An A/G-rich motif in the rat fibroblast growth factor-2 gene confers enhancer activity on a heterologous promoter in neonatal rat cardiac myocytes.
We have cloned the rat fibroblast growth factor-2 (FGF-2) promoter region including 1058 base pairs (bp) of 5'-flanking DNA. Complete sequencing of this promoter region revealed a 74 bp domain between nucleotides-793 and-720 that was greater than 97% A/G-rich. A repeat of the sequence 5'-AGGGAGGG-3' separated by 11 bp was located at the core of this domain. A 37 bp A/G-rich oligonucleotide containing these AGGG-repeat sequences was synthesised, and tested for function on a minimal herpes simplex virus thymidine kinase (TK) promoter, fused to the firefly luciferase gene (TKp.luc), in transiently transfected neonatal rat cardiac myocytes. Promoter activity was stimulated approximately 3 fold in the presence of AGGG-repeat sequences. This effect was neither tissue or species-specific since TK promoter activity was increased approximately 11 fold in both rat and human glial tumor cells. Four specific complexes (Cl-4) were detected between neonatal rat heart nuclear proteins and the 37 bp A/G-rich oligonucleotide by gel mobility shift assay. Competition with excess unlabelled 37 bp A/G-rich oligonucleotide revealed that two complexes represented very high affinity/specificity interactions (C2 > C4) while Cl and C3 were of lower affinity. As a result, competition with up to a 25 fold molar excess of 37 bp A/G-rich oligonucleotide led to the loss of C2 and C4, and a corresponding and transient increase in the levels of Cl and C3, which themselves were reduced with more competitor oligonucleotide. The AGGG-repeat resembles the 5'-gGGGAGGG-3' sequence previously implicated in the response of the atrial natriuretic factor promoter to the alpha-adrenergic agonist, phenylephrine. Although an additional 1.5 fold increase in TK promoter activity was detected in the presence of the 37 bp A/G-rich oligonucleotide with phenylephrine treatment of transfected myocytes, this effect was not statistically significant. Furthermore, there was no difference in the gel mobility shift (Cl-4) pattern obtained with the 37 bp A/G-rich oligonucleotide and nuclear protein isolated from neonatal rat cardiac myocytes grown in the presence or absence of norepinephrine. These data suggest that the A/G rich sequences in the rat FGF-2 gene 5'-flanking DNA, including the AGGG-repeat, are able to confer stimulatory activity on a promoter in a tissue- and species-independent manner, but alone are not able to induce a significant phenylephrine response in neonatal rat cardiac myocytes.
Detillieux
K A
KA
Gene Technology Group, University of Manitoba, Winnipeg, Canada.
Meyers
A F
AF
Meij
J T
JT
Cattini
P A
PA
D000241:Adenosine / Q000235:genetics*; D000818:Animals; D000831:Animals, Newborn; D001482:Base Composition; D001483:Base Sequence; D002478:Cells, Cultured; D004742:Enhancer Elements, Genetic; D016222:Fibroblast Growth Factor 2 / Q000235:genetics* / Q000502:physiology*; D006151:Guanosine / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D009206:Myocardium / Q000166:cytology / Q000378:metabolism*; D009687:Nuclear Proteins / Q000378:metabolism; D009928:Organ Specificity / Q000235:genetics; D011401:Promoter Regions, Genetic; D051381:Rats; D017207:Rats, Sprague-Dawley; D012091:Repetitive Sequences, Nucleic Acid; D014407:Tumor Cells, Cultured
D009687:Nuclear Proteins; D016222:Fibroblast Growth Factor 2; D006151:Guanosine; D000241:Adenosine