Deciphering the Role of the SREBF1 Gene in the Transcriptional Regulation of Porcine Adipogenesis Using CRISPR/Cas9 Editing

Abstract

Sterol regulatory element-binding protein 1 (SREBP1) is an important transcription factor that controls lipid metabolism and adipogenesis. Two isoforms, SREBP1a and SREBP1c, are generated by alternative splicing of the first exon of the SREBF1 gene. The porcine SREBF1 gene has mainly been studied for its role in lipid metabolism in adipose tissues, but little is known about its involvement, and the role of its two isoforms, in adipogenesis. The aim of the present study was to introduce a deletion in the 5'-regulatory region of the SREBF1c gene, considered crucial for adipogenesis, using the Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein 9 (CRISPR/Cas9) method. This approach allows for the evaluation of how inhibiting SREBF1c transcription affects the expression of other genes essential for adipocyte differentiation, particularly PPARG, CEBPA, CEBPB, CEBPD, GATA2, and FABP4. It was observed that disrupting the SREBF1c isoform had no effect on the GATA2 gene but did result in a decrease in the expression of the CEBPA and CEBPD genes, an increase in the expression of CEBPB, and an inhibition in the expression of the PPARG and FABP4 genes. These changes in gene expression blocked adipogenesis, as could be seen by the failure of lipid droplets to accumulate. Our results provide evidence highlighting the pivotal role of the SREBP1c isoform in the regulation of porcine adipogenesis.

Keywords: CRISPR/Cas9; MSC; SREBF1; SREBP1; adipogenesis; isoforms; pig.

Figure 1

Simplified scheme of transcriptional regulation in adipogenesis, highlighting the transcription factors analyzed in this study. Adapted from [18].

Figure 2

The 5′-regulatory sequence of SREBF1c with marked transcription factor binding sites and positions of two sgRNAs (sgRNA1 and sgRNA3 in blue frames) used to excise the proximal promoter region.

Figure 3

Sanger sequencing of the PCR product confirmed the deletion of 567 bp in the SREBF1c 5′-regulatory region. The position of the joined DNA fragments in the AD-MSC cells and the partial sequences recognized by sgRNA1 and sgRNA3 are shown.

Figure 4

Location of the 567 bp deletion site in the genomic region covering SREBF1a and SREBF1c isoforms according to Sscrofa 11.1 assembly (NC_010454.4) chromosome 12 reference sequence. E1–E4: exons 1–4. Genomic position of the 567 bp deletion: 12: 60740868-60741434.

Figure 5

Relative transcript levels of SREBF1a (a) and SREBF1c (b) during subsequent days of adipogenesis. The error bars represent 95% confidence intervals. **: significantly higher in AD-MSC_WT than in AD-MSC_DEL, p < 0.001; ^^: significantly lower in AD-MSC_WT than in AD-MSC_DEL, p < 0.001; ^: significantly lower in AD-MSC_WT than in AD-MSC_DEL, p < 0.05.

Figure 6

Relative mRNA levels of SREBF1a (a) and SREBF1c (b) isoforms in the subcutaneous and visceral adipose tissue of wild-type pigs. The boxplots show the lower quartile (lower edge of the box), median (black line dividing the box into two parts), upper quartile (upper edge of the box), outliers (dots), and whiskers (values inside the lower and upper quartiles). **: significant differences between groups, p < 0.001, Wilcoxon’s signed rank test.

Figure 7

Measuring of lipid accumulation based on BODIPY 493/503 fluorescent intensity during successive days of adipocyte differentiation (a). Error bars represent 95% confidence intervals. D: day; **: significantly higher in AD-MSC_WT than in AD-MSC_DEL, p < 0.001; *: significantly higher in AD-MSC_WT than in AD-MSC_DEL, p < 0.05. Representative images showing adipocyte differentiation (b). Lipid droplets were stained with BODIPY 493/503 (green); the nuclei were counterstained with DAPI (blue). Scale bar: 50 µm.

Figure 8

Relative transcript levels of GATA2 (a), CEBPD (b), CEBPB (c), CEBPA (d), PPARG (e), and FABP4 (f) during subsequent days of adipogenesis in AD-MSC_WT and AD-MSC_DEL. Error bars represent 95% confidence intervals. **: significantly higher in AD-MSC_WT than in AD-MSC_DEL, p < 0.001; *: significantly higher in AD-MSC_WT than in AD-MSC_DEL, p < 0.01; ^^: significantly lower in AD-MSC_WT than in AD-MSC_DEL, p < 0.001; ^: significantly lower in AD-MSC_WT than in AD-MSC_DEL, p < 0.05.