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. Author manuscript; available in PMC: 2017 Nov 20.
Published in final edited form as: Nat Genet. 2017 Jan 16;49(3):367–376. doi: 10.1038/ng.3753

Figure 7. CDH2 and TOP2B are epigenetic targets of 6AN that selectively regulate tumorigenic potential.

Figure 7

(a) Real-time RT-qPCR (top panel) and ChIP-qPCR for H3K9me2 and H3K27ac showed that 6AN had minimal effect on CDH2 mRNA expression or epigenetic state (H3K9me2/H3K27ac) over the CDH2 locus in the peritoneal subclone. Numbers along the x-axis of ChIP panels correspond to coordinates of the ChIP-PCR primer positions across the 1.4Mb CDH2 locus (Chr18q 27Mb:primer position). (b) In contrast, RT-PCR (top panel) showed that the distant metastatic subclone (A38Lg) from the same patient over-expressed CDH2 relative to A38Per (compare y-axis values), and that expression was repressed by 6AN (n=4 technical PCR replicates from two biological replicate experiments, error bars: s.d.m., *p=0.002 by two tailed t-tests). ChIP assays further showed that 6AN induced enrichment of H3K9me2 across the 1.4Mb CDH2 locus with corresponding reductions of H3K27ac directly over the genic region (n=2 biological replicates, error bars: s.e.m.). (c) RNAi against both CDH2 and TOP2B (Supplementary Fig. 15c–d) selectively blocked 3D tumor formation in distant metastatic and precursor subclones that over-expressed these genes by RNA-seq, with no effect on A38Per or HPDE cells (n=4 technical replicates, error bars: s.d.m., *p<0.01 by two tailed t-tests). Scale bars: 400μm.