Mutant-IDH1-dependent chromatin state reprogramming, reversibility, and persistence

Abstract

Mutations in IDH1 and IDH2 (encoding isocitrate dehydrogenase 1 and 2) drive the development of gliomas and other human malignancies. Mutant IDH1 induces epigenetic changes that promote tumorigenesis, but the scale and reversibility of these changes are unknown. Here, using human astrocyte and glioma tumorsphere systems, we generate a large-scale atlas of mutant-IDH1-induced epigenomic reprogramming. We characterize the reversibility of the alterations in DNA methylation, the histone landscape, and transcriptional reprogramming that occur following IDH1 mutation. We discover genome-wide coordinate changes in the localization and intensity of multiple histone marks and chromatin states. Mutant IDH1 establishes a CD24⁺ population with a proliferative advantage and stem-like transcriptional features. Strikingly, prolonged exposure to mutant IDH1 results in irreversible genomic and epigenetic alterations. Together, these observations provide unprecedented high-resolution molecular portraits of mutant-IDH1-dependent epigenomic reprogramming. These findings have substantial implications for understanding of mutant IDH function and for optimizing therapeutic approaches to targeting IDH-mutant tumors.

Fig. 1. Characterization of IDH1-mutant-induced gene expression reversibility

a, Summary of the cellular models used to study IDH1 R132H function and the epigenomic and transcriptomic datasets produced in this study. b, IHAs transduced with inducible empty vector (EV) or vector encoding wild-type (WT) or R132H (MUT) IDH1 were grown for 30 passages (baseline) in the presence (+) or absence (−) of 1 µg/ml doxycycline. Doxycycline was withdrawn from the medium for either two (top) or ten (bottom) passages after baseline (off). Western blot analysis of inducible IHAs confirms expression of IDH1 R132H only in the presence of doxycycline. c, Total intracellular 2HG was measured by gas chromatography coupled with mass spectrometry and normalized to an internal standard (D5-2HG) and cell number. 2HG levels are increased upon doxycycline administration and are completely inhibited at 15 passages following doxycycline withdrawal after baseline. d, Schematic of the reversibility model for inducible expression of IDH1 R132H in astrocytes. P, passage. e, Hierarchical clustering of global gene expression profiles of IHAs as shown in d. f, Heat maps of downregulated (left) and upregulated (right) gene expression clusters following doxycycline withdrawal at baseline. Pers., persistent. The color scale shows normalized expression levels. g, Gene expression time course for several persistently upregulated (top) and downregulated (bottom) genes. h, Line plots show average expression levels of three Affymetrix probes targeting CD24 over time. i, Percentages of genomic features for the methylated loci exhibiting the most inverse correlation with gene expression data in CD24⁺ as compared to CD24⁻ IDH1 R132H–expressing astrocytes. Hyper/down, hypermethylated and downregulated genes; hypo/up, hypomethylated and upregulated genes. IGR, intergenic region. j, Average expression of example genes upregulated in CD24⁺ cells measured in BrainSpan Developmental Transcriptome data. RPKM, reads per kilobase of transcript per million mapped reads; pcw, post-conception weeks; mos, months; yrs, years.

Fig. 2. Remodeling of chromatin states and the epigenomic landscape by mutant IDH1

a, Genome coverage (left) and number of peaks (right) for histone marks in IHAs stably expressing IDH1 R132H at passages 2, 10, and 40. b, Genome coverage (left) and number of peaks (right) for histone marks in parental IHAs at passages 2, 10, and 40. c, Heat maps displaying log₂-transformed input-normalized ChIP signal for k-means clusters ( k = 6) of the union of H3K4me3 (left), H3K9me3 (middle), and H3K36me3 (right) peaks for parental IHAs (Par) and all IHAs stably expressing mutant IDH1 (Mut). Scaled regions from peak start to peak end are displayed. The color scale indicates ChIP signal ranging from no enrichment (dark blue) to high enrichment (dark red). d, H3K9me3 (top) and H3K36me3 (bottom) log₂-transformed input-normalized ChIP signal profiles for the clusters identified in c. Scaled regions from peak start to peak end are displayed. e, HOMER-derived annotations of the genomic loci covered by H3K4me3 peaks in parental IHAs and IHAs stably expressing IDH1 R132H at passages 2, 10, and 40. TTS, transcription termination site. f, H3K4me3 read coverage normalized to 1× sequencing depth at the PDGFRA TSS and DANCR locus for parental IHAs and IHAs with stable expression of mutant IDH1. The range of the y axis is indicated and fixed for both plots. g, Number of called peaks in inducible IHAs at baseline (passage 30, Dox⁻ and Dox⁺) and 40 passages following doxycycline withdrawal from baseline (Dox^off 40 P). h,i, Chromatin states learned jointly across all IHAs stably expressing IDH1 R132H (h) and inducible IHAs (Dox⁻, Dox⁺, Dox^off 40 P) (i). Plots show emission parameters (%) learned on the basis of combinations of chromatin marks, genomic annotation, genome coverage (%), and transition parameters. Emission parameters correspond to the probability of a given histone mark occurring in a particular chromatin state. Shading indicates relative fold enrichment. LAD, lamina-associated domains.

Fig. 3. Reversibility and kinetics of DNA methylation changes in astrocytes inducibly expressing IDH1 R132H

a, Hierarchical clustering of global methylation profiles in inducible IHAs. b, Distribution of Δβ values (compared to baseline without doxycycline) for hypo- and hypermethylated loci at baseline (top panel) and subsequent passages after doxycycline withdrawal. c, Heat maps of hypermethylated (left) and hypomethylated (right) clusters following doxycycline withdrawal. At passage 40 following doxycycline withdrawal, 20% of hypermethylated and 30% of hypomethylated loci within the persistent clusters maintain their altered methylation state with equal or greater absolute β-value differences as compared to baseline. The color scale shows normalized methylation values ranging from low (dark blue) to high (red). d, Number of hypomethylated (blue) and hypermethylated (red) loci falling within the transient, gradual, and persistent clusters. e, Table showing the total number of hypermethylated (top) and hypomethylated (bottom) loci binned by β-value differences (at baseline) across the transient, gradual, and persistent methylation clusters. f, Overlap of hypermethylated and hypomethylated loci in Dox⁺ versus Dox⁻ IHAs at baseline with the 15-state ChromHMM annotations of the Roadmap Epigenomics project. ENCODE state annotations are noted below for the 15 states. g, Distribution of Δβ values (Dox⁺ versus Dox⁻ at baseline) for the loci corresponding to up- and downregulated genes (Dox⁺ versus Dox⁻ at baseline) within the transient, gradual, and persistent clusters stratified by genomic feature. None of the persistently upregulated genes fall within 3′ UTRs or intergenic regions. h, Log₂-transformed input-normalized H3K4me3, H3K27me3, H3K36me3, and H3K9me3 ChIP signal profiles (top) and heat maps (bottom) ±1 kb around hypomethylated CpG sites within the transient (dark blue), gradual (light blue), and persistent (yellow) clusters for Dox⁻, Dox⁺, and Dox^off IHAs. The color scale indicates ChIP signal ranging from no enrichment (dark blue) to high enrichment (dark red). i, Overlap of hypermethylated and hypomethylated clusters with chromatin states in Dox⁻ IHAs. “Background” corresponds to overlap of all probes on the Illumina HumanMethylation450K array with Dox⁻ chromatin states.

Fig. 4. Characterization of H3K4me3 dynamics in IDH1 R132H models

a, HOMER annotation of genomic regions within chromatin state 5 in inducible IHAs. n indicates the number of genome segments in state 5. The color scale indicates the percentage of total segments within each annotation category and ranges from white (0%) to dark blue (100%). b, Chromatin state transitions from Dox⁻ to Dox⁺ IHAs. Numbers inside the cells correspond to the z score of the genomic region covered by each transitioned state in Dox⁺ cells. The color scale ranges from low coverage (dark blue) to high coverage (red). c, Sequence-depth-normalized H3K4me3 and RNA-seq coverage tracks for MAP1LC3A show persistent reduction of H3K4me3 at its promoter and loss of gene expression over the exonic regions. d, Profiles (top) and heat maps (bottom) of log₂-transformed input-normalized H3K4me3 ChIP–seq signal ±1 kb around the start and end sites of the top 11,443 regions with significant gains in H3K4me3 in Dox⁺ as compared to Dox⁻ IHAs. The color scale indicates ChIP signal ranging from no enrichment (dark blue) to high enrichment (dark red). Start, peak start; end, peak end. e, Distribution of the locations for the top 11,443 differentially enriched H3K4me3 peaks with respect to their nearest TSSs. f, Pie chart showing the distribution of HOMER-annotated genomic features for the top 11,443 differentially enriched H3K4me3 peaks. g, GSEA (using GREAT toolbox) of the top 2,000 TSSs with the highest H3K4me3 enrichment across parental IHAs and IHAs stably expressing IDH1 R132H (Mutant) at passage 40, in inducible Dox⁻ and Dox⁺ IHAs, in Dox^off passage 40 IHAs, and in TS603 tumorspheres. Color corresponds to the multiple-testing-adjusted q value of enrichment. h, Profiles (top) and heat maps (bottom) of log₂-transformed input-normalized H3K4me3 ChIP signal ± 1kb around the start and end sites of the top 1,150 significant regions in the IDH1-mutant models (compared to IDH-wild-type cells) along with Dox^off passage 40 IHAs for the same regions. The color scale indicates ChIP signal ranging from no enrichment (dark blue) to high enrichment (dark red).

Fig. 5. Upregulated viral response and genomic instability in cells expressing IDH1 R132H

a, GSEA of the IFN-α signaling profile showing that the interferon signaling pathway is significantly upregulated in Dox⁺ as compared to Dox⁻ IHAs. NES, normalized enrichment score; FDR, false discovery rate. b, GSEA of the IFN-α signaling profile showing that the interferon signaling pathway is significantly upregulated in Dox^off states as compared to Dox⁻ IHAs. c, Heat map of RNA-seq-derived ERV expression for astrocytes with empty vector (green) or inducible expression of IDH1 R132H (purple) at baseline (Dox⁻, Dox⁺), 40 passages following baseline (Dox⁻ 40 P, Dox⁺ 40 P), 1 passage or 40 passages following doxycycline withdrawal (Dox^off 1 P, Dox^off 40 P), and tumorspheres (TS543, TS603, TS667) (gray). The scale corresponds to the z scores of regularized log-transformed read counts. Regularized log-transformed (rlog) is a function that transforms RNA-seq read counts. d, qPCR validation of select ERVs (from c) with increased expression in Dox⁺ IHAs at passage 70. Error bars, s.d; n = 3; **P < 0.01, ***P < 0.001, ****P < 0.0001; n.s., not significant (unpaired t test). e, Heat map of viral defense signature genes for astrocytes with empty vector (green) or inducible expression of IDH1 R132H (purple) at baseline (Dox⁻, Dox⁺), 40 passages following baseline (Dox⁻ 40 P, Dox⁺ 40 P), 1 passage or 40 passages following doxycycline withdrawal (Dox^off 1 P, Dox^off 40 P), and tumorspheres (TS543, TS603, TS667) (gray). Scale corresponds to the z scores of regularized log-transformed read counts. f, qPCR analysis of select ERVs involved in antiviral response showing increased expression in Dox⁺ IHAs at passage 70. Error bars, s.d; n = 3; *P < 0.05, **P < 0.01, ***P < 0.001; n.s., not significant (unpaired t test). g, GSEA showing that genes on chromosome 19q13 are concordantly and significantly downregulated in Dox⁺ as compared to Dox⁻ IHAs. h, Copy number profile derived from Illumina HumanMethylation450K arrays for astrocytes expressing IDH1 R132H at baseline (compared to Dox⁻ astrocytes). Red denotes amplification, and blue denotes loss. i, Quantification of metaphases with relative loss of 19q in Dox⁺ and Dox⁻ inducible IHAs at baseline (P30) and five passages after baseline (P35).

Fig. 6. Phenotype and tumor growth dynamics associated with mutant IDH1

a, In vitro proliferation of inducible astrocytes (Dox⁻, Dox⁺, and Dox^off; the number of passages following doxycycline withdrawal is specified for each plot). Error bars, s.e.m; n = 2. b, Serial bioluminescence imaging tracking the growth of inducible astrocytes (Dox⁻, n = 10 mice; Dox⁺, n = 20 mice). The arrow indicates the point at which the Dox⁺ group was randomized into Dox^off (doxycycline withdrawn; n = 10) and Dox⁺ (continued on doxycycline diet; n = 10) mice. Each time point shows the average radiance across groups (mean ± s.e.m.). c, Inducible IHAs labeled with ZsGreen and imaged with STPT. Bright areas of coronal sections correspond to ZsGreen positivity; Allen Brain Atlas coordinates are shown at the bottom. d, Left, whole brain surface reconstructions from 280 brain sections imaged with STPT using 20% resolution for raw data; scale bar, 500 µm. Middle panels, 3D tumor reconstructions for ZsGreen-labeled cells showing tumor location and growth (sagittal (left) and horizontal (right) views). Right, reconstructed tumors; scale bar, 250 µm. e, Semiquantitative prediction of cell numbers in various brain regions, indicating spread of fluorescent cells from a representative IDH1 R132H–expressing (Dox⁺) tumor in comparison to a representative tumor where IDH1 R132H expression was eliminated (Dox^off). MO, somatomotor areas; SS, somatosensory areas; ILA, infralimbic area; CP, caudoputamen; ACB, nucleus accumbens; FS, fundus of striatum; OT, olfactory tubercle; AAA, anterior amygdalar area; Palv, pallidum, ventral region; LHA, lateral hypothalamic area; LPO, lateral preoptic area.