Neural retina leucine-zipper regulates the expression of
Ppp2r5c, the regulatory subunit of protein
phosphatase 2A, in photoreceptor development
Jung-Woong Kim, Sang-Min Jang, Chul-Hong Kim, Joo-Hee An, Eun-Jin Kang and
Kyung-Hee Choi
Department of Life Science (BK21 program), College of Natural Sciences, Chung-Ang University, Seoul, Korea
Introduction
Protein phosphatase 2A (PP2A) is a major cellular ser-
ine ⁄ threonine phosphatase that plays a critical role in
balancing phosphorylation signals that are important
for cellular proliferation and differentiation [1,2]. The
catalytic C-subunit of PP2A associates with the scaf-
folding A-subunit, and the A ⁄ C heterodimer also binds
to regulatory B-subunits to form a heterotrimeric holo-
enzyme [3]. B-subunits can be divided into four distinct
families on the basis of their homology, namely B
(B55 or PR55) [4–7], B¢ (B56 or PR61) [8–11], B¢¢
(PR48 ⁄ 59 ⁄ 72 ⁄ 130) [12,13] and B¢¢¢ (PR93 ⁄ 110) [14],
and the B56 family consists of at least five different
gene p roducts, a (PPP2R5A), b (PPP2R5B), c (PPP2R5C),
d (PPP2R5D), and e (PPP2R5E) [8]. The five B56 fam-
ily members have diverse functions, including a mitotic
checkpoint in Xenopus laevis and binding to APC pro-
tein, which acts as a scaffold for b-catenin, axin and
glycogen synthase kinase-b [15,16]. Moreover, B56e is
involved in Xenopus eye development through the insu-
lin-like growth factor–phosphoinositide 3-kinase–Akt
and hedgehog signaling pathways [17]. It is believed
that PP2A exercises regulatory flexibility and substrate
specificity through association of the core A ⁄ C hetero-
dimer with one of the regulatory B-subunits [1,18].

promoter region of Ppp2r5c.
Abbreviations
ChIP, chromatin immunoprecipitation; E, embryonic day; EMSA, electrophoretic mobility shift assay; GST, glutatione S-transferase;
NRE, neural retina leucine-zipper-response element; Nrl, neural retina leucine-zipper; NS, not significant; P, postnatal day; PP2A, protein
phosphatase 2A; siRNA, small interfering RNA; WT, wild type.
FEBS Journal 277 (2010) 5051–5060 ª 2010 The Authors Journal compilation ª 2010 FEBS 5051
precise molecular mechanisms underlying the transcrip-
tional control of PP2A genes and the effects of diverse
combinations of PP2A subunits have not yet been elu-
cidated.
Neural retina leucine-zipper (Nrl) belongs to the
basic motif leucine-zipper family of transcription
factors [19]. Nrl is conserved in vertebrates and is
specifically expressed in photoreceptors and the pineal
gland [19,20]. Nr1 is essential for rod differentia-
tion, and may act as a molecular switch in the deter-
mination of photoreceptor cell fate, as Nrl knockout
mice have a complete lack of rods but enhanced
S-cones [21]. In humans, missense mutations of NRL
are associated with autosomal dominant retinitis pig-
mentosa [22], and this disease may be a result of
altered transcriptional activity of the NRL [23]. Nrl
interacts with cone-rod homeobox [24], Flt-3-interact-
ing zinc-finger [25] and TATA box-binding protein
[26] to regulate the expression of rhodopsin [27],
NR2E3 [28], cGMP-phosphodiesterase-a, cGMP-phos-
phodiesterase-b [29,30] and rod-specific genes [21].
These observations have shown that Nrl plays a criti-
cal role in the differentiation of rod photoreceptors
that involves spatiotemporal regulation of its target

sites for MZF1, CREB, GATA1, Hsf1 ⁄ 2 and CdxA.
A
B
Fig. 1. A conserved region of the Ppp2r5c
promoter contains putative Nrl-binding
motifs. (A) The promoter sequences for
human, cow and mouse Ppp2r5c genes
were aligned using the multiple sequencing
alignment program
CLUSTAL W. Underlined
sequences represent putative NREs.
Asterisks are indicated within twenty
nucleotides. (B) The mouse Ppp2r5c
promoter (GeneID: 26931) was analyzed
with a motif searching program to identify
binding sites of transcription factors.
Consensus binding sites are underlined, the
Nrl-binding site is printed in bold, and the
transcription start site is shown as +1.
Regulation of Ppp2r5c expression by Nrl J W. Kim et al.
5052 FEBS Journal 277 (2010) 5051–5060 ª 2010 The Authors Journal compilation ª 2010 FEBS
Nrl increases the endogenous Ppp2r5c transcript
level and Ppp2r5c reporter gene activity
We first screened various cell lines to identify cells that
abundantly express Nrl mRNA and proteins (Figs S1B
and S2C). Mouse hippocampal HT22 cells showed
high-level expression of Nrl mRNA and protein. To
determine whether Nrl is truly a transcriptional regula-
tor of Ppp2r5c, HT22 cells were transiently transfected
with FLAG–CMV2–Nrl expression plasmids, and

contained NRE (NRE) (Fig. 2D, upper panel). As
expected, the NRE included full length of wild-type
promoter. (WT) and the NRE mutant significantly
induced luciferase reporter activity in an Nrl concen-
tration-dependent manner (Fig. 2D, lanes 3, 4, 7 and
8). However, the NRE-deleted mutant promoter con-
struct (DNRE) abolished the luciferase activity under
conditions of Nrl overexpression (Fig. 2D, lanes 5 and
Ppp2r5c
6 6
Rhodopsin
***
2
3
4
5
2
3
4
5
***
Fold increase
Fold increase
1
0
1
0
Mock
Mock
Flag-Nrl

8
10
**
***
***
Fold increase
2
0
Mock
Mock
Flag–Nrl Flag–Nrl
Fold increase
Ppp2r5c promoter
25
Luciferase
NRE
: WT
Luciferase
: ΔNRE
Luciferase
NRE
: NRE
–87
–800
–74
–260
**
***
5
10

used as an internal control. (C, D) HEK293
cells were cotransfected with Ppp2r5c
promoter–Luc and pCMV–b-galactosidase
with increasing amounts (1 lg and 2 lg) of
plasmids encoding Nrl cDNA. Forty-eight
hours after transfection, luciferase activity
was measured. All data were normalized to
b-galactosidase activity. Data are expressed
as the fold increase over relative luciferase
units, normalized to the control. The statisti-
cal significant levels were considered
significant at P < 0.05 (*), very significant at
P < 0.01 (**), obviously significant at
P < 0.001 (***), or not significant (NS).
J W. Kim et al. Regulation of Ppp2r5c expression by Nrl
FEBS Journal 277 (2010) 5051–5060 ª 2010 The Authors Journal compilation ª 2010 FEBS 5053
6). These results suggest that the NRE at )87 to )74
is responsible for the Nrl-mediated transcriptional acti-
vation of mouse Ppp2r5c.
Nrl binds to the Ppp2r5c promoter in vitro
As the Ppp2r5c promoter contains a putative NRE
and its transcripts were increased by Nrl, we con-
ducted an electrophoretic mobility shift assay (EMSA)
with glutatione S-transferase (GST)-fused recombinant
Nrl (Fig. S4) to determine whether Nrl induces
Ppp2r5c transcription through direct binding to the
proximal promoter region of Ppp2r5c. An oligonucleo-
tide containing the consensus Nrl-binding site at )154
to )143 of the Ppp2r5c promoter was used as a hot
probe. As shown in the left panel of Fig. 3, incubation

ina, we used postnatal day (P)10 mouse retina for the
quantitative ChIP assay, because Nrl expression was
highly upregulated after the P4 stage (data not shown).
The Ppp2r5c and rhodopsin promoters were specifically
precipitated with antibody against Nrl, but not with
rabbit control serum, in the mouse retina (Fig. 4B).
It was previously reported that rod–cone differentia-
tion is regulated by increases in the expression levels of
Nrl to modulate its specific target gene expression in
photoreceptor precursor cells [20,31]. To determine the
developmental stage-specific recruitment of Nrl to the
Ppp2r5c promoter, quantitative ChIP assays were con-
ducted with developing mouse retinas of various
stages, from embryonic day (E)15 to P42. Nrl binding
to the Ppp2r5c promoter increased approximately five-
fold from P10 to P14, and thereafter decreased to
basal levels until P21 (Fig. 4C). This sharp increase in
Fig. 3. Nrl directly binds to the Ppp2r5c pro-
moter consensus element in vitro. EMSA
showing the binding of Nrl to NRE sites in
the rhodopsin and Ppp2r5c promoters.
Lanes are as indicated below the autoradio-
graph. Two or four micrograms of purified
proteins was used for EMSA. For the
competition experiment, lane 8 included a
10-fold molar excess of unlabeled NRE
oligonucleotide. Lane 6 contains 0.1 lLof
antibody against Nrl, and lane 7 contains the
same quantity of rabbit serum as a negative
control for Nrl antibody. Arrowheads repre-

was enhanced between P10 and P14 (Fig. 4C).
It has recently been reported that PP2A may play
important roles in developing eyes, and the functions of
PP2A appear to be highly regulated by various regula-
tory subunits [17]. The mRNA isoforms of the PP2A A-
subunit and B-subunit (PP2A-Aa ⁄ b and PP2A-Ba ⁄ b ⁄ c)
have also been shown to be highly expressed in the
mouse retina [32]. In this study, we first attempted to
Rho promoter
Ppp2r5c promoter
Mouse retina
***
***
6
8
10
Input (%)
12
***
2
4
0
IP:
Serum Nrl
Serum Nrl
Ppp2r5c promoter
Rho promoter
HT22 cells
***
Input (%)

10
BA
E
F
Input (%)
0.4
0.6
0.8
1.0
1.2
ChIP: Ppp2r5c promoter
0.0
0.2
E15 E20 P0 P2 P4 P8 P10 P14 P21 P42
Input (%)
0.6
ChIP: Rho promoter
0.2
0.3
0.4
0.5
0.0
0.1
E15 E20 P0 P2 P4 P8 P10 P14 P21 P42
D
C
IP with Nrl antibody
IP with IgG
IP with Nrl antibody
IP with IgG

(**), obviously significant at P < 0.001 (***),
or not significant (NS).
J W. Kim et al. Regulation of Ppp2r5c expression by Nrl
FEBS Journal 277 (2010) 5051–5060 ª 2010 The Authors Journal compilation ª 2010 FEBS 5055
identify transcription factors that regulate PP2A gene
expression in retinogenesis. To accomplish this, we used
a motif searching program, and found several putative
transcription factors that can bind to the highly con-
served PP2A gene promoters. Among the various PP2A
genes, we found that the Ppp2r5c (PP2A-B56c), Ppp2r2b
(PP2A-B55b; data not shown) and Ppp3cc (protein
phosphatase 3, catalytic subunit c; data not shown)
genes have an NRE on their promoter region. These
findings suggest that Nrl plays an important role during
eye development through regulation of the expression of
PP2A genes, including Ppp2r5c. In a previous study,
Yoshida et al. evaluated the gene expression patterns of
developing and mature Nrl
) ⁄ )
mouse retina by using
microarray experiments, and found that Ppp2r5c was
downregulated 2.12-fold in Nrl
) ⁄ )
mouse retina when
compared to Nrl
+ ⁄ +
mouse retina [33]. The current
results of our in silico-based biochemical approaches
revealed that the in vivo observations reported by Yosh-
ida et al. in Nrl knockout mouse might have been

PP2A. These results support the potential benefits of
association between Nrl and Ppp2r5c as its target gene
during retinogenesis. Further investigations are needed
to define the crucial target substrate proteins of
Ppp2r5c and its molecular mechanisms in photorecep-
tor differentiation.
Experimental procedures
Cell culture and transfection
Mouse hippocampal HT22 cells were obtained from the
ATCC (Manassas, VA, USA). HT22 cells were maintained
in DMEM supplemented with 10% fetal bovine serum
(Invitrogen, Carlsbad, CA, USA) and penicillin–streptomy-
cin (50 units per mL). Transient transfection was conducted
using Lipofectamine 2000 (Invitrogen) according to the
manufacturer’s instructions.
Animal use
ICR strain mice (SAM IBRS#301) were originally purchased
from Samtaco (Osan, Korea), and bred and maintained at
the barrier facilities of Chun-Ang University (School of
Medicine) under a 12 h light ⁄ dark cycle. Mice were killed by
cervical dislocation, and the retinas were then excised rapidly
(with removal of the lens) on an ice plate, after which they
were stored at )70 °C. The Chung-Ang University Institu-
tional Review Board approved (approval No. 40) all proce-
dures involving mice and rabbits used in this study.
Plasmid constructs
The Nrl full-length coding region was amplified from E18
mouse eye cDNA generated by reverse transcriptase
(iNtRON Biotechnology, Sung-Nam, Korea), with the fol-
lowing primers: forward, 5¢-ATG GCT TTC CCT CCC

genomic DNA was removed from 5 lg of total RNA by
incubation with 10 Units of RNase-free DNase I (New
England Biolabs, Ipswich, MA, USA) and 2 Units of
RNase inhibitor (New England Biolabs) in diethylpyrocar-
bonate-treated water. The reaction mixture was incubated
for 1 h at 37 °C and then for 10 min at 60 °C. RNA con-
centrations were determined by spectrophotometric analy-
sis. All RNA isolates had an A
260 nm
⁄ A
280 nm
between 1.8
and 2.0, indicating that the isolated RNA was suitable for
subsequent analyses. Oligo-dT (Intron Biotechnology) was
used as the primer in the first step of cDNA synthesis.
Total RNA (1 lg) was combined with 0.5 lg of oligo-dT,
200 lm dNTPs and H
2
O, and then preheated at 75 °C for
5 min to denature the secondary structures. The mixture
was then cooled rapidly to 20 °C, after which 4 lLof5·
RT buffer, 10 mm dithiothreitol and 200 U of avian myelo-
blastosis virus reverse transcriptase (Intron Biotechnology)
were added to give a total volume of 20 lL. The RT mix
was incubated at 42 °C for 60 min, after which the reaction
was stopped by heating at 95 °C for 5 min. The expression
levels of mouse rhodopsin and Ppp2r5c mRNA were mea-
sured by quantitative real-time PCR with the following spe-
cific primers: rhodopsin, forward, 5¢-TCA AGC CGG AGG
TCA ACA AC-3¢; rhodopsin, reverse, 5¢-TCT TGG ACA

described by Hellman et al. [38]. The synthesized upper
oligonucleotides (1 lg) were incubated with [
32
P]ATP[cP]
(Perkin Elmer, Covina, CA, USA) and T4 polynucleotide
kinase (New England Biolabs, Ipswich, MA, USA) for 1 h at
37 °C for radiolabeling. To stop the kinase reaction, 10 mm
Tris (pH 7.5), 1 mm EDTA and 100 mm NaCl were added to
the tubes. Complementary strands were denatured at 100 °C
for 5 min and annealed at room temperature. The dsDNA
(oligonucleotides for the rho promoter, 5¢-ATC TCG CGG
ATG CTG AAT CAG CCT CTG GC-3¢ and 5¢-GCC
AGA GGC TGA TTC AGC ATC CGC GAG AT-3¢; oli-
gonucleotides for the Ppp2r5c promoter, 5¢-CCC TGA
AGC CAG GAT GAG CCG CAG GGA AAG-3¢ and
5¢-TGG AGC TC G CTG ATT GGC CAG AAG CTG CAA-
3¢) was used for the following EMSA assay. The DNAÆpro-
tein binding reaction was conducted in a mixture including
10· binding buffer [100 mm Tris ⁄ Cl (pH 7.5), 10 mm EDTA,
1 m KCl, 1 mm dithiothreitol, 50% (v ⁄ v) glycerol,
0.1 mgÆmL
)1
BSA), 4000 c.p.m. of
32
P-labeled oligonucleo-
tides and affinity purified GST–Nrl for 30 min at 30 °C. In
some cases, double-stranded cold oligomers were added as a
cold competitor. This mixture was incubated on ice for
10 min without antibody or for 20 min with antibody in the
absence of the radiolabeled probe, and then for 30 min at

of each experimental group was immunoprecipitated over-
night with antibodies against Nrl at 4 °C; this was followed
by incubation with 40 lL of protein A–agarose beads (Milli-
pore, Bedford, MA, USA) for an additional 1 h at 4 °C. The
immune complexes were eluted with 100 lL of elution buffer
(1% SDS and 0.1 m NaHCO
3
), and formaldehyde cross-links
were reversed by heating at 65 °C for 4 h. Proteinase K was
added to the reaction mixtures, which were then incubated at
45 °C for 1 h. DNA of the immunoprecipitates and control
input DNA were purified with the PCR purification kit (Qia-
gen, Valencia, CA, USA), and then analyzed by quantitative
real-time PCR with the rhodopsin and Ppp2r5c promoter-spe-
cific primers (rhodopsin, forward, 5¢-ATG AGA CAC CCT
TTC CTT TCT-3¢; rhodopsin, reverse, 5¢-GTA GAC AGA
GAC CAA GGC TGC-3¢; Ppp2r5c, forward, 5¢-CCC TCT
AAG AGC TGG GAT TCT-3¢; Ppp2r5c, reverse, 5¢-CAA
ACT GAA GCT CTC TGC AGC-3¢).
Statistical analysis
Statistical analysis of variances between two different exper-
imental groups was conducted with Tukey’s post hoc com-
parison test, using spss (Version 12). All experiments were
repeated at least three times. The levels were considered sig-
nificant at P < 0.05 (*), very significant at P < 0.01 (**),
obviously significant at P < 0.001 (***), or not significant
(NS).
Antibody production
Details are given in Doc. S1.
Western blotting

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