Genotypes and phenotypes of analyzed brains from GFAP-SB11 crosses.

a<p>Abbreviations used: GFAP-SB = GFAP-SB11 transposase, T2 = T2/onc, p19 = p19Arf, wt = wild-type.</p><p>Genotypes and phenotypes of analyzed brains from GFAP-SB11 crosses.</p

The mutational, copy number, methylation and mRNA expression status of gCIS/CIS human orthologs in TCGA GBM data.

a<p>For <i>Kpna4/Gm1647</i> data presented are for <i>KPNA4</i> as <i>Gm1647</i> does not have a human ortholog.</p>b<p>Abbreviations used: N/A = not applicable (no human ortholog, or data not available), ns = non-significant, fc = fold change.</p>c<p>The percentage of tumors harboring hypermethylation and evidence for decreased mRNA expression (epigenetic silencing) are shown in parentheses.</p><p>The mutational, copy number, methylation and mRNA expression status of gCIS/CIS human orthologs in TCGA GBM data.</p

<i>Sleeping Beauty</i> Mouse Models Identify Candidate Genes Involved in Gliomagenesis

<div><p>Genomic studies of human high-grade gliomas have discovered known and candidate tumor drivers. Studies in both cell culture and mouse models have complemented these approaches and have identified additional genes and processes important for gliomagenesis. Previously, we found that mobilization of <i>Sleeping Beauty</i> transposons in mice ubiquitously throughout the body from the <i>Rosa26</i> locus led to gliomagenesis with low penetrance. Here we report the characterization of mice in which transposons are mobilized in the Glial Fibrillary Acidic Protein (GFAP) compartment. Glioma formation in these mice did not occur on an otherwise wild-type genetic background, but rare gliomas were observed when mobilization occurred in a <i>p19Arf</i> heterozygous background. Through cloning insertions from additional gliomas generated by transposon mobilization in the <i>Rosa26</i> compartment, several candidate glioma genes were identified. Comparisons to genetic, epigenetic and mRNA expression data from human gliomas implicates several of these genes as tumor suppressor genes and oncogenes in human glioblastoma.</p></div

FLI1 is expressed in a subset of cells in a glioma with a <i>Fli1</i> insertion.

<p>Immunoreactivity for FLI1 is in brown and immunoreactivity for IBA1 is in green. Nuclei are counterstained blue. A) 40× image of the dorsal third ventricular region in a control mouse without mobilizing transposons. B) 100× image of the boxed area in A. Asterisk indicates a FLI1 immunoreactive cell with morphologic features of a red blood cell. C) 40× image of the dorsal third ventricular region surrounded by tumor in AR151. D) 100× image of the boxed area in C. Arrowhead points to a nucleus that is negative for FLI1 and an arrow indicates an example of strong nuclear FLI1 staining. E) 100× image of tumor in AR151 that is distant from the ventricle. F) A 40× image of secondary only controls is shown for comparison to verify specific primary antibody staining. Scale bars = 50 µm.</p

CISs and gCISs identified in SB-gliomas.

a<p>Abbreviations used: GB = GenBank, N/A = not applicable, p value = p value for gCIS.</p><p>CISs and gCISs identified in SB-gliomas.</p

Genotypes and phenotypes of analyzed brains from Rosa26-SB11 crosses.

a<p>Abbreviations used: T2LC = T2/onc LC, T2ATG = T2/oncATG, p19 = p19Arf, wt = wild-type, AA = anaplastic astrocytoma, GBM = glioblastoma, PNET = primitive neuroectodermal tumor, DD = differential diagnosis.</p><p>Genotypes and phenotypes of analyzed brains from Rosa26-SB11 crosses.</p

GFAP-SB11 transgenics express functional transposase in a subset of GFAP⁺ cells.

<p>Two lines (A and B) were established and used for these experiments. SB = SB transposase, T2 = T2/onc, LV = lateral ventricle. A) Immunofluorescence for GFAP (red) and SB (green). Nuclei are stained with DAPI. Arrows indicate examples of SB⁺ GFAP⁺ cells while asterisks indicate examples of SB⁻ GFAP⁺ cells. Scale bars are 20 µm. B) PCR based excision assay showing that transposons have mobilized in the brains of SB⁺T2⁺ but not SB⁻T2⁺ or SB⁺T2⁻ mice from each line. A control PCR demonstrates that genomic DNA is present for all samples.</p

Genome-wide methylomic and transcriptomic analyses identify subtype-specific epigenetic signatures commonly dysregulated in glioma stem cells and glioblastoma

<p>Glioma stem cells (GSCs), a subpopulation of tumor cells, contribute to tumor heterogeneity and therapy resistance. Gene expression profiling classified glioblastoma (GBM) and GSCs into four transcriptomically-defined subtypes. Here, we determined the DNA methylation signatures in transcriptomically pre-classified GSC and GBM bulk tumors subtypes. We hypothesized that these DNA methylation signatures correlate with gene expression and are uniquely associated either with only GSCs or only GBM bulk tumors. Additional methylation signatures may be commonly associated with both GSCs and GBM bulk tumors, i.e., common to non-stem-like and stem-like tumor cell populations and correlating with the clinical prognosis of glioma patients. We analyzed Illumina 450K methylation array and expression data from a panel of 23 patient-derived GSCs. We referenced these results with The Cancer Genome Atlas (TCGA) GBM datasets to generate methylomic and transcriptomic signatures for GSCs and GBM bulk tumors of each transcriptomically pre-defined tumor subtype. Survival analyses were carried out for these signature genes using publicly available datasets, including from TCGA. We report that DNA methylation signatures in proneural and mesenchymal tumor subtypes are either unique to GSCs, unique to GBM bulk tumors, or common to both. Further, dysregulated DNA methylation correlates with gene expression and clinical prognoses. Additionally, many previously identified transcriptionally-regulated markers are also dysregulated due to DNA methylation. The subtype-specific DNA methylation signatures described in this study could be useful for refining GBM sub-classification, improving prognostic accuracy, and making therapeutic decisions.</p