basic ⁄
PLZF 776-bp mut14, pGL3basic ⁄ PLZF 776-bp mut15,
pGL3basic ⁄ PLZF 776-bp mut13,15, pGL3basic ⁄ PLZF
776-bp mut13,14,15) and a fixed amount of either CMV-
Cux1 (murine) or peGFP-CUX1 (human) expression vec-
tors with a constant total DNA amount of 800 ng per
transfection in OptiMEM medium (Invitrogen); 5 ng of the
pRL SV40 Renilla luciferase vector (Promega, Madison,
WI, USA) was also included in each reaction as a control
for transfection efficiency. After 4 h, transfection medium
was replaced by DMEM containing 10% fetal bovine
serum. Luciferase and Renilla activities were determined
with the dual luciferase assay kit (Promega). Each experi-
ment was repeated at least three times in triplicate.
ChIP assays
ChIP assays were performed using the ChIP assay kit,
according to the manufacturer’s instructions (Upstate, Mil-

lipore, Billerica, MA, USA). Subconfluent (75%) Caco-
2 ⁄ 15 cells were cross-linked with 1% formaldehyde for
10 min at 37 °C and sonicated to obtain a DNA average
size of 500 bp in length. Chromatin was immunoprecipitat-
ed with a rabbit polyclonal antibody against CUX1 (Santa
Cruz Biotechnology). Rabbit IgG (Santa Cruz Biotechnol-
ogy) was also used as a negative control. Ten percent of
the lysate was kept to verify the amount of DNA used for
each immunoprecipitation. Immunoprecipitated DNA was
purified with a phenol–chloroform extraction and resus-
pended in 20 lL of ultrapure water before PCR amplifica-
tion with Chip1up primer (5¢-aagctccagagggtctgcac-3¢) and
Chip1dw primer (5¢-gaaaggcatcccgaacgcat-3¢); Chip2up
primer (5¢-aaatgtcttgaccagccgtc-3¢) and Chip2dw primer
(5¢-gaaacaaaggcctctcccag-3¢); Chip3up primer (5¢-gctttgcagt-
cagaatggtc-3¢) and Chip3dw primer (5¢-ctgagcactgactac-
gaaac-3¢). The Chip1up and Chip1dw oligonucleotides
amplified a 139-bp PLZF gene region that contained no
predicted CUX1 binding site; the Chip2up and Chip2dw
oligonucleotides amplified a 289-bp PLZF gene region
containing CUX1 binding sites 5, 6 and 7 (Chip2,
Fig. 3A); the Chip3up and Chip3dw oligonucleotides
amplified a 207-bp PLZF gene region containing the
CUX1 binding site 15 (Chip3, Fig. 3A). The program used
for amplification was a first cycle of 95 °C for 2 min (Hot-
Start), followed by 10 cycles of 95 °C for 30 s, 58.7 °C for
30 s and 72 °C for 2 min, followed by 25 cycles of 95 °C
for 30 s, 58.7 °C for 30 s and 72 °C for 2 min, with an
increasing elongation time of 10 s every cycle, and a final
incubation at 72 °C for 7 min. Amplified PCR products

were separated on a 1.5% agarose gel and visualized by
ethidium bromide staining. The immunoprecipitated DNA
was also amplified by real-time PCR using the QuantiTect
SYBR Green PCR kit (Qiagen, Valencia, CA, USA) with
the same primers as described above. Data are expressed
as the fold increase over background (negative control,
IgG) normalized to input, as proposed by SuperArray Bio-
sciences and adapted as described previously (http://
www.workingthebench.com).
CUX1 represses PLZF transcriptional activity I. Fre
´
chette et al.
4250 FEBS Journal 277 (2010) 4241–4253 ª 2010 The Authors Journal compilation ª 2010 FEBS
Western blot analysis
Cells were harvested in lysis buffer containing 50 mm
Tris ⁄ HCl, pH 7.5, 150 mm NaCl, 1% NP-40 and 0.5%
Na-deoxycolate; 40 lg of total protein extract were ana-
lyzed by 3–8% Tris acetate NuPAGE (Invitrogen) for the
detection of CUX1 and by 4–12% Bis-Tris NuPAGE for
the detection of PLZF. Gels were transferred to poly(vinyli-
dene difluoride) blotting membrane (Roche Diagnostics)
and western blotting was performed as described previously
[48]. The following antibodies were used: CUX1 affinity-
purified goat polyclonal antibody (C-20 sc-6327 from Santa
Cruz Biotechnology), CUX1 affinity-purified rabbit poly-
clonal antibody (M-222X sc-13024 from Santa Cruz Bio-
technology), PLZF affinity-purified goat polyclonal
antibody (EMD Biosciences, San Diego, CA, USA) and
actin affinity-purified goat polyclonal antibody (Santa Cruz
Biotechnology).

RNA analysis
Total RNA was isolated from human fetus, Colo205, Caco-
2 ⁄ 15 and DLD-1 cells, and from Caco-2 ⁄ 15 shcontrol and
shCUX1 cells, using the RNeasy mini kit (Qiagen) accord-
ing to the manufacturer’s instructions. Reverse transcrip-
tion reactions were carried out at 42 °C for 1 h in the
presence of 4 lg of RNA, 40 milliunits of poly-oligo(dT)
(Amersham Biosciences, Baie d’Urfe
´
, QC, Canada) and 40
units of reverse transcriptase (Roche Diagnostics, Laval,
QC, Canada). Quantitative PCR was performed on a Light-
Cycler PCR apparatus V2.0 (Roche Diagnostics) and rela-
tive mRNA expression was measured using lightcycler
software 4.0 according to the manufacturer’s protocol
(Roche Diagnostics). Tata-box binding protein (TBP) and
human b2-microglobulin (hb2mic) mRNA expression were
analysed as a reference. Double-stranded DNA amplifica-
tion during PCR was monitored using SYBR Green I
(QuantiTect SYBR Green PCR kit; Qiagen). The primers
hybridized at 59 °C were as follows: 5¢-hrmPLZF1923up,
5¢-agcacactcaagagccacaa-3¢; hrmPLZF2056dw, 5¢-tcaaag
ggcttctcacctgt-3¢; hrmTBP1009up, 5¢-ggggagctgtgatgtgaagt-3¢;
hrmTBP1139dw, 5¢-ggagaacaattctgggtttga-3¢; hB2MIC89up,
5¢-tcgcgctactctctctttctg-3¢; hB2MIC227dw, 5¢-tcaatgtcggatg
gatgaaa-3¢.
Acknowledgements
We thank Dr Peter G. Traber for the generous gift of
the microarray data analysis that was originally per-
formed at the Penn Microarray Facility (University of

Pennsylvania, Philadelphia, PA, USA). We also thank
Drs Angus M. Sinclair and Richard H. Scheuermann
(UT Southwestern Graduate School of Biomedical
Sciences, Dallas, TX, USA) for the gift of the eGFP ⁄
CUX1 retroviral construct, Dr Jean-Franc¸ ois Beaulieu
(Universite
´
de Sherbrooke, QC, Canada) for providing
the Caco-2 ⁄ 15 cell lines and Elizabeth Herring for crit-
ical reading of the manuscript. This work was sup-
ported by a grant from the Canadian Institutes of
Health Research (MOP-89770). FB is a senior scholar
from the ‘Fonds de la Recherche en Sante
´
du Que
´
bec’
(FRSQ) and member of the FRSQ-funded ‘Centre de
Recherche Clinique E
´
tienne Lebel’.
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Supporting information
The following supplementary material is available:
Table S1. Gene transcript variations among pooled
Cux1 mutant versus pooled control colon samples.
This supplementary material can be found in the
online version of this article.
Please note: As a service to our authors and readers,
this journal provides supporting information supplied
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should be addressed to the authors.
I. Fre
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chette et al. CUX1 represses PLZF transcriptional activity
FEBS Journal 277 (2010) 4241–4253 ª 2010 The Authors Journal compilation ª 2010 FEBS 4253