Cloning and transcriptional activity analysis of the porcine cofilin 2 gene promoter

• We cloned the 2.5 kb 5′-flanking region of the porcine CFL2 gene by genome walking.
• We inserted the promoter fragments into luciferase reporter vectors.
• Our data show that the active region of the CFL2 promoter is from − 1502 bp to − 1317 bp.
• The porcine CFL2 promoter sequence encompasses a typical type II eukaryotic promoter.

Cofilins (CFL), including CFL1 and CFL2, are members of the family of actin-binding proteins in eukaryote. CFL2 is predominantly expressed in mammalian skeletal muscle and heart and is important to muscle fiber formation and muscular regeneration. To study transcriptional regulation of porcine CFL2, a 2.5 kb upstream sequence starting from the major CFL2 transcriptional start site was cloned by genome walking. Twelve DNA fragments of the 5′ flank region of the porcine CFL2 gene were further isolated from porcine genomic DNA via PCR and inserted into the luciferase reporter vector pGL4.10 to make 12 CFL2 reporter constructs. All reporter vectors were transfected into C2C12, NIH3T3, or Hela cells and their relative luciferase activity measured after 48 h, respectively. Bioinformatics analysis suggested that there were two TATA-boxes at the − 508 bp and − 453 bp, as well as a GC-box and a CAAT-box in this sequence. Additional transcription factor binding sites including SP1, AP1, AP2, and GATA-1 sites were also predicted. The transcriptional activity of pGL4.10-1554 (1502 bp to + 51 bp) was the strongest, and the promoter's active region was mapped to a region from − 1502 bp to − 1317 bp. Our data provide a foundation for future studies into transcriptional regulation of CFL2.