Elucidation of ApoA1 gene regulatory mechanism through searching causal single-nucleotide variants

2021 Fiscal Year Final Research Report

• Project/Area Number: 19K20175
• Research Category: Grant-in-Aid for Early-Career Scientists
• Allocation Type: Multi-year Fund
• Review Section: Basic Section 59040:Nutrition science and health science-related
• Research Institution: University of Tsukuba
• Principal Investigator: Aita Yuichi 筑波大学, 医学医療系, 助教 (60752152)
• Project Period (FY): 2019-04-01 – 2022-03-31
• Keywords: 生活習慣病

Outline of Final Research Achievements

We searched for transcription factors binding to causal single-nucleotide variants (SNVs) to find the mechanism how causal SNVs around ApoA1 gene determine plasma HDL levels. 35 candidates for causal SNVs were selected on the basis of their allele frequency. Among these, SNV #33 nearly doubled the ratio of luciferase activities in HepG2 cells. Next, two rounds of TFEL scan revealed that two transcription factors out of 1588 TFs bound to SNV #33. After assessments of reproducibility, we identified TFEL clone #1193 as a novel transcription factor regulating the gene expression level of ApoA1. Overexpression of TFEL clone #1193 in HepG2 cells increased endogenous ApoA1 expression. We identified not only the causal SNV that affected the transcription of ApoA1 gene but also the transcription factor that bound to this causal SNV and determined the expression level of ApoA1 gene.

Free Research Field

応用健康科学

Academic Significance and Societal Importance of the Research Achievements

血中HDLレベルを規定するcausal SNVの探索を目的に、TFEL scan法を用いてApoA1のプロモーター領域のSNVを解析した。同定された転写因子をヒト細胞株で過剰発現したところ、内因性のApoA1遺伝子の発現が増加した。ApoA1のプロモーター領域にあり、転写活性に影響を与えるSNVと、この部位に結合してApoA1遺伝子の発現を制御する転写因子を同定した。また、同定されたSNVがもたらす肝細胞での効果を検討するために、CRISPR/Cas9による相同組換え修復を利用してSNVを導入する実験系の構築を進めた。