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

ABSTRACT

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.