Nuclear Respiratory Factor 1 Negatively Regulates the P1 Promoter of the Peroxisome Proliferator-Activated Receptor-γ Gene and Inhibits Chicken Adipogenesis

Sun

et al. 2021

Front. Cell Dev. Biol.

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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.

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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

Cui

Sun

et al. 2021

Front. Cell Dev. Biol.

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“…The genomic region corresponding to the 5 UTR is usually part of the promoter. Our previous study demonstrated that the 108-bp sequence downstream of the transcription start site of PPARγ1, which is part of the 5 UTR, had the highest promoter activity (Cui et al, 2018). To test whether the cloned PPARγ1 5 UTR had promoter activity and whether the uORF mutation reduced it, we cloned DNA sequences corresponding to wildtype and uORF-mutated 5 UTRs of PPARγ1 into luciferase reporter vector pGL3-Basic, named pGL3-PPARγ1-WT and pGL3-PPARγ1-Mut, respectively.…”

Section: The Effect Of Uorf Mutation On Promoter Activitymentioning

confidence: 99%

An Upstream Open Reading Frame Represses Translation of Chicken PPARγ Transcript Variant 1

Chu

et al. 2020

Front. Genet.

Self Cite

Peroxisome proliferator-activated receptor γ (PPARγ) is a master regulator of adipogenesis. The PPARγ gene produces various transcripts with different 5untranslated regions (5 UTRs) because of alternative promoter usage and splicing. The 5 UTR plays important roles in posttranscriptional gene regulation. However, to date, the regulatory role and underlying mechanism of 5 UTRs in the posttranscriptional regulation of PPARγ expression remain largely unclear. In this study, we investigated the effects of 5 UTRs on posttranscriptional regulation using reporter assays. Our results showed that the five PPARγ 5 UTRs exerted different effects on reporter gene activity. Bioinformatics analysis showed that chicken PPARγ transcript 1 (PPARγ1) possessed an upstream open reading frame (uORF) in its 5 UTR. Mutation analysis showed that a mutation in the uORF led to increased Renilla luciferase activity and PPARγ protein expression, but decreased Renilla luciferase and PPARγ1 mRNA expression. mRNA stability analysis using real-time RT-PCR showed that the uORF mutation did not interfere with mRNA stability, but promoter activity analysis of the cloned 5 UTR showed that the uORF mutation reduced promoter activity. Furthermore, in vitro transcription/translation assays demonstrated that the uORF mutation markedly increased the translation of PPARγ1 mRNA. Collectively, our results indicate that the uORF represses the translation of chicken PPARγ1 mRNA.

“…Our previous study demonstrated that the 108-bp sequence downstream of the transcription start site of PPARγ1, which is part of the 5′ UTR, had the highest promoter activity (Cui et al 2018). To test whether the cloned PPARγ1 5′ UTR had promoter activity and whether the uORF mutation reduced it, we cloned DNA sequences corresponding to wild-type and uORF-mutated 5′ UTRs of PPARγ1 into luciferase reporter vector pGL3-Basic, named pGL3-PPARγ1-WT and pGL3-PPARγ1-Mut, respectively.…”

Section: The Effect Of Uorf Mutation On Promoter Activitymentioning

confidence: 99%

An upstream open reading frame represses translation of chicken PPARγ transcript variant 1

Chu

et al. 2019

Preprint

Self Cite

Peroxisome proliferator-activated receptor γ (PPARγ) is a master regulator of adipogenesis. The PPARγ gene produces various transcripts with different 5′-untranslated regions (5′ UTRs) because of alternative promoter usage and splicing.The 5′ UTR plays important roles in posttranscriptional gene regulation. However, to date, the regulatory role and underlying mechanism of 5′ UTRs in the posttranscriptional regulation of PPARγ expression remain largely unclear. In this study, we investigated the effects of 5′ UTRs on posttranscriptional regulation using reporter assays. Our results showed that the five PPARγ 5′ UTRs exerted different effects on reporter gene activity. Bioinformatics analysis showed that chicken PPARγ transcript 1 (PPARγ1) possessed an upstream open reading frame (uORF) in its 5′ UTR. Mutation analysis showed that a mutation in the uORF led to increased Renilla luciferase activity and PPARγ protein expression, but decreased Renilla luciferase and PPARγ1 mRNA expression. mRNA stability analysis using real-time RT-PCR showed that the uORF mutation did not interfere with mRNA stability, but promoter activity analysis of the cloned 5′ UTR showed that the uORF mutation reduced promoter activity. Furthermore, in vitro transcription/translation assays demonstrated that the uORF mutation markedly increased the translation of PPARγ1 mRNA.Collectively, our results indicate that the uORF represses the translation of chicken PPARγ1 mRNA.