title: Differentiation of retinal pigment epithelial cells in vitro uncovers silencer activity in the FGF-5 gene promoter.
abstract: Differentiated retinal pigment epithelial (RPE) cells in vivo express basal levels of FGF-5, a secreted member of the FGF gene family. RPE cells proliferate in response to pathological events, resulting in a transient increase in FGF-5 gene expression. The goal of this study is to identify cis-acting sequences in the FGF-5 gene promoter which upregulate FGF-5 gene expression when differentiated RPE cells enter the cell cycle and proliferate. In vitro cultures of RPE cells were transfected with various FGF-5 promoter/luciferase deletion constructs, using methods specifically optimized for proliferating and differentiated RPE cells. A proximal promoter/enhancer whose activity is not cell-context dependent was identified between FGF-5 sequences -314 and +48. In addition, a silencer element (-1256/-883) was identified in the distal region which is active only in differentiated RPE cells. When tested in a heterologous system, the same element had silencer activity in differentiated cells. Two small regions in the distal FGF-5 gene promoter, -1195/-1173 and -984/-967 were able to specifically bind to nuclear proteins from differentiated RPE cells but not from proliferating RPE cells as evidenced by gel mobility shift assays. Therefore, FGF-5 gene expression in the RPE may be regulated by the formation of differentiation-specific complexes.
authors.affiliation: Section of Molecular and Cellular Biology, University of California, Davis, California 95616, USA.
authors.forename: C M
authors.initials: CM
authors.lastname: Gelfman
authors.forename: C M
authors.initials: CM
authors.lastname: Kelleher
authors.forename: L M
authors.initials: LM
authors.lastname: Hjelmeland
mesh_terms: D001483:Base Sequence; D018929:Cell Culture Techniques; D002454:Cell Differentiation / Q000235:genetics; D002455:Cell Division / Q000235:genetics; D002460:Cell Line; D053938:DNA Fragmentation; D051521:Fibroblast Growth Factor 5; D005346:Fibroblast Growth Factors / Q000235:genetics* / Q000378:metabolism; D005786:Gene Expression Regulation; D006801:Humans; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism; D010857:Pigment Epithelium of Eye / Q000166:cytology / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D015183:Restriction Mapping; D014162:Transfection
chemical_list: C496449:FGF5 protein, human; D009687:Nuclear Proteins; D051521:Fibroblast Growth Factor 5; D005346:Fibroblast Growth Factors; D008156:Luciferases