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Interphotoreceptor retinoid binding protein (IRBP) is a glycolipoprotein that is required for normal retinal function (1,2). It is only expressed in photoreceptors and pinealocytes. While the -1783 to +101 region of the mouse IRBP gene acts as a strong promoter, there is an area between -156 to -70 that is necessary for suppression of IRBP expression in non photoreceptor cells and tissues. Orphan retinoic acid receptor (RORA) and estrogen receptor consensus elements are found in the -115 to -108 region of the mouse IRBP gene, suggesting a prime target for a cell-type specific silencing element to bind. Our purpose was to clearly define the DNA target within the -133 to -101 region. Five different oligonucleotide probes were created based on the mouse IRBP -133 to -101 region. Two of the probes were mutated within the consensus element area and two were mutated in either the first or second half of the probe. The remaining probe has the wild-type sequence. The probes were radio-labeled with 32P and used in electrophoretic mobility shift assays (EMSA) to determine which area of the probe the suppressor transcription factor binds. The data indicate that binding occurs in an area spanning from -133 to -118, a region outside of the consensus element.
Radio-labeling and Annealing Oligonucleotides: Oligonucleotide probes were made by the Emory University Microchemical Facility. The corresponding sense and antisense strands were annealed and the double-stranded oligonucleotides were labeled 5’ with 32P by the forward reacting T4 polynucleotide kinase.
Electrophoretic Mobility Shift Assays (EMSA): Radio-labeled probes were combined with increasing concentrations of poly dI-dC and either WERI retinoblastoma cell or Neuro2a neuroblastoma cell nuclear protein extracts. The samples were run on a 7% polyacrylamide gel in a tris-glycine buffer. The gel was dried overnight and exposed to film in -80oC for a period ranging from 8 to 24 hours, depending on the radioactivity of the probes. The film was developed using a Konica SRX-101 developer.
Transient Transfections and CAT Assays: WERI retinoblastoma and Neuro2a neuroblastoma cells were transiently transfected with various IRBP promoter fragments that were inserted upstream of a chloramphenicol acetyltransferase (CAT) gene. One promoter fragment, named SVOAT, had a random sequence and was used as the negative control. The vectors were referred to as p70, p107, and p140 because of their position relative to transcription start. These vectors were used to transiently transfect either WERI or Neuro2a cells using Superfect and Effectene transfection reagents, respectively. After a 48-hour incubation period, the cells were harvested and used in a CAT assay to measure promoter activity. A Beckman LS6500 Multi-Purpose Scintillation Counter was used to measure CAT activity.
- EMSA: In EMSAs using both WERI and Neuro2a nuclear extract, with 28 ng and 80 ng of poly dI-dC, a band disappeared when the -133 to -118 region was mutated. There was no loss of a band when the area of the consensus elements was mutated.
- CAT Assay: CAT assays of WERI cells transiently transfected with PSVOAT, the negative control, showed little activity. Cells transfected with p70 and p140 had high activities. However, p107 had a slightly lower activity than the previous three mentioned. Transfected Neuro2a cells showed similar results except that p107 showed very little activity.
The consensus element region, -115 to -108, is not the DNA target for a cell-type specific silencing element. An area upstream of the consensus element region, -133 to -118, appears to be the target for a transcription factor. A promoter element containing -133 to -118 appears to be an enhancer element.
Funded by Howard Hughes Medical Institute under Grant No. 52003071, National Science Foundation REU Grant No. 9820356, RO1 EY09378, RO1 EY12514
Vi employed electrophoretic mobility shift assays (EMSA) and transient transfections to characterize the –133 to –101 region (relative to transcription start) of the IRBP promoter. Based on the –133 to –101 mouse IRBP promoter sequence, a wild-type oligonucleotide probe and 4 mutants of that probe were created and radio-labeled with 32P. The probes were combined with increasing concentrations of poly dI-dC and either WERI or Neuro2a nuclear protein extract and run on a 7% polyacrylamide gel. Contrary to her hypothesis, Vi found that specific binding occurred within the –133 to –118 region and not in the consensus element region. Through the transient transfection of both WERI and Neuro2a cells, Vi found evidence of a restrictive element between –107 and –70 and an enhancing element between –140 and –107, an area that included the site for specific binding.
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