While the methylation of DNA in 5′ promoters suppresses gene expression, the role of DNA methylation in gene bodies is unclear1–5. In mammals, tissue- and cell type-specific methylation is present in a small percentage of 5′ CpG island (CGI) promoters, while a far greater proportion occurs across gene bodies, coinciding with highly conserved sequences5–10. Tissue-specific intragenic methylation might reduce,3 or, paradoxically, enhance transcription elongation efficiency1,2,4,5. Capped analysis of gene expression (CAGE) experiments also indicate that transcription commonly initiates within and between genes11–15. To investigate the role of intragenic methylation, we generated a map of DNA methylation from 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 revealed to be in intragenic and intergenic regions, while 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 promoters16. The general and CpG-island-specific patterns of methylation are conserved in mouse tissues. An in-depth investigation of the human SHANK3 locus17,18 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.
Intragenic DNA methylation; alternate promoters; comparative epigenomics; SHANK3
Diffuse gliomas consist of both low- and high-grade varieties, each with distinct morphological and biological features. The often extended periods of relative indolence exhibited by low-grade gliomas (LGG; WHO grade II) differ sharply from the aggressive, rapidly fatal clinical course of primary glioblastoma (GBM; WHO grade IV). Nevertheless, until recently, the molecular foundations underlying this stark biological contrast between glioma variants remained largely unknown. The discoveries of distinctive and highly recurrent genomic and epigenomic abnormalities in LGG have both informed a more accurate classification scheme and pointed to viable avenues for therapeutic development. As such, the field of neuro-oncology now seems poised to capitalize on these gains to achieve significant benefit for LGG patients. This report will briefly recount the proceedings of a workshop held in January 2013 and hosted by Accelerate Brain Cancer Cure (ABC2) on the subject of LGG. While much of the meeting covered recent insights into LGG biology, its focus remained on how best to advance the clinical management, whether by improved preclinical modeling, more effective targeted therapeutics and clinical trial design, or innovative imaging technology.
clinical trials; genomics; low-grade glioma; personalized medicine
Somatic mutations in cancer are more frequent in heterochromatic and late-replicating regions of the genome. We report that regional disparities in mutation density are virtually abolished within transcriptionally silent genomic regions of cutaneous squamous cell carcinomas (cSCCs) arising in an XPC−/− background. XPC−/− cells lack global genome nucleotide excision repair (GG-NER), thus establishing differential access of DNA repair machinery within chromatin-rich regions of the genome as the primary cause for the regional disparity. Strikingly, we find that increasing levels of transcription reduce mutation prevalence on both strands of gene bodies embedded within H3K9me3-dense regions, and only to those levels observed in H3K9me3-sparse regions, also in an XPC-dependent manner. Therefore, transcription appears to reduce mutation prevalence specifically by relieving the constraints imposed by chromatin structure on DNA repair. We model this novel relationship between transcription, chromatin state and DNA repair, revealing a new, personalized determinant of cancer risk.
HOXA genes encode critical transcriptional regulators of embryonic development that have been implicated in cancer. In this study, we documented functional relevance and mechanism of activation of HOXA9 in glioblastoma (GBM), the most common malignant brain tumor. Expression of HOXA genes was investigated using RT-PCR in primary gliomas and glioblastoma cell lines and was validated in two sets of expression array data. In a subset of GBM, HOXA genes are aberrantly activated within confined chromosomal domains. Transcriptional activation of the HOXA cluster was reversible by a PI3K inhibitor through an epigenetic mechanism involving histone H3K27 trimethylation. Functional studies of HOXA9 showed its capacity to decrease apoptosis and increase cellular proliferation along with TRAIL resistance. Notably, aberrant expression of HOXA9 was independently predictive of shorter overall and progression-free survival in two GBM patient sets, and improved survival prediction by MGMT promoter methylation. Thus, HOXA9 activation is a novel, independent and negative prognostic marker in GBM that is reversible through a PI3K-associated epigenetic mechanism. Our findings suggest a transcriptional pathway through which PI3K activates oncogenic HOXA expression with implications for mTOR or PI3K targeted therapies.
glioblastoma; prognosis; PI3K; HOXA; MGMT
Both genetic and epigenetic mechanisms contribute to meningioma development by altering gene expression and protein function. To determine the relative contribution of each mechanism to meningioma development, we used an integrative approach measuring copy number and DNA methylation changes genomewide. We found that genetic alterations affected 1.9%, 7.4%, and 13.3% of the 691 loci studied, whereas epigenetic mechanisms affected 5.4%, 9.9%, and 10.3% of these loci in grade I, II, and III meningiomas, respectively. Genetic and epigenetic mechanisms rarely involved the same locus in any given tumor. The predilection for epigenetic rather than genetic silencing was exemplified at the 5′ CpG island of WNK2, a serine-threonine kinase gene on chromosome 9q22.31. WNK2 is known to negatively regulate epidermal growth factor receptor signaling via inhibition of MEK1 (mitogen-activated protein kinase kinase 1), and point mutations have been reported in WNK1, WNK2, WNK3, and WNK4. In meningiomas, WNK2 was aberrantly methylated in 83% and 71% of grade II and III meningiomas, respectively, but rarely in a total of 209 tumors from 13 other tumor types. Aberrant methylation of the CpG island was associated with decreased expression in primary tumors. WNK2 could be reactivated with a methylation inhibitor in IOMM-Lee, a meningioma cell line with a densely methylated WNK2 CpG island and lack of WNK2 expression. Expression of exogenous WNK2 inhibited colony formation, implicating it as a potential cell growth suppressor. These findings indicate that epigenetic mechanisms are common across meningiomas of all grades and that for specific genes such as WNK2, epigenetic alteration may be the dominant, grade-specific mechanism of gene inactivation.
epigenetic; genetic; meningioma; restriction landmark genome scanning; WNK2
Accurate detection of epimutations in tumor cells is crucial for understanding the molecular pathogenesis of cancer. Alterations in DNA methylation in cancer are functionally important and clinically relevant, but even this well-studied area is continually re-evaluated in light of unanticipated results, including a strong connection between aberrant DNA methylation in adult tumors and polycomb group profiles in embryonic stem cells, cancer-associated genetic mutations in epigenetic regulators such as DNMT3A and TET family genes, and the discovery of abundant 5-hydroxymethylcytosine, a product of TET proteins acting on 5-methylcytosine, in human tissues. The abundance and distribution of covalent histone modifications in primary cancer tissues relative to normal cells is a largely uncharted area, although there is good evidence for a mechanistic role of cancer-specific alterations in epigenetic marks in tumor etiology, drug response and tumor progression. Meanwhile, the discovery of new epigenetic marks continues, and there are many useful methods for epigenome analysis applicable to primary tumor samples, in addition to cancer cell lines. For DNA methylation and hydroxymethylation, next-generation sequencing allows increasingly inexpensive and quantitative whole-genome profiling. Similarly, the refinement and maturation of chromatin immunoprecipitation with next-generation sequencing (ChIP-seq) has made possible genome-wide mapping of histone modifications, open chromatin and transcription factor binding sites. Computational tools have been developed apace with these epigenome methods to better enable the accuracy and interpretation of the data from the profiling methods.
cancer epigenetics; DNA Methylation; MeDIP; Microarray; RRBS; Shotgun Bisulfite Sequencing; MRE; ChIP-seq
Activated STAT3 and increased expression of the histone methyltransferase EZH2 are independently associated with the most malignant subset of gliomas. In this issue of Cancer Cell, Kim and colleagues discover that EZH2 enhances STAT3 activation by trimethylatinglysine 180 in STAT3 and does so preferentially in glioma stem-like cells.
Epigenetic mechanisms involving DNA methylation, histone modifications and noncoding RNAs regulate and maintain gene-expression states. Similar to genetic mutations, alterations in epigenetic regulation can lead to uncontrolled cell division, tumor initiation and growth, invasiveness and metastasis. Research in brain cancer, particularly gliomas, has uncovered global and gene-specific DNA hypomethylation, local DNA hypermethylation of gene promoters and the de-regulation of microRNA expression. Understanding epigenetic dysregulation in brain cancers has provided new tools for prognostication, as well as suggesting new approaches to therapy. There is significant interest in new sequencing-based technologies that map genetic and epigenetic alterations comprehensively and at high resolution. These methods are being applied to brain tumors, and will better define the contribution of epigenetic defects to tumorigenesis.
DNA methylation; epigenetics; glioma; histone deacetylase; histone modification; microRNA; SAHA/vorinostat
We have extended our understanding of the molecular biology underlying adult glioblastoma over many years. In contrast, high-grade gliomas in children and adolescents have remained a relatively under-investigated disease. The latest large-scale genomic and epigenomic profiling studies have yielded an unprecedented abundance of novel data and revealed deeper insights into gliomagenesis across all age groups, highlighting key distinctions, but also some commonalities. As we are on the verge of dissecting glioblastomas into meaningful biological subgroups, this Review summarizes the hallmark genetic alterations associated with distinct epigenetic features and patient characteristics in both paediatric and adult disease, and examines the complex interplay between the glioblastoma genome and epigenome.
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 exomes 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, suggesting 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 and AKT-mTOR 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 suggest that there may be important differences between them. We performed a systematic comparison of human iPS cells generated from hepatocytes (representative of endoderm), skin fibroblasts (mesoderm) and melanocytes (ectoderm). All low passage iPS cells analyzed 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 reduces the efficiency of human iPS cell generation, suggesting that somatic memory genes may be functionally relevant during reprogramming.
Gliomas arise through genetic and epigenetic alterations of normal brain cells, though the exact cell of origin for each glioma subtype is unknown. The alteration-induced changes in gene expression and protein function allow uncontrolled cell division, tumor expansion and infiltration into surrounding normal brain parenchyma. The genetic and epigenetic alterations are tumor subtype and tumor-grade specific. Particular alterations predict tumor aggressiveness, tumor response to therapy and patient survival. Genetic alterations include deletion, gain, amplification, mutation and translocation which result in oncogene activation and tumor suppressor gene inactivation, or in some instances the alterations may simply be a consequence of tumorigenesis. Epigenetic alterations in brain tumors include CpG island hypermethylation associated with tumor suppressor gene silencing, gene-specific hypomethylation associated with aberrant gene activation, and genome-wide hypomethylation potentially leading to loss of imprinting, chromosomal instability and cellular hyperproliferation. Other epigenetic alterations, such as changes in the position of histone variants and changes in histone modifications are also likely important in the molecular pathology of brain tumors. Given that histone deacetylases are targets for drugs that are already in clinical trial, surprisingly little is known about histone acetylation in primary brain tumors. While a majority of epigenetic alterations are independent of genetic alterations, there is interaction on specific genes, signaling pathways and within chromosomal domains. Next-generation sequencing technology is now the method of choice for genomic and epigenome profiling, allowing more comprehensive understanding of genetic and epigenetic contributions to tumorigenesis in the brain.
Genomics; Epigenomics; Gliomas; Methylation; Acetylation
Transposable element (TE) derived sequences comprise half of our genome and DNA methylome, and are presumed densely methylated and inactive. Examination of the genome-wide DNA methylation status within 928 TE subfamilies in human embryonic and adult tissues revealed unexpected tissue-specific and subfamily-specific hypomethylation signatures. Genes proximal to tissue-specific hypomethylated TE sequences were enriched for functions important for the tissue type and their expression correlated strongly with hypomethylation of the TEs. When hypomethylated, these TE sequences gained tissue-specific enhancer marks including H3K4me1 and occupancy by p300, and a majority exhibited enhancer activity in reporter gene assays. Many such TEs also harbored binding sites for transcription factors that are important for tissue-specific functions and exhibited evidence for evolutionary selection. These data suggest that sequences derived from TEs may be responsible for wiring tissue type-specific regulatory networks, and have acquired tissue-specific epigenetic regulation.
Primary triple negative breast cancers (TNBC) represent approximately 16% of all breast cancers1 and are a tumour type defined by exclusion, for which comprehensive landscapes of somatic mutation have not been determined. Here we show in 104 early TNBC cases, that at the time of diagnosis these cancers exhibit a wide and continuous spectrum of genomic evolution, with some exhibiting only a handful of somatic aberrations in a few pathways, whereas others contain hundreds of somatic events and multiple pathways implicated. Integration with matched whole transcriptome sequence data revealed that only ~36% of mutations are expressed. By examining single nucleotide variant (SNV) allelic abundance derived from deep re-sequencing (median >20,000 fold) measurements in 2414 somatic mutations, we determine for the first time in an epithelial tumour, the relative abundance of clonal genotypes among cases in the population. We show that TNBC vary widely and continuously in their clonal frequencies at the time of diagnosis, with basal subtype TNBC2,3 exhibiting more variation than non-basal TNBC. Although p53 and PIK3CA/PTEN somatic mutations appear clonally dominant compared with other pathways, in some tumours their clonal frequencies are incompatible with founder status. Mutations in cytoskeletal and cell shape/motility proteins occurred at lower clonal frequencies, suggesting they occurred later during tumour progression. Taken together our results show that future attempts to dissect the biology and therapeutic responses of TNBC will require the determination of individual tumour clonal genotypes.
Human organic cation transporter 3 (OCT3, SLC22A3) mediates the uptake of many important endogenous amines and basic drugs in a variety of tissues. OCT3 is identified as one of the important risk loci for prostate cancer and is markedly under-expressed in aggressive prostate cancers. The goal of this study was to identify genetic and epigenetic factors in the promoter region that influence the expression level of OCT3. Haplotypes that contained the common variants, g.-81G>delGA (rs60515630) (minor allele frequency (MAF) 11.5% in African American) and g.-2G>A (rs555754) (MAF>30% in all ethnic groups) showed significant increases in luciferase reporter activities and exhibited stronger transcription factor binding affinity than the haplotypes that contained the major alleles. Consistent with the reporter assays, OCT3 mRNA expression levels were significantly higher in Asian (P<0.001) and Caucasian (P<0.05) liver samples from individuals who were homozygous for g.-2A/A in comparison with those homozygous for the g.-2G/G allele. Studies revealed that the methylation level in the basal promoter region of OCT3 was associated with OCT3 expression level and tumorigenesis capability in various prostate cancer cell lines. The methylation level of the OCT3 promoter was higher in 62% of prostate tumor samples compared with matched normal samples. Our studies demonstrate that genetic polymorphisms in the proximal promoter region of OCT3 alter the transcription rate of the gene and may be associated with altered expression levels of OCT3 in human liver. Aberrant methylation contributes to the reduced expression of OCT3 in prostate cancer.
SLC22A3; Polymorphism; Methylation and Prostate Cancer
This open-label, single-arm, phase II study combined enzastaurin with temozolomide plus radiation therapy (RT) to treat glioblastoma multiforme (GBM) and gliosarcoma. Adults with newly diagnosed disease and Karnofsky performance status (KPS) ≥ 60 were enrolled. Treatment was started within 5 weeks after surgical diagnosis. RT consisted of 60 Gy over 6 weeks. Temozolomide was given at 75 mg/m2 daily during RT and then adjuvantly at 200 mg/m2 daily for 5 days, followed by a 23-day break. Enzastaurin was given once daily during RT and in the adjuvant period at 250 mg/day. Cycles were 28 days. The primary end point was overall survival (OS). Progression-free survival (PFS), toxicity, and correlations between efficacy and molecular markers analyzed from tumor tissue samples were also evaluated. A prospectively planned analysis compared OS and PFS of the current trial with outcomes from 3 historical phase II trials that combined novel agents with temozolomide plus RT in patients with GBM or gliosarcoma. Sixty-six patients were enrolled. The treatment regimen was well tolerated. OS (median, 74 weeks) and PFS (median, 36 weeks) results from the current trial were comparable to those from a prior phase II study using erlotininb and were significantly better than those from 2 other previous studies that used thalidomide or cis-retinoic acid, all in combination with temozolomide plus RT. A positive correlation between O-6-methylguanine-DNA methyltransferase promoter methylation and OS was observed. Adjusting for age and KPS, no other biomarker was associated with survival outcome. Correlation of relevant biomarkers with OS may be useful in future trials.
adjuvant therapy; enzastaurin; glioblastoma multiforme; radiation therapy; temozolomide
To address the association between sequence variants within the MGMT promoter-enhancer region and methylation of MGMT in premalignant lesions from smokers and lung adenocarcinomas, their biological effects on gene regulation, and targeting MGMT for therapy.
SNPs identified through sequencing a 1.9kb fragment 5' of MGMT were examined in relation to MGMT methylation in 169 lung adenocarcinomas and 1731 sputum samples from smokers. The effect of promoter haplotypes on MGMT expression was tested using a luciferase reporter assay and cDNA expression analysis along with allele-specific sequencing for methylation. The response of MGMT methylated lung cancer cell lines to the alkylating agent temozolomide was assessed.
The A allele of rs16906252 and the haplotype containing this SNP were strongly associated with increased risk for MGMT methylation in adenocarcinomas (ORs ≥ 94). This association was observed to a lesser extent in sputum samples in both smoker cohorts. The A allele was selectively methylated in primary lung tumors and cell lines heterozygous for rs16906252. With the most common haplotype as the reference, a 20–41% reduction in promoter activity was seen for the haplotype carrying the A allele that correlated with lower MGMT expression. The sensitivity of lung cancer cell lines to temozolamide was strongly correlated with levels of MGMT methylation and expression.
These studies provide strong evidence that the A allele of a MGMT promoter-enhancer SNP is a key determinant for MGMT methylation in lung carcinogenesis. Moreover, temozolamide treatment may benefit a subset of lung cancer patients methylated for MGMT.
MGMT; allele specific methylation; single nucleotide polymorphism; sputum; lung cancer
Sequencing-based DNA methylation profiling methods are comprehensive and, as accuracy and affordability improve, will increasingly supplant microarrays for genome-scale analyses. Here, four sequencing-based methodologies were applied to biological replicates of human embryonic stem cells to compare their CpG coverage genome-wide and in transposons, resolution, cost, concordance and its relationship with CpG density and genomic context. The two bisulfite methods reached concordance of 82% for CpG methylation levels and 99% for non-CpG cytosine methylation levels. Using binary methylation calls, two enrichment methods were 99% concordant, while regions assessed by all four methods were 97% concordant. To achieve comprehensive methylome coverage while reducing cost, an approach integrating two complementary methods was examined. The integrative methylome profile along with histone methylation, RNA, and SNP profiles derived from the sequence reads allowed genome-wide assessment of allele-specific epigenetic states, identifying most known imprinted regions and new loci with monoallelic epigenetic marks and monoallelic expression.
DNA methylation; Sequencing; Bisulfite
The H3K9me3 histone modification is often found at promoter regions, where it functions to repress transcription. However, we have previously shown that 3′ exons of zinc finger genes (ZNFs) are marked by high levels of H3K9me3. We have now further investigated this unusual location for H3K9me3 in ZNF genes. Neither bioinformatic nor experimental approaches support the hypothesis that the 3′ exons of ZNFs are promoters. We further characterized the histone modifications at the 3′ ZNF exons and found that these regions also contain H3K36me3, a mark of transcriptional elongation. A genome-wide analysis of ChIP-seq data revealed that ZNFs constitute the majority of genes that have high levels of both H3K9me3 and H3K36me3. These results suggested the possibility that the ZNF genes may be imprinted, with one allele transcribed and one allele repressed. To test the hypothesis that the contradictory modifications are due to imprinting, we used a SNP analysis of RNA-seq data to demonstrate that both alleles of certain ZNF genes having H3K9me3 and H3K36me3 are transcribed. We next analyzed isolated ZNF 3′ exons using stably integrated episomes. We found that although the H3K36me3 mark was lost when the 3′ ZNF exon was removed from its natural genomic location, the isolated ZNF 3′ exons retained the H3K9me3 mark. Thus, the H3K9me3 mark at ZNF 3′ exons does not impede transcription and it is regulated independently of the H3K36me3 mark. Finally, we demonstrate a strong relationship between the number of tandemly repeated domains in the 3′ exons and the H3K9me3 mark. We suggest that the H3K9me3 at ZNF 3′ exons may function to protect the genome from inappropriate recombination rather than to regulate transcription.
The haploid human genome contains approximately 29 million CpGs that exist in a methylated, hydroxymethylated or unmethylated state, collectively referred to as the DNA methylome. The methylation status of cytosines in CpGs and occasionally in non-CpG cytosines influences protein–DNA interactions, gene expression, and chromatin structure and stability. The degree of DNA methylation at particular loci may be heritable transgenerationally and may be altered by environmental exposures and diet, potentially contributing to the development of human diseases. For the vast majority of normal and disease methylomes however, less than 1% of the CpGs have been assessed, revealing the formative stage of methylation mapping techniques. Thus, there is significant discovery potential in new genome-scale platforms applied to methylome mapping, particularly oligonucleotide arrays and the transformative technology of next-generation sequencing. Here, we outline the currently used methylation detection reagents and their application to microarray and sequencing platforms. A comparison of the emerging methods is presented, highlighting their degrees of technical complexity, methylome coverage and precision in resolving methylation. Because there are hundreds of unique methylomes to map within one individual and interindividual variation is likely to be significant, international coordination is essential to standardize methylome platforms and to create a full repository of methylome maps from tissues and unique cell types.
DNA methylation; MeDIP; methylation-sensitive restriction enzyme; microarray; MRE; next-generation sequencing; reduced representation bisulfite sequencing; RRBS
Much recent effort has focused on identifying and characterizing cellular markers that distinguish tumor propagating cells (TPCs) from more differentiated progeny. We report here an unusual promoter DNA methylation pattern for one such marker, the cell surface antigen CD133 (Prominin 1). This protein has been extensively used to enrich putative cancer propagating stem-like cell populations in epithelial tumors, and especially, glioblastomas. We find that, within individual cell lines of cultured colon cancers and glioblastomas, the promoter CpG island of CD133 is DNA methylated, primarily, in cells with absent or low expression of the marker protein whereas lack of such methylation is evident in purely CD133+ cells. Differential histone modification marks of active versus repressed genes accompany these DNA methylation changes. This heterogeneous CpG island DNA methylation status in the tumors is unusual in that other DNA hypermethylated genes tested in such cultures preserve their methylation patterns between separated CD133+ and CD133− cell populations. Furthermore, the CD133 DNA methylation seems to constitute an abnormal promoter signature since it is not found in normal brain and colon but only in cultured and primary tumors. Thus, the DNA methylation is imposed on the transition between the active versus repressed transcription state for CD133 only in tumors. Our findings provide additional insight for the dynamics of aberrant DNA methylation associated with aberrant gene silencing in human tumors.
CD133; DNA methylation; tumor propagating cells (TPC); histone modifications; cancer