A SPLICE VARIANT OF E2-2 BASIC HELIX-LOOP-HELIX PROTEIN REPRESSES THEBRAIN-SPECIFIC FIBROBLAST-GROWTH-FACTOR-1 PROMOTER THROUGH THE BINDING TO AN IMPERFECT E-BOX

We previously demonstrated that a cis-element (-489 to -467) in the br ain-specific fibroblast growth factor (FGF)-1 promoter (FGF-1.B) binds multiple nuclear factors, and this binding enhances transcriptional a ctivity of this promoter. Here we report the isolation of three cDNA c lones, VL1, VL2 and VL3, from a human brain stem cDNA expression libra ry using four tandem repeats of the 26-base pair sequence (-492 to -46 7) as the probe. These cDNA clones represent the variant of bHLH prote in E2-2/SEF2-1 in having 12 additional nucleotides encoding the amino acids RSRS, The glutathione S-transferase (GST) fusion proteins of VL1 , 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 RS RS in U1240MG glioblastoma cells resulted in repression of FGF-I,B pro moter activity. We further showed a significant repression of promoter activity (>40 fold) by E2-2 (lacking the amino acid sequence RSRS) wh en the E47 reporter construct, containing a hexameric E-box site, was used. In contrast, the E2-2 variant containing the RSRS sequence has n o significant effect on either the FGF-1 promoter or E47 promoter, The se results suggest that the relative abundance of the two splice varia nts of E2-2 in brain could be an important determinant for the express ion of FGF-1.