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]. This characteristic of PP2A contributes to its ability to regulate multiple cellular functions; however, the Keywords neural retina leucine-zipper; photoreceptor development; PP2A regulatory subunit; Ppp2r5c; target gene Correspondence K H. Choi, Department of Life Science (BK21 program), College of Natural Sciences, Chung-Ang University, 221 Heuksuk Dong, Dongjak Ku, Seoul 156-756, South Korea Fax: +82 2 824 7302 Tel: +82 2 820 5209 E-mail: khchoi@cau.ac.kr (Received 12 July 2010, revised 11 September 2010, accepted 11 October 2010) doi:10.1111/j.1742-4658.2010.07910.x Protein phosphatase 2A plays an important role in balancing phosphoryla- tion signals that are critical for cell proliferation and differentiation. Here, we report that Ppp2r5c (regulatory subunit of protein phosphatase 2A) expression was regulated by the transcription factor neural retina leucine- zipper (Nrl) through enhancement of its transcriptional activity on the Ppp2r5c promoter. Using electrophoretic mobility shift assays and chroma- tin immunoprecipitation, we also found that Nrl bound directly to the Nrl-response element on the Ppp2r5c promoter. The affinity of binding of Nrl to the Ppp2r5c promoter was tightly regulated during mouse photo- receptor development. Overall, these results suggest that Ppp2r5c expres- sion is regulated by Nrl during retinogenesis through direct binding to the 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 gene expression. In this study, we identified Nrl as a novel transcrip- tional factor that regulates Ppp2r5c gene expression in photoreceptor development. Furthermore, an unbiased motif search of Ppp2r5c promoter sequences revealed that the Ppp2r5c promoter has putative Nrl-binding sequences. Moreover, the functional roles of Nrl in Ppp2r5c transcription were examined in vitro and in vivo. We also present the association profiles of Nrl on the Ppp2r5c promoter during mouse photoreceptor development, with the goal of determining the critical stage for Nrl-mediated Ppp2r5c expression. Results Conserved sequences of the Ppp2r5c promoter contain Nrl-binding sites In the search for conserved putative regulatory elements, the sequence of the human Ppp2r5c pro- moter from )300 to )1 relative to the transcriptional start site was compared with corresponding regions of mouse and cow sequences, with clustal w. Highly conserved noncoding sequences were determined among these Ppp2r5c promoters with a minimal sequence similarity of 67% (Fig. 1A). To identify tran- scription factors that might bind to the Ppp2r5c pro- moter and regulate its expression, we used tfsearch software (Searching transcription factor binding sites, Version 1.3). As shown in Fig. 1B, the Nrl response element (NRE) was found at )154 to )143 from the transcription start site. Furthermore, the putative pro- moter region of the Ppp2r5c gene contained binding 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 Ppp2r5c mRNA expression was analyzed by quantita- tive real-time PCR. As shown in Fig. 2A, overexpres- sion of Nrl induced Ppp2r5c transcription (left panel), and rhodopsin expression by Nrl was also confirmed as a positive control (right panel). To further examine whether the increased expression of Ppp2r5c transcripts was specifically modulated by Nrl, we used Nrl small interfering RNA (siRNA) transfectants in which the expression of Nrl was approximately 60% abrogated (Fig. S3A,B). As shown in Fig. 2B, transfection of Nrl siRNA effectively decreased the levels of Ppp2r5c (Fig. 2B, right panel) and rhodopsin (Fig. 2B, left panel) mRNA. We then conducted luciferase reporter assays with Ppp2r5c promoters to test the Nrl-mediated transcriptional activity. The Ppp2r5c promoter frag- ment from 800 to )1 was cloned into luciferase repor- ter constructs that were transiently transfected with the Nrl expression plasmids into HEK293 cells, which do not express mRNA and protein of Nrl endoge- nously (Figs S1B and S2C). In the presence of exoge- nous Nrl, Ppp2r5c promoter activity was significantly increased by Nrl in a dose-dependent manner (Fig. 2C, left panel). Nrl also enhanced its known target rhodopsin promoter activity (Fig. 2C, right panel). To further investigate Nrl-mediated transcriptional activa- tion through a putative NRE, we performed luciferase reporter assays with various mutant forms of the mouse Ppp2r5c promoter in HEK293 cells. To accom- plish this, we cloned two different mutants, the NRE- deleted mutant (DNRE) and the truncated mutant that 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 Flag-Nrl Ppp2r5c 1.2 1.2 Rhodopsin *** *** 0.4 0.6 0.8 1.0 0.4 0.6 0.8 1.0 Fold increase Fold increase 0.2 0 0.2 0 Mock Mock Ppp2r5c 10 12 Rhodopsin *** 4 6 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 15 20 25 *** *** 0 5 Flag–Nrl Flag–Nrl Flag–Nrl Flag–Nrl Reporter : Mock WT ΔNRE NRE NS NS –1 A B C D Fig. 2. Nrl increases endogenous Ppp2r5c mRNA levels and Ppp2r5c reporter gene activity. (A, B) HT22 cells were transfected with plasmids expressing FLAG–Nrl and ⁄ or Nrl siRNA vectors. RNA was extracted, and quantitative real-time PCR analysis was con- ducted using primers specific for rhodopsin, Ppp2r5c and Gapdh. The Gapdh gene was 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 of hot probes with Nrl produced slower-migrating DNAÆprotein complexes in a dose-dependent manner (lanes 4 and 5), whereas the control GST protein alone did not form DNAÆprotein complexes (lanes 2 and 3). The presence of Nrl in the proteinÆDNA complex was verified with antibody against Nrl, which supershifted a portion of the Nrl–probe complex (lane 6), whereas the IgG negative control did not alter the binding pat- tern (lane 7). The Ppp2r5c probe–Nrl protein–Nrl anti- body triple complex disappeared when cold Ppp2r5c probe was added as a competitor (lane 8). The rhodop- sin promoter was used as a positive control (Fig. 3, right panel). These results show that Nrl binds directly to its response element ()154 to )143 from the tran- scriptional start site) located in the promoter region of Ppp2r5c in vitro. Nrl was recruited to the Ppp2r5c promoter during photoreceptor development We next conducted a chromatin immunoprecipitation (ChIP) assay with HT22 cells, to further examine the binding of Nrl to the Ppp2r5c promoter in vivo. Nrl binding to the Ppp2r5c and rhodopsin promoters was examined by quantitative real-time of ChIP samples with appropriate primers. As shown in Fig. 4A, Nrl antibody specifically immunoprecipitated regions of the Ppp2r5c and rhodopsin promoter containing the NRE, whereas normal rabbit serum, used as a negative control, did not precipitate the Ppp2r5c and rhodopsin promoters in the HT22 cell lines. To confirm the asso- ciation of Nrl with the Ppp2r5c promoter in mouse ret- 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- sent the specific shifted band (a, unbound probes; b and c, bands shifted by Nrl; d, supershifted band; e, nonspecific bands). These experiments were conducted at least three times, and similar results were obtained. Regulation of Ppp2r5c expression by Nrl J W. Kim et al. 5054 FEBS Journal 277 (2010) 5051–5060 ª 2010 The Authors Journal compilation ª 2010 FEBS the Nrl binding affinity for the Ppp2r5c promoter was tightly regulated during photoreceptor development when compared with the patterns of binding of Nrl to the rhodopsin promoter (Fig. 4C,D). The mRNA tran- script levels of Ppp2r5c and rhodopsin in mouse retina corresponded to the observed increase in Nrl binding affinity for their promoter regions (Fig. 4E,F). Taken together, these results clearly indicate that Nrl regu- lated the expression of Ppp2r5c transcripts through direct binding to the Ppp2r5c promoter during mouse photoreceptor development. Discussion In this study, we found that Ppp2r5c or B56c (PP2A regulatory subunit) expression was regulated by Nrl. Ectopic expression or knockdown of Nrl modulated the Ppp2r5c mRNA expression level and Nrl-mediated luciferase activity on the Ppp2r5c and rhodopsin pro- moters (Fig. 2). We also demonstrated that Nrl bound to NRE on the Ppp2r5c promoter both in vitro and in vivo (Figs 3 and 4). Furthermore, the binding affin- ity of Nrl for the Ppp2r5c promoter was tightly regu- lated during mouse photoreceptor development, and 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 (%) 3 4 5 6 ** 1 2 0 IP: Serum Nrl Serum Nrl Ppp2r5c 2 3 *** 1.5 2.5 1 0 Fold increase 0.5 Rhodopsin 20 40 Fold increase 60 *** 30 50 0 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 Fig. 4. Occupation of Nrl on its target gene promoters in vivo. (A, B) ChIP analysis was conducted on HT22 cells (A) and mouse retina (B). Primers specific for the promoter regions of the target genes were used to detect the presence of putative promoter regions in the immunoprecipitates (IP) by quantitative real-time PCR. Target genes examined included rhodopsin and Ppp2r5c. (C, D) An antibody against Nrl was used to immunoprecipitate the bound chromatin fragments from developmental mouse retina from mouse E15 to P42 to determine when each of the various proteins binds relative to transcription initiation. Primers specific to the promoter regions of the target genes [Ppp2r5c (C) and rhodopsin (D)] were used to detect the presence of the putative pro- moter regions by quantitative real-time PCR. Total fragmented sequences were detected by the specific target gene primer using quantitative real-time PCR as an input control. (E, F) E15 and P14 mouse retinal RNA was extracted, and quantitative real-time PCR analysis was performed using primers specific for rhodopsin, Ppp2r5c and Gapdh. The Gapdh gene was used as an internal control. The statistical 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 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 caused by direct binding of Nrl to the Ppp2r5c promoter as its direct downstream target gene. By the use of quantitative ChIP and quantitative real-time PCR assays in developing mouse retinas, we showed that Nrl was significantly associated with Ppp2r5c promoters in vivo (Fig. 4C). As in a study conducted by Peng [31], in which the association of Nrl with the rhodopsin promoter was shown, we also detected increased patterns of Nrl recruitment on the Ppp2r5c promoter from P10 to P14 in the mouse retina (Fig. 4C), as well as the induction of Ppp2r5c tran- scripts that corresponded to the transcription factor association (Fig. 4E). However, prior to the associa- tion of Nrl with the Ppp2r5c promoter in the early stage of development (between E15 and P8), we also detected the Ppp2r5c transcripts in the mouse retinas (data not shown). These findings raise the possibility that other important transcription factors in eye devel- opment, such as GATA1, CREB and Hsf1 [34–36], might regulate the expression of Ppp2r5c mRNA prior to the appearance of Nrl in the retina. Like the increase in Nrl expression during development, Ppp2r5c mRNA expression was precisely controlled by Nrl through direct binding to its target gene promoter. Despite intensive studies, the mechanisms of PP2A in photoreceptor cell physiology and development have yet to be fully elucidated. In this respect, we have dem- onstrated that Nrl, an important transcription factor in photoreceptor development, directly regulates the expression of Ppp2r5c, which is a regulatory subunit of 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 AGT CCC-3¢; reverse, 5¢-TCA GAG GAA GAG GTG TGT GTG-3¢. Amplified Nrl cDNA was introduced into the pCRII–TOPO vector (Invitrogen), and the Nrl clone was verified by DNA sequencing. Nrl-full length cDNA was subcloned into the pFLAG–CMV2 vector (Sigma- Aldrich, St. Louis, MO, USA) and the pGEX4T1 vector (Amersham Pharmacia Biotech, Uppsala, Sweden), and then verified by DNA sequencing. Luciferase reporter con- structs were generated by PCR amplification of the mouse rhodopsin and Ppp2r5c promoter sequences ()800 to )1) using mouse genomic DNA (rhodopsin promoter, forward, 5¢-ATG GTC ATC CCT CCC TGG-3¢; rhodopsin promoter, reverse, 5¢-CCA CGC CTG TGA CGT TGG-3¢; Ppp2r5c pro- moter (WT), forward, 5¢-TAG CAC TTC CTG ACT ATT- 3¢; Ppp2r5c promoter (WT), reverse, 5¢-AAA AAA AAG ACA AAC TGA-3¢; Ppp2r5c promoter (DNRE), forward, 5¢-TAG CAC TTC CTG ACT ATT-3¢; Ppp2r5c promoter (DNRE), reverse, 5¢-GAA GCT GCA ACT TAA AAT-3¢; Ppp2r5c promoter (NRE), forward, 5¢-AGC AGG TAC GGA TCA CTG-3¢; Ppp2r5c promoter (NRE), reverse, Regulation of Ppp2r5c expression by Nrl J W. Kim et al. 5056 FEBS Journal 277 (2010) 5051–5060 ª 2010 The Authors Journal compilation ª 2010 FEBS 5¢-AAA AAA AAG ACA AAC TGA-3¢). The PCR product was HindIII-digested, introduced into the pGL4.12 basic vector (Promega, Madison, WI, USA), and verified by DNA sequencing. RNA preparation and quantitative real-time PCR Total RNA was isolated from 100 mg of mouse retina and various cell lines with TRIzol solution (Invitrogen), accord- ing to the manufacturer’s specifications. Contaminated 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 CGG TAG CAG AG-3¢; Ppp2r5c, forward, 5¢-AGT ACC TGG GGA TTG GC-3¢; Ppp2r5c, reverse, 5¢-CAT GGC TTG ATA TAC AAC GC-3¢; Gapdh, forward, 5¢-GGG CAC TTA CGG GTG TTA GA-3¢; Gapdh, reverse, 5¢-CCC TGT CTG GTT TCC A CA GT-3¢. The primers were designed with primer 3, and cross-checked by a blast search of the NCBI da tabase. The specificity of each of the amplified prod ucts generated was confirmed by melting curve analysis. The iQ SYBR Green PCR Supermix (Bio-Rad, Hercules, CA, USA) and the CFX96 Real-Time PCR Detection System (Bio-Rad) were used to detect the real-time quantitative PCR products of reverse-transcribed cDNA samples, according to the manufac- turer’s instructions. T he Gapdh gene was used for normalization. The relative mRNA e xpression was calculated by the 2 )(DDCt) method, as previously described [37]. PCR was conducted in duplicate for each experimental condition tested. Luciferase assay HEK293 cells were cultured in 60 mm dishes and transfect- ed using Lipofectamine 2000, with the luciferase reporter constructs (0.1 lg), pCMV–b-galactosidase and FLAG–Nrl. The cells were lysed in reporter lysis buffer 48 h after trans- fection (Promega). Cell extracts were analyzed with the luciferase reporter assay system, using a glomax luminome- ter (Promega). Luciferase activities were normalized on the basis of the b-galactosidase activity of the cotransfected vector. All transfection experiments were repeated indepen- dently at least three times. EMSA Oligonucleotide labeling and EMSA were conducted as 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 30 °C in the presence of the radiolabeled probe, after which it was resolved on a 10% acrylamide gel that had been prerun at 100 V for 1 h with 400 mm Tris ⁄ acetic acid ⁄ EDTA buffer. The loaded gel was run at 200 V for 90 min, dried and then placed on Kodak X-ray film (Eastman Kodak, Rochester, NY, USA) to generate an autoradiogram. The film was developed after overnight exposure at )20 °C. ChIP A ChIP assay was conducted following the protocol provided by Upstate Biotechnology (Lake Placid, NY, USA). Briefly, the indicated mouse retinal tissues were cut into small pieces (1–3 mm 3 ), and the tissues were cross- linked with 1% paraformaldehyde (Sigma-Aldrich, St. Louis, MO, USA) in NaCl ⁄ P i for 15 min at 37 °C. HT22 cells were also treated with 1% paraformaldehyde. The cells were then washed with ice-cold NaCl ⁄ P i and resuspended in 200 lL of SDS sample buffer containing a protease 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 5057 inhibitor mixture. The suspension was sonicated three times for 10 s with a 1 min cooling period on ice, after which it was precleared with 20 lL of protein A–agarose beads blocked with sonicated salmon sperm DNA for 30 min at 4 ° C. The beads were then removed, after which the chromatin solution 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 Details are given in Doc. S2. 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Comp Biochem Physiol A Mol Integr Physiol 146, 131–140. 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 5059 37 Livak KJ & Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2[-Delta Delta C(T)] method. Methods 25, 402– 408. 38 Hellman LM & Fried MG (2007) Electrophoretic mobility shift assay (EMSA) for detecting protein– nucleic acid interactions. Nat Protoc 2, 1849–1861. Supporting Information The following supplementary material is available: Doc. S1. Antibody production. Doc. S2. Western blotting. Fig. S1. Expression of neural retina Nrl mRNA. Fig. S2. Generation of the polyclonal antibody for Nrl and expression of Nrl proteins in various cell lines. Fig. S3. Efficiency of Nrl siRNA on HT22 cells. Fig. S4. Affinity purification of GST–Nrl proteins. 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 by the authors. Such materials are peer-reviewed and may be re-organized for online delivery, but are not copy-edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors. Regulation of Ppp2r5c expression by Nrl J W. Kim et al. 5060 FEBS Journal 277 (2010) 5051–5060 ª 2010 The Authors Journal compilation ª 2010 FEBS . Neural retina leucine-zipper regulates the expression of Ppp2r5c, the regulatory subunit of protein phosphatase 2A, in photoreceptor development Jung-Woong. regulate the expression of Ppp2r5c mRNA prior to the appearance of Nrl in the retina. Like the increase in Nrl expression during development, Ppp2r5c mRNA expression