Additional file 1 of Mapping chromatin remodelling in glioblastoma identifies epigenetic regulation of key molecular pathways and novel druggable targets

Additional file 1: Figures S1-S12. Figure S1- Single track analysis of the ChIP Seq dataset to identify outliers. a) Schematic of the experimental workflow. EPSC: expanded potential stem cells, iNSC: induced neural stem cells, GIC: glioblastoma initiating cells. b) Number of ChIP Seq peaks found in the 10 GIC (top panel) and in the 10 iNSC (bottom panel) for the four HM. Arrows and italic bold font show a lower number of peaks for H3K36me3 GIC54 and H3K27me3 GIC61. c) Person correlation heatmap represents the 40 ChIP Seq tracks for the four HM of the ten patients. Red font highlights GIC19 H3K27ac clustering apart from other H3K27ac tracks. The two samples identified in Fig. S1b with a low number of peaks are shown in italic. Figure S2- Complete dataset used in the study. a) Person correlation heatmap represents the complete ChIP Seq tracks used in this study including the four HM of the patients’ GIC. b) Person correlation heatmap represents the complete ChIP Seq tracks used in this study including the four HM of the patients’ iNSC. Figure S3- Comparative analysis of iNSC and GIC for each HM at peak level. a) Principal component analysis (PCA) representing affinity sites in GIC and iNSC for the four histone modifications (H3K4me3, H3K36me3, H3K27ac and H3K27me3). b) Correlation heatmaps representing significantly differentially bound sites (FDR&lt;0.05) between the GIC (purple) and iNSC (pink) for the four histone modifications across genome. c) Venn diagram showing the overlapping peaks for each HM. Figure S4- Comparative analysis of iNSC and GIC for each HM at gene level. a-b) Visualization of pathways enriched from genes uniquely found linked to activating (a) and repressing (b) HM in GIC as compared to iNSC. Pathways are annotated based on pathway enrichment analysis performed with Reactome and represented as circle, colours represent each histone (see legend), size of the circle is proportional to the number of genes involved in the pathway, all pathways with FDR&lt;0.05 are shown. Figure S5- ChIP and RNA Seq datasets integration and pathway analysis. a) Pearson correlation heatmap of ChIP Seq dataset for each histone modifications and RNAseq dataset [23] integrative analysis for all genes in GIC (purple square) and iNSC (pink square). RNAseq data are represented as log fold change of Differentially Expressed (DE) genes between GIC and iNSC. LogFC DE&gt;1 and &lt;-1 for genes up and downregulated in GIC as compared to iNSC respectively. LogFC DE&gt;1 and &lt;-1 when genes are down and upregulated in iNSC as compared to GIC respectively. b-c) Volcano plot representing genes bound by each histone modification based on the fold change on the input. Among them, the upregulated genes (LogFC(DE)&gt;1) are shown in red and the downregulated (LogFC (DE)&lt;-1) are shown in blue in GIC (b) and iNSC (c). d) Venn diagram showing percentages of specific and common genes in GIC (purple) and iNSC (pink) identified by peak calling for each histone modification. e) Visualisation of the enriched pathways identified in GIC from genes upregulated in GIC as compared to iNSC and associated with a gain of activating HM (H3K4me3, H3K36me3 and H3K27ac) and a loss of repressing HM (H3K27me3). Pathways are annotated based on pathways enrichment analysis performed with Reactome and represented as circle, colours represent each HM (see legend), size of the circle is proportional to the number of genes involved in the pathway (FDR&lt;0.05). Figure S6- Pathways analysis and identification of GSC as differentially regulated and DE gene. a) Visualisation of the enriched pathways identified in GIC from genes downregulated in GIC as compared to iNSC and associated with the loss of activating HM and gain of repressing HM. Pathways are annotated based on pathways enrichment analysis performed with Reactome and represented as circle, colours represent each HM (see legend), size of the circle is proportional to the number of genes involved in the pathway (FDR&lt;0.05). b) Comparison of Chip Seq tracks of each modification in iNSC (pink) and GIC (purple) in the GSC locus. Tracks shown are an average of the 10 patients and the black square highlights the region with differential peaks. c-d) Representative images of GSC protein expression (green) in iNSC and GIC of patients 19 and 61 (c). Nuclei are countered staining with DAPI. Negative control shows the immunofluorescence without primary GSC antibody (d). Scale bars: 50µm (c)and 100µm (d). e) GBMap [97] analysis showing the expression of GSC in the different cell types of the brain. Figure S7- ChromHMM analysis. a) Percentages of overlap of the two models (trained on iNSC or on GIC) in GIC and iNSC. b) Heatmap of the emission parameters displaying the overlap fold enrichment for various external genomic annotations. c) Distribution of ChIP Seq peaks’ genomic annotations for each chromatin state in GIC and iNSC. Heatmap represent Fisher’s exact test statistical analysis and fold change of ChIP Seq peaks between GIC and iNSC. d) Venn diagram showing number of peaks specific to GIC (purple), overlapping with iNSC and specific to iNSC (pink) for each chromatin state defined by ChromHMM analysis. Figure S8- ChromHMM analysis without and with RNA Seq integration. a) Percentages of upregulated (red) and downregulated (blue) genes in GIC as compared to iNSC (left) and in iNSC as compared to GIC (right) in each chromatin state based on transcriptomic dataset from the SYNGN Cohort. Number of genes is also specified for each condition. b) Visualisation of the enriched pathways identified in GIC from genes upregulated in GIC as compared to iNSC and associated with a gain of activating chromatin state. Pathways are annotated based on pathways enrichment analysis performed with Reactome and represented as circle, colours represent each histone (see legend), size of the circle is proportional to the number of genes involved in the pathway (FDR&lt;0.05). c) mRNA expression of OAS1 and OAS3 in iNSC and GIC from the RNAseq dataset of the SYNGN cohort [23]. Results are expressed in log 2 (tpm) transcript per million (tpm). One-way ANOVA test.*p value&lt;0.05, **p value&lt;0.01 and ***p value&lt;0.001. d) Survival curve of primary glioblastoma patients with high and low expression of OAS1 (left panel) and OAS3 gene (right panel). Source: TCGA. Stat test: log-rank,*p value&lt;0.05, **p value&lt;0.01 and ***p value&lt;0.001. e) Spatial expression of OAS1 (middle) and OAS3 (right) in glioblastoma bulk samples, analysed on IvyGap. The left panel shows an example of histological anatomic structure identified in a sub-block and the right panel represents the expression of OAS1 and OAS3 in RNAseq data from anatomic structures shown as log2 normalised gene expression. Leading Edge is defined as the border of the tumour, where ratio of tumour to normal cells is 1-3 / 100. Infiltrating tumour is defined as the intermediate zone between leading edge and cellular tumour, where ratio of tumour to normal cells is 10-20 /100. Cellular tumour is defined as tumour core, where tumour to normal cells is 100-500 / 1. One-way ANOVA test.*p value&lt;0.05, **p value&lt;0.01 and ***p value&lt;0.001. f) Single cell RNAseq data from [31] showing OAS1 (top) and OAS3 (bottom) expression (left panels) in scRNAseq-defined clusters in glioblastoma samples (right panels). Data are plotted as tSNE, with logTPM expression ranging from light orange to dark. Figure S9- ChIP and RNA Seq integration and identification of differentially regulated and DE genes. a) Visualisation of the enriched pathways identified in GIC from genes downregulated in GIC as compared to iNSC and associated with the loss of activating HM and gain of repressing HM. Pathways are annotated based on pathways enrichment analysis performed with Reactome and represented as circle, colours represent each HM (see legend), size of the circle is proportional to the number of genes involved in the pathway (FDR&lt;0.05). b) Survival curve of primary glioblastoma patients with high and low expression of UBE2D. Source: TCGA. Stat test: log-rank, *p value&lt;0.05, **p value&lt;0.01 and ***p value&lt;0.001. c) Single cell RNAseq data from [31] showing UBE2D expression in scRNAseq-defined clusters in glioblastoma samples (right panels). Data are plotted as tSNE, with logTPM expression ranging from light orange to dark. d) Bar plot showing the down and upregulated genes associated with proximal enhancers only found in GIC and in iNSC. Stat test: Fisher’s exact test,***p value&lt;0.001. e) Gene ontology analysis showing the biological processes regulated by the upregulated genes associated with proximal enhancers found only in GIC (left) and downregulated genes associated with proximal enhancers found only in iNSC (right). Enrichment analysis was performed with GO Biological Processes, with an adjusted p value &lt; 0.05. Figure S10- Switching states as assessed by ChromHMM. a) Heatmap showing the number of peaks changing from each NSC state to each GIC state in at least 2 patients. b) Pie charts show percentages of peaks in each transitioning states between in GIC and iNSC. c) Distribution of genomic annotations of peaks switching from repressing in iNSC to activating state in GIC (top plots) and from activating in iNSC to repressing switches in GIC (bottom plots). Figure S11- SMOX is a novel target gene identified in transitioning states. a) Comparison of ChIP Seq tracks of each modification from iNSC (pink) and GIC (purple) in the SMOX locus. Tracks shown are an average of the 10 patients and the black squares highlight the regions with differential peaks. b) Survival curve of primary glioblastoma patients with high and low expression of SMOX. Source: TCGA. Stat test: log-rank, *p value&lt;0.05, **p value&lt;0.01 and ***p value&lt;0.001. c) Spatial expression of SMOX in glioblastoma bulk samples, analysed on Ivy -GAP. The left panel shows an example of histological anatomic structure identified in a sub-block and the right panel represents the expression of SMOX in RNAseq data from anatomic structures shown as log2 normalised gene expression. Leading Edge is defined as the border of the tumour, where ratio of tumour to normal cells is 1-3 / 100. Infiltrating tumour is defined as the intermediate zone between leading edge and cellular tumour, where ratio of tumour to normal cells is 10-20 /100. Cellular tumour is defined as tumour core, where tumour to normal cells is 100-500 / 1. One-way ANOVA test. *p value&lt;0.05, **p value&lt;0.01 and ***p value&lt;0.001. d) Single cell RNAseq data from [31] showing SMOX expression (top panel) in scRNAseq-defined clusters in [31] (bottom panel). Data are plotted as tSNE, with logTPM expression ranging from light orange to dark. e) GBMap [97] analysis showing the expression of SMOX in the different cell types present in the brain. f) mRNA expression of the SMOX in iNSC and GIC from three patients of the SYNGN cohort (19, 52 and 61) measured by RTqPCR. Results are expressed in two delta Ct (detailed in Material and Methods section). g) Representative images of SMOX staining with immunofluorescence (green) in iNSC and GIC of patients 19 and 61. Nuclei are counterstained with DAPI. Scale bar:100µm. h) Cell viability measured with CellTiter Glo Promega Kit in response to increasing doses of SMOX inhibitor MDL72527 (1, 10, 100µM). Results are expressed in fold change of the average from three patients (19, 52 and 61) standardised on the vehicle (0) for iNSC (pink border) and GIC (purple border). i) Representative images of GIC nuclei, counterstained with DAPI, on the bottom side of the transwell membrane after 24 hours of treatment with SMOX inhibitor MDL72527 (10µM). Scale bar:100µm. Figure S12- GABBR2 is a novel target gene identified in transitioning states. a) Comparison of ChIP Seq tracks of each modification from iNSC (pink) and GIC (purple) in the GABBR2 locus. Tracks shown are an average of the 10 patients and the black square highlights the region with differential peaks. b) mRNA expression of the GABBR2 in iNSC and GIC from three patients of the SYNGN cohort (19, 52 and 61) measured by RTqPCR. Results are expressed in two delta Ct (detailed in Material and Methods section). c) Survival curve of primary glioblastoma patients with high and low expression of GABBR2. Source: TCGA. Stat test: log-rank,*p value&lt;0.05, **p value&lt;0.01 and ***p value&lt;0.001. d) Spatial expression of GABBR2 in glioblastoma bulk samples, analysed on Ivy -GAP. The left panel shows an example of histological anatomic structure identified in a sub-block and the right panel represents the expression of GABBR2 in RNAseq data from anatomic structures shown as log2 normalized gene expression. Leading Edge is defined as the border of the tumour, where ratio of tumour to normal cells is 1-3 / 100. Infiltrating tumour defined as the intermediate zone between leading edge and cellular tumour, where ratio of tumour to normal cells is 10-20 /100. Cellular tumour defined as tumour core, where tumour to normal cells is 100-500/ 1. One-way ANOVA test. *p value&lt;0.05, **p value&lt;0.01 and***p value&lt;0.001. e) Single cell RNAseq data showing GABBR2 expression (left panel) in scRNAseq of glioblastoma samples in clusters defined in [31] (right panel). Data are plotted as tSNE, with logTPM expression ranging from light orange to dark. f) GBMap analysis showing the expression of GABBR2 in the different cell types present in the brain. g) Cell viability measured with CellTiter Glo Promega Kit in response to increasing doses of GABBR2 agonist Baclofen (red bars) and the antagonist CGP36742 (blue bars) (0, 10, 100, 200µM). Results are expressed in fold change of the average from three patients (19, 52 and 61) standardised on the vehicle (0) for iNSC (pink border) and GIC (purple border). h) Representative images of GIC nuclei, counterstained with DAPI, on the bottom side of the transwell membrane after 24 hours of treatment with GABBR2 agonist Baclofen (100µM) and the antagonist CGP36742 (200µM). Scale bar:100µm.