Shikonin Binds and Represses PPARγ Activity by Releasing Coactivators and Modulating Histone Methylation Codes
<p>Genome-wide RNA-seq analysis. (<b>A</b>) Clustering analysis of 2406 genes with 1.5-fold changes following shikonin treatment. (<b>B</b>) Gene Ontology (GO) analysis of genes with 1.5-fold changes. (<b>C</b>–<b>E</b>) Bedgraph analysis of three adipogenic genes downregulated by shikonin treatment. Enrichment of tag fragments from RNA-seq results are shown in the gene bodies of <span class="html-italic">Fabp4</span> (<b>C</b>), <span class="html-italic">Adipoq</span> (<b>D</b>), and <span class="html-italic">Lpl</span> (<b>E</b>).</p> "> Figure 2
<p>Genome-wide RNA-seq analysis of shikonin-regulated genes. (<b>A</b>) Gene sets significantly associated with PPARγ were targeted through GSEA, and the normalized enrichment score (NES) was measured. Gene sets with a nominal <span class="html-italic">p</span>-value of <0.05 and false discovery rate (FDR) <span class="html-italic">q</span>-value of <0.25 were considered significantly enriched. (<b>B</b>) Scatter plot analysis of selected gene sets. In total, 13,965 significant genes (log2 value of mRNA expression > 4.0 in RNA-seq of DMSO or shikonin) are displayed in gray. Among them, 2406 genes with a 1.5-fold change in expression level are shown in green. The distribution of genes associated with the indicated GO terms are displayed by brown, blue, and red spots. (<b>C</b>) Effects of shikonin on the mRNA expression of five PPARγ response genes. 3T3-L1 cells were differentiated and treated with DMSO or shikonin, then their transcript expressions were measured by RT-qPCR and normalized to GAPDH. Results are presented as the relative expression compared to DMSO controls. Values are represented as the means ± SDs from three independent experiments (* <span class="html-italic">p</span> < 0.05).</p> "> Figure 3
<p>Antagonistic effects of shikonin on PPARγ regulation. (<b>A</b>) Results of the luciferase reporter gene assay. HEK293 cells were cotransfected as described in the Materials and Method section and treated with rosiglitazone (Rosi, 0.5 μM) and the indicated concentrations of shikonin. Luciferase values were normalized to the β-galactosidase activity. The error bars represent means ± SDs of three independent experiments (** <span class="html-italic">p</span> < 0.01). (<b>B</b>) Effects of shikonin on Rosi-induced expression of the <span class="html-italic">Lpl</span> gene. 3T3-L1 cells were differentiated and treated with 1 μM of Rosi and the indicated concentrations of shikonin for 6 days. Expression of mRNA, measured by RT-qPCR, was normalized to the GAPDH expression level and indicated as fold change relative to that of the DMSO control. Bars represent means ± SDs of three independent experiments (* <span class="html-italic">p</span> < 0.05 and ** <span class="html-italic">p</span> < 0.01). (<b>C</b>) Microscale thermophoresis (MST) assays were performed as described in the Materials and Methods section. (<b>D</b>) Effects of shikonin on the rosiglitazone-induced interaction of PPARγ with the coactivator CBP. GST pull-down assays were performed as described in the Materials and Methods section. The immobilized GST-CBP (amino acids 1–460 or 602–1095) fusion protein was incubated with the indicated concentration of shikonin in the presence of 1 μM of Rosi and a His-PPARγ ligand-binding domain (LBD). Bound proteins were visualized by western blotting using an anti-His antibody. The input represents 5% of His-PPARγ used for the binding assays.</p> "> Figure 4
<p>Epigenetic regulation of PPARγ target genes by shikonin. Eight days after adipogenesis, 3T3-L1 cells were fixed and harvested for ChIP assays using the indicated antibodies against H3H4me3 and H3K27me3. Promoter occupancy was determined using qPCR and primer sets on the targeted promoters of three genes (<span class="html-italic">Hs11b1</span>, <span class="html-italic">Acsl1</span>, and <span class="html-italic">Retn</span>). Data are represented as means ± SDs for three independent experiments (* <span class="html-italic">p</span> < 0.05 and ** <span class="html-italic">p</span> < 0.01).</p> ">
Abstract
:1. Introduction
2. Materials and Methods
2.1. Chemicals and Reagents
2.2. Adipocyte Differentiation
2.3. Oil Red O (ORO) Staining
2.4. Real-Time Quantitative RT-PCR (RT-qPCR)
2.5. RNA-Sequencing and Gene Ontology (GO) Analysis
2.6. Gene Set Enrichment Analysis (GSEA)
2.7. Luciferase Reporter Gene Assays
2.8. Microscale Thermophoresis (MST) Analysis
2.9. Glutathione-S-Transferase (GST) Pull-Down
2.10. ChIP Assays
2.11. Statistical Analysis
3. Results
3.1. Shikonin Inhibits Adipogenesis and Downregulates Adipogenic Genes
3.2. Shikonin Reduces the Expression of PPARγ Target Genes
3.3. Shikonin Acts as an Antagonist by Directly Binding PPARγ
3.4. Shikonin Inhibits PPARγ Target Gene Expression through Enrichment of Active or Repressive Histone Codes on Target Promoters
4. Discussion
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
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Park, U.-H.; Youn, H.; Kim, E.-J.; Um, S.-J. Shikonin Binds and Represses PPARγ Activity by Releasing Coactivators and Modulating Histone Methylation Codes. Nutrients 2023, 15, 1797. https://doi.org/10.3390/nu15071797
Park U-H, Youn H, Kim E-J, Um S-J. Shikonin Binds and Represses PPARγ Activity by Releasing Coactivators and Modulating Histone Methylation Codes. Nutrients. 2023; 15(7):1797. https://doi.org/10.3390/nu15071797
Chicago/Turabian StylePark, Ui-Hyun, HyeSook Youn, Eun-Joo Kim, and Soo-Jong Um. 2023. "Shikonin Binds and Represses PPARγ Activity by Releasing Coactivators and Modulating Histone Methylation Codes" Nutrients 15, no. 7: 1797. https://doi.org/10.3390/nu15071797