Insulator dysfunction and oncogene activation in IDH mutant gliomas

Abstract

Gain-of-function IDH mutations are initiating events that define major clinical and prognostic classes of gliomas. Mutant IDH protein produces a new onco-metabolite, 2-hydroxyglutarate, which interferes with iron-dependent hydroxylases, including the TET family of 5'-methylcytosine hydroxylases. TET enzymes catalyse a key step in the removal of DNA methylation. IDH mutant gliomas thus manifest a CpG island methylator phenotype (G-CIMP), although the functional importance of this altered epigenetic state remains unclear. Here we show that human IDH mutant gliomas exhibit hypermethylation at cohesin and CCCTC-binding factor (CTCF)-binding sites, compromising binding of this methylation-sensitive insulator protein. Reduced CTCF binding is associated with loss of insulation between topological domains and aberrant gene activation. We specifically demonstrate that loss of CTCF at a domain boundary permits a constitutive enhancer to interact aberrantly with the receptor tyrosine kinase gene PDGFRA, a prominent glioma oncogene. Treatment of IDH mutant gliomaspheres with a demethylating agent partially restores insulator function and downregulates PDGFRA. Conversely, CRISPR-mediated disruption of the CTCF motif in IDH wild-type gliomaspheres upregulates PDGFRA and increases proliferation. Our study suggests that IDH mutations promote gliomagenesis by disrupting chromosomal topology and allowing aberrant regulatory interactions that induce oncogene expression.

Extended Data Figure 1. DNA methylation and CTCF binding at deregulated boundaries

(a) Box plots show DNA methylation levels over CTCF sites (200 bp window centered on the peak) within boundaries predicted by gene pair correlation analysis to be disrupted. All CTCF sites located within a 1 kb window centered on a disrupted boundary were considered. Methylation levels were determined from whole genome bisulfite data for three IDH mutant (red labels) and three IDH wildtype (black labels) tumors. (b) Bars show average normalized ChIP-seq signal over all CTCF sites located inside a 1 kb window centered on a disrupted boundary.

Extended Data Figure 2. Expression of FIP1L1 in mouse brain cells and survival effects of PDGFRA and FIP1L1

(a) Expression of FIP1L1 in isolated mouse brain cell types. (b) Kaplan-Meier Plot based on TCGA data3 indicates that combined FIP1L1 and PDGFRA expression is a negative prognostic factor in IDH1 mutant lower-grade gliomas. Multivariate analysis including the known prognostic factor 1p/19q deletion diminished this effect into non-significance, suggesting that other predictors of survival may also play a role in this model.

Extended Data Figure 3. CTCF anchored loop in the PDGFRA region

(a) Schematic depiction of a HiC interaction signature of a CTCF-anchored loop domain, compared to an ordinary domain, as described by Rao et al., Cell 2014. CTCF-anchored loop domains are characterized by an increased interaction score at the apex of the domain, representing a CTCF-CTCF dimeric interaction. (b) IMR90 HiC contact matrix for the PDGFRA/FIP1L1 locus, as presented in Figure 3a. Solid circle indicates CTCF dimer interaction point. Dashed circles indicate lack of CTCF dimeric anchor signature. (c) IMR90 HiC contact matrix as in (b), but with expanded heatmap scale, more clearly conveys the CTCF-anchored loop that insulates PDGFRA. (d,e) HiC contact matrix for GM12878 cells for the same region confirms a single CTCF-anchored loop (solid circle) between PDGFRA and FIP1L1. These data support the significance of this specific boundary in locus topology and PDGFRA insulation.

Extended Data Figure 4. Characterization of the FIP1L1 enhancer

(a) H3K27ac ChIP-seq track for GSC6 gliomaspheres reveals strong enrichment over the FIP1L1 enhancer. CTCF ChIP-seq track reveals location of the boundary element insulator (as in Figure 3a). FIP1L1 enhancer (i) and promoter (ii) are indicated. (b) H3K27ac ChIP-seq tracks for IDH mutant and wild-type gliomaspheres and glioma specimens reveal enrichment over the FIP1L1 enhancer. (c) ChIP-seq tracks for glioma master transcription factors and other histone modifications support the enhancer identity of the element (H3K27ac, H3K4me1, SOX2, OLIG2; lacks H3K4me3, lacks H3K27me3). In contrast, the FIP1L1 promoter has a distinct ‘promoter-like’ chromatin state.

Extended Data Figure 5. Interaction of the FIP1L1 enhancer with nearby promoters and PDGFRA quantified by reciprocal chromatin conformation capture (3C)

(top) The H3K27ac, CTCF and genetic architecture of the FIP1L1/PDGFRA locus is indicated, highlighting the 3C strategy. (bottom) Plots indicate the interaction signal of the indicated sites (black lines) with the common enhancer primer. The FIP1L1 enhancer interacts with local promoters in wild-type and mutant tumors and models. In IDH wild-type gliomas, it shows essentially no interaction with the PDGFRA promoter. In IDH mutant gliomas, it interacts with the PDGFRA promoter with comparable strength to the local interactions, despite the much larger intervening distance (900 kb). Error bars reflect standard deviations.

Extended Data Figure 6. Crenolanib reverses the increased growth of PDGFRA insulator disrupted cells

Insulator CRISPR-infected gliomaspheres exhibit a roughly 2-fold increase in proliferation rate, compared to control sgRNA infected gliomaspheres. This proliferative advantage is eliminated by treatment with the PDGFRα inhibitor Crenolanib. Crenolanib and Dasatinib both inhibit PDGFRα, but their other targets are non-overlapping. Hence, this sensitivity provides further support that PDGFRA induction drives the increased proliferation of the insulator CRISPR gliomaspheres. (Error bars reflect standard deviations).

Extended Data Figure 7. Signature of boundary deregulation in IDH mutant gliomas is robust

Volcano plot depicts the significance (y-axis) of gene pairs that are either more or less correlated in IDH mutant than IDH wild-type gliomas. This plot was generated by repeating the analysis in the main text and shown in Figure 1f, except that here the statistics were performed using only the 14,055 genes expressed at >1 TPM in at least half the samples. This indicates that the boundary deregulation signature in IDH mutant gliomas is not sensitive to noise from lowly expressed genes.

Figure 1. CTCF binding and gene insulation compromised in IDH mutant gliomas

(a) Binding profiles for the methylation-sensitive insulator CTCF are shown for a representative locus in IDH1 mutant and wildtype tumors, normalized by average signal. (b) Scatterplot compares CTCF binding signals between IDH mutant (y-axis) and IDH1 wildtype gliomas (x-axis) for all detected CTCF sites. A larger fraction of sites is commonly lost in all IDH1 mutants (n=625) than gained (n=300). (c) Histogram compares GC content between CTCF sites that are lost or retained. (d) Box plots show DNA methylation levels over lost CTCF sites, as determined from whole genome bisulfite data for three IDH wildtype and three IDH mutant tumors. (e) Plot depicts average correlation between gene pairs as a function of distance across RNA-seq profiles for human brain. Gene pairs separated by a constitutive CTCF-bound boundary per HiC have lower correlations. (f) Volcano plot depicts the significance (y-axis) of gene pairs that are more (or less) correlated in IDH mutant than IDH wildtype lower-grade gliomas. Gene pairs with significantly increased correlations in IDH mutants (right) tend to cross boundaries (orange), while those with decreased correlations (left) more likely reside in the same domain (blue). These data indicate that IDH mutant, G-CIMP gliomas have reduced CTCF binding and altered expression patterns suggestive of defective gene insulation.

Figure 2. Topological domain boundaries disrupted in IDH1 mutant gliomas

(a) Scatterplot depicts significance of deregulated boundaries in IDH mutant tumors (y-axis) against fold-change of most up-regulated gene in adjacent domains (x-axis). PDGFRA is adjacent to a significantly deregulated boundary and up-regulated in IDH mutants. (b) Boxplots compare PDGFRA expression (left) or copy number (right) for 443 glioblastoma tumors, classified by IDH status and expression subtype. IDH mutants (red) have elevated PDGFRA expression, despite normal copy number. (c) Plots compare PDGFRA (y-axis) and FIP1L1 (x-axis) expression in IDH wildtype (left) and mutant (right) gliomas. The genes correlate specifically in IDH mutants, consistent with deregulation of the intervening boundary/insulator.

Figure 3. Insulator loss allows PDGFRA to interact with a constitutive enhancer

(a) Contact domain structure shown for a 1.7 MB region containing PDGFRA. Heat depicts HiC interaction scores between triangulated loci in IMR90 cells. Domains are visible as triangle-shaped regions of high interaction scores. Convergent CTCF sites anchor a loop that separates PDGFRA and FIP1L (black circle). H3K27ac and CTCF profiles are aligned to the contact map. Interaction trace (below) depicts HiC signals between the PDGFRA promoter and all other positions in the region. Genes, FIP1L1 enhancer (per H3K27ac) and insulator (per HiC and CTCF binding) are indicated. (b) The right CTCF peak in the insulator contains a CTCF motif with a CpG at a methylation-sensitive position. (c,d) ChIP-qPCR data show that CTCF occupancy over the boundary is reduced in IDH mutant (red) gliomas and models, relative to wildtype (black). (e) Methylation levels of the CpG in the CTCF motif were measured in gliomaspheres by bisulfite sequencing, and plotted as percent of alleles protected from conversion. (f) Methylation levels of the CpG in the CTCF motif were measured in glioma specimens by methylation-sensitive restriction, and plotted as relative protection. (g) Expanded views of FIP1L1 enhancer locus and PDGFRA locus shown with H3K27ac tracks. Vertical black bars indicate the locations of the common PDGFRA promoter primer and four complementary primers tested in 3C. (h–k) Plots show normalized 3C interaction frequencies between PDGFRA promoter and indicated regions. A strong interaction between PDGFRA promoter and FIP1L1 enhancer is evident in IDH mutant tumors and models. (Error bars in all panels reflect standard deviations of triplicate observations).

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).