The maintenance of key germline derived DNA methylation patterns during preimplantation development depends on stores of DNA cytosine methyltransferase-1o (DNMT1o) provided by the oocyte. Dnmt1omat−/− mouse embryos born to Dnmt1Δ1o/Δ1o female mice lack DNMT1o protein and have disrupted genomic imprinting and associated phenotypic abnormalities. Here, we describe additional female-specific morphological abnormalities and DNA hypomethylation defects outside imprinted loci, restricted to extraembryonic tissue. Compared to male offspring, the placentae of female offspring of Dnmt1Δ1o/Δ1o mothers displayed a higher incidence of genic and intergenic hypomethylation and more frequent and extreme placental dysmorphology. The majority of the affected loci were concentrated on the X chromosome and associated with aberrant biallelic expression, indicating that imprinted X-inactivation was perturbed. Hypomethylation of a key regulatory region of Xite within the X-inactivation center was present in female blastocysts shortly after the absence of methylation maintenance by DNMT1o at the 8-cell stage. The female preponderance of placental DNA hypomethylation associated with maternal DNMT1o deficiency provides evidence of additional roles beyond the maintenance of genomic imprints for DNA methylation events in the preimplantation embryo, including a role in imprinted X chromosome inactivation.
During oocyte growth and maturation, vital proteins and enzymes are produced to ensure that, when fertilized, a healthy embryo will arise. When this natural process is interrupted, one or more of these essential elements can fail to be produced thus compromising the health of the future embryo. We are using a mouse model, lacking an enzyme (DNMT1o) produced in the oocyte and only required post-fertilization in the early embryo for the maintenance of inherited DNA methylation marks. Here, we reveal that oocytes lacking DNMT1o, when fertilized, generated conceptuses with a wide variety of placental abnormalities. These placental abnormalities were more frequent and severe in females, and showed specific genomic regions constantly deprived of their normal methylation marks. The affected genomic regions were concentrated on the X chromosome. Interestingly, we also found that a region important for the regulation of the X chromosome inactivation process was hypomethylated in female blastocysts and was associated with sex-specific abnormalities in the placenta, relaxation of imprinted X chromosome inactivation, and disruption of DNA methylation later in development. Our findings provide a novel unanticipated role for DNA methylation events taking place within the first few days of life specifically in female preimplantation embryos.
Aberrant DNA methylation and concomitant transcriptional silencing of death-associated protein kinase 1 (DAPK1) have been demonstrated to be key pathogenic events in chronic lymphocytic leukemia (CLL). In acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS), however, the presence of elevated DNA methylation levels has been a matter of continued controversy. Several studies demonstrated highly variable frequencies of DAPK1 promoter methylation by the use of methylation-specific PCR (MSP). By quantitative high resolution assessment, we demonstrate that aberrant DNA methylation is an extremely rare event in this region. We observed elevated levels just in one out of 246 (0.4%) AML patients, all 42 MDS patients were unmethylated. In conclusion, we present a refined DAPK1 methylation analysis in a large representative patient cohort of AML and MDS patients proofing almost complete absence of elevated DNA methylation. Our results highlight the importance of quantitative measurements particularly for translational research questions on primary patient specimens.
Increased ZAP-70 expression predicts poor prognosis in chronic lymphocytic leukemia (CLL). Current methods for accurately measuring ZAP-70 expression are problematic, preventing widespread application of these tests in clinical decision making. We therefore used comprehensive DNA methylation profiling of the ZAP-70 regulatory region to identify sites important for transcriptional control.
Patients and Methods
High-resolution quantitative DNA methylation analysis of the entire ZAP-70 gene regulatory regions was conducted on 247 samples from patients with CLL from four independent clinical studies.
Through this comprehensive analysis, we identified a small area in the 5′ regulatory region of ZAP-70 that showed large variability in methylation in CLL samples but was universally methylated in normal B cells. High correlation with mRNA and protein expression, as well as activity in promoter reporter assays, revealed that within this differentially methylated region, a single CpG dinucleotide and neighboring nucleotides are particularly important in ZAP-70 transcriptional regulation. Furthermore, by using clustering approaches, we identified a prognostic role for this site in four independent data sets of patients with CLL using time to treatment, progression-free survival, and overall survival as clinical end points.
Comprehensive quantitative DNA methylation analysis of the ZAP-70 gene in CLL identified important regions responsible for transcriptional regulation. In addition, loss of methylation at a specific single CpG dinucleotide in the ZAP-70 5′ regulatory sequence is a highly predictive and reproducible biomarker of poor prognosis in this disease. This work demonstrates the feasibility of using quantitative specific ZAP-70 methylation analysis as a relevant clinically applicable prognostic test in CLL.
Assessment of DNA methylation has become a critical factor for the identification, development and application of methylation based biomarkers. Here we describe a systematic comparison of a quantitative high-resolution mass spectrometry-based approach (MassARRAY), pyrosequencing and the broadly used methylation-specific PCR (MSP) technique analyzing clinically relevant epigenetically silenced genes in acute myeloid leukemia (AML). By MassARRAY and pyrosequencing, we identified significant DNA methylation differences at the ID4 gene promoter and in the 5′ region of members of the SFRP gene family in 62 AML patients compared with healthy controls. We found a good correlation between data obtained by MassARRAY and pyrosequencing (correlation coefficient R2 = 0.88). MSP-based assessment of the identical samples showed less pronounced differences between AML patients and controls. By direct comparison of MSP-derived and MassARRAY-based methylation data as well as pyrosequencing, we could determine overestimation of DNA methylation data by MSP. We found sequence-context dependent highly variable cut-off values of quantitative DNA methylation values serving as discriminator for the two MSP methylation categories. Moreover, good agreements between quantitative methods and MSP could not be achieved for all investigated loci. Significant correlation of the quantitative assessment but not of MSP-derived methylation data with clinically important characteristics in our patient cohort demonstrated clinical relevance of quantitative DNA methylation assessment. Taken together, while MSP is still the most commonly applied technique for DNA methylation assessment, our data highlight advantages of quantitative approaches for precise characterization and reliable biomarker use of aberrant DNA methylation in primary patient samples, particularly.
ID4; SFRP; AML; MSP; MassARRAY; biomarker; pyrosequencing; quantitative DNA methylation analysis
High-risk types of human papilloma virus (HPV) are increasingly associated with oropharyngeal squamous cell carcinoma (OPSCC). Strikingly, patients with HPV-positive OPSCC are highly curable with ionizing radiation and have better survival compared with HPV-negative patients, but the underlying molecular mechanisms remain poorly understood. We applied an array-based approach to monitor global changes in CpG island hypermethylation between HPV-negative and HPV-positive OPSCCs and identified a specific pattern of differentially methylated regions that critically depends on the presence of viral transcripts. HPV-related alterations were confirmed for the majority of candidate gene promoters by mass spectrometric, quantitative methylation analysis. There was a significant inverse correlation between promoter hypermethylation of ALDH1A2, OSR2, GATA4, GRIA4, and IRX4 and transcript levels. Interestingly, Kaplan-Meier analysis revealed that a combined promoter methylation pattern of low methylation levels in ALDH1A2 and OSR2 promoters and high methylation levels in GATA4, GRIA4, and IRX4 promoters was significantly correlated with improved survival in 3 independent patient cohorts. ALDH1A2 protein levels, determined by immunohistochemistry on tissue microarrays, confirmed the association with clinical outcome. In summary, our study highlights specific alterations in global gene promoter methylation in HPV-driven OPSCCs and identifies a signature that predicts the clinical outcome in OPSCCs.
Non-coding RNAs are much more common than previously thought. However, for the vast majority of non-coding RNAs, the cellular function remains enigmatic. The two long non-coding RNA (lncRNA) genes DLEU1 and DLEU2 map to a critical region at chromosomal band 13q14.3 that is recurrently deleted in solid tumors and hematopoietic malignancies like chronic lymphocytic leukemia (CLL). While no point mutations have been found in the protein coding candidate genes at 13q14.3, they are deregulated in malignant cells, suggesting an epigenetic tumor suppressor mechanism. We therefore characterized the epigenetic makeup of 13q14.3 in CLL cells and found histone modifications by chromatin-immunoprecipitation (ChIP) that are associated with activated transcription and significant DNA-demethylation at the transcriptional start sites of DLEU1 and DLEU2 using 5 different semi-quantitative and quantitative methods (aPRIMES, BioCOBRA, MCIp, MassARRAY, and bisulfite sequencing). These epigenetic aberrations were correlated with transcriptional deregulation of the neighboring candidate tumor suppressor genes, suggesting a coregulation in cis of this gene cluster. We found that the 13q14.3 genes in addition to their previously known functions regulate NF-kB activity, which we could show after overexpression, siRNA–mediated knockdown, and dominant-negative mutant genes by using Western blots with previously undescribed antibodies, by a customized ELISA as well as by reporter assays. In addition, we performed an unbiased screen of 810 human miRNAs and identified the miR-15/16 family of genes at 13q14.3 as the strongest inducers of NF-kB activity. In summary, the tumor suppressor mechanism at 13q14.3 is a cluster of genes controlled by two lncRNA genes that are regulated by DNA-methylation and histone modifications and whose members all regulate NF-kB. Therefore, the tumor suppressor mechanism in 13q14.3 underlines the role both of epigenetic aberrations and of lncRNA genes in human tumorigenesis and is an example of colocalization of a functionally related gene cluster.
Recent results suggest that genome regions not coding for proteins are read and transcribed into RNA. While the function for the majority of the resulting non-coding RNA molecules remains unclear, some of them are termed according to their length (typically 200–2,000 nucleotides) as long non-coding RNA (lncRNA) genes that play a role in regulating the activity of target genes. In most instances, this deregulation involves changes of so-called “epigenetic” marks associated with the DNA that are inherited to the cellular progeny without changes in the DNA sequence. Here we describe an example where two lncRNA genes (DLEU1 and DLEU2) are epigenetically deregulated together with a cluster of neighboring protein-coding tumor suppressor genes in almost all patients suffering from chronic lymphocytic leukemia. Such a common regulation suggests that the affected genes are involved in the same cellular pathway. In line with this notion, the 13q14.3 genes modulate the NF-kB signalling pathway, either inducing or repressing its activity. An activation of NF-kB has previously been shown to promote survival of the leukemic cells, underlining the importance of the 13q14.3 tumor suppressor locus for the pathomechanism of the disease.
Dilated cardiomyopathies (DCM) show remarkable variability in their age of onset, phenotypic presentation, and clinical course. Hence, disease mechanisms must exist that modify the occurrence and progression of DCM, either by genetic or epigenetic factors that may interact with environmental stimuli. In the present study, we examined genome-wide cardiac DNA methylation in patients with idiopathic DCM and controls. We detected methylation differences in pathways related to heart disease, but also in genes with yet unknown function in DCM or heart failure, namely Lymphocyte antigen 75 (LY75), Tyrosine kinase-type cell surface receptor HER3 (ERBB3), Homeobox B13 (HOXB13) and Adenosine receptor A2A (ADORA2A). Mass-spectrometric analysis and bisulphite-sequencing enabled confirmation of the observed DNA methylation changes in independent cohorts. Aberrant DNA methylation in DCM patients was associated with significant changes in LY75 and ADORA2A mRNA expression, but not in ERBB3 and HOXB13. In vivo studies of orthologous ly75 and adora2a in zebrafish demonstrate a functional role of these genes in adaptive or maladaptive pathways in heart failure.
biomarker; dilated cardiomyopathy; DNA methylation; epigenetics; heart failure
We previously reported a rare germline variant (c.1-6531) that resulted in allele–specific expression (ASE) of death-associated protein kinase 1 (DAPK1) and predisposition to chronic lymphocytic leukemia (CLL). We investigated a cohort of CLL patients lacking this mutation for the presence of ASE of DAPK1. We developed a novel strategy that combines single-nucleotide primer extension (SNuPE) with MALDI-TOF mass spectrometry, and detected germline DAPK1 ASE in 17 out of 120 (14.2%) CLL patients associated with a trend towards younger age at diagnosis. ASE was absent in 63 healthy controls. Germline cells of CLL patients with ASE showed increased levels of DNA methylation in the promoter region, however, neither genetic nor further epigenetic aberrations could be identified in the DAPK1 5′ upstream regulatory region, within distinct exons or in the 3′-UTR. We identified B-lymphoid malignancy related cell line models harboring allelic imbalance and found that allele-specific methylation in DAPK1 is associated with ASE. Our data indicate that ASE at the DAPK1 gene locus is a recurrent event, mediated by epigenetic mechanisms and potentially predisposing to CLL.
Aberrant expression of the homeodomain transcription factor CDX2 occurs in most cases of acute myeloid leukemia (AML) and promotes leukemogenesis, making CDX2, in principle, an attractive therapeutic target. Conversely, CDX2 acts as a tumor suppressor in colonic epithelium. The effectors mediating the leukemogenic activity of CDX2 and the mechanism underlying its context-dependent properties are poorly characterized, and strategies for interfering with CDX2 function in AML remain elusive. We report data implicating repression of the transcription factor KLF4 as important for the oncogenic activity of CDX2, and demonstrate that CDX2 differentially regulates KLF4 in AML versus colon cancer cells through a mechanism that involves tissue-specific patterns of promoter binding and epigenetic modifications. Furthermore, we identified deregulation of the PPARγ signaling pathway as a feature of CDX2-associated AML and observed that PPARγ agonists derepressed KLF4 and were preferentially toxic to CDX2+ leukemic cells. These data delineate transcriptional programs associated with CDX2 expression in hematopoietic cells, provide insight into the antagonistic duality of CDX2 function in AML versus colon cancer, and suggest reactivation of KLF4 expression, through modulation of PPARγ signaling, as a therapeutic modality in a large proportion of AML patients.
Acute myeloid leukemia (AML) is a disease characterized by uncontrolled proliferation of clonal neoplastic hematopoietic precursor cells. This leads to the disruption of normal hematopoiesis and bone marrow failure. Major breakthroughs in the past have contributed to our understanding of the genetic failures and the changed biology in AML cells that underlie the initiation and progression of the disease. It is now recognized that not only genetic but also epigenetic alterations are similarly important in this process. Since these alterations do not change the DNA sequences and are pharmacologically reversible, they have been regarded as optimal targets for what is now known as epigenetic therapy. In this review, we will discuss our current understanding of normal epigenetic processes, outline our knowledge of epigenetic alterations in AML, and discuss how this information is being used to improve current therapy of this disease.
Metastatic melanoma is a fatal disease due to the lack of successful therapies and biomarkers for early detection and its incidence has been increasing. Genetic studies have defined recurrent chromosomal aberrations, suggesting the location of either tumor suppressor genes or oncogenes. Transcription factor 21 (TCF21) belongs to the class A of the basic helix-loop-helix family with reported functions in early lung and kidney development as well as tumor suppressor function in the malignancies of the lung and head and neck. In this study, we combined quantitative DNA methylation analysis in patient biopsies and in their derived cell lines to demonstrate that TCF21 expression is downregulated in metastatic melanoma by promoter hypermethylation and TCF21 promoter DNA methylation is correlated with decreased survival in metastatic skin melanoma patients. In addition, the chromosomal location of TCF21 on 6q23–q24 coincides with the location of a postulated metastasis suppressor in melanoma. Functionally, TCF21 binds the promoter of the melanoma metastasis-suppressing gene, KiSS1, and enhances its gene expression through interaction with E12, a TCF3 isoform and with TCF12. Loss of TCF21 expression results in loss of KISS1 expression through loss of direct interaction of TCF21 at the KISS1 promoter. Finally, overexpression of TCF21 inhibits motility of C8161 melanoma cells. These data suggest that epigenetic downregulation of TCF21 is functionally involved in melanoma progression and that it may serve as a biomarker for aggressive tumor behavior.
Previous observation has shown that the wild-type Kras2 allele is a suppressor of lung cancer in mice. Here we report that loss of heterozygosity (LOH) of chromosome 12p was detected in ~50% of human lung adenocarcinomas and large cell carcinomas, and Kras2 mutations were detected at codon 12 in ~40% of adenocarcinomas and large cell carcinomas. Interestingly, all of the lung adenocarcinomas and large cell carcinomas containing a Kras2 mutation exhibited allelic loss of the wild-type Kras2 allele when a correlation between LOH of the region on chromosome 12p and Kras2 mutation was made. These results from human lung cancer tissues provide a strong evidence in support of our previous observation in mouse models that the wild-type Kras2 is a tumor suppressor of lung cancer.
wild-type Kras2; mutations: lung cancer; loss of heterozygosity; tumor suppressor
A recent study reports that histone deacetylase (HDAC) inhibitors, AR42 and MS- 275, upregulated H3K4 methylation marks in prostate cancer cells, leading to transcriptional activation of genes including those associated with roles in tumor suppression and cell differentiation (1). Evidence suggests that the crosstalk between histone deacetylation and histone H3K4 methylation is attributable to the ability of these HDAC inhibitors to repress the JARID1 family of histone H3 lysine 4 demethylases (H3K4DMs), including RBP2, PLU-1, SMCX, and LSD1, through the downregulation of Sp1 expression. This demonstrates the complexity of the functional roles of HDACs in the regulation of histone modifications as well as the activation of epigenetically silenced gene expression. Equally important is the ability of HDAC inhibitors to transcriptionally suppress H3K4DM gene expression which has therapeutic implications, in that several H3K4DMs such as LSD1 and PLU-1 have been implicated in the pathogenesis of many types of malignancies.
Histone deacetylase; H3K4 methylation; H3K4 demethylases; Histone codes; Histone deacetylase inhibitors; AR42, MS-275
Epigenetic alterations in cancer include changes in DNA methylation and associated histone modifications that influence the chromatin states and impact gene expression patterns. Due to recent technological advantages, the scientific community is now obtaining a better picture of the genome-wide epigenetic changes that occurs in a cancer genome. These epigenetic alterations are associated with chromosomal instability and changes in transcriptional control which influence the overall gene expression differences seen in many human malignancies. In this review, we will briefly summarize our current knowledge of the epigenetic patterns and mechanisms of gene regulation in healthy tissues and relate this to what is known for cancer genomes. Our focus will be on DNA methylation. We will review the current standing of technologies that have been developed over recent years. This field is experiencing a revolution in the strategies used to measure epigenetic alterations, which includes the incorporation of next generation sequencing tools. We also will review strategies that utilize epigenetic information for translational purposes, with a special emphasis on the potential use of DNA methylation marks for early disease detection and prognosis. The review will close with an outlook on challenges that this field is facing.
The family of Toll-like receptors (TLRs) is critical in linking innate and acquired immunity. Polymorphisms in the genes encoding TLRs have been associated with autoimmune diseases and cancer. We investigated the genetic variation of TLR genes and its potential impact on melanoma susceptibility and patient survival. The study included 763 cutaneous melanoma cases recruited in Germany and 736 matched controls that were genotyped for 47 single nucleotide polymorphisms (SNPs) in 8 TLR genes. The relationship between genotype, disease status and survival was investigated taking into account patient and tumor characteristics, and melanoma treatment. Analysis of 7 SNPs in TLR2, 7 SNPs in TLR3 and 8 SNPs in TLR4 showed statistically significant differences in distribution of inferred haplotypes between cases and controls. No individual polymorphism was associated with disease susceptibility except for the observed tendency for TLR2-rs3804099 (odds ratio OR = 1.15, 95% CI 0.99–1.34, p = 0.07) and TLR4-rs2149356 (OR = 0.85, 95% CI 0.73–1.00, p = 0.06). Both polymorphisms were part of the haplotypes associated with risk modulation. An improved overall survival (Hazard ratio HR 0.53, 95% CI 0.32–0.88) and survival following metastasis (HR 0.55, 95% CI 0.34–0.91) were observed in carriers of the variant allele (D299G) of TLR4-rs4986790. In addition various TLR2, TLR4 and TLR5 haplotypes were associated with increased overall survival. Our results point to a novel association between TLR gene variants and haplotypes with melanoma survival. Our data suggest a role for the D299G polymorphism in the TLR4 gene in overall survival and a potential link with systemic treatment at stage IV of the disease. The polymorphic amino acid residue, located in the ectodomain of TLR4, can have functional consequences.
Although most CpG islands are generally thought to remain unmethylated in all adult somatic tissues, recent genome-wide approaches have found that some CpG islands have distinct methylation patterns in various tissues, with most differences being seen between germ cells and somatic tissues. Few studies have addressed this among human somatic tissues and fewer still have studied the same sets of tissues from multiple individuals. In the current study, we used Restriction Landmark Genomic Scanning to study tissue specific methylation patterns in a set of 12 human tissues collected from multiple individuals. We identified 34 differentially methylated CpG islands among these tissues, many of which showed consistent patterns in multiple individuals. Of particular interest were striking differences in CpG island methylation, not only among brain regions, but also between white and grey matter of the same region. These findings were confirmed for selected loci by quantitative bisulfite sequencing. Cluster analysis of the RLGS data indicated that several tissues clustered together, but the strongest clustering was in brain. Tissues from different brain regions clustered together, and, as a group, brain tissues were distinct from either mesoderm or endoderm derived tissues which demonstrated limited clustering. These data demonstrate consistent tissue specific methylation for certain CpG islands, with clear differences between white and grey matter of the brain. Furthermore, there was an overall pattern of tissue specifically methylated CpG islands that distinguished neural tissues from non-neural.
Tissue specific methylation; CpG island methylation; neural; brain tissue; grey matter; white matter
Targeting aberrant DNA hypermethylation in chronic lymphocytic leukemia (CLL) and non-Hodgkin’s lymphoma (NHL) with decitabine may reverse epigenetic silencing in B-cell malignancies.
Twenty patients were enrolled in two phase I trials to determine the minimum effective pharmacologic dose (MEPD) of decitabine in patients with relapsed/refractory CLL (n=16) and NHL (n=4).
Patients received 1–3 cycles of decitabine. Dose limiting toxicity (DLT) was observed in 2 of 4 CLL and 2 of 2 NHL patients receiving decitabine at 15 mg/m2/d days 1–10, consisting of grade 3–4 thrombocytopenia and hyperbilirubinemia. Six patients with CLL received decitabine at 10 mg/m2/d days 1–10 without DLT; however, re-expression of methylated genes or changes in global DNA methylation were not observed. Therefore, a 5-day decitabine schedule was examined. With 15 mg/m2/d decitabine days 1–5, DLT occurred in 2 of 6 CLL and 2 of 2 NHL patients, consisting of grade 3–4 neutropenia, thrombocytopenia, and febrile neutropenia. Eight patients had stable disease. In 17 patients, there were no significant changes in genome-wide methylation or in target gene re-expression.
Dose-limiting myelosuppression and infectious complications prevents dose escalation of decitabine to levels associated with changes in global methylation or gene re-expression in CLL and NHL.
decitabine; methylation; chronic lymphocytic leukemia; non-Hodgkin’s lymphoma
Epigenetic inactivation of genes by DNA hypermethylation plays an important role in carcinogenesis. An in vitro model of human breast epithelial cell transformation was used to study epigenetic changes induced by estradiol during the neoplastic process. Different stages of tumor initiation and progression are represented in this model being MCF-10F the normal stage; trMCF cells, the transformed stage; bsMCF cells, the invasive stage and, caMCF cells, the tumor stage. Global methylation studies by restriction landmark genomic scanning (RLGS) showed an increased DNA-methylation during the in the invasive and tumor stages. Expression studies showed that NRG1 (neuregulin 1), CSS3 (chondroitin sulfate synthase 3) and SNIP (SNAP-25-interacting protein) were downregulated in the invasive and tumor cells. The transformed cells showed low expression of STXBP6 (amysin) compared to the parental cells MCF-10F. The treatment of these cells with the demethylating agent 5-aza-dC alone or in combination with the histone deacetylase inhibitor trichostatin increased the expression of NRG1, STXBP6, CSS3 and SNIP confirming that DNA methylation plays an important role in the regulation of the expression of these genes. The NRG1 exon 1 has a region located between −136 to +79 (considering +1, the translational initiation site) rich in CpG sites that was analyzed by methylation specific PCR (MSP). NRG1 exon 1 showed progressive changes in the methylation pattern associated with the progression of the neoplastic process in this model; NRG1 exon 1 was unmethylated in MCF-10F and trMCF cells, becoming hypermethylated in the invasive (bsMCF) and tumor (caMCF) stages. Studies of human breast tissue samples showed that NRG1 exon 1 was partially methylated in 14 out of 17 (82.4%) invasive carcinomas although it was unmethylated in normal tissues (8 out of 10 normal breast tissue samples). Furthermore, NRG1 exon 1 was partially methylated in 9 out of 14 (64.3%) morphologically normal tissue samples adjacent to invasive carcinomas.
Epigenetic changes; Breast cancer; DNA methylation; Neuregulin; Cell transformation
A diet lacking folic acid and choline and low in methionine (folate/methyl deficient diet, FMD diet) fed to rats is known to produce preneoplastic nodules (PNNs) after 36 weeks and hepatocellular carcinomas (tumors) after 54 weeks. FMD diet-induced tumors exhibit global hypomethylation and regional hypermethylation. Restriction landmark genome scanning analysis with methylation-sensitive enzyme NotI (RLGS-M) of genomic DNA isolated from control livers, PNNs and tumor tissues was performed to identify the genes that are differentially methylated or amplified during multistage hepatocarcinogenesis. Out of the 1250 genes analysed, 2 to 5 genes were methylated in the PNNs, whereas 5 to 45 genes were partially or completely methylated in the tumors. This analysis also showed amplification of 3 to 12 genes in the primary tumors. As a first step towards identifying the genes methylated in the PNNs and primary hepatomas, we generated a rat NotI–EcoRV genomic library in the pBluescriptKS vector. Here, we describe identification of one methylated and downregulated gene as the rat protein tyrosine phosphatase receptor type O (PTPRO) and one amplified gene as rat C-MYC. Methylation of PTPRO at the NotI site located immediate upstream of the trancription start site in the PNNs and tumors, and amplification of C-MYC gene in the tumors were confirmed by Southern blot analyses. Bisulfite genomic sequencing of the CpG island encompassing exon 1 of the PTPRO gene revealed dense methylation in the PNNs and tumors, whereas it was methylation free in the livers of animals on normal diet. Reverse transcription–polymerase chain reaction (RT–PCR) analysis showed significant decrease in the expression of PTPRO in the tumors and in a transplanted rat hepatoma. The expression of PTPRO mRNA in the transplanted hepatoma after demethylation with 5-azacytidine, a potent inhibitor of DNA methyltransferases, further confirmed the role of methylation in PTPRO gene expression. These results demonstrate alteration in methylation profile and expression of specific genes during tumor progression in the livers of rats in response to folate/methyl deficiency, and further implicate the potential role of PTPRO as a novel growth regulatory gene at least in the hepatocellular carcinomas.
DNA methylation; DNA amplification; folate deficiency; hepatocellular carcinoma; PTPRO; 5-azacytidine
Early stages in the development of chronic lymphocytic leukemia (CLL) have not been explored mainly due to the inability to study normal B-cells in route to transformation. In order to determine such early events of leukemogenesis, we have used a well established mouse model for CLL. Over-expression of human TCL1, a known CLL oncogene, in murine B-cells leads to the development of mature CD19+/CD5+/IgM+ clonal leukemia with a similar disease phenotype seen in human CLL. Herein, we review our recent study using this TCL1 murine model for CLL and corresponding human CLL samples in a cross-species epigenomics approach to address the timing and relevance of epigenetic events occurring during leukemogenesis. We were able to demonstrate that the mouse model recapitulates epigenetic events very similar to what has been reported for human CLL and thus provides an exciting new tool to study early epigenetic events. Epigenetic alterations are seen at a time of three month after birth, much earlier than the phenotypically visible disease which occurs around 11 month of age. An early event in gene silencing is the inactivation of transcription factor Foxd3 expression through an NF-κB mediated process in animals with one month of age.
CLL; genetics; methylation; epigenetics; TCL1
Gene amplification, a common mechanism for oncogene activation in cancers, has been used in the discovery of novel oncogenes. . Low level copy number gains are frequently observed in head and neck squamous cell carcinomas (HNSCCs) where numerous amplification events and potential oncogenes have already been reported. Recently, we applied restriction landmark genome scanning (RLGS) to study gene amplifications in HNSCC and located novel and uncharacterized regions in primary tumor samples. Gain on chromosome 8q22.3, the location of YWHAZ (14-3-3ζ), is found in 30-40% HNSCC cases. Data obtained from fluorescence in situ hybridization (FISH) and immunohistochemistry on HNSCC tissue microarrays confirmed frequent low-level YWHAZ copy number gain and protein overexpression. YWHAZ mRNA was frequently upregulated in patients’ tumor tissues. Furthermore, YWHAZ RNAi significantly suppressed the growth rate of HNSCC cell lines, and overexpression of YWHAZ in HaCaT immortalized human skin keratinocytes promotes overgrowth, as well as morphological changes. Reduced YWHAZ levels increased the G1/G0-phase proportion, decreased the S-phase proportion and the rate of DNA synthesis. Based on this evidence, we suggest that YWHAZ is a candidate proto-oncogene and deserves further investigation into its role in HNSCC carcinogenesis.
HNSCC; amplification; oncogene; YWHAZ/14-3-3ζ
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
A hallmark of cancer cells is hypermethylation of CpG islands (CGIs), which probably arises from upregulation of one or more DNA methyltransferases. The purpose of this study was to identify the targets of DNMT3B, an essential DNA methyltransferase in mammals, in colon cancer.
Chromatin immunoprecipitation with DNMT3B specific antibody followed by CGI microarray identified genes with or without CGIs, repeat elements and genomic contigs in RKO cells. ChIP-Chop analysis showed that the majority of the target genes including P16, DCC, DISC1, SLIT1, CAVEOLIN1, GNA11, TBX5, TBX18, HOXB13 and some histone variants, that harbor CGI in their promoters, were methylated in multiple colon cancer cell lines but not in normal colon epithelial cells. Further, these genes were reactivated in RKO cells after treatment with 5-aza-2′-deoxycytidine, a DNA hypomethylating agent. COBRA showed that the CGIs encompassing the promoter and/or coding region of DCC, TBX5, TBX18, SLIT1 were methylated in primary colorectal tumors but not in matching normal colon tissues whereas GNA11 was methylated in both. MassARRAY analysis demonstrated that the CGI located ∼4.5 kb upstream of HOXB13 +1 site was tumor-specifically hypermethylated in primary colorectal cancers and cancer cell lines. HOXB13 upstream CGI was partially hypomethylated in DNMT1−/− HCT cells but was almost methylation free in cells lacking both DNMT1 and DNMT3B. Analysis of tumor suppressor properties of two aberrantly methylated transcription factors, HOXB13 and TBX18, revealed that both inhibited growth and clonogenic survival of colon cancer cells in vitro, but only HOXB13 abolished tumor growth in nude mice.
This is the first report that identifies several important tumor suppressors and transcription factors as direct DNMT3B targets in colon cancer and as potential biomarkers for this cancer. Further, this study shows that methylation at an upstream CGI of HOXB13 is unique to colon cancer.
The tumor suppressor C/CAAT enhancer binding protein alpha (C/EBPα) is a transcription factor involved in cell cycle control and cellular differentiation. A recent study showed that C/EBPα is frequently downregulated in Head and Neck Squamous Cell carcinoma (HNSCC) by DNA methylation in an upstream regulatory region. Here we investigated how DNA methylation in the upstream regulatory region disrupts the transcriptional regulation of C/EBPα in HNSCC. The results reveal that aberrant methylation correlates with Methyl Binding Domain protein binding and repressive histone modifications. This methylated region contains previously uninvestigated AP2α binding sites. AP2α suppresses C/EBPα promoter activity and protein expression. Interestingly, silencing AP2α by shRNA increases the anti-proliferative isoform of C/EBPα (p42C/EBPα). Furthermore, growth analysis revealed that these two isoforms yield very different proliferative properties in HNSCC.
DNA methylation; AP2α; C/CAAT enhancer binding protein alpha (C/EBPα); 5-aza-2′-deoxycitidine (5-aza-dC); alternative translation; p30C/EBPα; p42C/EBPα; Head and Neck Squamous Cell Carcinoma (HNSCC)