Modulation of PLAGL2 transactivation by positive cofactor 2 (PC2), a component of
the ARC/Mediator complex
Sara J. Wezensky, Tracey S. Hanks, Michelle J. Wilkison, Mary Cloud Ammons,
Daniel W. Siemsen, Katherine A. Gauss⁎
Department of Veterinary Molecular Biology, Montana State University, Bozeman, MT 59717, USA
a b s t r a c ta r t i c l ei n f o
Received 8 September 2009
Received in revised form 6 December 2009
Accepted 8 December 2009
Available online 16 December 2009
Received by A. Rynditch
Positive cofactor 2
The pleomorphic adenoma gene (PLAG) family of transcription factors regulates a wide range of
physiological processes, including cell proliferation, tissue-specific gene regulation, and embryonic
development, although little is known regarding the mechanisms that regulate PLAG protein activity. In
this study, a yeast two-hybrid screen identified PC2, a component of the Mediator complex, as a PLAGL2-
binding protein. We show that PC2 cooperates with PLAGL2 and PU.1 to enhance the activity of a known
PLAGL2 target promoter (NCF2). The PLAGL2-binding element in the NCF2 promoter consisted of the core
sequence of the bipartite PLAG1 consensus site, but lacked the G-cluster motif, and was recognized by
PLAGL2 zinc fingers 5 and 6. Promoter and PLAGL2 mutants showed that PLAGL2 and PU.1 were required to
bind to their respective sites in the promoter, and PC2 knockdown demonstrated that PC2 was essential for
enhanced promoter activity. Co-immunoprecipitation and promoter-reporter studies reveal that the effect of
PC2 on PLAGL2 target promoter activity was conferred via the C-terminus of PLAGL2, the region that is
required for PC2 binding and contains the PLAGL2 activation domain. Importantly, chromatin immunopre-
cipitation analysis and PC2 knockdown studies confirmed that endogenous PC2 protein associated with the
NCF2 promoter in MM1 cells in the region occupied by PLAGL2, and was required for PLAGL2 target promoter
activity in TNF-α-treated MM1 cells, respectively. Lastly, the expression of another known PLAGL2 target
gene, insulin-like growth factor II (IGF-II), was greatly diminished in the presence of PC2 siRNA. Together, the
data identify PC2 as a novel PLAGL2-binding protein and important mediator of PLAGL2 transactivation.
Published by Elsevier B.V.
PLAGL2 is a member of the recently identified PLAG family of
transcription factors. The additional members include PLAG1 and
PLAGL1. PLAG proteins are highly homologous in the N-terminal zinc
finger domain (PLAGL1 and PLAGL2 are 73% and 79% identical to
PLAG1, respectively) with the C-terminalregion being more divergent
(Kas et al., 1998). Although they have been implicated in a range of
important physiological processes, including cancer initiation and
progression, little is known regarding the mechanisms whereby PLAG
proteins regulate these processes (reviewed by Abdollahi, 2007; Van
et al., 2007). To date, few PLAG target genes or regulatory cofactors
have been reported.
PLAG1 and PLAGL2 are considered oncogenic, while PLAGL1
appears to function as a tumor suppressor. PLAG1 was the initial
member identified due to its involvement in the t(3:8)(p21;q12)
chromosomal translocation associated with about 25% of all human
pleomorphic adenomas of the salivary glands (Kas et al., 1997).
Upregulation of PLAG1 has also been identified as the primary genetic
PLAGL2 has been demonstrated in leukaemogenesis in retroviral
promoter insertion studies with Cbfb-MYH11 knock-in chimeric mice
(Castilla et al., 2004). In addition, PLAG1 and PLAGL2 show increased
expression in 20% of human acute myeloid leukemia (AML) samples,
with PLAGL2 expression preferentially induced in human AML
samples with inv(16) (Landrette et al., 2005). Interestingly, PLAGL2
Gene 452 (2010) 22–34
Abbreviations: PC2, positive cofactor 2; NCF2, neutrophil cytosolic factor 2; PLAG,
pleomorphic adenoma gene; PLAG1, pleomorphic adenoma gene 1; PLAGL1, pleomor-
phic adenoma gene-like 1; PLAGL2, pleomorphic adenoma gene-like 2; IGF-II, insulin-
like growth factor II; AML, acute myeloid leukemia; CASTing, cyclic amplification and
selection of target sequences; NADPH, reduced nicotinamide-adenine dinucleotide
phosphate; Co-IP, co-immunoprecipitation; HEK293, human embryonic kidney 293
cells; MM1, MonoMac1 cells; RPMI, Roswell Park Memorial Institute; 3AT, 3-amino-
1,2,4-triazole; X-α-Gal, 5-bromo-4-chloro-3-indolyl α-D-galactopyranoside; BLAST,
Basic Local Alignment Search Tool; bp, base pair; NT, N-terminus; CT, C-terminus; GFP,
green fluorescent protein; TRR, TNF-α-responsive region; EMSA, electrophoretic
mobility shift assay; RT-PCR, reverse transcription polymerase chain reaction; TNF-α,
tumor necrosis factor α; ChIP, chromatin immunoprecipitation; PCR, polymerase chain
reaction; SREBP, sterol regulatory element binding protein; NLS, nuclear localization
⁎ Corresponding author. Tel.: +1 406 994 5721; fax: +1 406 994 4303.
E-mail addresses: email@example.com (S.J. Wezensky),
firstname.lastname@example.org (T.S. Hanks), email@example.com (M.J. Wilkison),
firstname.lastname@example.org (M.C. Ammons), email@example.com
(D.W. Siemsen), firstname.lastname@example.org (K.A. Gauss).
0378-1119/$ – see front matter. Published by Elsevier B.V.
Contents lists available at ScienceDirect
journal homepage: www.elsevier.com/locate/gene
has also been shown to regulate the pro-apoptotic factor, NIP-3,
demonstrating that PLAGL2 may also function as a tumor suppressor
(Mizutani et al., 2002). In contrast, PLAGL1 inhibits tumor cell growth
by controlling apoptosis and cell cycle progression (Spengler et al.,
1997), and the loss of PLAGL1 expression during spontaneous
transformation of ovary surface epithelial cells and transcriptional
silencing in a variety of human cancers strongly suggests PLAGL1
functions as a tumor suppressor (Abdollahi et al., 1997; Kamikihara et
al., 2005). Elucidating the mechanisms of PLAG activation will provide
important insight into the role of these proteins in tumorigenesis.
Thesimilarity in DNA-binding specificityof PLAGproteinssuggests
that there may be some functional redundancy in the family, as
implied by the normal expression of PLAG1 target genes in PLAG1-
deficient mice (Hensen et al., 2004; Declercq et al., 2003). CASTing
identified a bipartite consensus sequence for PLAG1 containing a core
sequence (GGRGGCC),recognizedby PLAG1zinc fingers 6 and7, and a
G-cluster (GGG) located six to eight nucleotides downstream,
recognized by zinc finger 3 (Voz et al., 2000). While PLAGL2 was
also shown to bind to the PLAG1 consensus sequence with analogous
zinc fingers (5, 6 and 2), PLAGL1 recognized a sequence that was also
GC rich (GGGGGGCCCC) but lacked the G-cluster. With relatively few
PLAG target genes identified, the full range of in vivo PLAG DNA-
binding sites, and the degree of functional redundancy or competition
for target sites between family members, remains unclear.
Post-translational modification has been shown to play an
important role in the activity of PLAG1 and PLAGL2 proteins. While
SUMOylation of PLAG1 and PLAGL2 reduces the transcriptional
activity of these proteins, acetylation at the same lysine residues
targeted for SUMOylation increases their transcriptional activity (Van
et al., 2004; Zheng and Yang, 2005). On the other hand, PLAGL1
activity appears to be regulated more through epigenetic means
affecting the expression of PLAGL1, including CpG methylation and
histone deacetylation (Arima et al., 2006; Abdollahi et al., 2003).
In recent studies, we observed that PLAGL2 plays a role in the
regulation of NCF2, the gene encoding the NADPH oxidase cytosolic
protein known as p67phox(Ammons et al., 2007). Although we
observed direct binding of PLAGL2 to the NCF2 promoter sequence,
overexpression of PLAGL2 alone was not capable of activating an NCF2
promoter-reporter plasmid (unpublished data), suggesting that other
factors were required. To further elucidate the mechanisms whereby
PLAGL2 regulates gene expression, a yeast two-hybrid screen, baited
with full-length human PLAGL2, was carried out to identify potential
PLAGL2co-activators. PC2,a componentof theMediatorcomplex, was
identified as a putative PLAGL2-binding protein, and in vivo co-
immunoprecipitation (Co-IP) studies confirmed this interaction. We
demonstrate that PC2 enhances the activity of a known PLAGL2 target
promoter (NCF2) in cooperation with PLAGL2 and PU.1, is required for
PLAGL2 regulation of NCF2 expression in TNF-α-treated MonoMac1
cells, and is important in PLAGL2-induced expression of IGF-II. The
data presented here identify PC2 as a PLAGL2-binding protein and an
important regulator of PLAGL2 transactivation.
2. Materials and methods
Oligonucleotide primers were synthesized by Integrated DNA
Technologies, and PLAGL2 and PC2 antibodies were from Proteintech
Group, Inc. Alexa Fluor 546 goat anti-rabbit antibodies were from
2.2. Cell culture
HEK293 cells were grown in Dulbecco's modified Eagle's medium
supplemented with 10% fetal bovine serum, penicillin, and strepto-
mycin. Human MonoMac1 (MM1) macrophage cells were grown in
RPM1 supplemented with 10% fetal bovine serum, penicillin, and
2.3. Yeast two-hybrid screen
The Matchmaker yeast two-hybrid system 2 (Clontech, Mountain
View, CA) was used according to the manufacturer's protocol with
some modifications. The yeast reporter vector pAS2-1, containing
full-length PLAGL2 cDNA, was used to transform the competent
yeast strain Y187 to generate the bait strain Y187[pAS2-1(PLAGL2)].
No autoactivity of the pAS2-1(PLAGL2) reporter was detected and
45 mM 3AT was sufficient to inhibit leaky HIS3 expression of the
bait strain. The AH109 [pACT2 (human leukocyte cDNA)] strain was
mated with the bait strain Y187 [pAS2-1 (PLAGL2)] generating
1.3×108library clones on medium lacking tryptophan and leucine,
sufficiently covering the library. Diploid clones were replica plated
onto plates lacking histidine, tryptophan, and leucine to allow leaky
HIS3 background for a histidine jump-start assay. Plates were
incubated for 6 days at 30 °C and colonies were replated on
medium plates lacking histidine, adenine, tryptophan, and leucine
and supplemented with 45 mM 3AT to screen for reporter
activation. One hundred ninety two diploid reporter-positive clones
were streaked onto fresh selection plates and assayed for β-
galactosidase activity using a filter lift assay and for α-galactosidase
activity using X-α-Gal medium plates. Prey plasmids recovered
from diploid clones positive for nutritional and colorimetric reporter
gene activity were amplified, sequenced, and analyzed using the
2.4. Plasmid constructs
The pGL3-NCF2(500) promoter-reporter plasmid contains 500 bp
of sequence proximal to the ATG translational start site of the NCF2
gene and was described previously (Gauss et al., 2002). PLAGL2,
PLAGL2 N-terminus (NT), and PLAGL2 C-terminus (CT) PCR amplified
cDNA was cloned into the pAcGFP1 vector (Clontech) or pcDNA3.1
vector (PLAGL2-NT and PLAGL2-CT) using pcDNA3.1-PLAGL2 as
template. PC2434–784 cDNA was subcloned from pACT2-PC2434–784
into the pProLabel-C vector using EcoRI and XhoI sites. Full-length PC2
was subcloned from pOTB7-PC2 into the pProLabel-C vector and the
pcDNA3.1 vector using EcoRI and XhoI sites. pcDNA3.1-PLAGL2 and
pAcGFP1-PLAGL2 zinc finger mutants were generated using a site
directed mutagenesis kit (Stratagene) with pcDNA3.1-PLAGL2 or
pAcGFP1-PLAGL2 as template. Zinc finger mutagenesis primers were
designed to replace the first histidine of the C2H2 motif with an
alanine. All recombinant plasmids were sequenced to confirm valid
nucleotide sequence and expressed in HEK293 cells, followed by
immunoblot analysis with the appropriate antibodies when available,
or expressed in rabbit reticulocytes, to confirm expression of proteins.
All recombinant proteins migrated to their predicted molecular mass
as follows: PLAGL2 (54.6 kD), PC2 (86.8 kD), GFP-PLAGL2 (81.5 kD),
GFP-PLAGL2-NT (56.0 kD), and GFP-PLAGL2-CT (52.6 kD).
HEK293 cells (2×104per well) were cultured on Permanox
chamber slides (Nalge Nunc International Corp.) and transfected with
100 ng of expression plasmid using Lipofectamine 2000. Immunos-
taining for PC2 was performed using the BD Cytofix/Cytoperm Kit (BD
Biosciences) as follows. Cells were fixed for 20 min at room
temperature (RT) with 250 μL fixation/permeabilization solution
per well, followed by two 10-min washes at RT in 250 μL 1× perm/
wash solution. Cells were then incubated with anti-PC2 antibodies
(1:100) in 1× perm/wash solution for 30 min at RT, followed by 2
washes. Cells were then incubate for 1 h at RT, in 125 μL of Alexa Fluor
S.J. Wezensky et al. / Gene 452 (2010) 22–34
546 goat anti-rabbit antibodies (Invitrogen) diluted 1:1000 in 3%
powdered milk/1× PBS, followed by 4 washes.
2.6. In vitro translation of protein in rabbit reticulocytes
Recombinant expressionplasmids were transcribed and translated
in vitro using the TNT Quick Coupled Transcription/Translation
Systems (Promega). [35S]Methionine labeled protein was run on a
10% SDS-PAGE gel, and the gel was subjected to autoradiography to
confirm molecular mass and similar expression levels.
2.7. Electrophoretic mobility shift assay (EMSA)
EMSAs were carried out essentially as previously described
(Ammons et al., 2007; Gauss et al., 2002). Briefly, double-stranded
wild-type or mutant NCF2 TRR (TNF-α-responsive region) or PLAGL2
consensus site DNA was incubated with wild-type or mutant PLAGL2
protein generated in rabbit reticulocyte lysate in 1× binding buffer
(Roche) for 20 min at 5 °C. Samples were run on a 5% non-denaturing
polyacrylamide gel (19:1) in 0.5× TBE electrophoresed at 170 V for
3.5 h. Gels were fixed, dried and subjected to autoradiography.
2.8. In vivo co-immunoprecipitation (Co-IP) assay
In vivo Co-IP was carried out using the Matchmaker Chemilu-
minescent Co-IP System according to the manufacturer's protocol
(Clontech). This system utilizes a fluorescent AcGFP1 tag and the
enzymatic ProLabel reporter for chemiluminescent detection of
physical interactions between proteins that are expressed in
mammalian cells. Following co-transfection of GFP-tagged bait and
ProLabel-tagged prey plasmids, cell lysates are subjected to Co-IP
using anti-GFP antibodies, and protein-protein interactions are
detected using the ProLabel enzyme complementation assay.
Initially, the functional reporter is split into two non-functional
fragments: the small ProLabel and the larger Enzyme Acceptor. In
the presence of the Enzyme Acceptor, the ProLabel and Enzyme
Acceptor combine and become an active enzyme that cleaves the
chemiluminescent substrate. This system has been demonstrated to
be highly sensitive and quantitative (Eglen and Singh, 2003; Eglen,
Prior to experimentation, all pAcGFP1 recombinant plasmids were
tested for expression of correct size protein by anti-GFP immunoblot,
and all ProLabel-tagged recombinant plasmids were tested to verify
activity as follows. Cells were seeded the day before transfection at
5×104cells in 24-well tissue culture plates. Cells were transfected
with 800 ng of DNA using Lipofectamine 2000. After 6 h, medium was
replaced with fresh complete medium. Twenty-four hours post-
transfection,cells wereharvestedfor immunoblot analysisorProLabel
For in vivo Co-IP, HEK293 cells were seeded at 1.5×106cells per
100-mm tissue culture plates 24 h prior to transfection. DNA (8 μg)
was transiently transfected using Lipofectamine 2000. Twenty-four
hours post-transfection, cells were harvested for Co-IP. Protein
concentration was determined by BCA and 250 μg of protein cell
lysate was added to each co-immunoprecipitation reaction and
incubated with anti-GFP antibodies (Clontech) in 500 μL cell lysis
buffer for 2 h at 5 °C. Protein G Plus/A agarose beads (Clontech) were
added, andthe reactions were incubated overnightat 5 °C. Beadswere
washed, and samples were assayed for ProLabel activity in triplicate at
10-min intervals over 180 min using a microplate luminometer
(Fluroscan Ascent FL, Thermo Electron, Waltham, MA).
For in vivo Co-IP of endogenous PC2 and GFP-PLAGL2, HEK293
cells were transfected as above and lysates were immunoprecipitated
with anti-GFP or anti-PC2 antibodies and protein G Plus/A agarose
beads (Clontech) overnight. Beads were washed and samples were
subjected to immunoblot analysis using anti-PC2 or anti-GFP
2.9. Transfections and luciferase assay
HEK293 and HeLa cells were transiently transfected as above using
Lipofectamine 2000. Briefly, cells were seeded 1 day prior to
transfections at 5×104per well in 24-well tissue culture plates. On
the day of the transfection, 50 ng of pGL3 reporter plasmid, 150 ng of
expression plasmids, and 50 ng of pRL-TK were co-transfected. Empty
pcDNA3.1 plasmid was added to appropriate samples to adjust the
total amount of DNA to 800 ng per transfection. Each transfection was
done in duplicate. At 24 h post-transfection, cells were assayed for
promoter-reporter activity (firefly luciferase) and Renilla luciferase
activity using the dual-luciferase reporter assay according to the
manufacture's protocol (Promega) and read on a Lumat LB 9507
luminometer (EG&G Berthold, Germany).
For PC2 siRNA studies using the NCF2 promoter-reporter plasmid,
HEK293 cells were mock transfected or transfected with 5 pmol of
PC2 siGENOME SMARTpool siRNA (CCAAGACCCGGGACGAAUA, CGA-
CAAGAACGAAGACAGA, CGUCAGUGAUCCUAUGAAU, GGUCAGU-
CAAAUCGAGGAU) (Dharmacon) or control siGLO RNA using
Lipofectamine 2000. At 24 h post-transfection, cells were replated
and allowed to grow 24 h. Cells were then transfected with
appropriate promoter-reporter and expression plasmids, followed
by luciferase detection as above at 72 h post-siRNA transfections. To
determine the effect of PC2 knockdown on insulin-like growth factor
II (IGF-II) mRNA levels, HEK293 cells were transfected as above, and
RNA was isolated 72 h post-siRNA transfection using an RNeasy kit
(Qiagen). RNA (100 ng) was subjected to RT-PCR using IGF-II-
specific (sense, 5′-GCTGTTTCCGCAGCTGTGA-3′; antisense, 5′-
CTGCTTCCAGGTGTCATATTGG-3′) and ribosomal 28S-specific
(sense, 5′-TTGAAAATCCGGGGGAGAG-3′; antisense, 5′-ACATTGTTC-
CAACATGCCAG-3′) primers and a One-Step RT-PCR kit (Invitrogen).
PCR products were visualized, at increasing cycle numbers to ensure
analysis during the linear phase of PCR amplification, on an agarose
gel stained with ethidium bromide.
The effect of PC2 knockdown on PC2 protein expression was
determined by immunoblot analysis as follows. HEK293 cells were
transfected as above, cells were harvested at 24 and 48 h post-
transfection, and cell lysate was subjected to SDS-PAGE analysis.
Proteins were transferred to nylon membrane and PC2 protein was
detected using anti-PC2 antibodies and chemiluminescent detection.
2.10. Chromatin immunoprecipitation (ChIP)
ChIP experiments were performed essentially as described
(Ammons et al., 2007). MM1 cells were treated with TNF-α
(20 ng/mL) for 24 h to induce endogenous PLAGL2 binding to the
endogenous NCF2 TRR, followed by chromatin isolation. Briefly, cells
were treated with formaldehyde and processed for ChIP experiments
using a ChIP-IT kit (Active Motif), according to the manufacturer's
protocol. A sample of precleared chromatin was reserved for the
input in PCR analysis. Protein/DNA complexes were immunopreci-
pitated overnight with the appropriate antibodies (negative control
antibodies provided with the ChIP-IT kit or anti-PLAGL2 or anti-PC2
antibodies) and processed for PCR amplification. PCR amplification
of each ChIP sample was performed with primers flanking the NCF2
TRR in the endogenous NCF2 promoter (sense, 5′-CATCTGGCCCA-
GAAAGTGA-3′; antisense, 5′-CTTCATTCCAGAGGCTGATGG-3′) and
GAPDH promoter-specific primers (sense, 5-’TACTAGCGGTTT-
TACGGGCG-3′; antisense, 5′-TCGAACAGGAGGAGCAGAGAGCG-3′).
PCR products were assayed at increasing cycle numbers to ensure
analysis during the linear phase of PCR amplification. PCR products
were subjected to agarose gel electrophoresis and stained with
S.J. Wezensky et al. / Gene 452 (2010) 22–34
2.11. Quantitative reverse transcriptase-polymerase chain reaction
RNA was isolated using the RNeasy Kit (Qiagen). The qRT-PCR
amplification was performed in an ABI PRISM 7500 Fast Real-Time
PCR System, using 12.5 mL SYBR Green PCR Master Mix (Qiagen),
1 μM gene-specific primer mix (QuantiTect Primer Assays), 0.25 μL
QuantiFast RT mix, 100 ng template RNA, and water to 25 μL total
volume. PCR amplification was carried out as follows: 10 min at 50 °C,
5 min at 95 °C, followed by 40 cycles of 10 s at 95 °C, 30 s at 60 °C.
Samples were analyzed in triplicate and were normalized to beta-
actin expression levels.
2.12. Statistical analysis
One-way ANOVA was performed on the indicated sets of data.
Post-test analysis used Tukey's pair-wise comparisons (GraphPad
Prism Software). Pair-wise comparisons with differences at Pb0.05
were considered to be statistically significant.
3.1. Yeast two-hybrid screen identifies PC2 as a putative PLAGL2-binding
To identify potential PLAGL2 cofactors, we utilized a yeast two-
hybrid screen baited with full-length human PLAGL2. The reporter
strain (AH109), carrying a human leukocytecDNAlibrary, and thebait
strain (Y187) were mated yielding 1.3×108library clones for
nutritional and colorimetric screening.
enzyme UBC9, was one of the putative PLAGL2-binding proteins
identified in the screen (data not shown). UBC9 has been previously
identified in a yeast two-hybrid screen using a partial murine PLAGL2
as bait (Van et al., 2004), demonstrating that the above screen
successfully identified a known PLAGL2 protein-protein interaction.
Another interesting clone retrieved from this screen was a partial
cDNA corresponding to positive cofactor 2 (PC2), also known as
ARC105, MED15, CTG7A, PCQAP, TIG1, and TNRC7 and a component of
Mediator, a multiprotein complex that is recruited to promoters by
and Napoli, 2007; Yang et al., 2006). The partial PC2 clone contained
amino acid residues 434–784, a region which includes a number of
phosphorylation sites, a nuclear localization signal, and part of a
proline rich region (Fig. 1A).
To confirm the in vivo interaction between PLAGL2 and PC2 in
yeast, the bait and prey plasmids were recovered from their
respective yeast strains and transformed into the opposite strain.
The bait and prey strains were mated, and screening for both
nutritional and colorimetric gene reporter activity confirmed the
interaction between PLAGL2 and PC2434–784 in yeast (data not
3.2. PC2 interacts with the C-terminal domain of PLAGL2
Full-length PLAGL2, as well as the N-terminus and C-terminus of
PLAGL2 (Fig. 1B), were expressed as GFP-tagged proteins from the
pAcGFP1 vector, while full-length PC2 and PC2434–784were expressed
as ProLabel-tagged proteins from the pProLabel-C vector. Transfection
of HEK293 cells with the pcDNA3.1-PLAGL2, pAcGFP1-PLAGL2, and
the ProLabel-PC2 expression plasmids, and subsequent immunoblot
analysis, showed expression of exogenous proteins migrating to their
predicted molecular weights (Fig. 2A). In addition, Fig. 2A showed
barely detectable levels of endogenous PC2 protein, consistent with a
previous report demonstrating endogenous PC2 expression in
HEK293 cells (Ishikawa et al., 2006). Fluorescence microscopy
showed nuclear localization of full-length GFP-PLAGL2 (Fig. 2B and
C), as previously demonstrated in HEK293 cells (Zheng and Yang,
2005), relative to the diffuse expression of GFP throughout the cell.
GFP-PLAGL2-NT (N-terminus) also localized to the nucleus (Fig. 2C),
suggesting that, like PLAG1 (Braem et al., 2002), the nuclear
localization signal (NLS) is located in the N-terminal domain of
PLAGL2. GFP-PLAGL2-CT (C-terminus) was diffusely expressed
throughout the cell, similar to GFP expression (Fig. 2C), which is
consistent with nuclear localization being conferred via the N-
terminus of PLAGL2.
Co-localization of GFP-PLAGL2 and PC2 was observed in HEK293
cells transiently transfected with pAcGFP1-PLAGL2 and pProLabel-
PC2 (full-length) expression plasmids and subsequently immunos-
tained for PC2 protein (Fig. 3D). Mock transfected cells (Fig. 3D, Mock
panels) demonstrated low levels of endogenous PC2 protein in all the
cells, while cells transfected with pProLabel-PC2 (Fig. 3D, PC2 panels)
showed much greater levels of exogenous PC2 protein in transfected
versus non-transfected cells in the same panel. When pAcGFP1-
PLAGL2 and pProLabel-PC2 were co-transfected (Fig. 3D, GFP-
PLAGL2/PC2 panels), co-localization of pAcGFP1-PLAGL2 and pPro-
Label-PC2 was apparent in the merged panels. The data demonstrate
that both PLAGL2 and PC2 localize to the nucleus in HEK293 cells, and
are consistent with the role of both proteins as transcriptional
regulators and a previous report demonstrating nuclear localization of
endogenous and exogenous PC2 protein in HEK293 cells (Ishikawa et
To confirm the interaction between PLAGL2 and PC2 in vivo,
HEK293 cells were transiently transfected with pAcGFP1-PLAGL2 and
Fig. 1. Diagram of PC2 and PLAGL2 proteins indicating functional domains and partial protein regions used for Co-IP and promoter-reporter studies. (A) PC2 full-length protein
(upper diagram) indicating the KIX domain, the proline rich domain and the nuclear localization signal (NLS). Lower diagram represents the partial PC2 yeast two-hybrid clone. (B)
PLAGL2 full-length protein (upper diagram) with the zinc finger and activation domains indicated. Middle and lower diagrams represent the N- and C-terminal domains of PLAGL2
used for experiments. Numbers on the right designate amino acids.
S.J. Wezensky et al. / Gene 452 (2010) 22–34
Fig. 2. Nuclear localization of PLAGL2 is supported via the N-terminal domain. (A) Immunoblot of PLAGL2, GFP-PLAGL2 full-length (FL), GFP-PLAGL2 N-terminal (NT), GFP-PLAGL2 C-
terminal (CT) and Prolabel-PC2 full-length. HEK293 cells were mock transfected (−) or transfected with pcDNA-PLAGL2 (+), pAcGFP1-PLAGL2-FL (FL), pAcGFP1-PLAGL2-NT (NT),
pAcGFP1-PLAGL2-CT (CT) or pProLabel-PC2 (+) plasmid DNA. Lysates were subjected to anti-PLAGL2, anti-GFP, or anti-PC2 immunoblot analysis as indicated above the blot.
Molecular mass is indicated on the left. (B and C) Cellular localization of full-length GFP-PLAGL2, GFP-PLAGL2-NT and GFP-PLAGL2-CT. HEK293 cells were transfected with pAcGFP1,
pAcGFP1-PLAGL2-FL, pAcGFP1-PLAGL2-NT, or pAcGFP1-PLAGL2-CT as indicated and cells were analyzed by phase or fluorescence microscopy. For nuclear staining, cells were
treated with DRAQ5 (B, panels 2 and 6). Phase contrast: B, panels 3 and 7, and C, upper panel. FITC filter: B, panels 1 and 5, and C, middle panel. Merged images: B, panels 4 and 8, and
C, lower panel. (D) Co-localization of GFP-PLAGL2 and PC2. HEK293 cells were transfected with pAcGFP1-PLAGL2-FL or pProLabel-PC2 plasmids, both plasmids, or mock transfected
as indicated. Cells were immunostained for PC2 and analyzed by microscopy. Mock, PC2, and GFP-PLAGL2 transfections: first panel, phase; second panel, fluorescence; third panel,
merge of phase and fluorescence. PC2 and GFP-PLAGL2 co-transfection: first panel, phase; second and third panels, fluorescence; fourth panel, merge of green and red panels; fifth
panel, merge of green, red and phase panels.
S.J. Wezensky et al. / Gene 452 (2010) 22–34
pProLabel-PC2 (full-length) for co-immunoprecipitation studies. As
seen in Fig. 3A (upper panel, lanes 2 and 3), both endogenous and
exogenous PC2 protein was co-immunoprecipitated from cells
expressing GFP-PLAGL2 and immunoprecipitated with anti-GFP
antibodies. Similarly, when analogous samples were immunoprecipi-
tated with anti-PC2 antibodies, GFP-PLAGL2 was co-immunoprecipi-
tated in the presence and absence of exogenously expressed PC2 (Fig.
3A, lower panel, lanes 2 and 3). The data demonstrate that GFP-
PLAGL2 interacts with endogenous PC2 protein, as well as exogenous
PC2, and provide additional evidence for a functional interaction
between PLAGL2 and PC2.
The Matchmaker Chemiluminescent Co-IP system (Clontech)
was used to further characterize the interaction between PLAGL2
and PC2 in vivo. As more fully explained in section 2.8, this system
incorporates the highly sensitive ProLabel enzyme complementa-
tion assay that allows a quantitative measure of physical interac-
tions between bait and prey proteins in mammalian cells (Eglen
and Singh, 2003; Eglen, 2002). HEK293 cells were transiently
transfected with pAcGFP1-PLAGL2 and pProLabel-PC2 (full-length)
or pProLabel-PC2434–784 for co-immunoprecipitation studies.
PLAGL2 protein-protein complexes were immunoprecipitated with
anti-GFP antibodies followed by detection of ProLabel activity over
180 min (Fig. 3B, upper panel). Both full-length PC2 and PC2434–784
interacted with PLAGL2, as demonstrated by the significant
ProLabel activity detected relative to Co-IPs performed with the
pAcGFP1 and pProLabel-PC2 (full-length) or pProLabel-PC2434–784
controls. The lower panel of Fig. 3B shows the fold change at
100 min. These results confirm the interaction between PLAGL2 and
PC2 and demonstrate that the C-terminus of PC2 is capable of
supporting this interaction.
Fig. 3. PC2 interacts with the C-terminal domain of PLAGL2. (A) Co-IP of endogenous PC2 and GFP-PLAGL2. pAcGFP (lane 1), pAcGFP1-PLAGL2-FL (lane 2), or pAcGFP1-PLAGL2-FL
and pProLabel-PC2 full-length (lane 3) transfected HEK293 cells were immunoprecipitated with anti-GFP antibodies (upper panel) or anti-PC2 antibodies (lower panel) and
subjected to immunoblot analysis using anti-PC2 antibodies (upper panel) or anti-GFP antibodies (lower panel). (B) HEK293 cells were transfected with negative controls pAcGFP1
and pProLabel vectors (solid circle), pAcGFP1 and pProLabel-PC2 full-length (solid square) or pProLabel-PC2434–784(solid diamond), pAcGFP1-PLAGL2-FL and pProLabel (solid
triangle), or samples pAcGFP1-PLAGL2-FL and pProLabel-PC2 full-length (open square) or pProLabel-PC2434–784(open diamond). Lysates were immunoprecipitated with anti-GFP
antibodies and ProLabel detection was performed in triplicate. Upper graph demonstrates kinetic response over 180 min, and lower graph shows fold change relative to
corresponding controls at 100 min. (B–D) Cells were transfected with pAcGFP1 and pProLabel plasmids, as indicated below the graphs. Data are presented as fold change relative to
corresponding controls (empty pAcGFP1 and pProLabel plasmids). Results are representative of at least three separate experiments and samples that are statistically significantly
different (PN0.05) from the PLAGL2/PC2 sample are indicated (⁎).
S.J. Wezensky et al. / Gene 452 (2010) 22–34
To demonstrate the specificity of the PLAGL2/PC2 interaction,
HEK293 cells were transfected with pAcGFP1-PLAGL2 and pProLabel-T,
a plasmidexpressinga ProLabel-tagged protein(SV40largeT antigen)
no significant ProLabel activity detected with the pAcGFP1-PLAGL2
and pProLabel-T sample relative to the pAcGFP-PLAGL2 and pProLa-
bel-PC2 sample (Fig. 3C). SV40 large T antigen (pProLabel-T) was,
however, efficiently co-immunoprecipitated with GFP-p53 (positive
bait for SV40 large T), as seen by the ∼55-fold change relative to the
control (pAcGFP1 and pProLabel-T).
PLAGL2 contains six C2H2 zinc fingers in the N-terminus and an
activation domain in the C-terminus (Fig. 1B). To determine if PC2
bound to the N- or C-terminus of PLAGL2, HEK293 cells were
transfected with pAcGFP1-PLAGL2, pAcGFP1-PLAGL2-NT or
pAcGFP1-PLAGL2-CT and pProLabel-PC2, followed by immunoprecip-
itation and detection of ProLabel activity. Figure 3D shows that the
interaction between PC2 and PLAGL2 is via the C-terminus of PLAGL2.
This result is consistent with the finding that mutagenesis of any
single PLAGL2 zinc finger in the N-terminus showed no significant
difference in ProLabel activity when compared to the full-length
PLAGL2/PC2 sample (data not shown).
3.3. PLAGL2, PC2 and PU.1 cooperate to enhance activation of the NCF2
In a previous study, we determined that PLAGL2 plays a role in
regulation of the NCF2 gene (Ammons et al., 2007); however,
overexpression of PLAGL2 alone was not sufficient to activate an
NCF2 promoter-reporter plasmid, suggesting the requirement for
other factors. To determine if PC2 expression could modulate PLAGL2
transactivation, HEK293 cells were transfected with an NCF2
promoter-reporter plasmid along with pcDNA3.1 expressionplasmids
for PLAGL2 and PC2. As seen in Fig. 4 (upper graph), there was no
significant increase in promoter-reporter activity in the sample co-
expressing PLAGL2 and PC2, as compared to the control (promoter-
reporter and empty expression plasmid DNA). Transfection of PU.1,
another factor previously shown to be required for NCF2 basal activity
(Li et al., 2001; Gauss et al., 2002), consistently showed a slight
increase in promoter activity that was not enhanced by co-
transfection of either PLAGL2 or PC2. However, when PLAGL2 and
PC2 plasmids were co-transfected with PU.1, there was enhanced
activity (∼2 fold) from the NCF2 promoter-reporter plasmid relative
to samples transfected with any single or double combination of
expression plasmids. Similar results were observed with HeLa cells
(Fig. 4, lower graph), although the small increase in promoter activity,
consistently observed in HEK293 cells in the presence of PU.1, was not
detected. These results are consistent with a previous report
demonstrating that, although PC2 is a component of larger protein
complex, overexpression of PC2 alone is capable of stimulating a
promoter-reporter plasmid containing a binding elementfor Smad3, a
factor demonstrated to directly interact with PC2 (Kato et al., 2002).
This effect was not a result of PC2 affectively increasing the expression
of PLAGL2 or PU.1, as observed by Western blot analysis during the
characterization of these expression plasmids and respective anti-
bodies (data not shown). The requirement for PU.1, and the close
juxtaposition of PU.1 and PLAGL2 on the promoter, raised the
possibility of an interaction between PU.1 and PLAGL2 or PC2 or
both; however, Co-IP studies supported no such interactions (data not
shown). Taken together, the data show that PLAGL2, PC2, and PU.1
cooperate to enhance NCF2 promoter-reporter activity.
3.4. PLAGL2 recognizes a partial PLAG1 consensus site in the NCF2
promoter via C2H2 zinc fingers 5 and 6
The data suggest a model for PLAGL2 transactivation whereby
PLAGL2 and PU.1 bind directly to the NCF2 promoter, via their
respective DNA recognition sites, and that PC2 associates with the
promoter through PLAGL2 interactions, resulting in cooperative
functional PU.1 sites in the NCF2 promoter (Li et al., 2001; Gauss et al.,
2002), and although the exact PLAGL2-binding site was not deter-
mined, we did determine that PLAGL2 binds to the TNF-α-responsive
region (TRR) of the NCF2 promoter, although with approximately 50-
fold less affinity relative to the PLAG consensus site (Ammons et al.,
2007). To further test the proposed model, we needed to first
characterize the PLAGL2 recognition sequence in the NCF2 TRR.
The nucleotide sequence required for PLAGL2 binding to the NCF2
TRR was determined by subjecting a series of mutant NCF2 TRR
double-strand oligonucleotides to EMSA. The sequence of the NCF2
TRR is shown in Fig. 5A along with the altered nucleotides of the
various TRR mutants. Figure 5B shows that TRR mutants 10 and 11
both prevent PLAGL2 binding to the TRR relative to the wild-type TRR
and TRR mutants 1 through 9, thus identifying the PLAGL2-binding
site. The sequence altered by mutants 10 and 11 (GGAGGCC), when
(GGRGGCC) of the bipartite PLAG1 consensus site (Voz et al., 2000).
Although there is no obvious G-cluster six to eight nucleotides
downstream from this core sequence, the remaining TRR sequence is
relativelyGC rich.Since previousstudies haveshownthatPLAGL2zinc
fingers 5 and 6 bind to the core sequence of the PLAG1 consensus site,
and zinc finger 2 binds the G-cluster (Hensen et al., 2002), the
Fig. 4. PC2 cooperates with PLAGL2 and PU.1 to enhance activity from the NCF2
promoter-reporter. HEK293 (upper graph) and HeLa (lower graph) cells were
transfected with the pGL3-NCF2(500) promoter-reporter plasmid and pcDNA3.1-
PLAGL2, pcDNA3.1-PU.1, and pcDNA3.1-PC2 expression plasmids as indicated and
lysates were assayed for luciferase activity. pRL-TK plasmid was used to control for
transfection efficiency. Activity is shown as fold change relative to empty pGL3 vector
transfected with similar plasmids for each sample. Transfections were done in duplicate
and results are representative of at least three separate experiments. The difference
between the PLAGL2, PU.1, and PC2 sample and other samples was statistically
significant (Pb0.05) as indicated (⁎).
S.J. Wezensky et al. / Gene 452 (2010) 22–34
differences in relative binding of PLAGL2 to the PLAG1 consensus
binding site and TRR shown here (Fig. 5B, Con versus TRR), and in our
of the G-cluster motif of the bipartite PLAG1 consensus site in the TRR.
Absence of the G-cluster in the NCF2 TRR suggested that PLAGL2
zinc finger 2 would not play an important role in PLAGL2 recognition
of the TRR sequence. To determine which zinc fingers were required
for PLAGL2 binding to the NCF2 TRR relative to the PLAG1 consensus
Fig. 5. PLAGL2 binds the NCF2 promoter via a GC-rich sequence. (A) Sequence (uppercase) of wild-type NCF2 TRR and PLAG1 consensus binding site are indicated. Sequential
mutations are designated 1 through 11, and substituted nucleotides are shown (lower case). The core and cluster sequences of the PLAG1 consensus binding site are underlined. (B)
EMSA of PLAGL2 protein binding to the wild-type and mutant NCF2 TRR. PLAGL2 protein was incubated with no DNA (−), or DNA containing the PLAG1 consensus (Con), non-
specific sequence (NS), wild-type NCF2 TRR (TRR), or NCF2 TRR mutants 1 through 11, as indicated and analyzed by EMSA. (C) PLAGL2 wild-type (WT) or zinc finger mutants (M1-
M6) were generated in rabbit reticulocytes and subjected to SDS-PAGE analysis. V represents sample generated with empty pcDNA3.1 vector. Molecular mass is indicated on the
right. (D and E) EMSA using wild-type PLAGL2 protein (WT) and zinc finger mutants (M1-M6) and NCF2 TRR (D) or PLAG1 consensus DNA (E). Densitometry of EMSA is to the right
with wild-type PLAGL2 set to 100%.
S.J. Wezensky et al. / Gene 452 (2010) 22–34
sequence, each PLAGL2 zinc finger was mutated individually by
replacing the first histidine of the C2H2 motif with an alanine to
prevent formation of a functional zinc finger (Ikeda and Kawakami,
1995). The PLAGL2 zinc finger mutant proteins were produced in
rabbit reticulocytes to demonstrate that each mutant plasmid
generated approximately equal amounts of protein of the correct
molecular weight (Fig. 5C). EMSA was used to test the ability of the
zinc finger mutants to bind to the NCF2 TRR, as well as to the PLAG1
consensus site (Fig. 5D and E). As reported previously (Hensen et al.,
2002), we also found that zinc fingers 2, 5 and 6 were important for
PLAGL2 binding to the PLAG1 consensus sequence (Fig. 5E) which is
shown by the appreciable loss of binding with these specific zinc
finger mutants. Figure 5D shows the importance of zinc fingers 5 and
6 in binding to the TRR with less of a role for zinc finger 2. This was
consistent with results from the TRR mutagenesis analysis showing
that the PLAGL2-binding site in the TRR consisted of only the core
sequence of the PLAG1 consensus site, which is recognized by PLAGL2
zinc fingers 5 and 6.
3.5. Direct binding of PLAGL2 and PU.1 to their respective DNA
recognition sites, and the C-terminus of PLAGL2 are necessary for
enhanced NCF2 promoter-reporter activity
The data suggest that PLAGL2 and PU.1 are required at the NCF2
promoter, presumably through direct binding to their respective
recognition sites, and that PC2 is targeted to the promoter via the C-
terminal domain of PLAGL2 for increased promoter activity. To further
test this model, NCF2 promoter-reporter assays were performed using
a promoter containing a mutation in the PLAGL2-binding site in the
TRR analogous to mutant 10 in Fig. 5A. While there was still a slight
increase in promoter activity observed in the presence of PU.1, the
loss of the PLAGL2-binding site in the TRR completely abrogated the
enhanced activation of the promoter-reporter (Fig. 6A) relative to the
PU.1 expressing samples. Because the samples expressing PLAGL2, or
PC2, or PLAGL2 and PC2 consistently did not show any promoter-
reporter activity over basal levels, these controls were not included in
the remaining promoter-reporter studies. When the zinc fingers (5 or
6) required for TRR binding were mutated, there was, again, no
enhanced activation of the NCF2 promoter-reporter observed (Fig.
6B). Finally, when the plasmid (PLAGL2-NT) expressing only the N-
terminal zinc finger DNA-binding domain of PLAGL2 and lacking the
C-terminus required for PC2 binding was substituted for wild-type
PLAGL2, there was also no increase in promoter activity observed (Fig.
6C). The data show that activation of the NCF2 promoter-reporter by
PLAGL2, PU.1 and PC2 requires binding of PLAGL2 to the identified
PLAGL2-binding site in the NCF2 TRR via zinc fingers 5 and 6 and the
C-terminal domain of PLAGL2, which is involved in PC2 binding.
To determine the role of PU.1 in the enhanced NCF2 promoter
activity, a promoter-reporter construct with mutated PU.1 sites
(Gauss et al., 2002) was tested. As seen in Fig. 6D (middle bar), the
Fig. 6. PLAGL2 and PU.1 binding to the NCF2 promoter is required for the PLAGL2/PU.1/PC2 enhanced NCF2 promoter-reporter activity. HEK293 cells were transfected with pGL3-
NCF2(500) promoter-reporter plasmid along with the indicated pcDNA3.1 expression plasmids and lysates were assayed for luciferase activity. Activity is shown as fold change
relative to controls (empty pGL3 vector transfected with similar plasmids). (A) pGL3-NCF2(500) containing the NCF2 TRR mutation #10 (Fig. 5A) was transfected along with the
indicated expression plasmids. (B and C) Wild-type pGL3-NCF2(500) was transfected with either wild-type PLAGL2 or PLAGL2 zinc finger mutants M5 or M6 (B) or PLAGL2-NT (C)
and PU.1 and PC2 expression plasmids, as indicated. (D) pGL3-NCF2(500) containing three mutated PU.1 sites (Gauss et al., 2002) was transfected along with the indicated
expression plasmids. Transfections were done in duplicate and results are representative of at least three separate experiments. Samples that were statistically significantly different
(PN0.05) from the WT PLAGL2/PU.1 sample are indicated (⁎).
S.J. Wezensky et al. / Gene 452 (2010) 22–34
slight increase in promoter activity usually observed with PU.1
expression (Fig. 6A–C) was not detected in the absence of PU.1
binding sites. In addition, there was no enhanced promoter activity
with PLAGL2, PU.1 andPC2 expression,demonstrating the importance
of PU.1 in the cooperative activation of NCF2 promoter-reporter.
Together, the data demonstrate that PLAGL2 and PU.1 must bind their
respective sites in the promoter and that the C-terminal domain of
PLAGL2, which is essential for PC2 binding, is required for enhanced
3.6. PC2 knockdown completely abrogates the PLAGL2, PU.1, and PC2
cooperative activation of the NCF2 promoter-reporter plasmid
To establish the requirement for PC2 in enhanced activation of the
NCF2 promoter, promoter-reporter assays were repeated in the
presence of PC2 siRNA. HEK293 cells were mock transfected, or
transfected with control siRNA (siGLO) or PC2 siRNA, and 48 h post-
siRNA transfection, cells were transfected with expression plasmids.
As seen in Fig. 7A, there was a significant difference in promoter-
reporter activity when comparing the PLAGL2, PU.1, and PC2 sample
and the analogous sample transfected in the presence of PC2 siRNA,
demonstrating that PC2 is essential for this cooperative promoter
activation. These results were consistent with levels of PC2 observed
at 48 h post-siRNA transfection (Fig. 7C), where there was little to no
detectable levels of PC2 in the PC2 siRNA sample compared to samples
expressing exogenous PC2 in the absence or presence of control
siRNA. Importantly, there was no significant reduction in promoter
activity of a similar transfection in the presence of control siRNA
(siGLO), and consistent with previous findings (Yang et al., 2006), PC2
knockdown did not globally inhibit transcription, as the activity from
the thymidine kinase promoter of the pRL-TK plasmid was virtually
the same between samples (Fig. 7B). These data demonstrate that PC2
is required for the cooperative promoter-reporter activity.
3.7. Endogenous PC2 associates with the endogenous NCF2 promoter at
the NCF2 TRR and is required for PLAGL2 regulation of NCF2 in
TNF-α-treated MonoMac1 cells
Together, the data strongly suggest that the effects of PC2 on the
enhanced promoter activity are due to a physical association of PC2
with the promoter via an interaction with PLAGL2 at the NCF2 TRR.
The lack of activation of the endogenous NCF2 promoter in PLAGL2,
PU.1 and PC2 transfected HEK293 cells (data not shown), possibly due
to negative epigenetic regulation (Fuks, 2005; Chen et al., 2002),
prompted us to use MM1 cells, a model we used previously
demonstrating PLAGL2 association with the NCF2 TRR in response to
TNF-α (Ammons et al., 2007), to determine if endogenous PC2
localized to the endogenous NCF2 promoter in the same region
recognized by PLAGL2 (NCF2 TRR). ChIP analysis was performed using
chromatin isolated from TNF-α-treated MM1 cells. Figure 8A (upper
panel) shows that, relative to the no antibody and negative control
antibodies, the NCF2 TRR was specifically immunoprecipitated by
either PLAGL2 or PC2 antibodies, demonstrating that PC2 associates
with the endogenous NCF2 promoter in a similar region occupied by
PLAGL2. Importantly, no significant association of PLAGL2 or PC2 was
detected with the control GAPDH gene (Fig. 8A, lower panel).
Sequential ChIP analysis performed to further demonstrate co-
occupancy of PLAGL2 and PC2 on the NCF2 TRR was inconclusive,
possibly due to the low levels of these endogenous transcriptional
In a previous report (Ammons et al., 2007), we showed that
PLAGL2 bound to the NCF2 TRR and was required for increased
expression of NCF2 in response to TNF-α in MM1 cells. To determine if
PC2 was also necessary for PLAGL2 regulation of NCF2 in response to
TNF-α, MM1 cells were mock transfected, or transfected with control
siRNA (siGLO) or PC2 siRNA 48 h prior to TNF-α treatment. RNA was
isolated 24 h post TNF-α treatment and subjected to PC2- and NCF2-
specific qRT-PCR. As seen if Fig. 8B (upper graph), the ∼5 fold increase
in NCF2 mRNA with TNF-α treatment is affectively and significantly
reduced in the presence of PC2 siRNA, but not with control siRNA. The
lower graph shows that PC2 mRNA levels were lower in the presence
of PC2 siRNA, and although this change was not statically significant,
Fig. 7. PC2 is required for enhanced NCF2 promoter-reporter activity. HEK293 cells
were mock transfected or transfected with control siRNA (siGLO) or PC2 siRNA as
indicated, followed by transfection with pGL2-NCF2(500) and the indicated expression
plasmids. Lysates were assayed for luciferase activity. (A) Luciferase activity from the
NCF2 promoter-reporter is presented as fold change relative to controls as in Figs. 4
and 6. (B) Luciferase activity from the pRL-TK plasmid is presented as luminescent
units. Transfections were done in duplicate and results are representative of three
separate experiments. Samples that were statistically significantly different (PN0.05)
from the PLAGL2/PU.1/PC2 sample are indicated (⁎). (C) HEK293 cells were transfected
as above with PC2 expression plasmid, control siRNA (siGLO) or PC2 siRNA as indicated.
Cell lysates were harvested at 24 h (upper panel) and 48 h (lower panel) and subjected
to anti-PC2 immunoblot analysis.
S.J. Wezensky et al. / Gene 452 (2010) 22–34
the reduction in PC2 mRNA levels was consistent between experi-
ments. The inability to completely knockdown PC2 mRNA was likely
due to the relative inefficiency of MM1 cell transfection compared to
HEK293 cells. This also explains that, while the reduction in NCF2
mRNA was significant with PC2 knockdown, it was not down at basal
levels. Together, the data show that endogenous PLAGL2 and PC2
associate with the NCF2 TRR and that PC2 is required for PLAGL2
regulated expression of NCF2 in TNF-α-treated MM1 cells, thus
demonstrating the functional relevance of PC2 as a modulator of
3.8. PC2 knockdown inhibits PLAGL2 induced expression of IGF-II
To determine if PC2 played a role in the regulation of other PLAGL2
target genes, we evaluated the effect of PC2 knockdown on the
expression of IGF-II, a known PLAGL2 target gene in PLAGL2
expressing HEK293 cells (Hensen et al., 2002). HEK293 cells were
transfected with PLAGL2 expression plasmid in the presence or
absence of PC2 siRNA, followed by RT-PCR analysis of IGF-II mRNA
levels. As seen in Fig. 9, the increase in IGF-II mRNA levels with
PLAGL2 expression was greatly diminished in the presence of PC2
siRNA (Fig. 9, lane 6), relative to expression in the mock and negative
control siRNA (siGLO) transfections (Fig. 9, lanes 2 and 4). The data
demonstrate that PC2 is also important in the regulation of a second
PLAGL2 target gene, IGF-II.
All three members of the PLAG family have been shown to be
involved in tumorigenesis (reviewed by Van et al., 2007; Abdollahi,
2007); however, little is known about the mechanisms that regulate
their activity. In the present study, we identified PC2, a component of
the ARC/Mediator complex, as a novel PLAGL2-binding protein and an
important modulator of PLAGL2 transactivation.
The partial PC2434–784clone isolated from the yeast two-hybrid
screen demonstrated thatthe C-terminal domain of PC2 was sufficient
for PLAGL2 binding. In vivo Co-IP studies in HEK293 cells confirmed
the yeast two-hybrid results and show that PC2 binds to PLAGL2 via
the C-terminus of PLAGL2. This is consistent with previous reports
showing a direct interaction of the Mediator subunit, PC2, with other
transcriptional regulators, including the sterol regulatory element
binding protein (SREBP), and Smad factors 2/3 and 4 (Yang et al.,
2006; Kato et al., 2002). The ability of PC2 to interact with various
gene-specific transcription factors suggests that, as a component of
the ARC/Mediator complex, PC2 may play a key role in targeting
Mediator to a distinct set of promoters.
Full-length PLAGL2 expressed in the Co-IP studies was shown to be
nuclear, as previously reported (Zheng and Yang, 2005). We further
ascertained that, like PLAG1 (Braem et al., 2002), nuclear localization
was determined by the N-terminal domain of PLAGL2. Although we
did not attempt to specifically identify a nuclear localization signal,
PLAGL2 does contain a similar sequence (PRPR) at the same amino
acid location corresponding to theputative PLAG1nuclear localization
Fig. 8. Endogenous PC2 associates with the NCF2 promoter at the NCF2 TRR and is
required for PLAGL2 regulation of NCF2 in TNF-α-treated MM1 cells. (A) MM1 cells
were treated with TNF-α (20 ng/mL) prior to chromatin isolation (Ammons et al.,
2007). ChIP analysis was performed with no antibody (No Ab), negative control
antibodies (NegC), anti-PLAGL2, or anti-PC2 antibodies, as indicated. ChIP samples and
the input sample were subjected to NCF2 TRR-specific (upper panel) or GAPDH-specific
(lower panel) PCR amplification. Results are representative of three individual
experiments. (B) MM1 cells were mock transfected or transfected with control siRNA
(siGLO) or PC2 siRNA as indicated. Cells were treated with TNF-α (20 ng/mL) and RNA
was isolated and subjected to NCF2-specific (upper graph) and PC2-specific (lower
graph) qRT-PCR in triplicate. Data are presented as fold expression with mock
transfected, non-TNF-α-treated sample set to one. Results are representative of three
individual experiments. The PC2 siRNA, TNF-α-treated NCF2 sample was statistically
significantly different (PN0.05) from the mock transfected, TNF-α-treated sample and
is indicated (⁎).
Fig. 9. PC2 is important in PLAGL2 regulation of IGF-II. HEK293 cells were mock
transfected or transfected with control siRNA (siGLO) or PC2 siRNA as indicated,
followed by transfection with empty pcDNA3.1 vector (lanes 1, 3, and 5) or pcDNA3.1-
PLAGL2 expression plasmid (lanes 2, 4, and 6). RNA was isolated and subjected to RT-
PCR using IGF-II- and 28S-specific primers. Samples were separated on an agarose gel
(upper panel), and the IGF-II specific bands were subjected to densitometry (lower
panel). Results are representative of three individual experiments.
S.J. Wezensky et al. / Gene 452 (2010) 22–34
sequence (KRKR). Thus, it will be of interest to determine if the PRPR
sequence of PLAGL2 serves a similar function.
The binding of PLAGL2 to the NCF2 TRR in previous studies was
somewhat surprising, as there appeared to be no obvious binding site
for PLAGL2 within this sequence. In this report, mutagenesis of the
TRR showed that the nucleotide sequence required for PLAGL2
binding contained the PLAG1 consensus core sequence (GGRGGCC)
but lacked the G-cluster motif. Although this binding site is located on
the reverse strand, this arrangement is similar to the functional PLAG1
binding site characterized in the IGF-II promoter (Voz et al., 2000).
Mutagenesis of PLAGL2 zinc fingers demonstrated that zinc fingers 5
and 6 were important for TRR binding, with zinc finger 2 playing
much less of a role. These data are consistent with a previous report
demonstrating the importance of PLAGL2 zinc fingers 5 and 6 in
binding to the PLAG1 core sequence and of zinc finger 2 in binding to
the G-cluster (Hensen et al., 2002). The ability of PLAGL2 to bind
relatively efficiently to the core sequence alone demonstrates that,
like PLAGL1, PLAGL2 can also bind GC-rich DNA in the absence of the
G-cluster. These data support the idea that these family members can
recognize DNA-binding sites that are, in general, GC rich with some
variability in sequence, possibly leading to functional redundancy
between proteins and/or competition for similar target genes.
Elucidation of the complete set of PLAG target genes and DNA
recognition sites in their respective target promoters will be
important in addressing this issue.
As stated earlier, overexpression of PLAGL2 alone had little to no
affect on the activity of the NCF2 promoter-reporter plasmid in
HEK293 cells, although we demonstrated binding of endogenous
PLAGL2 to the NCF2 promoter in TNF-α-treated MM1 cells in a recent
study (Ammons et al., 2007). The lack of NCF2 promoter-reporter
activity by PLAGL2 alone, however, was not unprecedented, as similar
results were reported for PLAGL2 transactivation of an IGF-II
promoter-reporter (Ning et al., 2008). In those studies, overexpres-
sion of PLAGL2 had no significant effect on the activity of the IGF-II
promoter-reporter; however, when PLAGL2 was overexpressed with
Tip60, a PLAGL2-binding protein that modulates PLAGL2 transactiva-
tion through acetylation, the activity of the IGF-II promoter-reporter
was stimulated ∼2 fold. In contrast, another PLAGL2 target promoter,
SP-C, showed no enhanced activity in the presence of Tip60,
demonstrating that the effect of Tip60 on PLAGL2 transactivation
was promoter-specific. In this study, we show that PC2 enhanced
PLAGL2 transactivation of the NCF2 promoter 2- to 3-fold in
cooperation with PU.1. We also show the importance of endogenous
PC2 in PLAGL2-induced IGF-II expression, implying that the effect of
PC2 on PLAGL2 target promoter activity may be a general mechanism
of PLAGL2 transactivation. Additional studies, however, are required
to determine if PC2 is indeed being targeted to the IGF-II promoter via
PLAGL2 binding. Together, the findings presented here demonstrate
that PC2 is a modulator of PLAGL2 transactivation, and it will be of
interest to determine if PC2 plays a role in the regulation of all PLAGL2
responsive genes or, as suggested for Tip60 regulated PLAGL2
activation, if the effect is limited to a subset of PLAGL2 target
The data presented here support a model whereby PLAGL2, PU.1,
and PC2 physically associate with the NCF2 promoter directly, or
indirectly through protein-protein interactions, and that they
cooperate to enhance the activity of the NCF2 promoter-reporter.
The data suggest that PLAGL2 and PU.1 are required to bind to their
respective sites in the promoter, and that PC2 is targeted to the
promoter via the C-terminus of PLAGL2, the region required for
transactivation. We demonstrate that GFP-PLAGL2 interacts with
endogenous PC2 and that endogenous PC2 associates with a PLAGL2
target promoter in TNF-α-treated MM1 cells in a similar region
occupied by PLAGL2. In addition, we show that PLAGL2 regulation of
NCF2 in response to TNF-α is inhibited with PC2 knockdown, thus
demonstrating the physiological relevance of PC2 as an effector of
PLAGL2 transactivation. We cannot, however, rule out the possibility
that the interaction between PLAGL2 and PC2 is indirect, and that
there may be additional proteins acting as linkers between PLAGL2
and PC2, as suggested by the inconclusiveness of the sequential ChIP
studies (data not shown). The role of PC2 as a subcomponent of the
large multiprotein Mediator complex implies that the PC2-enhanced
NCF2 promoter activity is likely the result of Mediator recruitment to
the promoter via PLAGL2, allowing formation of a stable pre-
initiation complex. Additional studies are necessary to further
characterize this model and to determine if the effect of PC2 is
indeed through targeting Mediator to PLAGL2 target promoters.
Although there are many reports alluding to the role of the PLAG
proteins in key physiological processes, including oncogenesis, under-
standing the mechanisms whereby PLAG proteins regulate these
processes, including the identification of the complete set of target
genes and regulatory cofactors, is far from complete. This study further
contributes toour understandingofPLAGgeneregulation byestablish-
ing PC2 as a novel PLAGL2-binding protein and modulator of PLAGL2
transactivation. Considering the oncogenic potential of PLAG proteins,
elucidating their mechanisms of activation by identifying gene targets,
signaling pathways, and the functional relationship between family
members will be important in identifying avenues for early diagnosis
and novel therapeutic targets for treatment of diseases and/or
disorders associated with the aberrant expression of these proteins.
This work was supported in part by NIH grants RR-024237,
RR-020185, and RR-016455. Wilkison was supported by NIH grant
P20 RR16455-08 from the NCRR.
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