2023 Fiscal Year Research-status Report
Elucidation of the transcriptional pathways in which amino acids act as a nutritional signal
| Project/Area Number |
23K13900
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| Research Institution | University of Tsukuba |
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Principal Investigator |
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| Project Period (FY) |
2023-04-01 – 2025-03-31
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| Keywords | Nutrigenomics / Klf15 / High protein diet |
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| Outline of Annual Research Achievements |
Regarding the Klf15 independent pathway, based on the RNA-Seq analysis of mice fed HPD vs LPD and Klf15 knockout experiment we determined which of the genes involved in amino acid metabolism have altered regulation in response to lack of Klf15 gene while receiving HPD. The genes that their regulation pattern has not been changed are considered Klf15 independent. Regarding in vivo Ad-Luc analysis of candidate genes that estimated promoter site got linked to luciferase reporter gene and formed and Ad-promotrt-Luc plasmid. The adenovirus was applied in the liver of mice fed HPD. The results showed the activity of Ad-promoter-Luc in response to protein intake, and we acquired the genomic region that functions independently from regulatory role of Klf15.
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| Current Status of Research Progress |
Current Status of Research Progress
2: Research has progressed on the whole more than it was originally planned.
Reason
According to the submitted research plan, progress has been smooth. Regarding KLF15 independent pathways of gene regulation, we determined which of the genes have altered regulation in response to lack of Klf15 gene while receiving HPD. Construction of the Ad-promoter-Luc plasmid was successful. Regarding the in vivo Ad-luc analysis of KLF15 promoter, the experimental methods has been established and the process is expected to go well.
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| Strategy for Future Research Activity |
Regarding the in vivo Ad-luc analysis of KLF15 promoter, the mechanism of KLF15 expression regulation induced by HPD intake will be analyzed by the in vivo Ad-luc method, and the amino acid-responsive enhancer region that recognizes changes in amino acid composition will be identified. Next, we will identify expression regulators that bind to amino acid-responsive enhancers by the TFEL scan method. The molecular interaction will be clarified by trans omics analysis. By proteome analysis, we will proceed with the elucidation of the identified complex centered on the expression regulator and its molecular modification, and at the same time, superimpose the metabolome analysis results of the amino acid metabolites in the nucleus to regulate the expression.
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| Causes of Carryover |
We had planned to conduct an experiment to confirm the reproducibility of the IVIS method in February 2024, but due to a malfunction of analytical equipment, the experiment could not be carried out, and the amount to be used in the next fiscal year was incurred.
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