Transcription factor organic cation transporter 1 (OCT-1) affects the expression of porcine Klotho (KL) gene
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Abstract
Klotho (KL), originally discovered as an aging suppressor, was a membrane protein that shared sequence similarity with the β-glucosidase enzymes. Recent reports showed Klotho might have a role in adipocyte maturation and systemic glucose metabolism. However, little is known about the transcription factors involved in regulating the expression of porcine KL gene. Deletion fragment analysis identified KL-D2 (-418 bp to -3 bp) as the porcine KL core promoter. MARC0022311 in KL intron 1 appeared a polymorphism (A or G) in Landrace × DIV pigs, and relative luciferase activity of pGL3-D2-G was significantly higher than pGL3-D2-A. This was possibly the result of a change in KL binding ability with transcription factor organic cation transporter 1 (OCT-1), which was confirmed using electrophoretic mobility shift assays (EMSA) and chromatin immunoprecipitation (ChIP). Moreover, OCT-1 regulated endogenous KL expression by RNA interference. Our study indicates SNP MARC0022311 affects porcine KL expression by regulating its promoter activity via OCT-1.
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2016. Transcription factor organic cation transporter 1 (OCT-1) affects the expression of porcine Klotho (KL) gene. PeerJ Preprints 4:e1800v1 https://doi.org/10.7287/peerj.preprints.1800v1note This preprint is not peer-reviewed. You may wish to reference the subsequent peer-reviewed version of this article.
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Supplemental Information
Transcription factor binding site prediction of the procine KL intron 1 containing MARC0022311 (KL g.1474 A>G)
Quadrilateral frame indicated the substitutions and extra binding site of OCT-1. (A) Predicted by BIOBASE online software. (B) Predicted by TFserach online software.
Genotyping results of MARC0022311
(A) PK cells. (B) ST cells. MARC0022311 was marked in gray backgound.
OCT-1 binding sites in the porcine KL intron 1
(A) Frequency distribution of the predicted OCT-1 binding sites. X-axis indicated the length of the porcine KL intron 1 in bp. Y-axis was the frequency of the predicted OCT-1 binding sites. (B) ChIP analysis of three candidate OCT-1 binding sites (1395 bp to 1525 bp, 14322 bp to 14436 bp, 30970 bp to 31141 bp) in KL intron 1 in PK cells. Primers used for ChIP-PCR was shown in Table 1. Input and R were positive control, while IgG was the negative control.
Additional Information
Competing Interests
The authors declare that they have no competing interests.
Author Contributions
Yan Li performed the experiments, analyzed the data, contributed reagents/materials/analysis tools, wrote the paper, prepared figures and/or tables, reviewed drafts of the paper.
Lei Wang performed the experiments, analyzed the data, contributed reagents/materials/analysis tools, wrote the paper, prepared figures and/or tables, reviewed drafts of the paper.
Jiawei Zhou analyzed the data, contributed reagents/materials/analysis tools, reviewed drafts of the paper.
Fenge Li conceived and designed the experiments, wrote the paper, reviewed drafts of the paper.
Animal Ethics
The following information was supplied relating to ethical approvals (i.e., approving body and any reference numbers):
All animal procedures were performed according to protocols approved by the Biological Studies Animal Care and Use Committee of Hubei Province, PR China. Sample collection was approved by the ethics committee of Huazhong Agricultural University (No.30700571 for this study).
Data Deposition
The following information was supplied regarding data availability:
The raw data has been supplied as a Supplemental Dataset.
Funding
This work was supported financially by Key Projects in National Science R&T Program (2015BAD03B02, 2014BAD20B01), Hubei Science R&T Program (2014BBB008, 2014BBA194), and Fundamental Research Funds for the Central Universities. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
