Functional identification in human RPE cells of cis-acting sequences in the FGF-5 gene promoter

C. M. Gelfman, C. M. Kelleher, Leonard M Hjelmeland

Research output: Contribution to journalArticle

Abstract

Purpose. FGF-5 is a secreted member of the FGF gene family which has been shown to be expressed in the outer retina where it may be involved in trophic support of photoreceptors or pathological neovascularization. The goal of this project is identify cis-acting sequences in the FGF-5 5′ flanking region important in the regulation of cell type-specific FGF-5 gene expression in human RPE cells. Methods. A 1650 bp DNA fragment from the FGF-5 5′ flanking region was subcloned into the luciferase reporter gene vector, pGL2-Basic, and sequenced. A set of nested deletions was constructed using Exonuclease III, and the 5′ endpoints were mapped. Luciferase activity was measured following transient transfection of reporter gene contructs into ARPE-19 cells, a human RPE cell line developed and characterized in our laboratory. Results. We have shown that the 1650 bp fragment from the FGF-5 5′ flanking region has promoter activity in human RPE cells. AP-1, AP-2, TEF-1, and SP1 sites were identified within this region by sequence analysis. Deletion studies revealed a negative regulatory element between positions -634 and -446. Upon further examination of this fragment, a consensus binding site for FSE2, a negative regulator of adipocyte differentiation, was found. Conclusions. We have cloned and sequenced a fragment which contains the functional FGF-5 gene promoter and have begun to identify key cis-acting regulatory elements within this fragment in human RPE cells. This region is currently being examined in a variety of cell types in order to demonstrate cell type-specific gene expression.

Original languageEnglish (US)
JournalInvestigative Ophthalmology and Visual Science
Volume37
Issue number3
StatePublished - Feb 15 1996

ASJC Scopus subject areas

  • Ophthalmology

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