Abstract
Four different transcripts encoding fibroblast growth factor 1 (FGF-1, also known as aFGF) have been previously identified in our laboratory. Among them, FGF-1.B is the major transcript expressed specifically in the neuronal cells in brain tissue. Using the transient transfection experiment in a glioblastoma cell line, U1240MG, that expresses 1.B, we previously identified two regulatory regions (RR1 and RR2) in the brain-specific promoter, FGF-1.B. In the present study, we showed that the minimal region required for the DNA-protein interaction in RR2 resides in an 18-base pair (-484 to -467) sequence, by using DNase I footprinting and methylation interference studies and electrophoretic mobility shift assays. This minimal cis-acting element was found to be sufficient in enhancing the reporter activity driven by the heterologous herpes simplex virus thymidine kinase promoter in the 1.B-positive U1240MG cell line. This enhancing effect, however, was not detected in a glioblastoma cell line, U1242MG, which is negative for 1.B expression. By electrophoretic mobility shift assays, we also identified a specific DNA-protein complex, namely complex I, which is specific for 1.B-positive cell lines and human brain tissue. By in situ UV cross-linking experiment, we further showed that complex I contains two major DNA-binding proteins of apparent molecular masses of 37 and 98 kDa. Our results suggest that the formation of complex I, resulting from the heterodimerization of a 37-kDa protein (1.B-specific) and a 98-kDa protein (ubiquitous) may likely be a prerequisite for the enhanced expression of 1.B transcript in neuronal cells.
Highlights
The FGF1 family consists of 14 structurally related polypeptide growth factors [1, 2]
The study of the tissue- or cell-specific distribution of FGF-1 transcripts has led to the identification of four different transcripts having the same protein coding exons but different 5Ј-untranslated exons [27,28,29,30,31]
FGF-1.B is the predominant transcript in brain, gliomas, and some glioblastoma cell lines (e.g. U1240MG and U251MG) [26, 32]
Summary
The FGF1 family consists of 14 structurally related polypeptide growth factors [1, 2]. FGF-1, a prototype member, was originally identified as a mitogen for endothelial cells [3] and subsequently for a variety of mesoderm- and neuroectodermderived cells [4, 5] This growth factor is involved in a variety of physiological and pathological function such as tissue growth, wound healing, neovascularization, mesoderm development, and angiogenesis (6 –10). We have previously identified a 41-bp DNA sequence (Ϫ507 to Ϫ467) in the brain-specific FGF-1.B promoter, designated as regulatory region 2 (RR2), through luciferase reporter assays [34]. We have narrowed down the minimal cis-acting sequence to an 18-bp region (Ϫ484 to Ϫ467) which can form more than one DNA-protein complex including the 1.B-specific complex, complex I This 18-bp sequence is sufficient to enhance the luciferase reporter gene expression driven by a heterologous thymidine kinase (tk) promoter in a cell line-specific manner. UV cross-linking analysis of this 1.B-specific complex re-
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