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J Biol Chem. 1998 Jul 24;273(30):19269-76.

A splice variant of E2-2 basic helix-loop-helix protein represses the brain-specific fibroblast growth factor 1 promoter through the binding to an imperfect E-box.

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  • 1Department of Internal Medicine and Comprehensive Cancer Center, Ohio State University, Columbus, Ohio 43210, USA.


We previously demonstrated that a cis-element (-489 to -467) in the brain-specific fibroblast growth factor (FGF)-1 promoter (FGF-1.B) binds multiple nuclear factors, and this binding enhances transcriptional activity of this promoter. Here we report the isolation of three cDNA clones, VL1, VL2 and VL3, from a human brain stem cDNA expression library using four tandem repeats of the 26-base pair sequence (-492 to -467) as the probe. These cDNA clones represent the variant of bHLH protein E2-2/SEF2-1 in having 12 additional nucleotides encoding the amino acids RSRS. The glutathione S-transferase (GST) fusion proteins of VLl, VL2, and VL3 immunologically react with anti-E2-2 antibody and anti-GST-VL2 antibody. Electrophoretic mobility shift assay and methylation interference assay revealed that the GST fusion proteins specifically bind to an imperfect E-box sequence (GACCTG) present in the 26-base pair sequence. Transient expression of the full-length E2-2 without RSRS in U1240MG glioblastoma cells resulted in repression of FGF-1.B promoter activity. We further showed a significant repression of promoter activity (>40 fold) by E2-2 (lacking the amino acid sequence RSRS) when the E47 reporter construct, containing a hexameric E-box site, was used. In contrast, the E2-2 variant containing the RSRS sequence has no significant effect on either the FGF-1 promoter or E47 promoter. These results suggest that the relative abundance of the two splice variants of E2-2 in brain could be an important determinant for the expression of FGF-1.

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