DNA 甲基化通过防止 NRF1 结合到鸡 PPARγ 基因启动子 P1 上，至少部分促进了 PPARγ 转录本 1 的表达。

DNA methylation promotes the expression of PPARγ transcript 1 at least in part by preventing NRF1 binding to the promoter P1 of chicken PPARγ gene.

机构信息

College of Animal Science and Technology, Northeast Agricultural University, Harbin 150030, China; College of Life Science and Agriculture Forestry, Qiqihar University, Qiqihar, 161006, China; Key Laboratory of Chicken Genetics and Breeding, Ministry of Agriculture and Rural Affairs; Key Laboratory of Animal Genetics, Breeding and Reproduction, Education Department of Heilongjiang Province, Harbin 150030, China.

College of Animal Science and Technology, Northeast Agricultural University, Harbin 150030, China; Key Laboratory of Chicken Genetics and Breeding, Ministry of Agriculture and Rural Affairs; Key Laboratory of Animal Genetics, Breeding and Reproduction, Education Department of Heilongjiang Province, Harbin 150030, China.

出版信息

Poult Sci. 2024 May;103(5):103559. doi: 10.1016/j.psj.2024.103559. Epub 2024 Feb 16.

DOI:10.1016/j.psj.2024.103559

PMID:38430780

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC10912915/

Abstract

Peroxisome proliferator-activated receptor gamma (PPARγ) is a master regulator of adipogenesis. Our previous study revealed that chicken PPARγ has 3 alternative promoters named as P1, P2, and P3, and the DNA methylation of promoter P3 was negatively associated with PPARγ mRNA expression in abdominal adipose tissue (AAT). However, the methylation status of promoters P1 and P2 is unclear. Here we assessed promoter P1 methylation status in AAT of Northeast Agricultural University broiler lines divergently selected for abdominal fat content (NEAUHLF). The results showed that promoter P1 methylation differed in AAT between the lean and fat lines of NEAUHLF at 7 wk of age (p < 0.05), and AAT expression of PPARγ transcript 1 (PPARγ1), which was derived from the promoter P1, was greatly higher in fat line than in lean line at 2 and 7 wk of age. The results of the correlation analysis showed that P1 methylation was positively correlated with PPARγ1 expression at 7 wk of age (Pearson's r = 0.356, p = 0.0242), suggesting P1 methylation promotes PPARγ1 expression. To explore the underlying molecular mechanism of P1 methylation on PPARγ1 expression, bioinformatics analysis, dual-luciferase reporter assay, pyrosequencing, and electrophoresis mobility shift assay (EMSA) were performed. The results showed that transcription factor NRF1 repressed the promoter activity of the unmethylated P1, but not the methylated P1. Of all the 4 CpGs (CpG48, CpG49, CpG50, and CpG51), which reside within or nearby the NRF1 binding sites of the P1, only CpG49 methylation in AAT was remarkably higher in the fat line than in lean line at 7 wk of age (3.18 to 0.57, p < 0.05), and CpG49 methylation was positively correlated with PPARγ1 expression (Pearson's r = 0.3716, p = 0.0432). Furthermore, EMSA showed that CpG49 methylation reduced the binding of NRF1 to the P1. Taken together, our findings illustrate that P1 methylation promotes PPARγ1 expression at least in part by preventing NRF1 from binding to the promoter P1.

摘要

过氧化物酶体增殖物激活受体 γ（PPARγ）是脂肪生成的主调节因子。我们之前的研究表明，鸡 PPARγ 有 3 个替代启动子，分别命名为 P1、P2 和 P3，启动子 P3 的 DNA 甲基化与腹部脂肪组织（AAT）中 PPARγmRNA 的表达呈负相关。然而，启动子 P1 和 P2 的甲基化状态尚不清楚。本研究评估了在东北农业大学肉鸡品系中，腹部脂肪含量（NEAUHLF）差异选择的品系中 AAT 中启动子 P1 的甲基化状态。结果表明，7 周龄时，瘦型和肥胖型 NEAUHLF 之间 AAT 的启动子 P1 甲基化存在差异（p<0.05），并且源自启动子 P1 的 PPARγ 转录物 1（PPARγ1）的 AAT 表达在肥胖型品系中显著高于瘦型品系。相关性分析结果表明，7 周龄时 P1 甲基化与 PPARγ1 表达呈正相关（皮尔逊相关系数=0.356，p=0.0242），提示 P1 甲基化促进 PPARγ1 表达。为了探讨 P1 甲基化对 PPARγ1 表达的潜在分子机制，进行了生物信息学分析、双荧光素酶报告基因检测、焦磷酸测序和电泳迁移率变动分析（EMSA）。结果表明，转录因子 NRF1 抑制未甲基化 P1 的启动子活性，但不抑制甲基化 P1 的启动子活性。在位于 P1 的 NRF1 结合位点内或附近的 4 个 CpG（CpG48、CpG49、CpG50 和 CpG51）中，只有 AAT 中的 CpG49 甲基化在 7 周龄时肥胖型品系显著高于瘦型品系（3.18 至 0.57，p<0.05），且 CpG49 甲基化与 PPARγ1 表达呈正相关（皮尔逊相关系数=0.3716，p=0.0432）。此外，EMSA 显示 CpG49 甲基化降低了 NRF1 与 P1 的结合。综上所述，本研究结果表明，P1 甲基化通过防止 NRF1 结合到启动子 P1 上来促进 PPARγ1 的表达。