Localization of upstream silencer elements involved in the expression of cone transducin alpha-subunit (GNAT2).

PURPOSE: To localize cis-acting elements involved in the expression of the cone-specific G-protein, cone transducin alpha-subunit (GNAT2). METHODS: In this study, the authors used a genomic clone, HGLG3, to sequence 3139 base pairs of the upstream region of the GNAT2 gene and to localize cis-acting elements involved in the expression of GNAT2. Upstream elements were localized functionally by transfection of chloramphenicol acetyltransferase gene constructs containing nested deletions of this upstream region into WERI-Rb1 cells. Cell specificity of the localized elements was determined by transfection of the HeLa cells. Trans-acting factor-binding sites to functional cis-acting elements were determined by DNasel footprinting. Cell specificity of protein interaction with footprinted regions was tested by electrophoretic mobility shifts with nuclear extracts from WERI-Rb1 and HeLa cells. RESULTS: Transfection of WERI-Rb1 and HeLa cells revealed the presence of a strong, noncell-specific silencer region between -1130 and -23, a weak, cell-specific promoter between -151 and -10, and a stronger, noncell-specific element between +143 and +167. DNaseI footprinting showed three major footprints (S1, S2, and S3) between -807 and -176, indicating the binding sites for putative negative trans-acting factors. Individual footprinted sequences had similar electrophoretic mobility shifts when they were incubated with nuclear extracts from either WERI-Rb1 or HeLa cells, suggesting that these cells express the same negative factors. CONCLUSIONS: The expression of the GNAT2 gene is controlled by a strong silencer region, a weak upstream cell-specific promoter, and a strong downstream element. The silencer region interacts with similar proteins from retina- and nonretina-derived cell lines.

Elements regulating the transcription of human interstitial retinoid-binding protein (IRBP) gene in cultured retinoblastoma cells.

PURPOSE: To identify cis-acting elements and trans-acting factors involved in the expression of human IRBP gene. METHODS: Transient transfection of WERI-Rb1 and HeLa cells, DNase 1 footprinting, gel mobility-shift assay and yeast one-hybrid system were used to study the regulatory elements that are involved in the expression of human IRBP gene. RESULTS: A region between -1620 and -1411 was shown to have enhancer properties. Using nuclear extracts from WERI-Rb1 and HeLa cells, four footprints were identified in the proximal promoter region (-206 to +68). The core promoter element IP1 binds to OTX2 in the yeast one-hybrid system. By cotransfecting HeLa cells, OTX2 could transactivate the irbp promoter. The functions of IP2 (from -119 to -86) and IP3 (from -183 to -147) remain to be determined. The region containing the HeLa cell-specific footprint IP4 (from -202 to -180) could silence the OTX2 transactivation of the irbp promoter. CONCLUSION: The 5'-flanking region of irbp contains an enhancer sequence. The possible silencer upstream from the core promoter may serve to suppress expression of irbp in HeLa cells. When the proximal promoter is used to identify binding proteins in a human retina library by the yeast one hybrid system, nine of the identified clones contained the cDNA sequence for the homeodomain protein OTX2. Since no clones for the homeodomain protein CRX were found, and since OTX2 can transcriptionally activate irbp in normally non-expressing HeLa cells, it is possible that OTX2 rather than CRX is the transcriptional activator for irbp in human photoreceptors.

A CAT reporter construct containing 277bp GNAT2 promoter and 214bp IRBP enhancer is specifically expressed by cone photoreceptor cells in transgenic mice.

PURPOSE: The alpha-subunit of human cone transducin plays an important role in interacting with visual pigment and activating the cGMP-dependent phosphodiesterase (cGMP-PDE). The human GNAT2 gene (cone transducin alpha-subunit) has been cloned and characterized by Fong et al. In this report, we describe the use of transgenic mice to characterize the tissue specificity of the GNAT2 promoter. METHODS: A chimeric reporter gene construct which consists of a 277 bp 5'-flanking fragment of the GNAT2 gene at 5' end of the chloramphenicol acetyltransferase (CAT) gene and a 214 bp enhancer region from the human interphotoreceptor retinoid-binding protein (IRBP) gene at the 3' end of the CAT gene was used to generate transgenic mice. Transgenic mice were identified by Southern blot hybridization and polymerase chain reaction (PCR) analysis using tail DNA from experimental animals. Immunostaining was used to study the developmental expression of CAT and the endogenous GNAT2 gene. RESULTS: Analysis of four transgenic mouse lines revealed that three lines had low CAT activity in the retina. The CAT gene, along with the endogenous GNAT2 gene, was expressed at high levels in cone photoreceptor cells in the fourth transgenic mouse line as determined by CAT enzyme assays and immunostaining. CONCLUSION: The results show that the 277 bp 5'-flanking sequence from the human GNAT2 gene coupled with the 214 bp IRBP enhancer can direct a tissue-specific expression pattern of CAT reporter gene in mouse retina, which parallels the expression pattern of endogenous GNAT2.

Characterization and comparative structural features of the gene for human interstitial retinol-binding protein.

We have cloned the gene for human interstitial retinol-binding protein (IRBP) and compared its nucleotide sequence with that of the corresponding cloned cDNA. The human IRBP gene is approximately 9.5 kilobase pairs (kbp) in length and consists of four exons separated by three introns. The introns are 1.6-1.9 kbp long. The gene is transcribed by photoreceptor and retinoblastoma cells into an approximately 4.3-kilobase mRNA that is translated and processed into a glycosylated protein of 135,000 Da. The amino acid sequence of human IRBP can be divided into four contiguous homology domains with 33-38% identity, suggesting a series of gene duplication events. In the gene, the boundaries of these domains are not defined by exon-intron junctions, as might have been expected. The first three homology domains and part of the fourth are all encoded by the first large exon, which is 3,180 base pairs long. The remainder of the fourth domain is encoded in the last three exons, which are 191, 143, and approximately 740 base pairs long, respectively. This unusual structure is shared with the bovine IRBP gene. A large (1.7 kbp) fragment appears to have been lost from the 3'-noncoding region of the last human exon. We conclude that the human and bovine genes have similar evolutionary histories.