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Insulin-response epigenetic activation of Egr-1 and JunB genes at the nuclear periphery by A-type lamin-associated pY19-Caveolin-2 in the inner nuclear membrane.

Jeong K, Kwon H, Lee J, Jang D, Pak Y - Nucleic Acids Res. (2015)

Bottom Line: Insulin controls transcription to sustain its physiologic effects for the organism to adapt to environmental changes added to genetic predisposition.INM-targeted pY19-Cav-2 in response to insulin associates specifically with the A-type lamin, disengages the repressed Egr-1 and JunB promoters from lamin A/C through disassembly of H3K9me3, and facilitates assembly of H3K9ac, H3K18ac and H3K27ac by recruitment of GCN5 and p300 and the subsequent enrichment of RNA polymerase II (Pol II) on the promoters at the nuclear periphery.Our findings show that Cav-2 is an epigenetic regulator of histone H3 modifications, and provide novel mechanisms of insulin-response epigenetic activation at the nuclear periphery.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Division of Applied Life Science (BK21 Plus Program), PMBBRC, Gyeongsang National University, Jinju 660-701, Korea.

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Lamin A/C-associated pY19-Cav-2-mediated recruitment of GCN5 and p300 precedes the subsequent enrichment of H3K9ac and H3K18ac and RNA Pol II on the Egr-1 and JunB promoters in response to insulin. (A and B) Insulin-induced enrichment of H3K9ac and H3K18ac on the Egr-1 and JunB promoters (−0.5 to −0.1 kb). Hirc-B cells treated with or without 100 nM insulin for 30 min were subjected to ChIP with anti-H3K9ac (A) or anti-H3K18ac (B) antibody on Egr-1 and JunB genes. qPCR data in all figures are presented (mean ± SE, n = 4). (C and D) Regulation of the enrichment of H3K9ac on Egr-1 and H3K18ac on JunB promoters and the RNA Pol II recruitment to the promoters (-0.1 kb) by lamin A/C-associated pY19-Cav-2. Control shRNA vector-expressed Hirc-B cells or pcDNA vector-expressed Cav-2 shRNA-, pcDNA-Cav-2-expressed Cav-2 shRNA-, pcDNA-Δ47–86-Cav-2-expressed Cav-2 shRNA- or pcDNA-Y19A-Cav-2-expressed Cav-2 shRNA-stable Hirc-B cells treated with or without 100 nM insulin for 30 min were subjected to ChIP with anti-H3K9ac or anti-H3K18ac antibody (C) or anti-RNA Pol II (D) antibody on the promoters of Egr-1 and JunB genes. qPCR data are presented (mean ± SE, n = 4). (E and F) Recruitment of GCN5 and p300 on the Egr-1 and JunB promoters by lamin A/C-associated pY19-Cav-2. Control shRNA vector-expressed Hirc-B cells or pcDNA vector-expressed Cav-2 shRNA-, pcDNA-Cav-2-expressed Cav-2 shRNA-, pcDNA-Δ47–86-Cav-2-expressed Cav-2 shRNA- or pcDNA-Y19A-Cav-2-expressed Cav-2 shRNA-stable Hirc-B cells treated with or without 100 nM insulin for 30 min were subjected to ChIP with anti-GCN5 (E) or anti-p300 (F) antibody on the promoters of Egr-1 and JunB genes. qPCR data are presented (mean ± SE, n = 4).
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Figure 6: Lamin A/C-associated pY19-Cav-2-mediated recruitment of GCN5 and p300 precedes the subsequent enrichment of H3K9ac and H3K18ac and RNA Pol II on the Egr-1 and JunB promoters in response to insulin. (A and B) Insulin-induced enrichment of H3K9ac and H3K18ac on the Egr-1 and JunB promoters (−0.5 to −0.1 kb). Hirc-B cells treated with or without 100 nM insulin for 30 min were subjected to ChIP with anti-H3K9ac (A) or anti-H3K18ac (B) antibody on Egr-1 and JunB genes. qPCR data in all figures are presented (mean ± SE, n = 4). (C and D) Regulation of the enrichment of H3K9ac on Egr-1 and H3K18ac on JunB promoters and the RNA Pol II recruitment to the promoters (-0.1 kb) by lamin A/C-associated pY19-Cav-2. Control shRNA vector-expressed Hirc-B cells or pcDNA vector-expressed Cav-2 shRNA-, pcDNA-Cav-2-expressed Cav-2 shRNA-, pcDNA-Δ47–86-Cav-2-expressed Cav-2 shRNA- or pcDNA-Y19A-Cav-2-expressed Cav-2 shRNA-stable Hirc-B cells treated with or without 100 nM insulin for 30 min were subjected to ChIP with anti-H3K9ac or anti-H3K18ac antibody (C) or anti-RNA Pol II (D) antibody on the promoters of Egr-1 and JunB genes. qPCR data are presented (mean ± SE, n = 4). (E and F) Recruitment of GCN5 and p300 on the Egr-1 and JunB promoters by lamin A/C-associated pY19-Cav-2. Control shRNA vector-expressed Hirc-B cells or pcDNA vector-expressed Cav-2 shRNA-, pcDNA-Cav-2-expressed Cav-2 shRNA-, pcDNA-Δ47–86-Cav-2-expressed Cav-2 shRNA- or pcDNA-Y19A-Cav-2-expressed Cav-2 shRNA-stable Hirc-B cells treated with or without 100 nM insulin for 30 min were subjected to ChIP with anti-GCN5 (E) or anti-p300 (F) antibody on the promoters of Egr-1 and JunB genes. qPCR data are presented (mean ± SE, n = 4).

Mentions: We further investigated if lamin A/C-associated pY19-Cav-2 in the INM participates in the histone H3 modifications for insulin-response epigenetic activation of Egr-1 and JunB at the nuclear periphery by ChIP analysis. Insulin stimulation decreased H3K9me3 and increased AcH3 around the TSSs of the genes (Supplementary Figure S2C and D), and specifically increased H3K9ac on Egr-1 and H3K18ac on JunB promoter regions (Figure 6A and B). H3K27ac was increased around the TSS of JunB in response to insulin (Supplementary Figure S3A). Insulin treatment had little effect on the levels of H3K14ac and H3K4ac within the regions of both genes (Supplementary Figure S3B and C). Total histone H3 signals on Egr-1 and JunB genes were not influenced by insulin treatment (Supplementary Figure S3D). ChIP with anti-IgG antibody showed no signal (Supplementary Figure S3E). No enrichment of Cav-2 or pY19-Cav-2 around the TSSs of a housekeeping gene, Actb was detected in response to insulin (Supplementary Figure S3F). Insulin-induced enrichment of H3K9ac on Egr-1 and H3K18ac on JunB promoters was abrogated in Cav-2-deficient cells (Figure 6C). Reexpression of WT Cav-2 restored the H3K9ac and H3K18ac enrichment but Δ47–86-Cav-2 or Y19A-Cav-2 mutant had no effect (Figure 6C). In parallel, Cav-2 depletion prevented insulin-induced enrichment of AcH3 and RNA Pol II on the promoters and the inhibition was rescued by ectopic WT Cav-2 but not Δ47–86-Cav-2 or Y19A-Cav-2 mutant (Figure 6D and Supplementary Figure S4A).


Insulin-response epigenetic activation of Egr-1 and JunB genes at the nuclear periphery by A-type lamin-associated pY19-Caveolin-2 in the inner nuclear membrane.

Jeong K, Kwon H, Lee J, Jang D, Pak Y - Nucleic Acids Res. (2015)

Lamin A/C-associated pY19-Cav-2-mediated recruitment of GCN5 and p300 precedes the subsequent enrichment of H3K9ac and H3K18ac and RNA Pol II on the Egr-1 and JunB promoters in response to insulin. (A and B) Insulin-induced enrichment of H3K9ac and H3K18ac on the Egr-1 and JunB promoters (−0.5 to −0.1 kb). Hirc-B cells treated with or without 100 nM insulin for 30 min were subjected to ChIP with anti-H3K9ac (A) or anti-H3K18ac (B) antibody on Egr-1 and JunB genes. qPCR data in all figures are presented (mean ± SE, n = 4). (C and D) Regulation of the enrichment of H3K9ac on Egr-1 and H3K18ac on JunB promoters and the RNA Pol II recruitment to the promoters (-0.1 kb) by lamin A/C-associated pY19-Cav-2. Control shRNA vector-expressed Hirc-B cells or pcDNA vector-expressed Cav-2 shRNA-, pcDNA-Cav-2-expressed Cav-2 shRNA-, pcDNA-Δ47–86-Cav-2-expressed Cav-2 shRNA- or pcDNA-Y19A-Cav-2-expressed Cav-2 shRNA-stable Hirc-B cells treated with or without 100 nM insulin for 30 min were subjected to ChIP with anti-H3K9ac or anti-H3K18ac antibody (C) or anti-RNA Pol II (D) antibody on the promoters of Egr-1 and JunB genes. qPCR data are presented (mean ± SE, n = 4). (E and F) Recruitment of GCN5 and p300 on the Egr-1 and JunB promoters by lamin A/C-associated pY19-Cav-2. Control shRNA vector-expressed Hirc-B cells or pcDNA vector-expressed Cav-2 shRNA-, pcDNA-Cav-2-expressed Cav-2 shRNA-, pcDNA-Δ47–86-Cav-2-expressed Cav-2 shRNA- or pcDNA-Y19A-Cav-2-expressed Cav-2 shRNA-stable Hirc-B cells treated with or without 100 nM insulin for 30 min were subjected to ChIP with anti-GCN5 (E) or anti-p300 (F) antibody on the promoters of Egr-1 and JunB genes. qPCR data are presented (mean ± SE, n = 4).
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Figure 6: Lamin A/C-associated pY19-Cav-2-mediated recruitment of GCN5 and p300 precedes the subsequent enrichment of H3K9ac and H3K18ac and RNA Pol II on the Egr-1 and JunB promoters in response to insulin. (A and B) Insulin-induced enrichment of H3K9ac and H3K18ac on the Egr-1 and JunB promoters (−0.5 to −0.1 kb). Hirc-B cells treated with or without 100 nM insulin for 30 min were subjected to ChIP with anti-H3K9ac (A) or anti-H3K18ac (B) antibody on Egr-1 and JunB genes. qPCR data in all figures are presented (mean ± SE, n = 4). (C and D) Regulation of the enrichment of H3K9ac on Egr-1 and H3K18ac on JunB promoters and the RNA Pol II recruitment to the promoters (-0.1 kb) by lamin A/C-associated pY19-Cav-2. Control shRNA vector-expressed Hirc-B cells or pcDNA vector-expressed Cav-2 shRNA-, pcDNA-Cav-2-expressed Cav-2 shRNA-, pcDNA-Δ47–86-Cav-2-expressed Cav-2 shRNA- or pcDNA-Y19A-Cav-2-expressed Cav-2 shRNA-stable Hirc-B cells treated with or without 100 nM insulin for 30 min were subjected to ChIP with anti-H3K9ac or anti-H3K18ac antibody (C) or anti-RNA Pol II (D) antibody on the promoters of Egr-1 and JunB genes. qPCR data are presented (mean ± SE, n = 4). (E and F) Recruitment of GCN5 and p300 on the Egr-1 and JunB promoters by lamin A/C-associated pY19-Cav-2. Control shRNA vector-expressed Hirc-B cells or pcDNA vector-expressed Cav-2 shRNA-, pcDNA-Cav-2-expressed Cav-2 shRNA-, pcDNA-Δ47–86-Cav-2-expressed Cav-2 shRNA- or pcDNA-Y19A-Cav-2-expressed Cav-2 shRNA-stable Hirc-B cells treated with or without 100 nM insulin for 30 min were subjected to ChIP with anti-GCN5 (E) or anti-p300 (F) antibody on the promoters of Egr-1 and JunB genes. qPCR data are presented (mean ± SE, n = 4).
Mentions: We further investigated if lamin A/C-associated pY19-Cav-2 in the INM participates in the histone H3 modifications for insulin-response epigenetic activation of Egr-1 and JunB at the nuclear periphery by ChIP analysis. Insulin stimulation decreased H3K9me3 and increased AcH3 around the TSSs of the genes (Supplementary Figure S2C and D), and specifically increased H3K9ac on Egr-1 and H3K18ac on JunB promoter regions (Figure 6A and B). H3K27ac was increased around the TSS of JunB in response to insulin (Supplementary Figure S3A). Insulin treatment had little effect on the levels of H3K14ac and H3K4ac within the regions of both genes (Supplementary Figure S3B and C). Total histone H3 signals on Egr-1 and JunB genes were not influenced by insulin treatment (Supplementary Figure S3D). ChIP with anti-IgG antibody showed no signal (Supplementary Figure S3E). No enrichment of Cav-2 or pY19-Cav-2 around the TSSs of a housekeeping gene, Actb was detected in response to insulin (Supplementary Figure S3F). Insulin-induced enrichment of H3K9ac on Egr-1 and H3K18ac on JunB promoters was abrogated in Cav-2-deficient cells (Figure 6C). Reexpression of WT Cav-2 restored the H3K9ac and H3K18ac enrichment but Δ47–86-Cav-2 or Y19A-Cav-2 mutant had no effect (Figure 6C). In parallel, Cav-2 depletion prevented insulin-induced enrichment of AcH3 and RNA Pol II on the promoters and the inhibition was rescued by ectopic WT Cav-2 but not Δ47–86-Cav-2 or Y19A-Cav-2 mutant (Figure 6D and Supplementary Figure S4A).

Bottom Line: Insulin controls transcription to sustain its physiologic effects for the organism to adapt to environmental changes added to genetic predisposition.INM-targeted pY19-Cav-2 in response to insulin associates specifically with the A-type lamin, disengages the repressed Egr-1 and JunB promoters from lamin A/C through disassembly of H3K9me3, and facilitates assembly of H3K9ac, H3K18ac and H3K27ac by recruitment of GCN5 and p300 and the subsequent enrichment of RNA polymerase II (Pol II) on the promoters at the nuclear periphery.Our findings show that Cav-2 is an epigenetic regulator of histone H3 modifications, and provide novel mechanisms of insulin-response epigenetic activation at the nuclear periphery.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Division of Applied Life Science (BK21 Plus Program), PMBBRC, Gyeongsang National University, Jinju 660-701, Korea.

Show MeSH