Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)
Article Title: Epigenetic Regulation of Hepatic Lipid Metabolism by DNA Methylation.
doi: 10.1002/advs.202206068
Figure Lengend Snippet: Figure 6. Methylation of the Klb promoter is enhanced by HFD. A) RRBS analysis reveals an enhanced DNA methylation rate at the Klb promoter by HFD as shown in UCSC Genome Browser on Mouse (NCBI37/mm9) Assembly. B) Pyrosequencing analysis of the DNA methylation at the CpG sites at the Klb promoter in the liver of HFD- and LFD-fed mice (n = 8). C) Quantitative RT-PCR analysis of Klb mRNA expression in liver of HFD- and LFD-fed mice (n = 6–8). D) The association of the peaks of the chromatin accessibility at the Klb promoter was analyzed by ATAC-seq and the reads of the Klb mRNA expression analyzed by RNA-seq in the liver of HFD- and LFD-fed mice. () Luciferase activity of the Klb promoter regulated by DNA methylation (n = 8). F) DNMT1 binding to the Klb promoter was measured by ChIP assays (n = 4). G) DNMT3A binding to the Klb promoter measured by ChIP assays (n = 4). 6-week-old male C57BL/6J mice were fed either LFD or HFD for 12 weeks. All data are expressed as mean±SEM. *p < 0.05 versus LFD. H) Heatmap of Klb and Dnmt1 expression across the three hepatocyte zones (periportal zone, midzone, and pericentral zone) in LD1KO mice and fl/fl mice with snRNA-seq analysis. I) Quantitative RT-PCR analysis of Klb mRNA expression in Dnmt1-deficient liver (left), Dnmt3a-deficient liver (middle), or Tet2-deficient liver (right) (n = 5-9). J) Pyrosequencing analysis of DNA methylation rate in the CpG sites of the Klb promoter in the liver of LD1KO mice and fl/flmice (n = 8). K) Pyrosequencing analysis of DNA methylation rate in the CpG sites of the Klb promoter in the liver of LT2KO mice and fl/fl mice (n = 4-8). All data are expressed as mean ± SEM. *p < 0.05 versus fl/fl.
Article Snippet: Antibodies: The antibodies used in immunoblotting, chromatin immunoprecipitation (ChIP) assays, and immunoprecipitation included KLB (AF2619, R&D Systems), DNMT1 (IMG-261A, IMGENEX), DNMT3a (IMG-268A, IMGENEX), GAPDH (6C5) (sc-32233, Santa Cruz)), E6AP (sc166689, Santa Cruz), and UB (sc-8017, Santa Cruz).
Techniques: Methylation, DNA Methylation Assay, Quantitative RT-PCR, Expressing, RNA Sequencing, Luciferase, Activity Assay, Binding Assay
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