Figure 4. Core TFs reprogram the epigenetic landscape of DGCs.
(A) Left: heatmap depicts H3K27ac signals for TPC-specific, DGC-specific or shared regulatory elements defined in Figure 1E. Relative to control vector infected DGCs, iTPCs gain H3K27ac over TPC-specific elements and lose H3K27ac over DGC-specific elements, consistent with genome-wide reprogramming of the epigenetic landscape. Right: pie charts show fraction of regulatory elements (dark cyan) in each set with H3K27ac in iTPC. (B) RNA-Seq expression and promoter H3K27ac levels at promoter are shown for TPC-specific TFs defined in Figure 2A (NES: Nestin). (C) Hierarchical clustering of MGG8 DGCs, TPCs and replicate iTPCs (iTPC1/2) by H3K27ac ChIP-Seq signal. (D) RNA-Seq tracks show that core TF mRNAs in iTPCs include 3′UTRs (shaded in gray). This indicates the endogenous loci are reactivated in iTPCs as the exogenous vectors lack 3′UTRs. (E) H3K27ac signal tracks for loci encoding core TFs show that endogenous regulatory elements (highlighted with grey shading) are reactivated in iTPCs. (F) Serum-induced differentiation leads iTPCs to convert to an adherent phenotype, up-regulate differentiation markers GFAP, beta III tubulin, MAP-2, GalC and (G) to lose CD133 expression. (H) Western blots confirm serum-induced differentiation of iTPCs leads to down-regulation of core TFs. Lower panels: tubulin loading control. These data indicate that the core TFs can reprogram DGCs into stem-like GBM cells, which have an epigenetic landscape similar to TPCs that is sustained by endogenous regulatory programs. See also Supplemental Figures S2.