Interphotoreceptor Retinoid-binding Protein Promoter Research Articles

Triple Vectors Expand AAV Transfer Capacity in the Retina.

Retinal gene transfer with adeno-associated viral (AAV) vectors holds great promise for the treatment of inherited retinal degenerations (IRDs). One limit of AAV is its transfer capacity of about 5 kb, which can be expanded to about 9 kb, using dual AAV vectors. This strategy would still not suffice for treatment of IRDs such as Usher syndrome type 1D or Alström syndrome type I (ALMS) due to mutations in CDH23 or ALMS1, respectively. To overcome this limitation, we generated triple AAV vectors, with a maximal transfer capacity of about 14 kb. Transcriptomic analysis following triple AAV transduction showed the expected full-length products along a number of aberrant transcripts. However, only the full-length transcripts are efficiently translated in vivo. We additionally showed that approximately 4% of mouse photoreceptors are transduced by triple AAV vectors and showed correct localization of recombinant ALMS1. The low-photoreceptor transduction levels might justify the modest and transient improvement we observe in the retina of a mouse model of ALMS. However, the levels of transduction mediated by triple AAV vectors in pig retina reached 40% of those observed with single vectors, and this bodes well for further improving the efficiency of triple AAV vectors in the retina.

28. Mouse and Pig Photoreceptor Transduction Mediated by Triple AAV Vectors

Purpose: The majority of inherited blinding diseases are caused by mutations in genes expressed specifically in photoreceptors (PRs). However the size of several of these genes exceeds the DNA cargo capacity of AAV vectors, to date the safest and most effective gene therapy vectors in the retina. We have previously demonstrated that dual AAV vectors expand AAV PR transfer capacity to about 9 kb. Our aim is to further expand this to around 13.5 kb using a triple AAV vector system. Methods: To assess the percentage of PR transduction mediated by three independent vectors, we initially injected subretinally in 4 week-old C57/BL6 mice single AAV8 vectors encoding either EGFP, dsRed or beta-gal reporter proteins. Then, in order to evaluate the PR transduction efficiency of triple AAV vectors we generated a reporter EGFP-dsRed fusion protein under the control of either the ubiquitous CMV or the PR-specific IRBP(interphotoreceptor retinoid-binding protein) promoters. The corresponding EGFP-dsRed expression cassettes were either included in single or triple AAV8 vectors (Fig1Fig1) which were administered by subretinal injection to C57/BL6 mice or large white pigs. Direct fluorescence, Western blot, and ELISA analysis were used to evaluate transgene expression. Results: In mice injected with three independent AAV8 vectors encoding for the different EGFP, dsRed and beta-gal reporter proteins, the percentage of PR co-transduced was 12%. In mouse and pig eyes injected with triple and single AAV8 vectors encoding EGFP-dsRed, full length protein expression was confirmed by Western Blot analysis independently of the CMV or IRBP promoters used; importantly, 75% of mouse and 65% of pig eyes injected with triple IRBP vectors showed bands of the expected size. Quantification in mice of EGFP-dsRed protein expression from triple AAV8 vectors mediated by either the CMV or the IRBP promoter shows 30% and 1%, respectively, of the levels observed with a single AAV8. Interestingly, in pigs, the levels of EGFP-dsRed protein expression from triple AAV8-IRBP vectors were 40% ratio of those observed with a single AAV8. Conclusions: Our results show that triple AAV vectors, which increase AAV transfer capacity up to 13.5 kb, transduce mouse and pig PR at levels that are 1% and 40% respectively of those obtained with single AAV vectors. This bode well for further testing this platform in animal models of inherited retinal degenerations.View Large Image | Download PowerPoint Slide

P53 Selectively Regulates Developmental Apoptosis of Rod Photoreceptors

Retinal cells become post-mitotic early during post-natal development. It is likely that p53, a well-known cell cycle regulator, is involved in regulating the genesis, differentiation and death of retinal cells. Furthermore, retinal cells are under constant oxidative stress that can result in DNA damage, due to the extremely high level of metabolic activity associated with phototransduction. If not repaired, this damage may result in p53-dependent cell death and ensuing vision loss. In this study, the role of p53 during retinal development and in the post-mitotic retina is investigated. A previously described super p53 transgenic mouse that expresses an extra copy of the mouse p53 gene driven by its endogenous promoter is utilized. Another transgenic mouse (HIP) that expresses the p53 gene in rod and cone photoreceptors driven by the human interphotoreceptor retinoid binding protein promoter was generated. The electroretinogram (ERG) of the super p53 mouse exhibited reduced rod-driven scotopic a and b wave and cone-driven photopic b wave responses. This deficit resulted from a reduced number of rod photoreceptors and inner nuclear layer cells. However, the reduced photopic signal arose only from lost inner retinal neurons, as cone numbers did not change. Furthermore, cell loss was non-progressive and resulted from increased apoptosis during retinal developmental as determined by TUNEL staining. In contrast, the continuous and specific expression of p53 in rod and cone photoreceptors in the mature retinas of HIP mice led to the selective loss of both rods and cones. These findings strongly support a role for p53 in regulating developmental apoptosis in the retina and suggest a potential role, either direct or indirect, for p53 in the degenerative photoreceptor loss associated with human blinding disorders.

The effect of retinoids and butyrate on the expression of CRX and IRBP in retinoblastoma cells

We sought to determine whether differentiation agents such as retinoids and butyrate regulate transcript levels of interphotoreceptor retinoid binding protein (IRBP) and cone rod homeobox (CRX), a homeodomain transcription factor that regulates IRBP promoter activity. WERI-Rb1 retinoblastoma cells were treated with all-trans retinol, all-trans retinoic acid, or butyrate. IRBP and CRX mRNA levels were determined by quantitative RT-PCR. Butyrate at low concentrations increased both mRNA levels but suppressed them at higher concentrations. Retinoic acid had minimal effects. Retinol increased CRX mRNA over four fold. IRBP and CRX transcript levels are sensitive to butyrate and CRX expression is sensitive to retinol.

Endogenous CRX expression and IRBP promoter activity in retinoblastoma cells

Purpose: To determine whether antisense oligonucleotides (AODNs) targeted against CRX, a photoreceptor-specific trans-acting factor, suppress CRX expression and interphotoreceptor retinoid binding protein (IRBP) promoter activity. Methods: Cultures of human retinoblastoma cells were transfected with chloramphenicol acetyltransferase (CAT) reporter plasmids containing a mouse IRBP promoter and AODNs directed against CRX. RT–PCR using primers specific to CRX, OTX2, GAPDH, or RNase H was conducted on total RNA isolated from retinoblastoma cells at various times following transfection with AODNs. Results: Transfection of retinoblastoma cells with IRBP promoter CAT constructs alone produced high activity. Co-transfection with AODNs suppressed IRBP promoter activity in a concentration-dependent manner, with half-maximal effect produced at about 2 nM AODN concentration. Transfection with CAT constructs containing an SV40 promoter produced high activity that was unaffected by co-transfection with AODNs. RT–PCR products were obtained for all target sequences. CRX RT–PCR product from cells transfected with AODNs was greatly diminished following transfection with an AODN whereas control transcripts, including that of OTX2, were relatively unaffected. Conclusions: The CRX-specific AODNs specifically and potently suppressed CRX expression and IRBP promoter activity, as measured by RT–PCR and transient transfection assays, respectively. Little or no effect was seen on controls. These data suggest that endogenous CRX is required for IRBP promoter activity in retinoblastoma cells.

Evidence of a tissue-restricting DNA regulatory element in the mouse IRBP promoter

The expression of interphotoreceptor retinoid binding protein (IRBP) is limited to photoreceptor cells of the retina and pinealocytes of the pineal gland. We sought to define cis-elements of the mouse IRBP 5′ flanking region that are required for this restricted activity. In vitro transient transfections of retinoblastoma and neuroblastoma cells and in vivo experiments with transgenic Xenopus laevis indicate that −1783/+101 and −156/+101 IRBP gene fragments directed expression predominantly to the retina and pineal, with minor neuronal expression elsewhere. In contrast, a −70/+101 fragment was less restrictive in controlling expression, exhibiting activity not only in retina, but also in forebrain, hindbrain, spinal cord, and motor neurons innervating gills.

Structural characterization and comparison of promoter activity of mouse and bovine interphotoreceptor retinoid-binding protein (IRBP) gene 5' flanking regions in WERI, Y79, chick retina cells, and transgenic mice

Purpose. To determine the sequences of the mouse and bovine interphotoreceptor retinoid-binding protein (IRBP) 5' flanking regions and whether these 5' flanking regions contain functional IRBP promoter activity in multiple cell types using both quantitative and statistical analyses. Methods. We sequenced the bovine and mouse 5' flanking regions of the IRBP gene and compared these sequences to the human gene sequence. To test for functional activity of this region, we used the same DNA construct, p1783, in four different cell types. Mobility shift, DNase footprints, and southwestern blots were used to determine where nuclear protein complexes bind the IRBP 5' flanking region. Results. The 5' flanking regions of the bovine, human, and mouse IRBP genes exhibit sequence similarity in regions immediately adjacent to the start of transcription (roughly 350 bases in length) and also over a 220 base sequence about 1.25 to 1.50 kb upstream of the transcription start site. Two different statistical approaches showed that the IRBP 5' flanking region possesses promoter activity in four different cell types. By using mobility shift, DNase I-protection experiments, and southwestern blotting, a region of about 45 bases at position -300 was identified that specifically binds a protein from the nuclei of bovine retina and Y79 cells. Conclusions. Specific DNA binding events are an essential part of IRBP promoter activity. The conservation of sequences far upstream of the transcription start suggest that unknown physiological processes remain to be understood in IRBP transcriptional regulation.

Modulation of CRX transactivation activity by phosducin isoforms.

Phosducin (Phd) and Phd-like proteins (PhLPs) selectively bind guanine nucleotide protein (G protein) betagamma subunits (Gbetagamma), while Phd-like orphan proteins (PhLOPs) lack the major functional domain for the binding of Gbetagamma. A retina- and pineal gland-specific transcription factor, cone-rod homeobox (CRX), was identified by a yeast two-hybrid screen using PhLOP1 as the bait. Direct protein-protein interactions between Phd or PhLOP1 and CRX were demonstrated using a beta-galactosidase quantitative assay in the yeast two-hybrid system and were confirmed by an in vitro binding assay and a glutathione S-transferase (GST) pull-down assay. To determine if the interaction with Phd or PhLOP1 affected CRX transactivation, a 120-bp interphotoreceptor retinoid binding protein (IRBP) promoter-luciferase reporter construct containing a CRX consensus element (GATTAA) was cotransfected into either COS-7 or retinoblastoma Weri-Rb-1 cells with expression constructs for CRX and either Phd or PhLOP1. Phd and PhLOP1 inhibited the transcriptional activation activity of CRX by 50% during transient cotransfection in COS-7 cells and by 70% in Weri-Rb-1 cells and COS-7 cells stably transfected with CRX. Phd inhibited CRX transactivation in a dose-dependent manner. Whereas Phd is a cytoplasmic phosphoprotein, coexpression of Phd with CRX results in Phd being localized both in the cytoplasm and nucleus. By contrast, PhLOP1 is found in the nucleus even without CRX coexpression. To address the physiological relevance of these potential protein interacting partners, we identified immunoreactive proteins for Phd and CRX in retinal cytosolic and nuclear fractions. Immunohistochemical analysis of bovine retinas reveals colocalization of Phd isoforms with CRX predominantly in the inner segment of cone cells, with additional costaining in the outer nuclear layer and the synaptic region. Our findings demonstrate that both Phd and PhLOP1 interact directly with CRX and that each diminishes the transactivation activity of CRX on the IRBP promoter. A domain that interacts with CRX is found in the carboxyl terminus of the Phd isoforms. Phd antibody-immunoreactive peptides are seen in light-adapted mouse retinal cytosolic and nuclear extracts. Neither Phd nor PhLOP1 affected CRX binding to its consensus DNA element in electrophoretic mobility shift assays. A model that illustrates separate functional roles for interactions between Phd and either SUG1 or CRX is proposed. The model suggests further a mechanism by which Phd isoforms could inhibit CRX transcriptional activation.

OTX2 homeodomain protein binds a DNA element necessary for interphotoreceptor retinoid binding protein gene expression

Transcription of the human interphotoreceptor retinoid binding protein (IRBP) gene is strictly tissue specific, being restricted to retinal photoreceptors and pinealocytes. We have previously demonstrated that a sequence named A element, in the IRBP promoter is essential for IRBP gene transcription in vivo. Here we demonstrate that the human homeodomain protein OTX2 is present in nuclear extracts of IRBP expressing cells and specifically interacts with the IRBP A promoter element in vitro. OTX2, as well as CRX, a homeodomain protein very similar to OTX2, activates the human IRBP promoter in co-transfection experiments.

Identification of a Retina-Specific Footprint Within the Retina-Specific Regulatory Region of the Human Interphotoreceptor Retinoid-Binding Protein Gene

Regulation of the retina-specific expression of the interphotoreceptor retinoid-binding protein (IRBP) gene in transgenic mice requires sequences between nucleotide (nt) −156 and +19. DNAse I footprinting and electrophoretic mobility shift assays identified a region between nt −69 and −40 that interacts with some nuclear proteins from bovine and mouse retinas but not from a variety of other neural and non-neural tissues. The selectivity of this sequence to elicit IRBP promoter activity is indicated by their failure to bind some nuclear proteins following mutagenesis. Therefore, IRBP promoter activity is linked to nuclear protein binding at a regulatory motif between nt −69 and −40.

Timing of Interphotoreceptor Retinoid-Binding Protein (IRBP) Gene Expression and Hypomethylation in Developing Mouse Retina

We determined during photoreceptor development if there is a retina-specific hypomethylation of the mouse gene encoding interphotoreceptor retinoid-binding protein (IRBP) that is associated with its activation. Second, the role of IRBP gene and protein expression in development was assessed by determining if their expression occurs before that of opsin. Retina-specific hypomethylation of the IRBP promoter region started on Embryonic (E) Day 11, at the time of cone formation, increased from E12 to E14, at the time of rod formation, and reached a peak on Postnatal (P) Day 4, which was followed thereafter by a slow decrease. Starting on E11, IRBP and opsin mRNA levels were quantitated relative to that of the β-actin gene with RNase protection analysis. β-Actin and IRBP transcripts were readily detected on E11. β-actin levels remained constant during embryonic and early postnatal stages and decreased slightly afterward. On the other hand, beginning on E13, when the rods are formed, the IRBP level markedly increased. In contrast, the opsin transcript first appeared later on P0 and then increased from P3 onward. After P6, the opsin and IRBP transcript levels became comparable and by P20 their levels reached constancy. The timing of the onset of protein expression for the IRBP and opsin genes was determined during the last proliferative cycle of the rod precursor cells before their differentiation. Mice at P2 or P3 were injected with bromodeoxyuridine (BrdU) and their retinal cells were dissociated and then double-labeled with antibodies against BrdU and either IRBP or opsin. Cells positive for both IRBP and BrdU were always observed as soon as 2 hr after injection but it took at least 40 hr before they became positive for both opsin and BrdU. Taken together, these results indicate that IRBP gene activation is associated with hypomethylation during the last mitosis before photoreceptor cell differentiation.

Different morphological types of retinal astrocytes in rabbit retina

Transcription of the human interphotoreceptor retinoid binding protein (IRBP) gene is strictly tissue specific, being restricted to retinal photoreceptors and pinealocytes. We have previously demonstrated that a sequence named A element, in the IRBP promoter is essential for IRBP gene transcription in vivo. Here we demonstrate that the human homeodomain protein OTX2 is present in nuclear extracts of IRBP expressing cells and specifically interacts with the IRBP A promoter element in vitro. OTX2, as well as CRX, a homeodomain protein very similar to OTX2, activates the human IRBP promoter in co-transfection experiments.

A family of multiple phosducin (33 kDa protein) genes

Transcription of the human interphotoreceptor retinoid binding protein (IRBP) gene is strictly tissue specific, being restricted to retinal photoreceptors and pinealocytes. We have previously demonstrated that a sequence named A element, in the IRBP promoter is essential for IRBP gene transcription in vivo. Here we demonstrate that the human homeodomain protein OTX2 is present in nuclear extracts of IRBP expressing cells and specifically interacts with the IRBP A promoter element in vitro. OTX2, as well as CRX, a homeodomain protein very similar to OTX2, activates the human IRBP promoter in co-transfection experiments.

Photoreceptor-specific activity of the human interphotoreceptor retinoid-binding protein (IRBP) promoter in transgenic mice

In order to define the cellular specificity of the interphotoreceptor retinoid-binding protein (IRBP) promoter in the retina, we linked the human IRBP promoter to the β-galactosidase ( lacZ) gene and made five lines of transgenic mice. In three of the five transgenic mouse lines, retinas showed positive staining upon incubation with 5-bromo-4-chloro-3-indolyl-β- d-galactoside (X-gal). Mice from one line (OVE278B) showed positive X-gal staining throughout the retina except for the most peripheral regions. Interestingly, the staining was heterogeneous throughout the retina. Heavily stained regions were interspersed with lightly stained areas. Mice in two other lines showed highly mosaic X-gal staining patterns. Histological examination demonstrated that staining was confined to photoreceptor cells in all three expressing families. Furthermore, electron microscopy showed that the promoter is active in both rod and cone cells. Our results demonstrate that the human IRBP promoter can be used to obtain photoreceptor-specific gene expression in transgenic mice.

Tissue-specific expression in transgenic mice directed by the 5'-flanking sequences of the human gene encoding interphotoreceptor retinoid-binding protein.

Interphotoreceptor retinoid-binding protein (IRBP) is an extracellular protein that has been suggested to participate in the visual process as a carrier for visual retinoids. A chimeric gene composed of the human IRBP promoter fused to the bacterial reporter gene chloramphenicol acetyltransferase (CAT) was used to generate transgenic mice. Analysis of six transgenic families revealed that the CAT gene, concomitant with the endogenous IRBP gene, was expressed primarily in the retina and, to a lesser extent, in the pineal gland. These results establish that a 1.3-kilobase fragment from the 5' end of the human IRBP gene is sufficient to direct transgene expression to a visual subdivision of the central nervous system.

Hypomethylation of the interphotoreceptor retinoid-binding protein (IRBP) promoter and first exon is linked to expression of the gene

The interphotoreceptor retinoid-binding protein (IRBP) is limited in expression to retinal photoreceptor cells and a subset of pinealocytes. We have obtained a genomic clone containing the entire coding region and 7 kb of 5' flanking sequence. As a first step in studying IRBP gene regulation we have examined the CpG methylation patterns of the entire IRBP gene in expressing and non-expressing human cells. This has been done by isolation of high molecular weight DNA from Y-79 cells grown in suspension or attached to poly-D-lysine, which synthesize IRBP at different levels, and from human lymphocytes, which were shown by northern analysis to lack IRBP message. The DNA was digested by either Hpa II, Msp I, or Hha I. Southern blots were prepared with these digests and hybridized with probes made from fragments covering the complete genomic clone. Probes from the first exon, the introns and the 3' end gave banding patterns which showed no differences between the expressing cells and the lymphocytes. A probe from the very 5' end did not give a clear banding pattern, probably due to the presence of repetitive elements in the probe. However, a Hind III probe covering the 5' flanking 3 kb and the beginning of the first exon hybridized with a 1.8 kb band in Hpa II digests of Y-79 cells which was not present in Hpa II digests of lymphocyte DNA. In addition, a 2.1-2.3 kb Hha I band was found only in the Y-79 DNA digests. Sequence analysis of the promoter region indicated that these bands were due to hypomethylation of sites within a CpG rich island from -1578 to -1108 in the promoter and hypomethylation of sites in the beginning of the first exon. A Hha I site between the CpG island and the first exon was not hypomethylated in the expressing Y-79 cells. We propose that hypomethylation of the CpG rich island of the IRBP promoter and the first exon is linked to the expression of this gene.