Transcriptional Regulation Associated with Subcutaneous Adipogenesis in Porcine ACSL1 Gene

Figures

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  Figure 1. C/EBPα upregulates the expression of ACSL1 through binding to two motifs in the promoter. (A) Illustration of putative binding sites of C/EBPα and CREB in the promoter of ACSL1. F and R indicate forward and reverse primer, respectively; (B) Promoter activity of the upstream sequence and a series of 5′ truncated fragments; (C) Overexpression efficiencies of vectors containing the coding sequences of porcine C/EBPα as revealed with western blotting; (D) Ectopic C/EBPα enhances the promoter activity; (E) Deletion of the binding sites of C/EBPα decreases the promoter activity. Scheme for deleting the two binding sites of C/EBPα is given above, and ^ indicates a nucleotide deletion; (F) Effects of C/EBPα on the promoter activity were abolished by deletion of the binding sites; (G) Ectopic C/EBPα increases the mRNA level of ACSL1 gene; (H) Ectopic C/EBPα increases the protein level of ACSL1 gene; (I) C/EBPα binds to the putative motifs as revealed by electrophoretic mobility shift assay. Differences identified: *, a, b, and c, p < 0.05; **, A, B, C, p < 0.01.

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  Figure 2. CREB upregulates the promoter activities of ACSL1 through two binding sites. (A) Overexpression efficiencies of vectors containing the coding sequences of the two isoforms of porcine CREB; (B) Overexpression of both isoforms of CREB enhances promoter activity. (C) The putative binding sites of CREB positively regulate promoter activity. Scheme for deleting the binding sites of CREB is provided below, ^ indicates the nucleotide was deleted; (D) The promoting effects of CREB on promoter activity were reversed by inactivation of the binding sites. The values were compared with that in cells cotransfected with a wild type reporter gene and plasmids overexpressing CREB; (E) Ectopic CREB increases the mRNA level of ACSL1. (F) Ectopic CREB increases the protein level of ACSL1. * p < 0.05, ** p < 0.01.

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  Figure 3. ACSL1 functions as a downstream gene of C/EBPα to promote adipogenesis. (A) Efficiencies of plasmids overexpressing C/EBPα and siRNA against C/EBPα; (B) Effects of ACSL1 on the differentiation of preadipocytes as revealed by Oil Red O staining and quantification of triglyceride; (C) ACSL1 mediates the effects of C/EBPα on adipogenesis. * p < 0.05, ** p < 0.01.

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  Figure 4. SNPs in the promoter are associated with the expression of ACSL1. (A) Sequencing diagram of SNPs exemplified with heterozygotes; (B) SNPs affect the promoter activity. The values were compared with that in cells transfected with wild type plasmid, pF2/R0; (C) Haplotype mutation decreases the expression of ACSL1 in vivo. * p < 0.05, ** p < 0.01.

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  Figure 5. Schematic structure of transcript variants identified. RT, transcripts identified with RT-PCR method; PB, transcripts identified with PacBio sequencing; RF, reference sequence (GenBank No. NM_001167629.2). Open boxes indicate that there are deletions compared with the RF sequence. The red vertical lines indicate sequence insertion, while white vertical lines indicate single base substitution. The bright blue lines indicate the initiation and termination codon of RF sequence, respectively.