KLF2 Inhibits Chicken Preadipocyte Differentiation at Least in Part via Directly Repressing PPARγ Transcript Variant 1 Expression

Abstract

Peroxisome proliferator-activated receptor gamma (PPARγ) is the master regulatory factor of preadipocyte differentiation. As a result of alternative splicing and alternative promoter usage, PPARγ gene generates multiple transcript variants encoding two protein isoforms. Krüppel-like factor 2 (KLF2) plays a negative role in preadipocyte differentiation. However, its underlying mechanism remains incompletely understood. Here, we demonstrated that KLF2 inhibited the P1 promoter activity of the chicken PPARγ gene. Bioinformatics analysis showed that the P1 promoter harbored a conserved putative KLF2 binding site, and mutation analysis showed that the KLF2 binding site was required for the KLF2-mediated transcription inhibition of the P1 promoter. ChIP, EMSA, and reporter gene assays showed that KLF2 could directly bind to the P1 promoter regardless of methylation status and reduced the P1 promoter activity. Consistently, histone modification analysis showed that H3K9me2 was enriched and H3K27ac was depleted in the P1 promoter upon KLF2 overexpression in ICP1 cells. Furthermore, gene expression analysis showed that KLF2 overexpression reduced the endogenous expression of PPARγ transcript variant 1 (PPARγ1), which is driven by the P1 promoter, in DF1 and ICP1 cells, and that the inhibition of ICP1 cell differentiation by KLF2 overexpression was accompanied by the downregulation of PPARγ1 expression. Taken together, our results demonstrated that KLF2 inhibits chicken preadipocyte differentiation at least inpart via direct downregulation of PPARγ1 expression.

DNA methylation promotes the expression of PPARγ transcript 1 at least in part by preventing NRF1 binding to the promoter P1 of chicken PPARγ gene

“…Among these 8 TFBSs, NRF1 caught our attention. We previously demonstrated showed that NRF1 inhibited the P1 promoter activity and PPARγ1 expression ( Cui et al, 2021 ). In addition, it has been shown that NRF1 is a methyl-sensitive transcription factor and DNA methylation inhibits its DNA binding and transactivation activity ( Wang et al, 2017 ).…”

“…The pCMV-HA-NRF1 and pGL3P1-327/+108 vectors were constructed previously by our laboratory ( Cui et al, 2018 ). Both unmethylated and methylated pGL3P1-327/+108 vectors were prepared as described previously ( Cui et al, 2021 ).…”

KLF2/PPARγ axis contributes to trauma‐induced heterotopic ossification by regulating mitochondrial dysfunction

“…There are two possible mechanisms, both direct and indirect. Studies found that KLF2 was able to exert an inhibitory effect on PPARγ at the promoter level 36,37 . Two‐tandem Kruèppel binding sites, 5‐CCCACCTCTCCCA‐3 (base pairs 82–93), are present in the proximal promoter region of PPARγ.…”

“…Studies found that KLF2 was able to exert an inhibitory effect on PPARγ at the promoter level. 36 , 37 Two‐tandem Kruèppel binding sites, 5‐CCCACCTCTCCCA‐3 (base pairs 82–93), are present in the proximal promoter region of PPARγ. Electrophoretic mobility gel shift showed that KLF2 was able to bind this sequence and directly repress the PPARγ promoter, resulting in a 70% inhibition of promoter activity.…”