Analysis of upstream regulatory regions required for the activities of two promoters of the rat aldolase A gene

Abstract

Rat aldolase A gene has 2 promoters with different tissue specificities (M-and AH promoters). The M promoter is active only in adult skeletal muscle and induced during myogenesis, whereas the AH promoter is active ubiquitously in many tissues, including various cancer cells. Regulatory sequences for these promoters were investigated through assays for transient expression after introduction into myogenic and nonmyogenic cells. When M promoter-CAT fusion genes were transfected into primary cultures of chicken myoblasts, expression of CAT activity was drastically induce d during myotube formation. The region comprising 202 to 85 base pairs (bp) upstream from the transcription initiation site was found to be necessary for the induction and an enhancer activity whose region includes the AT-rich recognition sequence (MEF-2 binding site). On the other hand. 2 upstream regions were found to be responsible for AH promoter activity expressed in HepG2 cells. The distal region (-280 to -260) of the promoter includes the API binding sequence, whereas the proximal region (-207 to -180) contains a novel inverted repeat consisting of 22 bp but does not contain known promoter and enhancer sequences.

The MEF-3 Motif is Required for MEF-2-Mediated Skeletal Muscle-Specific Induction of the Rat Aldolase A Gene

“…MEF-2 plays an essential role in muscle-specific induction of the rat aldolase A gene. Recent studies have indicated that the region of the M promoter from -202 to -85 exhibits the properties of a muscle-specific enhancer in chicken primary myotubes differentiated in vitro (14). This region contains a single A/T-rich sequence, CTAAATATAG, which closely matches (9 of 10) the MEF-2 consensus sequence (41).…”

“…Plasmid constructions. The construction of pM202CAT and pM85CAT was described previously (14). The M promoter sequence in pM202CAT extends from 202 bp up- FIG.…”

“…Cell culture, DNA transfection, and CAT assay. Primary myoblast cells were prepared from the breast muscles of 11-day-old chicken embryos as described previously (14). The cells were cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum.…”

“…The cells were cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum. Transfection analyses were performed by the method of calcium phosphate coprecipitation as described previously (14) with 10 ,ug of test plasmid and 5 F±g of the simian virus 40-0-galactosidase vector pCH110 as the internal standard. After 4 h of incubation at 37°C, the medium containing the precipitates was removed and replaced with fresh medium.…”