Reactivation of telomerase reverse transcriptase (TERT) expression enables cells to overcome replicative senescence and escape apoptosis, fundamental steps in the initiation of human cancer. Multiple cancer types, including up to 83% of glioblastomas (GBM), harbor highly recurrent TERT promoter mutations of unknown function but specific to two nucleotide positions. We identify the functional consequence of these mutations in GBM to be recruitment of the multimeric 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 ETS motifs critically cooperate with these mutations to activate TERT, likely by facilitating GABP heterotetramer binding. GABP thus directly links TERT promoter mutations to aberrant expression in multiple cancers.

Summary 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.

Telomerase (TERT) activation is fundamental step in tumorigenesis. By maintaining telomere length, telomerase relieves a main barrier on cellular lifespan, enabling limitless proliferation driven by oncogenes. The recently discovered, highly recurrent mutations in the promoter of TERT are found in over 50 cancer types, and are the most common mutation in many cancers. Transcriptional activation of TERT, via promoter mutation or other mechanisms, is the rate-limiting step in production of active telomerase. While TERT is expressed in stem cells, it is naturally silenced upon differentiation. Thus, the presence of TERT promoter mutations may shed light on whether a particular tumor arose from a stem cell or more differentiated cell type. It is becoming clear that TERT mutations occur early during cellular transformation, and activate the TERT promoter by recruiting transcription factors that do not normally regulate TERT gene expression. This review highlights the fundamental and widespread role of TERT promoter mutations in tumorigenesis, including recent progress on their mechanism of transcriptional activation. These somatic promoter mutations, along with germline variation in the TERT locus also appear to have significant value as biomarkers of patient outcome. Understanding the precise molecular mechanism of TERT activation by promoter mutation and germline variation may inspire novel cancer cell-specific targeted therapies for a large number of cancer patients.

The majority of common variants associated with common diseases, as well as an unknown proportion of causal mutations for rare diseases, fall in noncoding regions of the genome. Although catalogs of noncoding regulatory elements are steadily improving, we have a limited understanding of the functional effects of mutations within them. Here, we perform saturation mutagenesis in conjunction with massively parallel reporter assays on 20 disease-associated gene promoters and enhancers, generating functional measurements for over 30,000 single nucleotide substitutions and deletions. We find that the density of putative transcription factor binding sites varies widely between regulatory elements, as does the extent to which evolutionary conservation or integrative scores predict functional effects. These data provide a powerful resource for interpreting the pathogenicity of clinically observed mutations in these disease-associated regulatory elements, and comprise a rich dataset for the further development of algorithms that aim to predict the regulatory effects of noncoding mutations.

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 role of intermediate methylation states in DNA is unclear. Here, to comprehensively identify regions of intermediate methylation and their quantitative relationship with gene activity, we apply integrative and comparative epigenomics to 25 human primary cell and tissue samples. We report 18,452 intermediate methylation regions located near 36% of genes and enriched at enhancers, exons, and DNase I hypersensitivity sites. Intermediate methylation regions average 57% methylation, are predominantly allele-independent, and are conserved across individuals and between mouse and human, suggesting a conserved function. At enhancers, these regions have an intermediate level of active chromatin marks and their associated genes have intermediate transcriptional activity. Exonic intermediate methylation correlates with exon inclusion at the level between that of fully methylated and unmethylated exons, highlighting gene context-dependent functions. We conclude that intermediate DNA methylation is a conserved signature of gene regulation and exon usage.

Mutations in the isocitrate dehydrogenase gene IDH1 are common in lower-grade glioma where they result in the production of 2-hydroxyglutarate (2HG), disrupted patterns of histone methylation and gliomagenesis. IDH1 mutations also co-segregate with mutations in the ATRX gene and the TERT promoter, suggesting that IDH mutation may drive the creation or selection of telomere-stabilizing events as part of immortalization/transformation process. To determine if and how this may occur, we investigated the phenotype of pRb/p53-deficient human astrocytes engineered with IDH1 wild-type (WT) or R132H mutant (IDH1mut) genes as they progressed through their lifespan. IDH1mut expression promoted 2HG production and altered histone methylation within 20 population doublings (PD), but had no effect on telomerase expression or telomere length. Accordingly, cells expressing either IDH1 WT or IDH1mut entered a telomere-induced crisis at PD 70. In contrast, only IDH1mut cells emerged from crisis, grew indefinitely in culture and formed colonies in soft agar and tumors in vivo. Clonal populations of post-crisis IDH1mut cells displayed shared genetic alterations, but no mutations in ATRX or the TERT promoter were detected. Instead, these cells reactivated telomerase and stabilized their telomeres in association with increased histone lysine methylation (H3K4me3) and c-Myc/Max binding at the TERT promoter. Overall, these results show that while IDH1mut does not create or select for ATRX or TERT promoter mutations, it can indirectly reactivate TERT, and in doing so contribute to astrocytic immortalization and transformation.

Studies of coat color mutants have greatly contributed to the discovery of genes that regulate melanocyte development and function. Here, we generated Yy1 conditional knockout mice in the melanocyte-lineage and observed profound melanocyte deficiency and premature gray hair, similar to the loss of melanocytes in human piebaldism and Waardenburg syndrome. Although YY1 is a ubiquitous transcription factor, YY1 interacts with M-MITF, the Waardenburg Syndrome IIA gene and a master transcriptional regulator of melanocytes. YY1 cooperates with M-MITF in regulating the expression of piebaldism gene KIT and multiple additional pigmentation genes. Moreover, ChIP–seq identified genome-wide YY1 targets in the melanocyte lineage. These studies mechanistically link genes implicated in human conditions of melanocyte deficiency and reveal how a ubiquitous factor (YY1) gains lineage-specific functions by co-regulating gene expression with a lineage-restricted factor (M-MITF)—a general mechanism which may confer tissue-specific gene expression in multiple lineages.