Transcriptional Regulation Study Of The Bovine ACSL1 Gene

The nutritional value and eating qualities of beef are enhanced when the unsaturated fatty acid content of fat is increased. Longer chain（n-3） polyunsaturated fatty acids such as docosahexaenoic acid（DHA; 22:6, n-3） and eicosapentaenoic acid（EPA, 20:5, n-3） also have well-recognized beneficial effects in terms of reducing the risk of cardiovascular disease, cancer, and type-2 diabetes, as well as critical roles in brain function, visual development in the fetus, and the lifelong maintenance of neural and visual tissues 1-4. Long-chain acyl-Co A synthetase 1（ACSL1） has been identified as a plausible functional and positional candidate gene for manipulation of fatty acid composition in bovine skeletal muscle. Polymorphisms of ACSL1 gene have the most significant associations with the relative contents of distinct fractions and the ratios of fatty acids（e.g., n-3 fatty acids, polyunsaturated,（n-3） long-chain polyunsaturated fatty acids, and trans-vaccenic acid） in bovine skeletal muscle. However, the mechanism regulating bovine ACSL1 transcription remaim elusive. The aim of this study was investigate the transcriptional regulatory mechamism of bovine ACSL1 gene ralted with meat quality traits, which providing the theory basis for molecular breeding in illuminating the molecular mechanisms involved in regulating polyunsaturated fatty acid composition of bovine skeletal muscle, improving eating qualities and enhancing the nutritional value of beef. The main contents were as follows:（1） Applying 5?-rapid amplification of c DNA end analysis（RACE）, we identified multiple transcriptional start sites（TSSs） in its promoter region, which was located 20659, 20656, 20608, and 3 bp upstream of the translational start site. The guanine residue（G） in the proximal 5? UTR, which was located 20659 bp upstream of the translational start site, was verified as the most upstream the translational start site（TSS-1） and designated as +1. TSS-1 was located 7 bp upstream of the 5? end of the published ACSL1 m RNA sequence（NM₀01076085）. The Real-time PCR analysis demonstrated that the basal expression level of ACSL1 was relatively high in subcutaneous adipose tissue and longissimus thoracis, but low in the rumen, reticulum, and lung tissue, thereby indicating that the ACSL1 gene might play a functional role in mediating the fatty acid composition of bovine skeletal muscle.（2） We cloned and sequenced a 2-kb fragment of the ACSL1 5’flanking region from bovine genomic DNA. Deletion analysis revealed a putative core promoter region located in-325/-140 bp upstream of the transcription site and it was also located in the predicted Cp G island. Multiple alignment analysis demonstrated that the putative core promoter region is more conserved across species, in particular the binding motifs for transcription regulation. The binding sites of E2F1、SP1、KLF15 and E2F4 were identified in bovine ACSL1 core promoter. In addition, there was a TATA-box at-36 bp of transcription starat site（+1）.（3）We cotransfected the plasnid p GL-325/+21 together with E2F1, SP1, KLF15,E2F4 and Control si RNA（1⁹ pmol） in C2C12. Repression of endogenus expression of E2F1 transcription factor induced a dose-dependent decrease in the promoter activity of ACSL1 gene in C2C12 cells（25%⁶2%）. Repression of endogenus expression of SP1 transcription factor induced a dose-dependent decrease in the promoter activity of ACSL1 gene in C2C12 cells（36% 56%）. Repression of endogenus expression of KLF15 transcription factor induced a dose-dependent decrease in the promoter activity of ACSL1 gene in C2C12 cells（36%⁵6%）. Repression of endogenus expression of E2F4 transcription factor induced a dose-dependent decrease in the promoter activity of ACSL1 gene in C2C12 cells（20%³9%）,（ p<0.01）.（4）To investigate the roles of these sites in the regulation of ACSL1, we constructed a series of DNA plasmids with 3-bp point mutations in the transcription factor binding sites and transiently transfected them into C2C12 cells. Mutation of the Sp1 binding site in the construct p GL-325/+12 resulted in a significant increase in the promoter activity（145%）, whereas the mutations of E2F1, KLF15, or E2F4 led to ca 58–71% reductions in the ACSL1 promoter activity. Double mutations of the E2F1 and KLF15 sites resulted in additional reductions in the transcriptional activity compared with the mutations of the E2F1, Sp1, KLF15, or E2F4 sites. In addition, EMSA assays also demonstrated that these transcription factors could specifically bind sequences in the proximal promoter of ACSL1.These results indicate that the E2F1 site（at position –197 to –181）, Sp1 site（at position –165 to –149）, KLF15 site（at position –164 to –146）, and E2F4 site（at position –157 to –141） were essential for basal transcriptional activity of the ACSL1 proximal minimal promoter, as well as as well as providing new insights into the regulatory mechanisms and biological functions of the ACSL1 gene in mediating the lipid composition of beef.