Figure 3. G9a silences Ifnβ and IFN-inducible gene expression in fibroblasts. (A) G9af/f and G9a−/− MEFs were stimulated with poly I:C and Ifnβ mRNA levels were determined by qPCR (top). The number and levels of genes in control and G9a-deficient MEFs treated with poly I:C were determined by microarray analysis (bottom). Venn diagrams show the differences in the number of genes that were up-regulated more than twofold in G9af/f and G9a−/− MEFs. Red, G9af/f; blue, G9a−/−. The hierarchical clustered heat maps show expression of the greater than twofold poly I:C–induced genes in G9a−/− as compared with G9af/f MEFs. Color bar represents log2 scale of raw signal values for each time point. Data represent triplicate samples. (B) Abundance of the chromatin marks associated with active gene transcription at Ifnb1 (left) and ISG (right) loci. The distribution and levels of epigenetic marks representing active transcription at Ifnb1 were measured by ChIP-Seq in G9af/f and G9a−/− MEFs before and after poly I:C stimulation. Scatter plots display differential abundance of transcription-associated histone marks at poly I:C–induced genes 6 h after stimulation. X- and y-axes are FPKM values for epigenetic marks in G9af/f and G9a−/− MEFS 6 h after stimulation. (C) G9af/f and G9a−/− splenic DCs were stimulated with poly I:C and Ifnβ mRNA levels were determined by qPCR. The number and levels of genes in control and G9a-deficient splenic DCs treated with poly I:C were determined by microarray analysis (bottom). Venn diagrams show the number of genes that were up-regulated more than twofold in G9af/f and G9a−/− DCs. Red, G9af/f; blue, G9a−/−. The hierarchical clustered heat maps show expression of the greater than twofold poly I:C–induced genes in G9a−/− as compared with G9af/f MEFs. Color bar represents log2 scale of raw signal values for each time point. Data represent duplicate samples. (D) WT MEFS were treated with pharmacological inhibitors of G9a, UNC0638 (1 µM), or BIX1294 (1 µM) for 7 d. The abundance and distribution of H3K9me2 was determined by ChIP-Seq. Y-axes represent the number of reads per million mapped reads. (E) MEFs or primary mouse keratinocytes were treated with DMSO, or with G9a inhibitors BIX1294 and UNC0638 for 7 d. Control or inhibitor-treated cells were stimulated with poly I:C and Ifnβ mRNA levels were determined by qPCR. Data are representative of three independent experiments. Error bars are SD.
Figure 3.

G9a silences Ifnβ and IFN-inducible gene expression in fibroblasts. (A) G9af/f and G9a−/− MEFs were stimulated with poly I:C and Ifnβ mRNA levels were determined by qPCR (top). The number and levels of genes in control and G9a-deficient MEFs treated with poly I:C were determined by microarray analysis (bottom). Venn diagrams show the differences in the number of genes that were up-regulated more than twofold in G9af/f and G9a−/− MEFs. Red, G9af/f; blue, G9a−/−. The hierarchical clustered heat maps show expression of the greater than twofold poly I:C–induced genes in G9a−/− as compared with G9af/f MEFs. Color bar represents log2 scale of raw signal values for each time point. Data represent triplicate samples. (B) Abundance of the chromatin marks associated with active gene transcription at Ifnb1 (left) and ISG (right) loci. The distribution and levels of epigenetic marks representing active transcription at Ifnb1 were measured by ChIP-Seq in G9af/f and G9a−/− MEFs before and after poly I:C stimulation. Scatter plots display differential abundance of transcription-associated histone marks at poly I:C–induced genes 6 h after stimulation. X- and y-axes are FPKM values for epigenetic marks in G9af/f and G9a−/− MEFS 6 h after stimulation. (C) G9af/f and G9a−/− splenic DCs were stimulated with poly I:C and Ifnβ mRNA levels were determined by qPCR. The number and levels of genes in control and G9a-deficient splenic DCs treated with poly I:C were determined by microarray analysis (bottom). Venn diagrams show the number of genes that were up-regulated more than twofold in G9af/f and G9a−/− DCs. Red, G9af/f; blue, G9a−/−. The hierarchical clustered heat maps show expression of the greater than twofold poly I:C–induced genes in G9a−/− as compared with G9af/f MEFs. Color bar represents log2 scale of raw signal values for each time point. Data represent duplicate samples. (D) WT MEFS were treated with pharmacological inhibitors of G9a, UNC0638 (1 µM), or BIX1294 (1 µM) for 7 d. The abundance and distribution of H3K9me2 was determined by ChIP-Seq. Y-axes represent the number of reads per million mapped reads. (E) MEFs or primary mouse keratinocytes were treated with DMSO, or with G9a inhibitors BIX1294 and UNC0638 for 7 d. Control or inhibitor-treated cells were stimulated with poly I:C and Ifnβ mRNA levels were determined by qPCR. Data are representative of three independent experiments. Error bars are SD.

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