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CCAAT/enhancer-binding protein α is a crucial regulator of human fat mass and obesity associated gene transcription and expression.

Ren W, Guo J, Jiang F, Lu J, Ding Y, Li A, Liang X, Jia W - Biomed Res Int (2014)

Bottom Line: Site-directed mutagenesis of the putative C/EBP α binding sites decreased FTO promoter activity.Chromatin immunoprecipitation (ChIP) experiment was carried out and the result shows direct binding of C/EBP α to the putative binding regions in the FTO promoter.Collectively, our data suggest that C/EBP α may act as a positive regulator binding to FTO promoter and consequently, activates the gene transcription.

View Article: PubMed Central - PubMed

Affiliation: Department of Endocrinology and Metabolism, Shanghai Jiao Tong University Affiliated Sixth People's Hospital, Shanghai Diabetes Institute, Shanghai Key Laboratory of Diabetes Mellitus, Shanghai Clinical Center for Diabetes, Shanghai 200233, China.

ABSTRACT
Several susceptibility loci have been reported associated with obesity and T2DM in GWAS. Fat mass and obesity associated gene (FTO) is the first gene associated with body mass index (BMI) and risk for diabetes in diverse patient populations. FTO is highly expressed in the brain and pancreas, and is involved in regulating dietary intake and energy expenditure. While much is known about the epigenetic mutations contributing to obesity and T2DM, less is certain with the expression regulation of FTO gene. In this study, a highly conserved canonical C/EBP α binding site was located around position -45~-54 bp relative to the human FTO gene transcriptional start site. Site-directed mutagenesis of the putative C/EBP α binding sites decreased FTO promoter activity. Overexpression and RNAi studies also indicated that C/EBP α was required for the expression of FTO. Chromatin immunoprecipitation (ChIP) experiment was carried out and the result shows direct binding of C/EBP α to the putative binding regions in the FTO promoter. Collectively, our data suggest that C/EBP α may act as a positive regulator binding to FTO promoter and consequently, activates the gene transcription.

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Related in: MedlinePlus

Identification of the sequence motif responsible for activation of the FTO promoter by C/EBPα. ChIP assay in HEK 293 cells showing that C/EBPα can bind to the human FTO promoter site. Amounts of coprecipitated DNA (Anti-C/EBPα) and the corresponding amounts in the input chromatin samples (input 1 : 10) were measured by PCR.
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fig4: Identification of the sequence motif responsible for activation of the FTO promoter by C/EBPα. ChIP assay in HEK 293 cells showing that C/EBPα can bind to the human FTO promoter site. Amounts of coprecipitated DNA (Anti-C/EBPα) and the corresponding amounts in the input chromatin samples (input 1 : 10) were measured by PCR.

Mentions: ChIP analysis was performed to test if C/EBPα binds to the FTO promoter. Nuclear lysates prepared from HEK 293 were subjected to sonication to shear DNA to lengths between 200 and 1000 bp on ice followed by phenol/chloroform extraction to recover protein/DNA complexes. C/EBPα antibody was used to pull down the complexes as instructed. The resultant precipitates were then used as templates for PCR amplification of FTO promoter sequence containing the C/EBPα binding motif. As shown in Figure 4, precipitates resulting from C/EBPα antibody yielded a corresponding band as that of amplification with input (1 : 10), an aliquot of chromatin that was not incubated with an antibody, while no band was showed in the negative control (IgG). These results clearly demonstrate an in vivo recruitment of C/EBPα binding element on the human FTO promoter. Taken together, our results suggest that C/EBPα specifically binds to the predicted motifs to regulate FTO transcription and expression directly.


CCAAT/enhancer-binding protein α is a crucial regulator of human fat mass and obesity associated gene transcription and expression.

Ren W, Guo J, Jiang F, Lu J, Ding Y, Li A, Liang X, Jia W - Biomed Res Int (2014)

Identification of the sequence motif responsible for activation of the FTO promoter by C/EBPα. ChIP assay in HEK 293 cells showing that C/EBPα can bind to the human FTO promoter site. Amounts of coprecipitated DNA (Anti-C/EBPα) and the corresponding amounts in the input chromatin samples (input 1 : 10) were measured by PCR.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4022073&req=5

fig4: Identification of the sequence motif responsible for activation of the FTO promoter by C/EBPα. ChIP assay in HEK 293 cells showing that C/EBPα can bind to the human FTO promoter site. Amounts of coprecipitated DNA (Anti-C/EBPα) and the corresponding amounts in the input chromatin samples (input 1 : 10) were measured by PCR.
Mentions: ChIP analysis was performed to test if C/EBPα binds to the FTO promoter. Nuclear lysates prepared from HEK 293 were subjected to sonication to shear DNA to lengths between 200 and 1000 bp on ice followed by phenol/chloroform extraction to recover protein/DNA complexes. C/EBPα antibody was used to pull down the complexes as instructed. The resultant precipitates were then used as templates for PCR amplification of FTO promoter sequence containing the C/EBPα binding motif. As shown in Figure 4, precipitates resulting from C/EBPα antibody yielded a corresponding band as that of amplification with input (1 : 10), an aliquot of chromatin that was not incubated with an antibody, while no band was showed in the negative control (IgG). These results clearly demonstrate an in vivo recruitment of C/EBPα binding element on the human FTO promoter. Taken together, our results suggest that C/EBPα specifically binds to the predicted motifs to regulate FTO transcription and expression directly.

Bottom Line: Site-directed mutagenesis of the putative C/EBP α binding sites decreased FTO promoter activity.Chromatin immunoprecipitation (ChIP) experiment was carried out and the result shows direct binding of C/EBP α to the putative binding regions in the FTO promoter.Collectively, our data suggest that C/EBP α may act as a positive regulator binding to FTO promoter and consequently, activates the gene transcription.

View Article: PubMed Central - PubMed

Affiliation: Department of Endocrinology and Metabolism, Shanghai Jiao Tong University Affiliated Sixth People's Hospital, Shanghai Diabetes Institute, Shanghai Key Laboratory of Diabetes Mellitus, Shanghai Clinical Center for Diabetes, Shanghai 200233, China.

ABSTRACT
Several susceptibility loci have been reported associated with obesity and T2DM in GWAS. Fat mass and obesity associated gene (FTO) is the first gene associated with body mass index (BMI) and risk for diabetes in diverse patient populations. FTO is highly expressed in the brain and pancreas, and is involved in regulating dietary intake and energy expenditure. While much is known about the epigenetic mutations contributing to obesity and T2DM, less is certain with the expression regulation of FTO gene. In this study, a highly conserved canonical C/EBP α binding site was located around position -45~-54 bp relative to the human FTO gene transcriptional start site. Site-directed mutagenesis of the putative C/EBP α binding sites decreased FTO promoter activity. Overexpression and RNAi studies also indicated that C/EBP α was required for the expression of FTO. Chromatin immunoprecipitation (ChIP) experiment was carried out and the result shows direct binding of C/EBP α to the putative binding regions in the FTO promoter. Collectively, our data suggest that C/EBP α may act as a positive regulator binding to FTO promoter and consequently, activates the gene transcription.

Show MeSH
Related in: MedlinePlus