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. 2006 Apr 20;25(10):2096–2106. doi: 10.1038/sj.emboj.7601090

Figure 4.

Figure 4

The antisense transcription silences the overlapping gene prior to the nonoverlapping gene. Bar graphs showing the activity of H19 and hygromycin genes in PS4polyA9.2 (A) and NS11 (B) plasmids, propagated for 2, 4 and 8 days post-transfection. The H19 and hygromycin gene activity was assessed using RPA. The percentage activity of the H19 and hygromycin genes was calculated after normalization against total input RNA and episomal copy number, as described in the legend of Figure 2. (C) Schematic representation of PS4polyA9.2 episomal construct. The dashed lines indicate the position of the primers used for quantitative PCR reactions. (D, E) Quantitative analyses of ChIP material (bar graphs showing percentage enrichment, the data represent means±s.d. of three independent ChIP experiments and quantification was performed as described in Figure 3E) using antibodies such as H3K9Me1, H3K9Me2 and H3K9Me3, H3K27Me3, H3K4Me2 and H3K9Ac on the crosslinked chromatin obtained from PH19- and PS4polyA9.2-transfected cells harvested at 2 and 8 days post-transfection. The ChIP analyses revealed that the H19 overlapping promoter (D) is more enriched with inactive chromatin-specific histone modifications in the 2-day sample as compared to the nonoverlapping hygromycin promoter (E). The bar graphs showing the real-time quantification of enrichment of H3K9Me3 and H3K9Ac in the EBNA (Ec, F) and H19 (H19c1 and H19c2, G) coding regions of PS4polyA9.2 and PH19 constructs. Real-time quantification was performed as described in Figure 3E. The data represent means±s.d. of three independent ChIP experiments. (H, I) Antisense transcription interferes with the occupancy of RNAPolII preinitiation complex. Quantitative PCR analysis of ChIP material obtained using RNAPolII and TFIIB antibodies on crosslinked chromatin from JEG-3 cells transfected with PS4polyA9.2 and PH19 for 2 and 8 days over the promoters of the overlapping H19 (H) and the nonoverlapping hygromycin gene (I). The graphs show percentage enrichment. Real-time quantification was performed as described in Figure 3E. The data represent means±s.d. of three independent ChIP experiments.