Figure 4. Boundary methylation and CTCF occupancy affect PDGFRA expression and proliferation.

(a) Schematic depicts chromatin loops and boundaries in the PDGFRA locus. In IDH wildtype cells (left), intact boundary insulates oncogene. Disruption of boundary by removing CTCF motif should activate the oncogene. In IDH mutant (right), hyper-methylation blocks CTCF, compromising boundary and allowing enhancer to activate oncogene. Demethylation should restore CTCF-mediated insulation. (b) Plot compares methylation of the CpG in the CTCF motif in IDH wildtype gliomaspheres (black), IDH mutant gliomaspheres (red) and IDH mutant gliomaspheres treated with 5μM 5-aza for 8 days (purple). (c) Plot compares CTCF occupancy over the boundary. (d) Plot compares PDGFRA expression. Demethylation restores PDGFRA insulation in IDH mutant gliomaspheres. (e) CTCF binding shown for the FIP1L1/PDGFRA region. Expanded view shows CTCF motif in the insulator targeted for CRISPR-based deletion. gRNA and protospacer adjacent motif (PAM) direct Cas9 nuclease to the motif. (f) Surveyor assay detects target site alterations in GSC6 gliomaspheres infected with Cas9 and sgRNA (but not in control cells infected with GFP-targeting sgRNA). (g) Sequencing of target site reveals the indicated deletions. CTCF motif disrupted on ~25% of alleles (compare to <0.01% in control). (h) Plot depicts fraction of reads in insulator CRISPR cells with a deletion of indicated size. (i) qPCR reveals increased PDGFRA expression in insulator CRISPR cells. (j) Flow cytometry reveals ~2-fold greater PDGFRa in insulator CRISPR cells. (k) Plot depicts gliomasphere growth. Insulator CRISPR cells exhibit ~2-fold increased proliferation, relative to control. This proliferation advantage is eliminated by PDGFRa inhibition. These results indicate that genetic or epigenetic disruption of the boundary compromises insulation of this oncogene. (Error bars in all panels reflect standard deviations of triplicate observations).