Knockout and Restoration Reveal Differential Functional Roles of PPARγ1 and PPARγ2 in Chicken Adipogenesis.

Peroxisome proliferator-activated receptor γ (PPARγ) is the master regulator of adipogenesis and is expressed as two isoforms, PPARγ1 and PPARγ2. Our previous lentiviral overexpression study showed that PPARγ1 and PPARγ2 differentially regulated proliferation, differentiation, and apoptosis of the immortalized chicken preadipocyte cell line (ICP2). However, we cannot rule out the possibility that the endogenous expression of PPARγ isoforms may compromise our findings. In this study, using the dual sgRNA-directed CRISPR/Cas9 system, we generated γ (γ) and γ-specific knockout (γ) ICP2 cell lines and investigated the differences in proliferation and differentiation among γ, γ, and wild-type ICP2 cells. EdU proliferation assay showed that both γ-specific and γ knockouts significantly increased the proliferation rates. Consistently, real-time RT-PCR analysis showed that both γ-specific and γ knockouts significantly upregulated the expression of proliferation marker genes PCNA and cyclinD1. FACS analysis revealed that γ knockout significantly increased the number of cells accumulating in the S phase and decreased the number of cells accumulating in the G/G phase. Oil Red O staining and gene expression analysis showed both γ-specific and γ knockouts dramatically reduced capacity for adipogenic differentiation. To corroborate our previous findings, PPARγ1 and PPARγ2 expression were restored in γ cells by using the lentiviruses expressing chicken PPARγ1 (LV-PPARγ1) and PPARγ2 (LV-PPARγ2), respectively. Subsequent assays showed that restoration of expression of either PPARγ1 or PPARγ2 suppressed proliferation and stimulated differentiation of the γ cells. By comparison, PPARγ2 had stronger anti-proliferative and pro-adipogenic effects than PPARγ1. To understand the molecular mechanism underlying their differential effects on differentiation of the γ cells, we performed RNA-seq in the γ cells in which individual PPARγ isoform expression was restored at 72 h of differentiation. Transcriptomic analysis revealed that restoring PPARγ1 expression caused far more differentially expressed genes (DEGs) than restoring PPARγ2 expression. GO and KEGG pathway enrichment analyses indicated that PPARγ1 and PPARγ2 had distinct and overlapping functions in adipogenesis. Taken together, our results clearly indicate that PPARγ1 and PPARγ2 differentially impact chicken adipogenesis.