Changes in chromatin state reveal ARNT2 at a node of a tumorigenic transcription factor signature driving glioblastoma cell aggressiveness

Although a growing body of evidence indicates that phenotypic plasticity exhibited by glioblastoma cells plays a central role in tumor development and post-therapy recurrence, the master drivers of their aggressiveness remain elusive. Here we mapped the changes in active (H3K4me3) and repressive (H3K27me3) histone modifications accompanying the repression of glioblastoma stem-like cells tumorigenicity. Genes with changing histone marks delineated a network of transcription factors related to cancerous behavior, stem state, and neural development, highlighting a previously unsuspected association between repression of ARNT2 and loss of cell tumorigenicity. Immunohistochemistry confirmed ARNT2 expression in cell sub-populations within proliferative zones of patients’ glioblastoma. Decreased ARNT2 expression was consistently observed in non-tumorigenic glioblastoma cells, compared to tumorigenic cells. Moreover, ARNT2 expression correlated with a tumorigenic molecular signature at both the tissue level within the tumor core and at the single cell level in the patients’ tumors. We found that ARNT2 knockdown decreased the expression of SOX9, POU3F2 and OLIG2, transcription factors implicated in glioblastoma cell tumorigenicity, and repressed glioblastoma stem-like cell tumorigenic properties in vivo. Our results reveal ARNT2 as a pivotal component of the glioblastoma cell tumorigenic signature, located at a node of a transcription factor network controlling glioblastoma cell aggressiveness. Electronic supplementary material The online version of this article (10.1007/s00401-017-1783-x) contains supplementary material, which is available to authorized users.


Introduction
De novo glioblastoma, the most common and malignant primary brain tumor in adults, is a paradigmatic example of heterogeneous tumors [11,49,54,64]. This heterogeneity stems from clonal selection of genomic and phenotypic variants, which arises not only from the accumulation of mutations but also from dynamic changes in cell states [27,28]. As a result, cells with different functional properties co-exist such as proliferative versus non-proliferative, migratory versus static, stem-like versus non-stem, pro-angiogenic versus non-pro-angiogenic. Understanding the basis for this heterogeneity is of importance to efficiently target pivotal tumor cells, especially in glioblastoma that exhibits a dismal prognosis despite aggressive therapies.
Studies of glioblastoma cells endowed with stem-like and tumor-initiating properties (GBM stem-like cells) have shown that aside from the heterogeneity linked to distinct mutational loads, cancer cell diversification can be achieved at the functional level within an unchanged genomic background [16]. Glioblastoma cells have been shown to adopt distinct transcriptomic profiles combined 1 3 with potentially distinct phenotypes and functional behaviors in response to environmental cues, which either favor acquisition of stem-like and tumorigenic properties [3,24,52] or in contrast induce their loss [41,57]. Epigenetic plasticity has been shown to accompany GBM stem-like cell adaptations to their changing microenvironment [21,22,52,71].
An important source of epigenetic plasticity is brought by post-translational histone modifications, such as methylation, acetylation, phosphorylation or ubiquitinylation of histone lysine (K) and arginine (R) residues [45]. These histone modifications alter either the affinity between DNA and histones or create binding sites for chromatin remodeling factors, thereby controlling DNA compaction and accessibility, subsequent transcription and hence ultimately functional outcomes [7,65]. Pioneer studies in embryonic stem cells (ESC) first revealed the link between histone H3 K4 and K27 trimethylation (H3K4me3 and H3K27me3) with transcriptional expression and repression, respectively [50,53,78], the importance of which has been confirmed by large scales epigenomic studies notably in the brain [12]. In addition, these studies reported the existence of bivalent genes bearing both H3K4me3 and H3K27me3 histone marks in ESC [4,6] as well as in adult multipotent/somatic stem cells [13,51]. These bivalent genes are associated with RNA polymerase II at their transcription start sites and are thought to be in a "poised" state ready to be fully activated or repressed during differentiation [1,10,37,50].
Here, we focused on the H3K4me3 and H3K27me3 marks to gain insights into the transcription factor network that sustains glioblastoma cell tumorigenic properties through a bottom-up approach schematized in Fig. 1a. We used as a starting paradigm human glioblastoma cells expressing or not expressing the micro-RNA cluster miR-302-367. Indeed our previous studies have shown that the expression of miR-302-367 represses the stem-like, and most importantly, tumor-initiating properties of human glioblastoma cells [22]. Mapping the genes epigenetically modified in glioblastoma cells following repression of their tumorigenic properties, allowed the modeling of an interrelated array of transcription factors implicated in pathways important for malignancy, stem cell state, and neural development. Most importantly, our results pinpointed a previously unsuspected involvement of the hypoxia-inducing factor (HIF) family member aryl hydrocarbon receptor nuclear translocator 2 (ARNT2) in the control of glioblastoma cell aggressiveness. We then verified and extended our findings using a combination of bioinformatics analysis of independent glioblastoma datasets, analysis of patients' tumor tissues, genetic manipulations of independent additional glioblastoma cell cultures and in vivo experiments. Our results demonstrate that ARNT2 controls the expression of several transcription factors associated with the stem-like properties of glioblastoma cells, and is essential for full tumorigenicity of glioblastoma cells.

Viable cell counting
Trypan blue exclusion test was used to determine the numbers of viable cells (Trypan blue solution, ThermoFisher, 0.4% v/v, 3 min incubation at room temperature). Blue and white cells (dead and alive, respectively) were counted with the Countess automated cell counter (Thermo Fisher, France).

Extreme limiting dilution assays (ELDA)
Cells were plated in 96-well plates at 1, 5, and 10 cells/ well/100 μl as previously described [2]. The percentage of wells with cell spheres was determined after 7 days. The analysis of the frequency of sphere-forming cells, a surrogate property of brain cancer stem-like cells [24] was performed with software available at http://bioinf.wehi.edu.au/ software/elda/ [34].

ChIP-seq sample preparation and analysis
ChIP assays were performed using ChIP-IT Express Magnetic Chromatin Immunoprecipitation kit following the manufacturer's protocol (Active motif, France) and 2 × 10 6 cells per sample and per epitope. Briefly, TG1 and TG1-miR-302-367 cells were cross-linked in 0.5% formaldehyde/PBS for 10 min at room temperature and then treated with 0.125 M glycine in PBS pH 7.4 for 5 min at room temperature. Samples were subsequently washed twice with icecold PBS and once with ice-cold PBS supplemented with protease inhibitors cocktail prior to be lysed.  [76] with input control libraries from the corresponding cell types. Peaks were then annotated using a window of ± 20 kb with respect to the coordinates of the beginning and end of RefSeq transcripts. More than 150 million short reads were obtained for all samples. These short reads were uniquely aligned to the human genome, resulting in a 77 and 76% of the genome covered in TG1 and TG1-miR, respectively. The data have been deposited in NCBI's Gene Expression Omnibus [20] and are accessible through GEO Series accession number GSE98330 (https://www.ncbi.nlm.nih.gov/geo/query/acc. cgi?acc=GSE98330). ARNT2 ChIP was performed as described above using anti-ARNT2 antibodies (Santa Cruz, Cliniscience sc-5581X, France) and 100-1000 bp 5706** chromatin fragments. QPCR analysis was performed on total (input) and immunoprecipitated chromatin, and results normalized over the corresponding input signal. Enhanced representation of the regions of interest was compared to TBP promoter negative control. Sequences of all primers used for ChIP-qPCR are listed in Online Resource 1.

Gene expression analysis
Total RNA was prepared using the RNeasy Plus Universal kit (Qiagen, France) according to the manufacturer's instruction. An on-column DNase digestion was performed during the extraction to yield a pure RNA fraction (RNase-Free DNase Set, Qiagen). cDNA was prepared using the

Expression profiling
Statistical and graphical analyses of ChIP-seq and microarray data were performed using the R software version 3.2.3 (http://cran.r-project.org/). Gene ontology (GO) analysis was performed with DAVID software (version 6.8, http://david. abcc.ncifcrf.gov/). GO analysis of all genes changing histone marks in TG1-miR compared to TG1 was achieved using all human genes as background (Homo Sapiens from DAVID). GO analyses of genes exchanging an active for a repressive histone mark and vice versa between TG1 and TG1-miR were achieved using as background all the genes with differing histone marks in TG1-miR and TG1. Genes encoding transcription factors were retrieved using the KEGG (http:// www.genome.jp/kegg/), and Genomatix databases (Genomatix, Germany). Interactions between the retrieved set of 202 transcription factors were analyzed with the STRING database (version 10.0, http://string-db.org/). Heat maps and z scores were downloaded from the IVY dataset (http:// glioblastoma.alleninstitute.org), and analyzed with XLSTAT version 1.2. z-score graphs were generated with Prism 6.0 software (GraphPad). ARNT2 mRNA expression was analyzed using publicly available data using the R2 Genomics Analysis and Visualization Platform (http://r2.amc.nl) (

Immunohistochemistry
Morphologic examination of patients' glioblastoma resections was performed on Hematoxylin and Eosin stained sections (3-4 μm). Immunolabeling was performed using an automated system (Autostainer Dako, Glostrup Denmark). Deparaffinization, rehydration and antigen retrieval were performed using the pretreatment module PTlink (Dako). ARNT2 immunostaining was achieved using anti-ARNT2 (Santa Cruz, 1:50) and anti-Ki67 antibodies (MIB-1, Dako, prediluted). Immunostaining was scored by a pathologist (FBV). Xenografted mouse brains were dissected after killing of the mice at 45 days post-graft of 6240** or 42 days postgraft of 5706** GBM stem-like cells expressing shControl or shARNT2. The brains were fixed in 4% paraformaldehyde in PBS for 48 h at 4 °C, cryoprotected in 30% sucrose in PBS at 4 °C until the tissue sank, frozen in isopentane at -40 °C, and stored at -80 °C. Cryostat sections of 30-µm-thickness were cut in the frontal plane. Thirteen sections, from the olfactory bubs to the posterior end of cerebellum were selected for the analysis. Sections were incubated with DAPI (Sigma, France) for 10 min at room temperature. Sections staining was analyzed with a fluorescent microscope (Axioplan 2, Zeiss). Images were acquired on digital camera (DXM 1200, Nikon, USA) using Zen 2 software (Zeiss) and prepared using Adobe Photoshop software (Adobe Systems, San José, USA).

Statistical analysis
R version 3.2.3, XLSTAT version 1.2 or Prism 6.0 software (GraphPad) were used for statistical analyses. The level of significance was set at p < 0.05. The type of statistical test used is provided in the figure legends. All experiments were performed using independent biological samples with the exception of the ChIP-seq. All experiments were repeated at least three times in an independent manner with the exception of the microarray experiment. PCA analysis was performed on XLSTAT version 1.2, based on a Pearson correlation matrix. First and second component (F1 and F2 axis) were used to generate a correlation circle where the variables (genes) were plotted as vectors according to their correlation with F1 and F2 axis.
The figures were prepared using Adobe Illustrator (Adobe Systems).

Repression of GBM stem-like cell properties is accompanied by discrete changes in epigenetic profiles
Lentiviral expression of miR-302-367 in the TG1 human GBM stem-like cell line (referred to as TG1-miR) resulted in loss of their stem-like and tumorigenic properties [22]. H3K4me3 and H3K27me3 profiling of TG1 and TG1-miR was performed by ChIP followed by deep sequencing (data accessible at https://www.ncbi.nlm.nih.gov/geo/query/acc.

3
cgi?acc=GSE98330). For each cell type analyzed, approximately 16,000 genes (~ 70% of the complete human exome) were found to be associated with the H3K4me3 and/or H3K27me3 mark (Online Resource 3). This analysis revealed a predominance of genes (~ 48%) associated with the active H3K4me3 mark in TG1 and in TG1-miR (Fig. 1b). Only ~ 10% were associated with the repressive H3K27me3 mark. An equivalent proportion (~ 10%) was associated with the bivalent mark (H3K4me3 and H3K27me3) (Fig. 1b). Western blot assays further demonstrated similar H3K27me3 and H3K4me3 protein levels in TG1 and TG1-miR (Fig. 1c). As described in other cell types [5,78], both marks were enriched in TG1 and TG1-miR at the level of the TSS, with the H3K27me3 mark being in addition spread along the gene bodies (Online Resource 4). Furthermore, as expected, the highest transcript levels were observed in the group of genes associated with the H3K4me3 mark, the lowest transcript levels in the group of genes associated with the H3K27me3 mark, whereas genes associated with the bivalent mark had intermediate expression levels (Fig. 1d). The mean transcript level of the group of genes associated with none of the marks was slightly above the mean expression level of the genes carrying the H3K27me3 mark, suggesting that these genes tended to be repressed. Altogether, these results show that miR-302-367 does not alter global levels of H3K4me3 and H3K27me3, or the proportion of genes associated with either modification or the repartition of the histone marks along the genes.
While the proportion of genes associated with each histone mark was globally unchanged, further analysis revealed a set of 5151 genes exhibiting a change in histone modifications between TG1 and TG1-miR. This number corresponded to 22% of the total number of sequenced genes. The overlap of genes differentially expressed between TG1 and TG1-miR is depicted with a Venn diagram (Fig. 2a). Detailed analysis pointed to the H3K4me3 mark as the most conserved mark following miR-302-367-induced repression of the cells' properties (Fig. 2b, Online Resource 3). Of the 11,080 genes enriched for H3K4me3 mark in TG1, only 92 (~ 0.8%) switched to H3K27me3, whereas 945 (~ 8.5%) acquired a bivalent chromatin state and 391 (~ 3.5%) lost the mark. Of the 2512 genes associated with H3K27me3, 112 genes (~ 4.5%) switched to H3K4me3 marks. Of the 2336 bivalent genes, 788 (34%) turned into H3K4me3 only, whereas 272 (~ 11.5%) retained only the H3K27me3 mark. In summary, close to half of the repressed (H3K27me3, 44%) and poised (bivalent, 49%) genes underwent a change in their epigenetic marks, whereas only a minority of active genes (H3K4me3, 13%) underwent epigenetic modifications in TG1-miR. Altogether, these results show that the repressive effects of miR-302-367 are accompanied by changes in the chromatin state of a subset of genes while the proportional repartition of each histone mark across the genome is conserved.

Changes in histone modifications highlights ARNT2 as a core member of a transcription factor network associated with maintenance of GBM stem-like cell properties
To identify the function of the genes whose chromatin state is modified following repression of the properties of GBM stem-like cells, we performed functional enrichment analysis using DAVID toolbox [14,15]. In a first step, we performed a gene ontology (GO) analysis using the whole set of the 5151 genes associated with different histone modifications in TG1 and TG1-miR (Fig. 2c, Online Resource 5). Several terms related to the central nervous system were significantly enriched as expected for cells derived from the brain. Consistent with the drastic change in cell functional state induced by the miR-302-367 cluster [22], we also found terms grouping genes located at the core of cell behavior (such as transcription, metabolism), and related to development and differentiation. We also found categories associated with cell motility (cell adhesion, differentiation, and chemotaxis) consistent with the propensity of TG1-miR to adhere to a permissive plastic support and with the loss of their invasive capacity [22]. Functional enrichment analysis restricted to genes that permute from an active to a repressive histone mark showed enrichments in terms related to the maintenance of the undifferentiated features of the cells (Notch and Wnt signaling pathways, negative regulation of neuron differentiation, Fig. 2d, Online Resource 5). Conversely, genes that changed from the repressive H3K27me3 to the active H3K4me3 mark showed enrichments in terms related to neural cell differentiation (Fig. 2e, Online Resource 5).
TG1 cells overexpressing miR-302-367 cluster exhibit a drastic change in their functional state, from tumorigenic cells with stem cell-like features to non-tumorigenic cells lacking stem-like properties [22]. To further decipher the molecular basis of this phenotypic change, we focused our analysis on transcription factors, which are likely to play a pivotal role in driving the changes in functional state. Genes encoding transcription factors were retrieved using the KEGG (http://www.genome.jp/kegg/), and Genomatix databases (Genomatix, Germany). Interactions between the set of 202 transcription factors retrieved from the 5151 genes presenting a change in epigenetic mark (Online Resource 6) were then analyzed using the STRING software (http:// string-db.org/ version 10.0, [69]. Only interactions with a high confidence level (0.7) were selected. Addition of three supplementary transcription factors, which were associated with the active H3K4me3 mark in both TG1 and TG1-miR (hypoxia inducible factor 1α/HIF1A, hypoxia inducible 1 3 factor 2α/EPAS1 and beta-catenin/CTNNB1), allowed optimization of the modeling of a network including a maximal number of elements. This analysis yielded a densely connected network gathering 91 transcription factors whose genes are associated with changing histone modifications following expression of the miR-302-367 cluster ( Fig. 3a  and b). The network included five nodes grouping transcription factors not only involved in cancer, but also in stemness based on the source of information: known interactions from curated databases (light blue), known interactions experimentally determined (pink), co-expression (black), protein homology (purple) and text mining (gray). Note that HIF1A, EPAS1 (HIF2A) and CTNNB1 (beta-catenin) have the same histone mark (H3K4me3) in TG1 and TG1-miR 1 3 H3K27me3 mark, namely LEF1 and ARNT2. LEF1 is a key component of the Wnt/β-catenin signaling pathway, a pathway known to contribute to the maintenance of the properties of GBM stem-like cells [38,77,79]. ARNT2 is considered, like its paralog ARNT, as an accessory partner required for full transcriptional activity of several proteins including HIF1α, HIF2α, AHR, NPAS4, and SIM1 [17,29,61,66]. Analysis of mRNA levels showed that ARNT2 was the only transcription factor in the hypoxia pathway (ARNT, ARNT2, HIF1α/HIF1A, HIF2α/EPAS1, HIF3α/HIF3A, and HIF1α inhibitor/HIF1AN), to exhibit a drastic reduction of its mRNA levels in TG1-miR compared to TG1 (Fig. 4a, Online Resource 7). This finding was coherent with changes in histone modifications at the ARNT2 locus from H3K4me3 in TG1 to H3K27me3 in TG1-miR (Fig. 4b). We did not find miR-302-367 target sites within the ARNT2 mRNA (MIR-Base, http://www.mirbase.org/), indicating that decreased ARNT2 expression in TG1-miR does not stem from direct targeting by miR-302-367. Immunoblot analysis showed that reduced transcription of ARNT2 was associated with a decrease in ARNT2 protein levels (Fig. 4c). These results together with the scant information currently available on the role of ARNT2 in cancer, led us to investigate further the possible implication of ARNT2 in the regulation of glioblastoma cell properties.

ARNT2 is functionally associated with a molecular signature linked to glioblastoma cell tumorigenicity within the patients' tumors
We first analyzed ARNT2 expression in two published independent transcriptome datasets of glioblastoma cells either devoid of or endowed with tumor-initiating properties [41,73]. In agreement with our observations, we found that ARNT2 expression was downregulated in nontumorigenic cells compared to tumorigenic cells in both datasets (Fig. 4d, e). Further, the analysis of the TCGA transcriptome dataset of 481 surgical tissue samples of untreated primary glioblastoma using the GlioVis Platform [9] showed lower ARNT2 mRNA levels in glioblastoma tissues than in non-tumoral brain tissues (Online Resource 8A). The finding of higher ARNT2 mRNA levels in normal brain tissues than in GBM tissues from which neurons are absent is coherent with the high ARNT2 expression in mature neurons [18,19,32]. Analysis of the TCGA glioblastoma dataset and the French glioma dataset gse16011  Fig. 4 Decreased ARNT2 expression is associated with non-tumorigenic glioblastoma cells. a Decreased ARNT2 mRNA levels in TG1-miR compared to TG1. QPCR assay. **p < 0.01, unpaired t test with Welch's correction, mean ± SD, n = 3 independent biological samples. b Loss of the active H3K4me3 mark and gain of the repressive H3K27me3 mark around the ARNT2 transcription start site in TG1-miR. c Decreased ARNT2 protein levels in TG1-miR compared to TG1. Western blot analysis. **p < 0.01, unpaired t test with Welch's correction, mean ± SD, n = 3 independent biological samples. d Analysis of published transcriptome dataset of early passage (P3) glioblastoma cells isolated from four human tumors, either endowed with self-renewing and tumor-initiating properties or devoid of them following serum-treatment [41]. ***p < 0.001, unpaired t test with Welch's correction, mean ± SD, n = 4. e Analysis of the publicly available HGCC transcriptome dataset of glioblastoma cells isolated from distinct patients' tumors and characterized for their ability to initiate tumors [73]. *p < 0.05, unpaired t test with Welch's correction, mean ± SD, n = 15 (Tumorigenic), n = 9 (non-Tumorigenic) 1 3 [26], showed no variation in ARNT2 expression according to MGMT status, IDH1 mutation or EGFR amplification (not shown). In accordance with the narrow distribution of ARNT2 expression levels across glioblastoma samples (Online Resource 8A), no correlation could be disclosed using the R2 Genomics Analysis and Visualization Platform (http://r2.amc.nl) between variations in ARNT2 mRNA levels and the overall survival of patients (Online Resource 8B).
Glioblastoma are characterized by the intermingling of differing tumor tissues including dense tumor areas where cancer cells predominate ("cellular tumor areas"), necrotic and perinecrotic areas with sparser tumor cells, areas more or less angiogenic, and infiltrated areas where tumor cells are distributed through the brain parenchyma. To refine the analysis of ARNT2 and its family kin in glioblastoma, we explored its expression using the IVY dataset, which provides gene mRNA levels in distinct glioblastoma zones (http://glioblastoma.alleninstitute.org). The results of this analysis singled out ARNT2 among the other HIF family members. ARNT2 mRNA levels were higher in the cellular areas of the tumors than in the perinecrotic zones and barely detectable in tumor blood vessels. HIF3A expression was evenly distributed in the different tumor zones, while HIF1A, HIF2A, and ARNT expressions were enriched in the perinecrotic zones and/or in blood vessels of the tumor (Fig. 5a, Online Resource 9). Coherent with the profile of ARNT2 mRNA distribution across glioblastoma areas, immunohistochemical analysis of neurosurgical samples of patient's tumors revealed enrichment in ARNT2-expressing cells with increased distance from necrosis (Online Resource 9B). Importantly, this analysis revealed that ARNT2-expressing cells were especially enriched in the proliferative zones of the tumor (Fig. 5b).
To further explore the relevance of ARNT2 expression in the context of the human tumors, we compared its expression in the tumor core areas of glioblastomas (IVY dataset) with the expression of genes associated with glioblastoma cells endowed with tumorigenic and stem-like properties. We used a 28 molecules' signature delineated by the Bernstein laboratory from the combined analysis of single cell transcriptome profiles of cultured GBM stem-like cells and of 254 cells sampled from five different glioblastomas [55]. We retrieved expression data from 27 of the 28 components of the signature from the IVY dataset (Online Resource 10). Principal component analysis (PCA) showed that ARNT2 expression co-varied with the tumorigenic/stem signature along the first principal component axis (F1 axis) (Fig. 5c). We verified whether this co-variation occurred also at the single cell level using the published transcriptome profiles of 254 glioblastoma cells [55]. This dataset contains 25 of the 28 signature's components (Online Resource 10). Principal component analysis showed that ARNT2 expression co-varied also with the stem signature at the single cell level (Online Resource 11).
To probe the functional relevance of the co-variations disclosed by analysis of glioblastoma tissues and single cells transcriptomes, we determined the effect of ARNT2 knockdown on the expression of SOX9, POU3F2 and OLIG2 in independent GBM stem-like cell cultures (6240** and 5706**) distinct from TG1 and TG1-miR. These three genes were selected with respect to their previously demonstrated role in glioblastoma cell tumorigenicity [31,44,68]. An 80 to 90% decrease in ARNT2 mRNA level was observed in cells expressing shARNT2 (Fig. 6a, b, Online Resource 13). We observed decreased SOX9, POU3F2 and OLIG2 mRNA levels following ARNT2 knockdown using lentiviral transduction of small hairpin RNA (shControl or shARNT2, Fig. 6a, b, Online Resource 12). Similar results were obtained in TG1 cells expressing shARNT2 (Online Resource13). In addition, we verified that ARNT2, OLIG2, POU3F2 and SOX9 expressions did not change in conditions of reduced oxygen levels (Online Resource 14). Finally, we verified whether ARNT2 binding sites are present within OLIG2, POU3F2 and SOX9 regulatory regions by ChIP-qPCR experiments using ARNT2 antibodies. This analysis showed an enrichment in ARNT2 binding sites in one or more of the SOX9, POU3F2 and OLIG2 regulatory regions tested, compared to the house keeping gene TBP (Online Resource 15), indicating that OLIG2, POU3F2 and SOX9 can be directly regulated through ARNT2 binding to their regulatory regions. Altogether these results confirmed the functional relevance of the co-variation of ARNT2 expression with the tumorigenic/stem signature identified by tumor tissue and single cell transcriptome analysis. Our analysis further showed that ARNT2 down-regulation was accompanied by a decrease in LEF1 mRNA levels (Fig. 6a, b, Online Resource 13A), whereas the expression of the HIF family members HIF1A, HIF2A and ARNT varied from one cell line to another (Fig. 6a, b, Online Resource 13A).
Taken together, these results demonstrate that ARNT2 is expressed at the mRNA and protein level within the tumors of patients with glioblastoma. Further, they show that ARNT2 is part of a tumorigenic/stem signature of glioblastoma cells, and regulates the expression of transcription factors previously shown to be involved in the control of glioblastoma cell tumorigenicity.

ARNT2 is essential for the maintenance of glioblastoma cell tumorigenic properties
The above results, associated with our initial finding of ARNT2 down-regulation in glioblastoma cells deprived of tumorigenic properties, led us to evaluate the role of ARNT2 in the control of glioblastoma cell tumorigenicity using orthotopic xenografts of GBM stem-like cells (6240**, 1 3 13B and C). Of note, our observation of increased ARNT mRNA levels upon ARNT2 knockdown (Fig. 6a, b) indicates that ARNT cannot compensate for ARNT2 knockdown. Orthotopic xenografts of 1×, 2×, 4× or 14 × 10 4 6240** or 5706** GBM stem-like cells stably expressing luciferase and either shControl or shARNT2 were used to follow tumor development with bioluminescent imaging. Results showed a striking reduction in tumor incidence in mice grafted with 6240**-and 5706**-shARNT2 compared to mice grafted with 6240**-and 5706**-shCTL cells (Fig. 6f, i, Online Resource 16A-D). Bioluminescence imaging 42 days postgraft revealed tumor formation in six out of seven, and in six out of six mice engrafted with 14 × 10 4 6240**-shControl and 5706**-shControl, respectively (Fig. 6f, i). In contrast, no bioluminescent signal was detected in the mice grafted with 6240**-shARNT2 or 5706**-shARNT2 (Fig. 6f, i). Similar results were obtained when grafting smaller numbers of cells (Online Resource 13). The reduced tumor development in the mice grafted with 6240** and 5706**-shARNT2 was confirmed by immunohistochemistry (Online Resource 16E-F). Survival assays revealed differing long-term consequences of ARNT2 knockdown according to the GBM stem-like cells grafted. Although we observed a significant improvement in the survival of the mice grafted with either 6240** or 5706** cells expressing shARNT2 (Kaplan-Meier analysis, Fig. 6h, k), only mice grafted with 6240**-shARNT2 eventually developed tumors. Determination of human ARNT2 mRNA levels by QPCR in these tumors showed ARNT2 as well as OLIG2, POU3F2 and SOX9 transcripts levels similar to tumors of the shCTL group (Online Resource 16G). This result indicates that the 6240** cells that formed the tumors escaped ARNT2 inhibition, further pointing to an essential role of this transcription factor for glioblastoma cell aggressiveness. Taken together, these results show that ARNT2 participates in the control of the tumorigenicity of glioblastoma cells.

Discussion
Understanding the molecular basis of the varying functional cell states that co-exist within glioblastoma and participate in tumor resistance to treatments is of great importance to improve current therapeutic management. Differences in the ability of glioblastoma cells from the same tumor to initiate neoplasms has notably been highlighted by grafting cells sorted from glioblastoma surgical resections in immune-deficient mouse brains [35,58,73]. Recent studies have also shown the striking phenotypic plasticity of glioblastoma cells, which can adopt more or less aggressive states during the course of the tumor evolution and treatment [3,33]. Here, we identified changes in the chromatin state of transcription factors, which accompany the passage of GBM stem-like cells from a highly aggressive to a poorly tumorigenic state. We uncovered a novel transcription factor controlling glioblastoma cell tumorigenicity, which is localized at a node of a transcription factor network controlling glioblastoma cell aggressiveness, and which clusters with a tumorigenic/stem signature of glioblastoma cells at both tissue and single cell levels.
Using the human glioblastoma cell line TG1 expressing or not expressing the micro-RNA cluster miR-302-367 as a model system [22], we profiled histone modifications. The results of our analyses uncovered a subset of genes showing changes in H3K4me3 or H3K27me3 between TG1 cells and TG1-miR cells in which the stem-like and tumorigenic properties have been repressed by expression of the miR-302-367. In agreement with the previously reported association of miR-302-367 with differentiation of GBM stem-like cells [22], [23], ontological pathway analysis of the subset of genes with changes in histone marks showed enrichment in ontological gene groups related to development and engagement in differentiation pathways. Enrichments in terms related to nervous system were also obtained (neuron, neurogenesis, synapse, forebrain), indicating conservation in the tumor cells of an imprint of their tissue of origin. Retrieval of the 202 transcription factors undergoing a change in histone marks further highlighted molecular pathways already Fig. 6 ARNT2 down-regulation impairs tumor initiation and development. a, b Consequences of ARNT2 down-regulation on the expression of HIF family members (HIF1A, HIF2A, ARNT), on core components of the tumorigenic/stem signature of glioblastoma cells (OLIG2, SOX9, POU3F2), and on the effector of the Wnt signaling pathway, LEF1. QPCR assay. Results are presented as fold changes in mRNA levels detected in GBM stem-like cells expressing shARNT2 compared to shControl (shCTL). *p < 0.05, **p < 0.01, ***p < 0.001, unpaired t test with Welch's correction, mean ± SD, n = 3 independent biological samples. c Down-regulation of ARNT2 is accompanied with decreased cell proliferation. shARNT2 versus shControl. *p < 0.05, ***p < 0.001, unpaired t test with Welch's correction, mean ± SD, n = 3 independent biological samples. d, e Knocking-down of ARNT2 inhibits the sphere-forming capability of GBM stem-like cells. Extreme limiting dilution assays. Sphere formation was scored 7 days after seeding 6240** (d) and 5706** (e) GBM stem-like cells expressing shControl or shARNT2. The percentage of tumor incidence was monitored for 6 (6240**) and 8 (5706**) weeks. g, j Bioluminescent analyses of tumor growth initiated by grafting 6240** (g) and 5706** GBM stem-like cells transduced with a luciferase construct and either a shControl or a shARNT2 construct. 28 days post-graft. Quantification of the bioluminescent signals. Mean ± SD, n = 7 mice per group for 6240** (g) and n = 6 per group for 5706** (j). h, k Kaplan-Meier survival curves demonstrating a significant survival benefit of mice grafted with GBM stem-like cells expressing shARNT2 compared to mice grafted with GBM stem-like cell expressing shControl. 6240** shCTL and shARNT2, each n = 6 (h). 5706*** shCTL, n = 5, 5706** shARNT2, n = 4 (k). Log-rank Mantel-Cox test ◂ 1 3 identified as important players in the regulation of neural stem/progenitor cell but also of ESC and GBM stem-like cell behaviors, illustrating the pertinence of mapping histone epigenetic marks for identifying regulators of glioblastoma cell properties. For example, we observed an increased H3K27me3 associated with the gene encoding Nanog, a key factor in ESC pluripotency, and which has also been implicated in the maintenance of GBM stem-like cell properties [22,52,75]. Similarly, changes were observed for LEF1, an effector of the Wnt signaling pathway known to be involved in neurogenesis [8] and maintenance of GBM stem-like cell [38,77,79], TCF3 and TCF7 that are transcriptional regulators of the Wnt pathway in neural stem cells and ESC [40,74] and the HES bHLH genes and FOXCs transcription factors implicated in the Notch signaling pathway, which is activated in neural stem cells and GBM stem-like cells [36,72].
Mapping known and predicted protein-protein interactions between transcription factors exhibiting changes in histone marks in GBM stem-like cell lacking tumorigenic properties generated a network articulated around ARNT2. ARNT2, like its paralog ARNT, is considered to act as a dimerization partner of HIF1/2α, the heterodimers triggering the expression of hypoxia-related genes [46,61]. ARNT2 expression is especially abundant in the central nervous system and kidney, while that of ARNT is ubiquitous [18,32]. In the central nervous system, ARNT2 mRNA and protein are enriched in neurons [18]. Although ARNT2/HIFs and ARNT/HIFs heterodimers are equally efficient to ensure neuronal responses to hypoxia [46], ARNT2 protein levels do not increase under hypoxic conditions unlike those of ARNT and HIF1/2α [42,47]. The role of ARNT2 in cancer is poorly explored. ARNT2 has been associated with increased as well as decreased growth of non-cerebral cancers [39,43,46,48,59,60]. In glioblastoma HIF1 and 2α, but not ARNT2, have been associated to adaptation of cancer cells to hypoxic conditions [30,42]. We found that the profile of ARNT2 expression in glioblastoma does not correspond with that expected for a hypoxia-related molecule. ARNT2 expression was highest in glioblastoma core zones rather than in the hypoxic necrotic and pseudopalisading zones. This observation favors a hypoxia-independent transcriptional role for ARNT2.
Of note, ARNT2 is one of the few transcription factors switching from the active H3K4me3 to the repressive H3K27me3 mark in GBM stem-like cells expressing miR-302-367. Analysis of publically available data sets indicated that down-regulation of ARNT2 mRNA occurs not only in the TG1-miR cell line, but importantly is also observed in non-tumorigenic glioblastoma cells either directly sorted from patients' tumors [73] or following serum-induced differentiation of GBM stem-like cell [41]. This was confirmed at the protein level by immunohistochemistry of ARNT2 expression in sub-populations of cells within proliferative zones of patients' glioblastoma. Furthermore, we demonstrated that ARNT2 knockdown inhibits tumor-initiating properties in vivo, supporting a role of ARNT2 in the tumorigenicity of glioblastoma cells.
Examination of tumor patients' transcriptome datasets further associated ARNT2 with a tumorigenic/stem signature of glioblastoma cells [55] at both the tissue and single cell levels. To ascertain the functional relevance of this association, we focused on three transcription factors members of this stem signature SOX9, POU3F2 and OLIG2, since the knockdown of these factors has previously been reported to inhibit glioblastoma cell tumorigenicity in vivo [31,44,68]. We found that ARNT2 knockdown not only impaired the cell tumorigenicity in vivo but also resulted in decreased expression of SOX9, POU3F2 and OLIG2, hence placing ARNT2 at the core of transcriptional regulations of glioblastoma cell tumorigenicity.
In conclusion, our results uncover a novel transcription factor essential for glioblastoma cell tumorigenic properties, show its functional relevance within the context of the patients' tumor, and shed new lights on the combinatorial organization of the transcription factor networks that regulate glioblastoma cell aggressiveness.

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