Reactivation of telomerase reverse transcriptase (TERT) expression enables cells to overcome replicative senescence and escape apoptosis, which are fundamental steps in the initiation of human ...cancer. Multiple cancer types, including up to 83% of glioblastomas (GBMs), harbor highly recurrent TERT promoter mutations of unknown function but specific to two nucleotide positions. We identified the functional consequence of these mutations in GBMs to be recruitment of the multimeric GA-binding protein (GABP) transcription factor specifically to the mutant promoter. Allelic recruitment of GABP is consistently observed across four cancer types, highlighting a shared mechanism underlying TERT reactivation. Tandem flanking native E26 transformation-specific motifs critically cooperate with these mutations to activate TERT, probably by facilitating GABP heterotetramer binding. GABP thus directly links TERT promoter mutations to aberrant expression in multiple cancers.
Tumor recurrence is a leading cause of cancer mortality. Therapies for recurrent disease may fail, at least in part, because the genomic alterations driving the growth of recurrences are distinct ...from those in the initial tumor. To explore this hypothesis, we sequenced the exornes of 23 initial low-grade gliomas and recurrent tumors resected from the same patients. In 43% of cases, at least half of the mutations in the initial tumor were undetected at recurrence, including driver mutations in TP53, ATRX, SMARCA4, and BRAF; this suggests that recurrent tumors are often seeded by cells derived from the initial tumor at a very early stage of their evolution. Notably, tumors from 6 of 10 patients treated with the chemotherapeutic drug temozolomide (TMZ) followed an alternative evolutionary path to high-grade glioma. At recurrence, these tumors were hypermutated and harbored driver mutations in the RB (retinoblastoma) and Akt-mTOR (mammalian target of rapamycin) pathways that bore the signature of TMZ-induced mutagenesis.
Human induced pluripotent stem (iPS) cells are remarkably similar to embryonic stem (ES) cells, but recent reports indicate that there may be important differences between them. We carried out a ...systematic comparison of human iPS cells generated from hepatocytes (representative of endoderm), skin fibroblasts (mesoderm) and melanocytes (ectoderm). All low-passage iPS cells analysed retain a transcriptional memory of the original cells. The persistent expression of somatic genes can be partially explained by incomplete promoter DNA methylation. This epigenetic mechanism underlies a robust form of memory that can be found in iPS cells generated by multiple laboratories using different methods, including RNA transfection. Incompletely silenced genes tend to be isolated from other genes that are repressed during reprogramming, indicating that recruitment of the silencing machinery may be inefficient at isolated genes. Knockdown of the incompletely reprogrammed gene C9orf64 (chromosome 9 open reading frame 64) reduces the efficiency of human iPS cell generation, indicating that somatic memory genes may be functionally relevant during reprogramming.
TERT promoter mutations reactivate telomerase, allowing for indefinite telomere maintenance and enabling cellular immortalization. These mutations specifically recruit the multimeric ETS factor GABP, ...which can form two functionally independent transcription factor species: a dimer or a tetramer. We show that genetic disruption of GABPβ1L (β1L), a tetramer-forming isoform of GABP that is dispensable for normal development, results in TERT silencing in a TERT promoter mutation-dependent manner. Reducing TERT expression by disrupting β1L culminates in telomere loss and cell death exclusively in TERT promoter mutant cells. Orthotopic xenografting of β1L-reduced, TERT promoter mutant glioblastoma cells rendered lower tumor burden and longer overall survival in mice. These results highlight the critical role of GABPβ1L in enabling immortality in TERT promoter mutant glioblastoma.
•The β1L tetramer-forming isoform of GABP activates the mutant TERT promoter•β1L disruption induces telomere loss and death only in TERT promoter mutant cells•Disruption of β1L reduces tumor growth and prolongs survival in xenografted mice•GABPβ1L is a potential therapeutic target for TERT promoter mutant glioblastoma
TERT promoter mutations generate a binding site for GABP and reactivate TERT expression. Mancini et al. show that GABPβ1L, among GABP subunits, is specifically required for the function of TERT promoter mutants, and disrupting GABPβ1L causes telomere loss and cell death exclusively in TERT promoter mutant cells.
Although it is known that the methylation of DNA in 5' promoters suppresses gene expression, the role of DNA methylation in gene bodies is unclear. In mammals, tissue- and cell type-specific ...methylation is present in a small percentage of 5' CpG island (CGI) promoters, whereas a far greater proportion occurs across gene bodies, coinciding with highly conserved sequences. Tissue-specific intragenic methylation might reduce, or, paradoxically, enhance transcription elongation efficiency. Capped analysis of gene expression (CAGE) experiments also indicate that transcription commonly initiates within and between genes. To investigate the role of intragenic methylation, we generated a map of DNA methylation from the human brain encompassing 24.7 million of the 28 million CpG sites. From the dense, high-resolution coverage of CpG islands, the majority of methylated CpG islands were shown to be in intragenic and intergenic regions, whereas less than 3% of CpG islands in 5' promoters were methylated. The CpG islands in all three locations overlapped with RNA markers of transcription initiation, and unmethylated CpG islands also overlapped significantly with trimethylation of H3K4, a histone modification enriched at promoters. The general and CpG-island-specific patterns of methylation are conserved in mouse tissues. An in-depth investigation of the human SHANK3 locus and its mouse homologue demonstrated that this tissue-specific DNA methylation regulates intragenic promoter activity in vitro and in vivo. These methylation-regulated, alternative transcripts are expressed in a tissue- and cell type-specific manner, and are expressed differentially within a single cell type from distinct brain regions. These results support a major role for intragenic methylation in regulating cell context-specific alternative promoters in gene bodies.
The evolutionary history of tumor cell populations can be reconstructed from patterns of genetic alterations. In contrast to stable genetic events, epigenetic states are reversible and sensitive to ...the microenvironment, prompting the question whether epigenetic information can similarly be used to discover tumor phylogeny. We examined the spatial and temporal dynamics of DNA methylation in a cohort of low-grade gliomas and their patient-matched recurrences. Genes transcriptionally upregulated through promoter hypomethylation during malignant progression to high-grade glioblastoma were enriched in cell cycle function, evolving in parallel with genetic alterations that deregulate the G1/S cell cycle checkpoint. Moreover, phyloepigenetic relationships robustly recapitulated phylogenetic patterns inferred from somatic mutations. These findings highlight widespread co-dependency of genetic and epigenetic events throughout brain tumor evolution.
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•Dramatic loss of DNA methylation during the progression of low-grade gliomas to GBM•Phenotypic convergence of genomic and epigenomic evolution on cell cycle defects•Phyloepigenetics recapitulates the phylogenetics of tumor evolution•Robust model of gliomagenesis from tumor initiation through malignant progression
Mazor et al. show that spatial and temporal patterns of DNA methylation and somatic mutations during the progression of low-grade gliomas to high-grade tumors produce remarkably similar evolutionary histories and both converge to deregulate the cell cycle.
Recent advancements in sequencing-based DNA methylation profiling methods provide an unprecedented opportunity to map complete DNA methylomes. These include whole-genome bisulfite sequencing (WGBS, ...MethylC-seq, or BS-seq), reduced-representation bisulfite sequencing (RRBS), and enrichment-based methods such as MeDIP-seq, MBD-seq, and MRE-seq. These methods yield largely comparable results but differ significantly in extent of genomic CpG coverage, resolution, quantitative accuracy, and cost, at least while using current algorithms to interrogate the data. None of these existing methods provides single-CpG resolution, comprehensive genome-wide coverage, and cost feasibility for a typical laboratory. We introduce methylCRF, a novel conditional random fields-based algorithm that integrates methylated DNA immunoprecipitation (MeDIP-seq) and methylation-sensitive restriction enzyme (MRE-seq) sequencing data to predict DNA methylation levels at single-CpG resolution. Our method is a combined computational and experimental strategy to produce DNA methylomes of all 28 million CpGs in the human genome for a fraction (<10%) of the cost of whole-genome bisulfite sequencing methods. methylCRF was benchmarked for accuracy against Infinium arrays, RRBS, WGBS sequencing, and locus-specific bisulfite sequencing performed on the same human embryonic stem cell line. methylCRF transformation of MeDIP-seq/MRE-seq was equivalent to a biological replicate of WGBS in quantification, coverage, and resolution. We used conventional bisulfite conversion, PCR, cloning, and sequencing to validate loci where our predictions do not agree with whole-genome bisulfite data, and in 11 out of 12 cases, methylCRF predictions of methylation level agree better with validated results than does whole-genome bisulfite sequencing. Therefore, methylCRF transformation of MeDIP-seq/MRE-seq data provides an accurate, inexpensive, and widely accessible strategy to create full DNA methylomes.
Temozolomide (TMZ) increases the overall survival of patients with glioblastoma (GBM), but its role in the clinical management of diffuse low-grade gliomas (LGG) is still being defined. DNA ...hypermethylation of the
O
6
-
methylguanine
-
DNA methyltransferase
(
MGMT)
promoter is associated with an improved response to TMZ treatment, while inactivation of the DNA mismatch repair (MMR) pathway is associated with therapeutic resistance and TMZ-induced mutagenesis. We previously demonstrated that TMZ treatment of LGG induces driver mutations in the RB and AKT–mTOR pathways, which may drive malignant progression to secondary GBM. To better understand the mechanisms underlying TMZ-induced mutagenesis and malignant progression, we explored the evolution of
MGMT
methylation and genetic alterations affecting MMR genes in a cohort of 34 treatment-naïve LGGs and their recurrences. Recurrences with TMZ-associated hypermutation had increased
MGMT
methylation compared to their untreated initial tumors and higher overall
MGMT
methylation compared to TMZ-treated non-hypermutated recurrences. A TMZ-associated mutation in one or more MMR genes was observed in five out of six TMZ-treated hypermutated recurrences. In two cases, pre-existing heterozygous deletions encompassing
MGMT
, or an MMR gene, were followed by TMZ-associated mutations in one of the genes of interest. These results suggest that tumor cells with methylated
MGMT
may undergo positive selection during TMZ treatment in the context of MMR deficiency.
Mutations in the TERT promoter represent the genetic underpinnings of tumor cell immortality. Beyond the two most common point mutations, which selectively recruit the ETS factor GABP to activate ...TERT, the significance of other variants is unknown. In seven cancer types, we identify duplications of wildtype sequence within the core promoter region of TERT that have strikingly similar features including an ETS motif, the duplication length and insertion site. The duplications recruit a GABP tetramer by virtue of the native ETS motif and its precisely spaced duplicated counterpart, activate the promoter and are clonal in a TERT expressing multifocal glioblastoma. We conclude that recurrent TERT promoter duplications are functionally and mechanistically equivalent to the hotspot mutations that confer tumor cell immortality. The shared mechanism of these divergent somatic genetic alterations suggests a strong selective pressure for recruitment of the GABP tetramer to activate TERT.
DNA methylation plays key roles in diverse biological processes such as X chromosome inactivation, transposable element repression, genomic imprinting, and tissue-specific gene expression. ...Sequencing-based DNA methylation profiling provides an unprecedented opportunity to map and compare complete DNA methylomes. This includes one of the most widely applied technologies for measuring DNA methylation: methylated DNA immunoprecipitation followed by sequencing (MeDIP-seq), coupled with a complementary method, methylation-sensitive restriction enzyme sequencing (MRE-seq). A computational approach that integrates data from these two different but complementary assays and predicts methylation differences between samples has been unavailable. Here, we present a novel integrative statistical framework M&M (for integration of MeDIP-seq and MRE-seq) that dynamically scales, normalizes, and combines MeDIP-seq and MRE-seq data to detect differentially methylated regions. Using sample-matched whole-genome bisulfite sequencing (WGBS) as a gold standard, we demonstrate superior accuracy and reproducibility of M&M compared to existing analytical methods for MeDIP-seq data alone. M&M leverages the complementary nature of MeDIP-seq and MRE-seq data to allow rapid comparative analysis between whole methylomes at a fraction of the cost of WGBS. Comprehensive analysis of nineteen human DNA methylomes with M&M reveals distinct DNA methylation patterns among different tissue types, cell types, and individuals, potentially underscoring divergent epigenetic regulation at different scales of phenotypic diversity. We find that differential DNA methylation at enhancer elements, with concurrent changes in histone modifications and transcription factor binding, is common at the cell, tissue, and individual levels, whereas promoter methylation is more prominent in reinforcing fundamental tissue identities.
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