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.
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.
Department of Internal Medicine and Comprehensive Cancer Center, Ohio State University, Columbus, Ohio 43210, USA.
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Liu
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Ray
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Yang
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Luntz-Leybman
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Chiu
D017398:Alternative Splicing; D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; D001933:Brain Stem / Q000378:metabolism*; D002478:Cells, Cultured; D004247:DNA / Q000378:metabolism*; D019175:DNA Methylation; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D016220:Fibroblast Growth Factor 1 / Q000235:genetics* / Q000378:metabolism; D015723:Gene Library; D018257:Helix-Loop-Helix Motifs; D006801:Humans; D011401:Promoter Regions, Genetic; D012091:Repetitive Sequences, Nucleic Acid; D012097:Repressor Proteins / Q000378:metabolism*; D051820:TCF Transcription Factors; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism*; D000073940:Transcription Factor 4; D058118:Transcription Factor 7-Like 2 Protein; D014157:Transcription Factors
D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; D004268:DNA-Binding Proteins; D012097:Repressor Proteins; D051820:TCF Transcription Factors; C532096:TCF4 protein, human; C544894:TCF7L2 protein, human; D015534:Trans-Activators; D000073940:Transcription Factor 4; D058118:Transcription Factor 7-Like 2 Protein; D014157:Transcription Factors; D016220:Fibroblast Growth Factor 1; D004247:DNA