Fig 1: G9a is downregulated in GBM and anti-correlated with HIF-1 target gene expression and survival of patients with GBM. (A–C) U251MG (A), LN229 (B) and U87MG (C) cells were exposed to 20% or 1% O2 for indicated time. IP assays were performed with anti-HIF-1α or anti-G9a antibody, followed by immunoblot assays (A). Immunoblot assays were performed with antibodies against K674me1, K674me2, G9a, GLP, HIF-1α or actin. (D) Parental, HIF-1α KO, HIF-2α KO or HIF-1/2α DKO U251MG cells were exposed to 20% or 1% O2 for 48 h. Immunoblot assays were performed with antibodies against G9a, GLP, HIF-1α, HIF-2α or actin. (E–J) Analysis of G9a/GLP mRNA expression in normal brain and human GBM. The data were retrieved from the TCGA (E and H) (30), GSE4536 (F and I) (32), and GSE4290 (G and J) (31) databases. **P < 0.01; ****P < 0.0001. ns, not significant. (K–M) Immunoblot assays were performed with antibodies against G9a, GLP, or actin in human GBM tissues and normal brain tissues (K). G9a (L) and GLP (M) bands were quantified by densitometry and normalized to actin. (N-P) Analysis of PTGS1 mRNA expression in normal brain and human GBM. The data were retrieved from the TCGA (N) (30), GSE4536 (O) (32), and GSE4290 (P) (31) databases. ***P < 0.001. (Q) Negative correlation between G9a and PTGS1 mRNA expression in human GBM. The data were retrieved from the TCGA (30). (R-U) Kaplan-Meier survival analysis for patients with GBM using the PROGgene program (34). Patients were divided by median expression levels of G9a (R and S) or GLP mRNA (T and U). HR, hazard ratio.
Fig 2: Analysis of dex-regulated genes that are dependent or independent of G9a, GLP and HP1γ.a Ingenuity pathway analysis of cellular functions for the 67 dex-regulated genes that are dependent on G9a, GLP and HP1γ (from Fig. 1d). The threshold for significance is indicated by the vertical orange line. b NALM-6 cells expressing shRNA against G9a, GLP, HP1γ or a non-specific sequence (shNS) were treated for 8 h with 100 nM dex or equivalent volume of vehicle ethanol (Eth). mRNA levels for the indicated G9a/GLP/HP1γ-dependent genes (left side, chosen from the 67 genes in the overlap of Fig. 1d) and the indicated G9a/GLP/HP1γ-independent genes (right side, chosen from the 82 genes in the white sector of Fig. 1e) were measured by RT-qPCR and normalized to β-actin mRNA levels. Results shown are mean ± SEM for six independent experiments. The p value was calculated using a paired t-test comparing results for dex-treated cells expressing shRNA against G9a, GLP or HP1γ to the dex-treated shNS sample, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ns not significant. c Model for regulation of lymphoblast death in B-ALL cells. Methylated G9a or GLP recruit HP1γ which facilitates the recruitment of RNA polymerase II and activates the expression of genes involved in cell death. G9a and GLP methylation is removed by a specific demethylase (KDM). Theoretically, inhibiting this KDM would increase the methylation and subsequently the GC-induced cell death
Fig 3: HP1γ is required for expression of most dex-regulated genes that are G9a/GLP dependent.Genome-wide RNA-sequencing analysis was performed on Nalm6 cells to identify the genes dependent on G9a, GLP and HP1γ for dex-regulated expression. a Hypothetical results of gene expression profiles for a given gene illustrate how specific pairwise comparisons between datasets for individual samples were performed. Numbered bars represent hypothetical mRNA levels from RNA-seq data for cells expressing the indicated shRNA and treated for 8 h with ethanol (Eth) or 100 nM dex. Colored letters represent pairwise comparisons performed to determine sets of genes for which the mRNA levels were significantly different between the bracketed cell samples: i.e., comparison A = set of dex-regulated genes (fold change ≥2, adjusted p ≤ 0.05); comparison B = set of G9a-dependent genes (no fold change cutoff, adjusted p ≤ 0.05); C = set of GLP-dependent genes (no fold change cutoff, adjusted p ≤ 0.05); D = set of HP1γ-dependent genes (no fold change cutoff, adjusted p ≤ 0.05). b White Venn diagram represents the dex-regulated genes in cells expressing shNS (comparison A); green Venn diagram, G9a-dependent genes in dex-treated cells (comparison B); blue Venn diagram, GLP-dependent genes in dex-treated cells (comparison C). Overlap areas indicate the number of genes shared among sets. c White Venn diagram represents the dex-regulated genes in cells expressing shNS (comparison A); purple Venn diagram, HP1γ-dependent genes in dex-treated cells (comparison D). Overlap areas indicate the number of genes shared among sets. d Red Venn diagram represents the dex-regulated genes that are dependent on both G9a and GLP, identified in the red overlap area of (b); purple Venn diagram, dex-regulated genes that are HP1γ dependent, identified in the light purple overlap area of (c). The percentages of dex-regulated, G9a/GLP-dependent genes that are HP1γ dependent or HP1γ independent are represented in the donut graph. e White Venn diagram represents the dex-regulated genes that are independent of both G9a and GLP, identified in the white sector of (b); purple Venn diagram, dex-regulated genes that are HP1γ dependent, identified in the light purple overlap area of (c). The percentages of dex-regulated, G9a/GLP-independent genes that are HP1γ dependent or HP1γ independent are represented in the donut graph. f Boxplot of the absolute value of log2 fold change in mRNA levels due to depletion of G9a, GLP or HP1γ. Gene expression changes caused by depletion of G9a, GLP, and HP1γ in dex-treated cells are shown for all dex-regulated genes that are G9a/GLP dependent (red) and G9a/GLP independent (white)
Fig 4: PHF20 is required for the deposition of histone H4K16ac on ERα target genes.(a) PHF20 is required for the E2-induced accumulation of histone H4K16ac on the promoters of GREB1 and PR. qPCR analysis of histone H4K16ac ChIP in control and PHF20 knockdown cells 15 and 45 min after E2 treatment. The H4K16ac ChIP was calculated as a ratio relative to total H3 ChIP. (b) PHF20 is required for ERα chromatin recruitment. qPCR analysis of ERα ChIP in the cells as in a. ERα ChIP is shown as a ratio relative to input. (c) G9a is required for the E2-induced accumulation of histone H4K16ac on the promoters of GREB1 and PR. qPCR analysis of histone H4K16ac ChIP in control and G9a knockdown cells 15 and 45 min after E2 treatment. (d) G9a is required for ERα chromatin recruitment. qPCR analysis of ERα ChIP in the cells as in c. In a–d, all error bars indicate the mean ±s.e.m. of three experiments. *P<0.05, **P<0.01 (Student's t-test). (e) Working model of G9a-mediated ERα methylation in hormonal response. Left panel: G9a methylates ERα at K235 in the nucleus in response to E2 stimulation, and ERαK235me2 is recognized by the Tudor domain of PHF20, which recruits the MOF complex to acetylate histone H4K16, thereby promoting the expression of ERα target genes GREB1 and PR. Middle panel: depletion of G9a abolishes ERαK235 methylation and the methylation-dependent ERα–PHF20 interaction, thereby reducing the recruitment of PHF20/MOF and ERα to the EREs of ERα target genes GREB1 and PR, consequently reducing their expression. Right panel: depletion of PHF20 reduces the H4K16ac levels on chromatin and the recruitment of ERα to EREs via feedback regulation, thereby reducing the expression of ERα target genes GREB1 and PR.
Fig 5: KDM4 family demethylates G9a and GLP.a Recombinant full-length (FL) hG9a was self-methylated with SAM, separated from residual SAM, then incubated with the indicated recombinant KDMs. Upper panel: Immunoblot with pan methyllysine antibody. Lower panel: After immunoblot analysis, the membrane was stained with Coomassie brilliant blue to detect quantities of assay protein components. Results shown are representative of three independent experiments. b Recombinant GST-hGLP N was methylated by GST-hGLP ΔN, separated from SAM, then incubated with the indicated recombinant KDMs. Upper panel: Immunoblot with pan methyllysine antibody. Lower panel: After detection, the membrane was Coomassie stained. Results shown are representative of three independent experiments. c Cos-7 cells were transfected with plasmids encoding flag-hG9a FL and HA-KDM4A, HA-KDM4B or HA-KDM4C. Lysates were immunoprecipitated with HA antibodies and analyzed by immunoblot using antibodies listed. Results shown are representative of three independent experiments
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