Surgical specimens of GBM tumors were collected at Massachusetts General Hospital with approval by the Institutional Review Board. Mechanically minced tissue was triturated and then cells were grown as gliomaspheres in serum-free neural stem cell medium [Neurobasal medium (Invitrogen) supplemented with 3 mmol/L L-glutamine (Mediatech), 1X B27 supplement (Invitrogen), 0.5X N2 supplement (Invitrogen), 2 ug/mL heparin (Sigma), 20 ng/mL recombinant human EGF (R & D systems), 20 ng/mL recombinant human FGF2 (R & D systems), and 0.5X penicillin G/streptomycin sulfate], as previously described (Wakimoto et al., 2009
). Genomic copy number alterations and tumor xenograft histopathology for these CSCs have been described (Wakimoto et al., 2011
). From the same tumors, traditional GBM cells lines, grown as adherent monolayer in DMEM 10% FCS were derived as previously described (Wakimoto et al., 2009
). GBM CSC differentiation was induced using 5% FCS and withdrawal of growth factors for 7 days on poly-L-ornithin and laminin coated plates (see below for details). Staining was performed for nestin (Santa Cruz, 1: 400), MAP-2 (Chemicon, 1: 150), and GFAP (Sigma, 1: 400).
Human ES-derived neural stem (NS) cells generated from H9 ES cells were obtained from Millipore and grown and passaged in neural stem-cell medium consisting of a 1:1 mix of DMEM/F12:Neurobasal (Invitrogen), 0.5X N2 (Invitrogen), 0.5X B27 (Invitrogen), 1X Glutamax, and 0.1 mM beta-mercaptoethanol, which was supplemented with 20 ng/ul of both EGF and FGF-2 (R&D). NS cells were grown on poly-L-ornithin and laminin coated plates. Poly-L-ornithine/laminin plates were generated as such: A 20 ug/ml solution of poly-L-ornithine (Sigma) in water was added to plates and plates were incubated at 37°C for 1 hour. The poly-L-ornithine solution was then removed, plates were washed three times with PBS, and then 5 ul/ml solution of laminin (Sigma) in PBS was added to plates and plates were incubated at 37°C for at least 3 hours. Cells were passaged using manual dissociation.
For differentiation into astrocytes, when cells were 80-90% confluent, the media was changed to NS medium with 3% FCS and without EGF or FGF-2. After 4 days, cells were fixed for immunofluorescence. For differentiation into neurons, NS cells were grown to 90% confluency. Then, medium was changed to either NS medium but without N2 and supplemented with 1X B27 or ENStem-A Neuronal Differentiation Medium (Chemicon) supplemented with L-glutamine (2mM). After 2 weeks cells were fixed for immunofluorescence.
For immunofluorescence cells were washed 1X with PBS and then fixed with 4% paraformaldehyde for 30 minutes. Cells were washed 3X with PBS and blocked for 2 hours (5% normal goat serum, 0.3% Triton X-100, PBS). Then primary antibodies in blocking solution were added and cells were incubated overnight at 4°C. The next day, cells were washed twice with PBS and then 2X with blocking solution. Secondary antibody in blocking solution was added and cells were incubated for 2 hours at room temperature. Cells were then washed 3-5 times with PBS, then counterstained with DAPI/1XPBS solution. Primary antibodies include anti-nestin 1:500 (Chemicon Cat SCR060), anti-Sox2 1:200 (Chemicon Cat SCR019), anti-BLBP 1:500 (Chemicon Cat AB9558), anti-GFAP (Chemicon Cat SCR019), anti-betaIII tubulin 1:500 (Chemicon Cat SCR060), and anti-MAP2 1:200 (Chemicon SCR019). Secondary antibodies were Alexa Fluor 488 (goat anti-rabbit IgG, Invitrogen Cat A-11008) at 1:200 and Alexa Fluor 555 (goat anti-mouse IgG, Invitrogen Cat A-21422) at 1:200.
Normal human astrocytes (NHA) were obtained from Lonza and propagated according to the manufacturer's specifications.
CD133 (Miltenyi Biotec CD133/1 PE cat # 130-080-801 and SSEA-1 (BD Biosciences cat # 560127) antibodies were used according to manufacturer's instruction. For TF staining in primary tumor, primary human glioblastomas were obtained from patients operated at Massachusetts General Hospital in accordance with an approved IRB protocol (2005-P-001609/16). Briefly, tumors were dissociated to single cell suspension using a Brain Tumor Dissociation Kit (Miltenyi Biotec), depleted for CD45-positive immune cells using microbeads and a MACS separator (Miltenyi Biotec). Cells were stained with SOX2 (R+D Systems) and ASCL1 (BD Pharmingen) antibodies conjugated to Alexa Fluor 647 or Alexa Fluor 488 using Alexa Fluor Protein Labeling kits (Invitrogen). Flow cytometric analysis was conducted with an LSR II flow cytometer (BD Biosciences) and analysis was performed with FlowJo software (Treestar).
ChIP assays were carried out on cultures of approximately 1 × 106
cells per sample and epitope, following the general procedures outlined in (Ku et al., 2008
; Mikkelsen et al., 2007
). Immunoprecipitation was performed using antibodies against H3K4me3 (Millipore 07-473), H3K27me3 (Millipore 07449), H3K36me3 (Abcam 9050), H3K4me1 (Abcam 8895), ASCL1 (Epitomics T091) or LEF1 (Abcam 53293). ChIP DNA samples were used to prepare sequencing libraries, which were then sequenced on the Illumina Genome Analyzer II or HiSeq by standard procedures. We sequenced an input control for each cell type for use in normalization. Read alignment to the hg19 human reference genome was performed with Bowtie (Langmead et al., 2009
) and density maps were generated with read extension to 200 bp with IGVtools (Robinson et al., 2011
; Thorvaldsdottir et al., 2013
). When several reads with same start position and direction were present, only one was kept for further analysis. Two replicates that were available for MGG8 GBM CSCs were merged into a single track. Visualization was performed with IGV. ChIP-Seq dataset statistics are summarized in Table S1
and data are available for viewing at http://www.broadinstitute.org/cgi-bin/epigenomics/public/prod/cloneportal.cgi?data=rheinbay_GBMCSC
DNA methylation assay and analysis
For each sample, about 1 × 106 cells were harvested and genomic DNA was isolated using the QiaAMP DNA mini kit following manufacturer's instructions. DNA was eluted in 100 uL water, treated with RNase (37°C for 30 mins), cleaned up again with the QiaAMP DNA mini kit.
Data were processed using the Illumina BeadStudio software. Probes with p-value calls above 0.05 were discarded and β values for two replicates for each sample were averaged. Probes with β≥0.75 were classified as “hypermethylated”. Data are available through GEO (GSE46016).
Detection of regions enriched for histone modifications
Genomic regions enriched for a histone mark were identified using a sliding window approach as previously described (Mikkelsen et al., 2007
) with several modifications. We adapted the previous approach for the highly copy-number variant genomes of the GBM CSCs with the help of an unenriched input sequencing track generated from whole-cell extract (WCE). In short, a fixed size window of 1 kb was used to scan the genome in 100 bp steps for local enrichment of ChIP signal. Significance of signal in each window was assessed based on the assumption that random read alignment would follow a Poisson distribution with parameter λChIP
was adjusted for local variation in genome copy number by multiplication with the observed-to-expected ratio (O/E) for unenriched input reads in this region (Mikkelsen et al., 2010). To increase numeric stability in regions of heterozygous deletions, we calculated this O/E ratio based on input reads in the scoring window as well as in a 10 kb and a 100 kb region centered at the current window, and used the maximal value of these three. When all three input O/E ratios were zero, λadjusted
was set to equal λChIP
. Poisson p-values were then calculated for each window with λadjusted
. P-values were corrected for multiple-hypothesis testing using the Benjamini-Hochberg procedure (Benjamini and Hochberg, 1995
) and only windows with significance p<10−5
were kept. Finally, adjacent (distance ≤1kb) enriched windows were merged into a single interval. For histone modifications in NS and NHA cells, we applied the same algorithm and parameters albeit without background correction. Genomic regions enriched for ASCL1 or LEF1 were identified with MACS (Zhang et al., 2008
) No background correction as described above was applied; instead, peaks identified in the WCE track served to remove spurious TF peaks.
Transcription start sites (TSS) for genes from the hg19 human genome assembly were defined as region from 500 bp upstream to 2 kb downstream as previously described (Mikkelsen et al., 2007
). Chromatin state calls for transcription start sites were then made based on a minimal overlap of 500 bp of enriched intervals with the 2.5 kb TSS region. A consensus set of TSS chromatin states in the CSCs was generated with the chromatin state of the majority (≥3 out of 4 CSC lines) assigned to summarized “CSC” cell type. 87% (n=20,422) of TSS satisfied the majority criterion and were thus included in the consensus set.
Functional gene enrichment analysis
Functional enrichment in gene sets was determined using the DAVID functional annotation tool with “FAT” GeneOntology terms (Dennis et al., 2003
; version 6.7). Benjamini-Hochberg p-values correcting for multiple hypothesis testing were used for further interpretation.
Generation of aberrantly active TF set
TFs were identified as those aberrantly active genes that were contained in the GO:0003700 transcription factor activity set or in the set of human TFs defined in (Vaquerizas et al., 2009
). We manually removed TFs whose chromatin state was incorrectly of ambiguously called by our algorithm to generate the final list of TFs. MYCN
was also removed because of focal amplification in the MGG8 cell line. For the TF chromatin state heatmap, we extracted H3K4me3 and H3K27me3 signal, respectively, in 40 bins covering a 5 kb region around the annotated TSS from density maps. For both H3K4me3 and H3K27me3, several control genes with similar chromatin state in all samples were chosen (Table S4
) and served to scale signal for each sample. TFs were ordered based on their H3K27me3 signal using hierarchical clustering (R, 2008
), and H3K4me3 and H3K27me3 maps were overlayed to generate the final map. Cells exceeding 15% of maximum signal for H3K4me3 and H3K27me3 (“bivalent”) were additionally enhanced with orange color.
RNA extraction and gene expression analyses
Total RNA was isolated from cells using Trizol Reagent (Invitrogen) and purified using the RNeasy Kit (Qiagen). Gene expression data were acquired with Affymetrix Human Genome U133 2.0 Arrays. CEL files were normalized with RMA and multiple probe sets per gene were collapsed by taking the maximum expression value using the GenePattern package (Reich et al., 2006
). Gene expression data for NHA was included from Balani et al., 2009
. (GSE12305). Normal brain and astrocytoma transcriptome profiles were used from (Sun et al., 2006
; GSE4290) and processed as described above. TCGA combined expression data and subtype information was obtained from (Verhaak et al., 2010
We used the HOMER software package (Heinz et al., 2010
) to search for de novo
enriched motifs in TF peak regions.
Overexpression and knockdown experiments
Human cDNA for ASCL1, OLIG1, OLIG2, HEY2, LHX2
were cloned into a lentiviral plasmid and sequence verified. Primers used are listed in Table S6
. For knockdown experiments, ASCL1
lentiviral shRNA set from Open Biosystems was used (RHS4533-NM_004316) of which TRCN0000013551 (CCCGAACTGATGCGCTGCAAA) yielded sufficient knockdown. The same sequence was also used in vector pGIPZ (RHS4430-101103529) to allow for GFP sorting. Lentiviruses were produced as previously described (Barde et al., 2010
). Briefly, cDNA coding or shRNA plasmids were cotransfected with GAG/POL
plasmids into 293T packaging cells to produce the virus used to infect the target cells (NHA or GBM CSC). Viral supernatant was concentrated by ultracentrifugation using an SW41Ti rotor (Beckman Coulter) at 28,000 rpm for 120 min. Using GFP control, efficiency of infection was estimated as greater than 90% (data not shown). For maximal homogeneity, NHA were selected using 0.75 ug/ml puromycin for 5 days and GBM CSC were either selected using 2 ug/ml puromycin or sorted for GFP depending on vector used. After selection, RNA was extracted (Qiagen RNeasy kit) following manufacturer's instructions.
Real-time quantitative reverse transcriptase-PCR
For gene expression assays, cDNA was obtained using Moloney murine leukemia virus reverse transcriptase and RNase H minus (Promega). Typically, 250 ng of template total RNA and 250 ng of random hexamers were used per reaction. Real-time PCR amplification was performed using Power SYBR mix and specific PCR primers, in a 7500 Fast PCR instrument (Applied Biosystems). Relative quantification of each target, normalized to an endogenous control (GAPDH), was performed using the comparative Ct method (Applied Biosystems). Error bars indicate standard error of the mean. Primer sequences are listed in Table S6
TOPFLASH-Firefly and FOPFLASH-Renilla plasmids were co-transfected with ASCL1
lentivirus or control vector in 293T cells using Fugene6, as previously described (Firestein et al., 2008
; Veeman et al., 2003
). When indicated, Wnt3a was added at 100ng/ml (R&D 5036-WN-010). Luciferase activity was measured after 48 hours using a Dual-Luciferase Reporter assay System (Promega E1910) according to manufacturer's instructions.
GFP-sorted GBM CSC spheres, infected either with lentiviral control vector or with ASCL1 shRNA vector, were mechanically dissociated into single cells and plate at a density of one cell per well in 96 well plates, in triplicate. Sphere number was assessed 2 weeks later under a fluorescence microscope. For sphere diameter quantification, five pictures were taken per condition at 100x magnification. At least 60 spheres per conditions were measured with Image J.
Intracranial injections of 5000 cells from acutely dissociated gliomaspheres were performed with a stereotactic apparatus (Kopf Instruments) at coordinates 2.2 mm lateral relative to Bregma point and 2.5 mm deep from dura mater. Four severe combined immunodeficient (SCID) mice were used per condition. Kaplan-Meier curves and statistical significance (log-rank test) were calculated with the R survival package (R, 2008
). Animal experiments were approved by protocol number 2009N000061.
RNA in-situ hybridization (ISH)
mRNA was detected in FFPE (formalin-fixed, paraffin-embedded) tissue sections using Quantigene ViewRNA (Affymetrix, CA). Probes for ASCL1 (type 1, red, VA1-11908, Affymetrix, CA) and Sox2 (type 6, blue, VA-11765) were used following the manufacturer's instructions for two-color chromogenic ISH. Tissue sections were prepared for hybridization by fixation in 10% formaldehyde, deparaffinization, boiling for 10 minutes, and digestion with protease for 20 minutes. Hybridization was performed for 2 hours at 40°C. Signal amplification and detection were performed using standard conditions for Fast Red and Fast Blue substrates. Tissues were counterstained with hematoxylin and visualized with a standard bright field microscope.