Cloning and characterization of mr-s, a novel SAM domain protein, predominantly expressed in retinal photoreceptor cells
- Tatsuya Inoue†1, 2, 3,
- Koji Terada†1,
- Akiko Furukawa1, 4,
- Chieko Koike1,
- Yasuhiro Tamaki3,
- Makoto Araie3 and
- Takahisa Furukawa1, 2Email author
© Inoue et al; licensee BioMed Central Ltd. 2006
Received: 01 November 2005
Accepted: 16 March 2006
Published: 16 March 2006
Sterile alpha motif (SAM) domains are ~70 residues long and have been reported as common protein-protein interaction modules. This domain is found in a large number of proteins, including Polycomb group (PcG) proteins and ETS family transcription factors. In this work, we report the cloning and functional characterization of a novel SAM domain-containing protein, which is predominantly expressed in retinal photoreceptors and the pineal gland and is designated mouse mr-s (major retinal SAM domain protein).
mr-s is evolutionarily conserved from zebrafish through human, organisms through which the mechanism of photoreceptor development is also highly conserved. Phylogenetic analysis suggests that the SAM domain of mr-s is most closely related to a mouse polyhomeotic (ph) ortholog, Mph1/Rae28, which is known as an epigenetic molecule involved in chromatin modifications. These findings provide the possibility that mr-s may play a critical role by regulating gene expression in photoreceptor development. mr-s is preferentially expressed in the photoreceptors at postnatal day 3–6 (P3-6), when photoreceptors undergo terminal differentiation, and in the adult pineal gland. Transcription of mr-s is directly regulated by the cone-rod homeodomain protein Crx. Immunoprecipitation assay showed that the mr-s protein self-associates mainly through the SAM domain-containing region as well as ph. The mr-s protein localizes mainly in the nucleus, when mr-s is overexpressed in HEK293T cells. Moreover, in the luciferase assays, we found that mr-s protein fused to GAL4 DNA-binding domain functions as a transcriptional repressor. We revealed that the repression activity of mr-s is not due to a homophilic interaction through its SAM domain but to the C-terminal region.
We identified a novel gene, mr-s, which is predominantly expressed in retinal photoreceptors and pineal gland. Based on its expression pattern and biochemical analysis, we predict that mr-s may function as a transcriptional repressor in photoreceptor cells and in pinealocytes of the pineal gland.
In the development of the mammalian retina, a diverse range of cell types is generated from a pool of multipotent retinal progenitor cells. Among these cell types, photoreceptors account for over 70% of all cells in the retina. In vertebrates, there are two classes of photoreceptors, rods and cones. Rods are sensors of dim light, while cones function in bright light and are responsible for color vision. Phototransduction, a series of signal amplifications detecting a single photon of light, is initiated by the capture of light with 11-cis-retinal, a chromophore bound by the opsin proteins: rhodopsin in rods and cone opsins in cones. The proteins that carry out phototransduction are located in an elaborate and highly specialized membranous structure, the outer segment. The outer segment appears to be relatively fragile, degenerating in response to many environmental and/or genetic perturbations. In the rodent retina, the production of specific cell types during development progresses in a general order [1, 2]. Rod photoreceptor generation peaks around the time of birth. Cone photoreceptors, ganglion cells, horizontal cells and amacrine cells are generated earlier, while Müller glia and bipolar cells are generated later. This production of different cell types at different times appears to derive from differences in the intrinsic properties of progenitor cells involved in the transcription or chromatin modification. Recent studies identified several important transcription factors of photoreceptor development [3–7]. Two Otx family homeobox genes, Otx2 and Crx, play essential roles in early cell fate determination and terminal differentiation of photoreceptors [3, 8, 9]. In the absence of Otx2 function, differentiating photoreceptor cells are converted to amacrine-like neurons . Crx, a downstream target of Otx2, controls the transcription of various photoreceptor cell-specific genes and is essential for the formation of outer segments, synaptic terminals, and phototransduction pathways [8, 10]. Crx transcripts begin to be expressed in developing photoreceptors at embryonic day 12.5 (E12.5) in the mouse and a strong upregulation of Crx transcription is apparent across the differentiating photoreceptors at postnatal day 6 (P6). Photoreceptor cells in the Crx knockout (KO) mice exhibit a dramatic reduction of many photoreceptor molecules including visual pigments and develop neither photoreceptor outer segments nor a synaptic terminus [8, 10]. In addition, mutations of human CRX have been demonstrated to be associated with three types of photoreceptor diseases: autosomal dominant cone-rod dystrophy 2, autosomal dominant-type retinitis pigmentosa, and Leber's congenital amaurosis (LCA) [11–14]. While Otx2 and Crx control general photoreceptor development, three other transcription factors, TRβ2, Nrl, and Nr2e3 regulate the specification of photoreceptor cell types [4, 5, 7].
SAM domains (also known as Pointed, HLH, or SPM domains) are ~70 residues long and have been reported as common protein-protein interaction modules [15–17]. This domain is found in a large number of proteins, including Polycomb group (PcG) proteins , serine threonine kinases , Eph family receptor tyrosine kinases , the p73 tumor suppressor , the RNA-binding protein Smaug , diacylglycerol kinases [23, 24], yeast mating type signaling proteins [19, 25] and ETS family transcription factors [26, 27]. The PcG proteins are transcriptional repressors that maintain gene silencing during development [28–30]. In mammals, PcG proteins are also implicated in Hox gene regulation. Their biological activity lies in stable silencing of specific sets of genes through chromatin modifications. A member of polycomb repressive complex 1 (PRC1), ph, contains a SAM domain at the C-terminal, and PRC1 complex is known to form helical, head-to-tail polymers through its SAM domain . These polymeric structures mediate the formation of a higher order chromatin structure and possibly stabilize the repressed state of Hox genes.
In this study, we identified a novel gene, mr-s, which encodes a SAM domain-containing protein. The SAM domain of mr-s is most closely related to that of ph mouse ortholog, MPH1/Rae28. mr-s is predominantly expressed in retinal photoreceptors when they undergo terminal differentiation, and adult pineal gland. The expression of mr-s is directly regulated by Crx. Moreover, mr-s is localized in the nucleus and can self-associate through its SAM domain-containing region. We also found that mr-s protein fused to GAL4 DNA-binding domain functions as a transcriptional repressor. These findings suggest that mr-s functions as a member of a transcriptional repressor complex in retinal photoreceptor development.
Cloning of mouse mr-s
Expression of mr-sin the developing retina and the pineal gland
To determine the tissue specificity of mr-s expression, the expression of the mr-s gene in various adult tissues was examined by Northern hybridization (Fig. 2H). As a control, P7 retinal RNA was used. Four bands corresponding to 7.2-kb, 4.0-kb, 2.5-kb and 2.2-kb were detected in P7 retina. The 2.2-kb band corresponds to the cDNA characterized in this study. The larger bands, possibly alternative spliced transcripts, have not yet been characterized. The mr-s probe did not detect a band in the adult tissues examined, indicating that these tissues do not express mr-s at a level comparable to that of the developing retina.
Previous reports revealed that many photoreceptor-specific genes are also expressed in the pineal gland . We examined the expression of mr-s transcripts in the whole embryo, whole body, retina, pineal gland, brain, liver and other organs at various stages by RT-PCR (Fig. 2I and data not shown). We amplified PCR fragments of 292 bp and 452 bp with primer pairs for genes encoding mouse mr-s and G3PDH, respectively. In E13 whole embryo and P0 whole body (except for the eye), no mr-s signal was detected. As expected, we observed that mr-s is expressed in the P7 and adult pineal gland. In the P7 and adult brain, liver and several other organs, the RT-PCR amplified band of mr-s was not detected (Fig. 2I and data not shown). Our data showed that mr-s is predominantly expressed in developing photoreceptors and the pineal gland.
Regulation of mr-stranscription by Crx homeodomain protein
To further examine whether Crx regulates mr-s transcription directly or not, we next performed a luciferase assay using the 1.2-kb proximal promoter region of mr-s fused to a luciferase gene as a luciferase reporter (Fig. 3D, Pro1.2k) and the Crx, Otx2, Nrl expression vectors, respectively. This 1.2-kb region of the mr-s upstream sequence contains three Crx binding consensus sequences. As shown in Fig. 3E, the luciferase activity was significantly upregulated when the Crx or Otx2 expression vector was co-introduced with Pro1.2k into HEK293T cells, while the luciferase activity was not altered when the Nrl expression vector was co-introduced. A previous report suggested that the transcriptional activity of Crx is augmented with Nrl when the rhodopsin promoter was used as a reporter gene . On the other hand, our present data showed that the luciferase gene expression was not upregulated when both Crx and Nrl expression vectors were co-introduced with Pro1.2k compared to the activity when the Crx only expression vector was introduced. This may be due to cell type differences because a cell type of retinal/pineal origin was not used in our luciferase assay. In addition, our present data showed that Otx2, which is reported to have the same binding consensus as Crx, also transactivated mr-s expression. As shown in Fig. 2A–F, the expression pattern of mr-s correlates with that of Crx. In contrast, the transcripts of Otx2 are mainly detected in the photoreceptor layer at embryonic stages. Therefore, we concluded that mr-s transcription is directly regulated mainly by Crx.
We also constructed reporter vectors in which mutations were introduced at the three Crx binding sites (Fig. 3D, mut1259, mut198, mut72, mut all). Then the Crx expression vector was co-introduced with mut1259, mut198, mut72 and mut all, respectively (Fig. 3F). The transactivaton activity by Crx was clearly reduced when mut198 or mut72 was co-introduced. On the other hand, when mut1259 was co-transfected, the transactivation activity by Crx was not altered. These results suggest that the Crx binding sites 72-bp and 198-bp upstream from the transcription initiation site are crucial for the direct regulation of mr-s transcription by Crx.
Self-association of mr-s protein
To investigate whether the mr-s protein self-associates mainly through the SAM domain, two site-directed mutations were generated in the SAM domain of mr-s (Fig. 1B, arrows). These mutations alter residues that are conserved in the SAM domain of ph and previous report indicates that these mutations of ph-SAM cause significant reduction in binding activity to the other SAM domain-containing protein, Sex comb on midleg (Scm) (41). Based on this result, we introduced two types of site-directed mutations, which correspond to the mutations introduced in ph protein, into Flag-tagged full-length mr-s (Flag-W404A and Flag-G453A). We found that Flag-W404A binding activity was significantly reduced and Flag-G453A binding activity was also slightly reduced compared to Flag-mrs (Fig. 5C). These results, together with yeast two-hybrid GAL4 assay, indicate that the mr-s protein self-associates strongly through its SAM domain and weakly through the N-terminus portion lacking SAM domain.
The subcellular localization of mr-s protein in mammalian cells
The GAL4-mr-s fusion protein functions as transcriptional repressor
Taken together, our findings suggest that DBD-mrs functions as a transcriptional repressor and that the repression activity of mr-s is not due to a homophilic interaction through its SAM domain but to the C-terminal region (amino acids 463 to 542).
In the present study, we identified a novel gene, mr-s, which is predominantly expressed in retinal photoreceptors and the pineal gland. The peak of mr-s expression in the developing retina is around P6. This expression pattern correlates with the rapid increase of Crx, rhodopsin and other photoreceptor genes around P6-P8. Around P6, the outer plexiform layer becomes visible and the outer layer of retina separates into two layers, ONL and INL. At the same time, photoreceptors begin to undergo terminal differentiation, forming the outer segment. We therefore hypothesized that mr-s is a key molecule in the late development of photoreceptors.
We previously reported that Otx2 and Crx have a critical role in photoreceptor development and that Otx2 directly regulates Crx transcription [3, 9]. In situ hybridization and RT-PCR showed significant reduction of mr-s signal in the Crx KO retina and pineal gland. Furthermore, the luciferase assay demonstrated that Otx2 and Crx may directly upregulate the transcription of mr-s in mammalian cells. In retinal photoreceptor cells, the Otx2 transcripts are not highly expressed at P6-P9, while the Crx transcripts are strongly detected around P6. Therefore, our results strongly suggest that mr-s transcription is directly regulated by Crx. In the present study, Nrl, a photoreceptor-specific transcription factor that is highly expressed in photoreceptors at the postnatal stage, did not affect the transcription of mr-s. This finding is actually consistent with the analysis of the Nrl KO mouse which was recently reported . The expression profiles of wild-type and Nrl KO retinas at P2, P10 and 2 months were analyzed and mr-s was not included in 161 differentially expressed genes in the Nrl KO retina.
Previous reports suggested that the SAM domain is a protein-protein interaction module. The SAM domain of the mr-s protein is closely related to that of ph and TEL, whose SAM domains can form a helical, head-to-tail polymeric structure and mediate the formation of a higher order chromatin structure. To characterize the biochemical function of mr-s, we performed yeast two-hybrid screening using full-length mr-s as the bait. As a result, the most frequent positive clones (5/28) in the screening were the cDNA fragments containing the SAM domain of mr-s. This strongly suggests that mr-s self-associates through its SAM domain. An immunoprecipitation assay, using two site-directed mutants of the SAM domain of mr-s, demonstrated that the mr-s protein can self-associate through its SAM domain in mammalian cells. While we did not address the question whether the SAM domain of mr-s forms a polymeric structure in the present study, the phylogenetic analysis of SAM domain of mr-s and other SAM domain-containing molecules suggests that mr-s can form head-to-tail polymer and mediate gene silencing by spreading repressive complexes along the chromatin similar to ph and/or TEL. Although our results in the immunoprecipitation assay demonstrated that the N-terminal constructs lacking a SAM domain still interact with each other, these results do not fit into the head-to-tail polymer model. We cannot exclude the possibility that the resulting protein-protein interaction of mr-s is an artifact of the overexpression conditions. The issue of whether or not mr-s forms a polymer awaits future analysis.
A previous report indicated that TEL contains a sequence-specific DNA binding domain, namely the ETS domain, and binds to specific sites via its ETS domain . TEL could serve to nucleate a polymer, which would spread by oligomerization of the SAM domain. In contrast to TEL, ph does not contain an obvious sequence-specific DNA binding motif (16). Therefore, its initial binding to the template may require protein-protein interactions with other sequence-specific transcriptional repressors. The segmentation gene-encoding transcriptional repressors such as Hunchback have a role in recruiting SAM domain-containing PcG proteins, which can spread along the template via polymerization . Since mr-s does not contain obvious DNA binding motifs, we suppose that there is a sequence-specific transcription factor(s) which interacts with mr-s. However, we did not find any transcription factors in the present yeast two-hybrid screening.
We also found that full-length mr-s fused to the GAL4 DNA binding domain (DBD-mrs) functions as a transcriptional repressor. This may support the idea that mr-s is involved in repressive complexes similar to other SAM domain-containing proteins. Our results, however, showed that the self-association of mr-s through its SAM domain is not essential for the transcriptional repressive activity. Polymerization of the SAM domain has been previously reported to be essential for the repressive functions of ph and TEL [43, 44]. On the other hand, human lethal(3) malignant brain tumor (H-L(3)MBT) protein, which also contains a SAM domain at the C-terminus, was reported as a transcriptional repressor and the repressor activity of H-L(3)MBT required mainly the presence of the MBT repeats but not the SAM domain . To determine the transcriptional repressor region of the mr-s protein, we performed a luciferase assay using site-directed mutants (DBD-W404A and DBD-G453A). The result showed that the reduced binding ability of self-association partially compromises the transcriptional repressive activity of mr-s (Fig. 7D). However, the repressive effect was more significant when DBD-tail, which does not contain SAM domain, was co-introduced with the 5xGAL4-pGL3 reporter plasmid. Therefore, we conclude that a region from amino acids 463 to 542 is mainly responsible for the repressor activity in the case of mr-s. Evolutionary conservation of the C-terminal 80 aa region of mr-s from zebrafish through human may underlie the functional importance of this region.
Two distinct multiprotein PcG complexes, PRC1 and PRC2, have been identified. PRC2 is involved in the initiation of silencing and contains histone deacetylases (HDACs) and histone methyltransferases, which can methylate histone H3 lysine 9 and 27, marks of silenced chromatin. PRC1, including ph, recognizes the histone H3 lysine 27 mark set by PRC2 and maintains a stable state of gene repression in which PRC1 blocks chromatin remodeling by the trithorax group-related SWI-SNF complex [46, 47]. Therefore, the mechanism of repression by PRC2 is thought to be HDAC-dependent while the mechanism of repression by PRC1 appears to be HDAC-independent. Our luciferase assay also showed that transcriptional repression of DBD-mrs was not affected by the addition of various concentrations of trichostatin A, a potent HDAC inhibitor (data not shown). Therefore, we speculate that the mechanism of repression of mr-s may be HDAC-independent and more similar to that of PRC1 complex.
In the present study, the biochemical experiments demonstrate that the mr-s protein functions as a transcriptional repressor and possibly down-regulates the spatial and temporal expression of the target genes during retinal photoreceptor development. Our data also revealed that the repressive activity of mr-s is mainly due to the C-terminal region (amino acids 463 to 542). However, downstream targets of mr-s still remain unclear. In situ hybridization showed that the peak of mr-s expression is around P6, when retinal photoreceptors undergo terminal differentiation. We hypothesize that the target genes of mr-s might be non-photoreceptor genes. In this case, mr-s may suppress the expression of non-photoreceptor genes in rod and cone photoreceptors. There may be another possibility that mr-s is involved in cell fate determination of rod photoreceptors versus cone photoreceptors. While cone photoreceptors are born during the early embryonic stages of mouse retinogenesis, rod photoreceptors are born primarily in the late embryonic and early postnatal period . The expression pattern of mr-s may suggest that mr-s is expressed in rod photoreceptors but not in cone photoreceptors as well as Nr2e3, which is known as a transcriptional repressor and is thought to down-regulate cone photoreceptor-specific genes. To clarify the biological function of mr-s, analysis of mice with targeted disruptions of mr-s will be very important.
Here we identified mouse mr-s, which is predominantly expressed in retinal photoreceptors and the pineal gland. mr-s is evolutionarily conserved from zebrafish through to human, suggesting a significant role of mr-s in photoreceptor development. Our present data suggest that mr-s protein localizes in the nucleus and can self-associated mainly through the SAM domain. Moreover, mr-s protein fused to GAL4 DBD functions as a transcriptional repressor. The repressive activity of mr-s is due to its C-terminal region (amino acid 463 to 542). Taken together, mr-s is a novel repressor molecule possibly involved in the development of retinal photoreceptors and pineal gland.
Isolation of mouse mr-scDNA
We used the bioinformatics method Digital Differential Display (NCBI, UniGene) to screen novel mouse genes expressed preferentially in the retina. Some of the clusters in the UniGene database were mainly from mouse retinal cDNAs. One clone in these clusters (#Mm. 246385) has a weak homology with the polyhomeotic family genes. A 735-bp cDNA fragment of this clone, encoding amino acids 140–384 was amplified by RT-PCR from mouse P0 retinal cDNA. This fragment was used as the probe for library screening, in situ hybridization and Northern hybridization. A mouse P0-P3 retinal cDNA library was screened using this mouse cDNA fragment. Positive bacteriophage clones were isolated and the full-length mr-s fragment was inserted into pBluescriptII (Stratagene). DNA sequencing was performed on both strands by the cycle sequencing method. The nucleotide sequence for mr-s gene has been deposited in the GenBank database under GenBank Accession Number #AY458844.
In situ hybridization
The 735-bp cDNA fragment of mr-s amplified by RT-PCR was used as a probe for in situ hybridization. In situ hybridization was performed as described previously .
Cell culture and transfection
HEK293T cells were maintained at 37°C in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (Sigma), 100 IU/ml penicillin and 100 μg/ml streptomycin. Transient transfection of HEK293T cells was carried out using calcium phosphate method or Fugene6 transfection reagent (Roche). Y79 retinoblastoma cells were maintained in Iscove's modified Dulbecco's medium with 4 mM L-glutamine adjusted to contain 1.5 g/L sodium bicarbonate, 20% FBS. Transient transfection was carried out using TransIt LT1 (Mirus).
RNA was extracted from the tissues of adult mice using Trizol (Invitrogen). A 5 μg of total RNA was electrophoresed in a 1.0% agarose-formaldehyde gel and transferred to a nylon membrane (Zeta-Probe GT, Bio-Rad). The 735-bp cDNA fragment encoding the SAM domain of mr-s was used as a probe for hybridization. Hybridization was performed according to the manufacturer's protocol. Washes with increasing stringency were performed, the last being at 50°C in 0.1× standard saline citrate/0.1% sodium dodecyl sulfate (SDS).
Total RNA was isolated from each tissue using Trizol. A 1 μg of total RNA was reverse transcribed using SuperscriptII (Invitrogen). RT-PCR primers, which span introns, for detection of mr-s cDNA were 5'-TGTCCAGCCCAGCCAACCCAAGGAGACGACA-3' and 5'-TGTGGTCTCCTCATCAGTGAAGA-3'. Product size was 292 bp (positions 965–1256 of Genbank Accession Number #AY458844). Primer pairs for mouse G3PDH were 5'-ACCACAGTCCATGCCATCAC-3' and 5'-TCCACCACCCTGTTGCTGTA- 3' which amplified a 452-bp product (positions 587–1038 of Genbank Accession Number #BC85275).
Yeast two-hybrid screening and GAL4 assay
We carried out yeast two-hybrid experiments using the MATCHMAKER GAL4 two-hybrid system 3 (BD Bioscience) as recommended by the manufacturer. We cloned the full-length mr-s into the pGBKT7 vector and used it to screen a library of mouse P0-P3 retinal cDNAs in the pGADT7 vector. Transformants that conferred growth were picked, isolated and re-introduced with the bait into AH109 to confirm interaction. Plasmid DNA was isolated from yeast using RPM Yeast Plasmid Isolation Kit (Qbiogene). In order to confirm the protein interactions, single colonies were picked and grown individually in synthetic complete media lacking leucine, tryptophan and containing X-gal. After 4–5 days, X-gal positive clones were selected and analyzed. To test self-interaction of mr-s, we subcloned the full-length, N-terminus (amino acids 1–400) and C-terminus (amino acids 391–542) of mr-s into pGBKT7 and pGADT7 vectors. We assessed interactions by scoring blue color on plates of medium containing X-gal.
For immunoprecipitation assay, 4× hemagglutinin (HA) or 3× Flag tagged cDNA fragment encoding full-length mr-s (full-HA and Flag-mrs), a cDNA fragment encoding amino acids 1–400 (ΔSAM-HA and Flag-ΔSAM), and a cDNA fragment encoding amino acids 400–542 (Flag-SAM) were subcloned into the pcDNA3 (Invitrogen) expression vector. We also constructed two site-directed mutants, Flag-W404A and Flag-G453A, using PCR. Each of these mutations was also introduced into Flag-mr-s. We transfected HEK293T cells with 5 μg of plasmid DNA per 6 cm dish by calcium phosphate method. Approximately 48 hr after transfection, cells were harvested in immunoprecipitation buffer (50 mM Tris-HCl [pH 7.5], 1 mM EDTA, 2 mM MgCl2, 150 mM NaCl, 1 mM phenylmethylsulfonyl fluoride [Wako], 1% Nonidet P-40, 10% glycerol) in the presence of protease inhibitor cocktail tablets (Roche). For each reaction, 1 mg of cell lysate was mixed with 0.5 μg of anti-Flag antibody (SIGMA, F3165) and 15 μl of protein G-Sepharose (Amersham) on a rotating wheel at 4°C for 2 hrs. Protein concentration was determined by the BCA protein assay system (Pierce). The beads were then washed three times with immunoprecipitation buffer and followed by three times with wash buffer (50 mM Tris-HCl [pH 7.5], 150 mM NaCl, 20 mM MgCl2). Proteins were then boiled for 5 min in SDS sample buffer (1% SDS, 1 mM Tris [pH 6.8], 40 mM DTT, 4% glycerol, 0.01% pyronine Y). The supernatants were fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to the nitrocellulose membrane (Trans-Blot Transfer Medium, Bio-Rad). Western blotting was performed with rabbit polyclonal anti-HA antibody (Santa Cruz, #sc-805). Signals were detected with horseradish peroxidase- conjugated goat anti-rabbit IgG and ECL plus Western Blotting Detection System (Amersham).
Subcellular localization analysis
We transfected the plasmid encoding HA-tagged full-length mr-s into HEK293T cells, seeded cells on coverslips coated with collagen 48 hr after transfection, fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) at room temperature for 20 min, and washed with PBS. Then we permeabilized cells in PBS containing 0.1% Triton X-100 for 15 min, washed again with PBS, and incubated in PBS containing 0.1% bovine serum albumin (BSA) for 30 min at room temperature. We incubated cells with anti-HA antibody overnight at 4°C (1:200 in PBS containing 0.2% BSA). The following day, we washed cells three times with PBS and incubated with a Cy3-conjugated goat IgG against rabbit IgG (1:400 in PBS, Jackson ImmunoReseach Laboratories) for 30 min. We rinsed cells three times with PBS, followed by observation using a confocal microscope FV300 (Olympus) equipped with a 60× objective lens.
For transcriptional analysis of the 1.2-kb promoter region of mr-s, we constructed a reporter plasmid (pro1.2k) by subcloning a 1.2-kb upstream genomic fragment of mr-s gene into a pGL3 luciferase reporter plasmid (Promega). The expression vectors were constructed by subcloning full-length Crx, Otx2, Nrl genes into a pMIK expression vector (a gift from Dr. K. Maruyama), respectively. For the "mut1259" vector, the Crx binding site at the -1259 bp position was mutated by replacing GGATTA with AGATCT. For the "mut198" vector, the Crx binding site at the -198 bp position was mutated by replacing TAATCC with GAATTC. For the "mut72" vector, the Crx binding site at the -72 bp position was mutated by replacing GGATTA with GAATTC. For the "mut all" vector, all of three Crx binding sites were mutated. 5xGAL4-pGL3 Control reporter plasmid, which contains five copies of the GAL4 DNA recognition sequence positioned immediately upstream of SV40 minimal promoter, was kindly gifted from Dr. T. Noguchi and used for analysis of the transcriptional activity of mr-s . To generate effector plasmids, various deletion fragments were produced by PCR reaction and subcloned into pGBKT7 vector, respectively. These fragments were digested with the sequence, which encodes GAL4 DNA binding domain, and inserted into pcDNA3. We transfected 0.1 μg of reporter plasmid DNA and 2 μg of the expression vector DNA per 6 cm dish into HEK293T cells using Fugene6 transfection reagent. We analyzed luciferase activity 48 hr after transfection.
List of abbreviations
orthodenticle-related homeobox 2
thyroid hormone receptor beta 2
neural retina leucine zipper
nuclear receptor subfamily 2 group E member 3
expressed sequence tag
ephrin receptor B2
ephrin receptor A4
translocation ETS leukemia.
We thank T. Noguchi for 5xGAL4-pGL3 reporter plasmid; A. Nishida for discussion and critical reading of the manuscript; A. Yoshimura for advice on confocal microscopy; A. Tani, Y. Kambara, M. Murai, Y. Hirao and H. Yoshii for technical assistance. This work was supported by Precursory Research for Embryonic Science and Technology (PRESTO), Dynamics of Development Systems Molecular Brain Science, Grant-in Aid for Scientific Research (B), Senri Life Science Foundation, and Uehara Foundation.
- Young RW: Cell differentiation in the retina of the mouse. Anat Rec. 1985, 212 (2): 199-205. 10.1002/ar.1092120215.View ArticlePubMedGoogle Scholar
- Cepko CL, Austin CP, Yang X, Alexiades M, Ezzeddine D: Cell fate determination in the vertebrate retina. Proc Natl Acad Sci U S A. 1996, 93 (2): 589-595. 10.1073/pnas.93.2.589.PubMed CentralView ArticlePubMedGoogle Scholar
- Furukawa T, Morrow EM, Cepko CL: Crx, a novel otx-like homeobox gene, shows photoreceptor-specific expression and regulates photoreceptor differentiation. Cell. 1997, 91 (4): 531-541. 10.1016/S0092-8674(00)80439-0.View ArticlePubMedGoogle Scholar
- Milam AH, Rose L, Cideciyan AV, Barakat MR, Tang WX, Gupta N, Aleman TS, Wright AF, Stone EM, Sheffield VC, Jacobson SG: The nuclear receptor NR2E3 plays a role in human retinal photoreceptor differentiation and degeneration. Proc Natl Acad Sci U S A. 2002, 99 (1): 473-478. 10.1073/pnas.022533099.PubMed CentralView ArticlePubMedGoogle Scholar
- Ng L, Hurley JB, Dierks B, Srinivas M, Salto C, Vennstrom B, Reh TA, Forrest D: A thyroid hormone receptor that is required for the development of green cone photoreceptors. Nat Genet. 2001, 27 (1): 94-98. 10.1038/83829.View ArticlePubMedGoogle Scholar
- Chen S, Wang QL, Nie Z, Sun H, Lennon G, Copeland NG, Gilbert DJ, Jenkins NA, Zack DJ: Crx, a novel Otx-like paired-homeodomain protein, binds to and transactivates photoreceptor cell-specific genes. Neuron. 1997, 19 (5): 1017-1030. 10.1016/S0896-6273(00)80394-3.View ArticlePubMedGoogle Scholar
- Mears AJ, Kondo M, Swain PK, Takada Y, Bush RA, Saunders TL, Sieving PA, Swaroop A: Nrl is required for rod photoreceptor development. Nat Genet. 2001, 29 (4): 447-452. 10.1038/ng774.View ArticlePubMedGoogle Scholar
- Furukawa T, Morrow EM, Li T, Davis FC, Cepko CL: Retinopathy and attenuated circadian entrainment in Crx-deficient mice. Nat Genet. 1999, 23 (4): 466-470. 10.1038/70591.View ArticlePubMedGoogle Scholar
- Nishida A, Furukawa A, Koike C, Tano Y, Aizawa S, Matsuo I, Furukawa T: Otx2 homeobox gene controls retinal photoreceptor cell fate and pineal gland development. Nat Neurosci. 2003, 6 (12): 1255-1263. 10.1038/nn1155.View ArticlePubMedGoogle Scholar
- Morrow EM, Furukawa T, Raviola E, Cepko CL: Synaptogenesis and outer segment formation are perturbed in the neural retina of Crx mutant mice. BMC Neurosci. 2005, 6 (1): 5-10.1186/1471-2202-6-5.PubMed CentralView ArticlePubMedGoogle Scholar
- Freund CL, Gregory-Evans CY, Furukawa T, Papaioannou M, Looser J, Ploder L, Bellingham J, Ng D, Herbrick JA, Duncan A, Scherer SW, Tsui LC, Loutradis-Anagnostou A, Jacobson SG, Cepko CL, Bhattacharya SS, McInnes RR: Cone-rod dystrophy due to mutations in a novel photoreceptor-specific homeobox gene (CRX) essential for maintenance of the photoreceptor. Cell. 1997, 91 (4): 543-553. 10.1016/S0092-8674(00)80440-7.View ArticlePubMedGoogle Scholar
- Sohocki MM, Sullivan LS, Mintz-Hittner HA, Birch D, Heckenlively JR, Freund CL, McInnes RR, Daiger SP: A range of clinical phenotypes associated with mutations in CRX, a photoreceptor transcription-factor gene. Am J Hum Genet. 1998, 63 (5): 1307-1315. 10.1086/302101.PubMed CentralView ArticlePubMedGoogle Scholar
- Sohocki MM, Daiger SP, Bowne SJ, Rodriquez JA, Northrup H, Heckenlively JR, Birch DG, Mintz-Hittner H, Ruiz RS, Lewis RA, Saperstein DA, Sullivan LS: Prevalence of mutations causing retinitis pigmentosa and other inherited retinopathies. Hum Mutat. 2001, 17 (1): 42-51. 10.1002/1098-1004(2001)17:1<42::AID-HUMU5>3.0.CO;2-K.PubMed CentralView ArticlePubMedGoogle Scholar
- Swaroop A, Wang QL, Wu W, Cook J, Coats C, Xu S, Chen S, Zack DJ, Sieving PA: Leber congenital amaurosis caused by a homozygous mutation (R90W) in the homeodomain of the retinal transcription factor CRX: direct evidence for the involvement of CRX in the development of photoreceptor function. Hum Mol Genet. 1999, 8 (2): 299-305. 10.1093/hmg/8.2.299.View ArticlePubMedGoogle Scholar
- Kim CA, Bowie JU: SAM domains: uniform structure, diversity of function. Trends Biochem Sci. 2003, 28 (12): 625-628. 10.1016/j.tibs.2003.11.001.View ArticlePubMedGoogle Scholar
- Ponting CP: SAM: a novel motif in yeast sterile and Drosophila polyhomeotic proteins. Protein Sci. 1995, 4 (9): 1928-1930.PubMed CentralView ArticlePubMedGoogle Scholar
- Qiao F, Song H, Kim CA, Sawaya MR, Hunter JB, Gingery M, Rebay I, Courey AJ, Bowie JU: Derepression by depolymerization; structural insights into the regulation of Yan by Mae. Cell. 2004, 118 (2): 163-173. 10.1016/j.cell.2004.07.010.View ArticlePubMedGoogle Scholar
- Kyba M, Brock HW: The Drosophila polycomb group protein Psc contacts ph and Pc through specific conserved domains. Mol Cell Biol. 1998, 18 (5): 2712-2720.PubMed CentralView ArticlePubMedGoogle Scholar
- Tu H, Barr M, Dong DL, Wigler M: Multiple regulatory domains on the Byr2 protein kinase. Mol Cell Biol. 1997, 17 (10): 5876-5887.PubMed CentralView ArticlePubMedGoogle Scholar
- Tessier-Lavigne M: Eph receptor tyrosine kinases, axon repulsion, and the development of topographic maps. Cell. 1995, 82 (3): 345-348. 10.1016/0092-8674(95)90421-2.View ArticlePubMedGoogle Scholar
- Chi SW, Ayed A, Arrowsmith CH: Solution structure of a conserved C-terminal domain of p73 with structural homology to the SAM domain. Embo J. 1999, 18 (16): 4438-4445. 10.1093/emboj/18.16.4438.PubMed CentralView ArticlePubMedGoogle Scholar
- Green JB, Gardner CD, Wharton RP, Aggarwal AK: RNA recognition via the SAM domain of Smaug. Mol Cell. 2003, 11 (6): 1537-1548. 10.1016/S1097-2765(03)00178-3.View ArticlePubMedGoogle Scholar
- Nagaya H, Wada I, Jia YJ, Kanoh H: Diacylglycerol kinase delta suppresses ER-to-Golgi traffic via its SAM and PH domains. Mol Biol Cell. 2002, 13 (1): 302-316. 10.1091/mbc.01-05-0255.PubMed CentralView ArticlePubMedGoogle Scholar
- Sakane F, Kai M, Wada I, Imai S, Kanoh H: The C-terminal part of diacylglycerol kinase alpha lacking zinc fingers serves as a catalytic domain. Biochem J. 1996, 318 ( Pt 2): 583-590.View ArticleGoogle Scholar
- Ramachander R, Kim CA, Phillips ML, Mackereth CD, Thanos CD, McIntosh LP, Bowie JU: Oligomerization-dependent association of the SAM domains from Schizosaccharomyces pombe Byr2 and Ste4. J Biol Chem. 2002, 277 (42): 39585-39593. 10.1074/jbc.M207273200.View ArticlePubMedGoogle Scholar
- Slupsky CM, Gentile LN, Donaldson LW, Mackereth CD, Seidel JJ, Graves BJ, McIntosh LP: Structure of the Ets-1 pointed domain and mitogen-activated protein kinase phosphorylation site. Proc Natl Acad Sci U S A. 1998, 95 (21): 12129-12134. 10.1073/pnas.95.21.12129.PubMed CentralView ArticlePubMedGoogle Scholar
- Jousset C, Carron C, Boureux A, Quang CT, Oury C, Dusanter-Fourt I, Charon M, Levin J, Bernard O, Ghysdael J: A domain of TEL conserved in a subset of ETS proteins defines a specific oligomerization interface essential to the mitogenic properties of the TEL-PDGFR beta oncoprotein. Embo J. 1997, 16 (1): 69-82. 10.1093/emboj/16.1.69.PubMed CentralView ArticlePubMedGoogle Scholar
- Simon JA, Tamkun JW: Programming off and on states in chromatin: mechanisms of Polycomb and trithorax group complexes. Curr Opin Genet Dev. 2002, 12 (2): 210-218. 10.1016/S0959-437X(02)00288-5.View ArticlePubMedGoogle Scholar
- Brock HW, van Lohuizen M: The Polycomb group--no longer an exclusive club?. Curr Opin Genet Dev. 2001, 11 (2): 175-181. 10.1016/S0959-437X(00)00176-3.View ArticlePubMedGoogle Scholar
- Francis NJ, Kingston RE: Mechanisms of transcriptional memory. Nat Rev Mol Cell Biol. 2001, 2 (6): 409-421. 10.1038/35073039.View ArticlePubMedGoogle Scholar
- Kim CA, Gingery M, Pilpa RM, Bowie JU: The SAM domain of polyhomeotic forms a helical polymer. Nat Struct Biol. 2002, 9 (6): 453-457.PubMedGoogle Scholar
- Kozak M: Interpreting cDNA sequences: some insights from studies on translation. Mamm Genome. 1996, 7 (8): 563-574. 10.1007/s003359900171.View ArticlePubMedGoogle Scholar
- Keen TJ, Mohamed MD, McKibbin M, Rashid Y, Jafri H, Maumenee IH, Inglehearn CF: Identification of a locus (LCA9) for Leber's congenital amaurosis on chromosome 1p36. Eur J Hum Genet. 2003, 11 (5): 420-423. 10.1038/sj.ejhg.5200981.View ArticlePubMedGoogle Scholar
- Hicks D, Barnstable CJ: Different rhodopsin monoclonal antibodies reveal different binding patterns on developing and adult rat retina. J Histochem Cytochem. 1987, 35 (11): 1317-1328.View ArticlePubMedGoogle Scholar
- Ahmad I, Redmond LJ, Barnstable CJ: Developmental and tissue-specific expression of the rod photoreceptor cGMP-gated ion channel gene. Biochem Biophys Res Commun. 1990, 173 (1): 463-470. 10.1016/S0006-291X(05)81081-2.View ArticlePubMedGoogle Scholar
- Ni M, Yamaki K, Kikuchi T, Ferrick M, Shinohara T, Nussenblatt RB, Chan CC: Developmental expression of S-antigen in fetal human and rat eye. Curr Eye Res. 1992, 11 (3): 219-229.View ArticlePubMedGoogle Scholar
- Stepanik PL, Lerious V, McGinnis JF: Developmental appearance, species and tissue specificity of mouse 23-kDa, a retinal calcium-binding protein (recoverin). Exp Eye Res. 1993, 57 (2): 189-197. 10.1006/exer.1993.1114.View ArticlePubMedGoogle Scholar
- Blackshaw S, Snyder SH: Developmental expression pattern of phototransduction components in mammalian pineal implies a light-sensing function. J Neurosci. 1997, 17 (21): 8074-8082.PubMedGoogle Scholar
- Peterson AJ, Kyba M, Bornemann D, Morgan K, Brock HW, Simon J: A domain shared by the Polycomb group proteins Scm and ph mediates heterotypic and homotypic interactions. Mol Cell Biol. 1997, 17 (11): 6683-6692.PubMed CentralView ArticlePubMedGoogle Scholar
- Yoshida S, Mears AJ, Friedman JS, Carter T, He S, Oh E, Jing Y, Farjo R, Fleury G, Barlow C, Hero AO, Swaroop A: Expression profiling of the developing and mature Nrl-/- mouse retina: identification of retinal disease candidates and transcriptional regulatory targets of Nrl. Hum Mol Genet. 2004, 13 (14): 1487-1503. 10.1093/hmg/ddh160.View ArticlePubMedGoogle Scholar
- Graves BJ, Gillespie ME, McIntosh LP: DNA binding by the ETS domain. Nature. 1996, 384 (6607): 322-10.1038/384322a0.View ArticlePubMedGoogle Scholar
- Kehle J, Beuchle D, Treuheit S, Christen B, Kennison JA, Bienz M, Muller J: dMi-2, a hunchback-interacting protein that functions in polycomb repression. Science. 1998, 282 (5395): 1897-1900. 10.1126/science.282.5395.1897.View ArticlePubMedGoogle Scholar
- Wood LD, Irvin BJ, Nucifora G, Luce KS, Hiebert SW: Small ubiquitin-like modifier conjugation regulates nuclear export of TEL, a putative tumor suppressor. Proc Natl Acad Sci U S A. 2003, 100 (6): 3257-3262. 10.1073/pnas.0637114100.PubMed CentralView ArticlePubMedGoogle Scholar
- Roseman RR, Morgan K, Mallin DR, Roberson R, Parnell TJ, Bornemann DJ, Simon JA, Geyer PK: Long-range repression by multiple polycomb group (PcG) proteins targeted by fusion to a defined DNA-binding domain in Drosophila. Genetics. 2001, 158 (1): 291-307.PubMed CentralPubMedGoogle Scholar
- Boccuni P, MacGrogan D, Scandura JM, Nimer SD: The human L(3)MBT polycomb group protein is a transcriptional repressor and interacts physically and functionally with TEL (ETV6). J Biol Chem. 2003, 278 (17): 15412-15420. 10.1074/jbc.M300592200.View ArticlePubMedGoogle Scholar
- Bannister AJ, Schneider R, Kouzarides T: Histone methylation: dynamic or static?. Cell. 2002, 109 (7): 801-806. 10.1016/S0092-8674(02)00798-5.View ArticlePubMedGoogle Scholar
- Shao Z, Raible F, Mollaaghababa R, Guyon JR, Wu CT, Bender W, Kingston RE: Stabilization of chromatin structure by PRC1, a Polycomb complex. Cell. 1999, 98 (1): 37-46. 10.1016/S0092-8674(00)80604-2.View ArticlePubMedGoogle Scholar
- Morrow EM, Furukawa T, Cepko CL: Vertebrate photoreceptor cell development and disease. Trends Cell Biol. 1998, 8 (9): 353-358. 10.1016/S0962-8924(98)01341-5.View ArticlePubMedGoogle Scholar
- Furukawa T, Kozak CA, Cepko CL: rax, a novel paired-type homeobox gene, shows expression in the anterior neural fold and developing retina. Proc Natl Acad Sci U S A. 1997, 94 (7): 3088-3093. 10.1073/pnas.94.7.3088.PubMed CentralView ArticlePubMedGoogle Scholar
- Tanaka T, Inazu T, Yamada K, Myint Z, Keng VW, Inoue Y, Taniguchi N, Noguchi T: cDNA cloning and expression of rat homeobox gene, Hex, and functional characterization of the protein. Biochem J. 1999, 339 ( Pt 1): 111-117. 10.1042/0264-6021:3390111.View ArticleGoogle Scholar
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