Investigating the potential role of genetic and epigenetic variation of DNA methyltransferase genes in hyperplastic polyposis syndrome

Background

Hyperplastic Polyposis Syndrome (HPS) is a condition associated with multiple serrated polyps, and an increased risk of colorectal cancer (CRC). At least half of CRCs arising in HPS show a CpG island methylator phenotype (CIMP), potentially linked to aberrant DNA methyltransferase (DNMT) activity. CIMP is associated with methylation of tumor suppressor genes including regulators of DNA mismatch repair (such as MLH1, MGMT), and negative regulators of Wnt signaling (such as WIF1). In this study, we investigated the potential for interaction of genetic and epigenetic variation in DNMT genes, in the aetiology of HPS.

Methods

We utilized high resolution melting (HRM) analysis to screen 45 cases with HPS for novel sequence variants in DNMT1, DNMT3A, DNMT3B, and DNMT3L. 21 polyps from 13 patients were screened for BRAF and KRAS mutations, with assessment of promoter methylation in the DNMT1, DNMT3A, DNMT3B, DNMT3L MLH1, MGMT, and WIF1 gene promoters.

Results

No pathologic germline mutations were observed in any DNA-methyltransferase gene. However, the T allele of rs62106244 (intron 10 of DNMT1 gene) was over-represented in cases with HPS (p<0.01) compared with population controls. The DNMT1, DNMT3A and DNMT3B promoters were unmethylated in all instances. Interestingly, the DNMT3L promoter showed low levels of methylation in polyps and normal colonic mucosa relative to matched disease free cells with methylation level negatively correlated to expression level in normal colonic tissue. DNMT3L promoter hypomethylation was more often found in polyps harbouring KRAS mutations (p = 0.0053). BRAF mutations were common (11 out of 21 polyps), whilst KRAS mutations were identified in 4 of 21 polyps.

Conclusions

Genetic or epigenetic alterations in DNMT genes do not appear to be associated with HPS, but further investigation of genetic variation at rs62106244 is justified given the high frequency of the minor allele in this case series.     

Non-germ Line Restoration of Genomic Imprinting for a Small Subset of Imprinted Genes in Ubiquitin-like PHD and RING Finger Domain-Containing 1 (Uhrf1) Null Mouse Embryonic Stem Cells

The underlying mechanism for the establishment and maintenance of differential DNA methylation in imprinted genes is largely unknown. Previous studies using Dnmt1 knock-out embryonic stem (ES) cells demonstrated that, although re-expression of DNMT1 restored DNA methylation in the non-imprinted regions, the methylation patterns of imprinted genes could be restored only through germ line passage. Knock-out of Uhrf1, an accessory factor essential for DNMT1-mediated DNA methylation, in mouse ES cells also led to impaired global DNA methylation and loss of genomic imprinting. Here, we demonstrate that, although re-expression of UHRF1 in Uhrf1^−/− ES cells restored DNA methylation for the bulk genome but not for most of the imprinted genes, it did rescue DNA methylation for the imprinted H19, Nnat, and Dlk1 genes. Analysis of histone modifications at the differential methylated regions of the imprinted genes by ChIP assays revealed that for the imprinted genes whose DNA methylation could be restored upon re-expression of UHRF1, the active histone markers (especially H3K4me3) were maintained at considerably low levels, and low levels were maintained even in Uhrf1^−/− ES cells. In contrast, for the imprinted genes whose DNA methylation could not be restored upon UHRF1 re-expression, the active histone markers (especially H3K4me3) were relatively high and became even higher in Uhrf1^−/− ES cells. Our study thus supports a role for histone modifications in determining the establishment of imprinting-related DNA methylation and demonstrates that mouse ES cells can be a valuable model for mechanistic study of the establishment and maintenance of differential DNA methylation in imprinted genes.     

Imprinting analysis of the mouse chromosome 7C region in DNMT1-null embryos

The mouse chromosome 7C, orthologous to the human 15q11–q13 has an imprinted domain, where most of the genes are expressed only from the paternal allele. The imprinted domain contains paternally expressed genes, Snurf/Snrpn, Ndn, Magel2, Mkrn3, and Frat3, C/D-box small nucleolar RNAs (snoRNAs), and the maternally expressed gene, Ube3a. Imprinted expression in this large (approximately 3–4 Mb) domain is coordinated by a bipartite cis-acting imprinting center (IC), located upstream of the Snurf/Snrpn gene. The molecular mechanism how IC regulates gene expression of the whole domain remains partially understood. Here we analyzed the relationship between imprinted gene expression and DNA methylation in the mouse chromosome 7C using DNA methyltransferase 1 (DNMT1)-null mutant embryos carrying Dnmt1^ps alleles, which show global loss of DNA methylation and embryonic lethality. In the DNMT1-null embryos at embryonic day 9.5, the paternally expressed genes were biallelically expressed. Bisulfite DNA methylation analysis revealed loss of methylation on the maternal allele in the promoter regions of the genes. These results demonstrate that DNMT1 is necessary for monoallelic expression of the imprinted genes in the chromosome 7C domain, suggesting that DNA methylation in the secondary differentially methylated regions (DMRs), which are acquired during development serves primarily to control the imprinted expression from the maternal allele in the mouse chromosome 7C.     

Assessment of global DNA methylation in peripheral blood cell subpopulations of early rheumatoid arthritis before and after methotrexate

IntroductionDNA methylation is an epigenetic mechanism regulating gene expression that has been insufficiently studied in the blood of rheumatoid arthritis (RA) patients, as only T cells and total peripheral blood mononuclear cells (PBMCs) from patients with established RA have been studied and with conflicting results.MethodFive major blood cell subpopulations: T, B and NK cells, monocytes, and polymorphonuclear leukocytes, were isolated from 19 early RA patients and 17 healthy controls. Patient samples were taken before and 1 month after the start of treatment with methotrexate (MTX). Analysis included DNA methylation with high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry-selected reaction monitoring (HPLC-ESI-MS/MS-SRM) and expression levels of seven methylation-specific enzymes by quantitative polymerase chain reaction (qPCR).ResultsDisease-modifying anti-rheumatic drug (DMARD)-naïve early RA patients showed global DNA hypomethylation in T cells and monocytes, together with a lower expression of DNA methyltrasnferase 1 (DNMT1), the maintenance DNA methyltransferase, which was also decreased in B cells. Furthermore, significantly increased expression of ten-eleven translocation1 (TET1), TET2 and TET3, enzymes involved in demethylation, was found in monocytes and of TET2 in T cells. There was also modest decreased expression of DNMT3A in B cells and of growth arrest and DNA-damage-inducible protein 45A (GADD45A) in T and B cells. Treatment with MTX reverted hypomethylation in T cells and monocytes, which were no longer different from controls, and increased global methylation in B cells. In addition, DNMT1 and DNMT3A showed a trend to reversion of their decreased expression.ConclusionsOur results confirm global DNA hypomethylation in patients with RA with specificity for some blood cell subpopulations and their reversal with methotrexate treatment. These changes are accompanied by parallel changes in the levels of enzymes involved in methylation, suggesting the possibility of regulation at this level.

Electronic supplementary material

The online version of this article (doi:10.1186/s13075-015-0748-5) contains supplementary material, which is available to authorized users.     

No Evidence for Mutations of CTCFL/BORIS in Silver-Russell Syndrome Patients with IGF2/H19 Imprinting Control Region 1 Hypomethylation

Background

Silver-Russell syndrome (SRS) is a genetically and clinically heterogeneous disease. Although no protein coding gene defects have been reported in SRS patients, approximately 50% of SRS patients carry epimutations (hypomethylation) at the IGF2/H19 imprinting control region 1 (ICR1). Proper methylation at ICR1 is crucial for the imprinted expression of IGF2, a fetal growth factor. CTCFL, a testis-specific protein, has recently been proposed to play a role in the establishment of DNA methylation at the murine equivalent of ICR1. A screen was undertaken to assess whether CTCFL is mutated in SRS patients with hypomethylation, to explore a link between the observed epimutations and a genetic cause of the disease.

Methodology/Principal Findings

DNA was obtained from 36 SRS patients with hypomethylation at ICR1. All CTCFL coding exons were sequenced and analyzed for duplications/deletions using both multiplex ligation-dependent probe amplification, with a custom CTCFL probe set, and genomic qPCR. Novel SNP alleles were analyzed for potential differential splicing in vitro utilizing a splicing assay. Neither mutations of CTCFL nor duplications/deletions were observed. Five novel SNPs were identified and have been submitted to dbSNP. In silico splice prediction suggested one novel SNP, IVS2-66A>C, activated a cryptic splice site, resulting in aberrant splicing and premature termination. In vitro splicing assays did not confirm predicted aberrant splicing.

Conclusions/Significance

As no mutations were detected at CTCFL in the patients examined, we conclude that genetic alterations of CTCFL are not responsible for the SRS hypomethylation. We suggest that analysis of other genes involved in the establishment of DNA methylation at imprinted genes, such as DNMT3A and DNMT3L, may provide insight into the genetic cause of hypomethylation in SRS patients.     

Role of DNMT3B in the regulation of early neural and neural crest specifiers

The de novo DNA methyltransferase DNMT3B functions in establishing DNA methylation patterns during development. DNMT3B missense mutations cause immunodeficiency, centromere instability and facial anomalies (ICF) syndrome. The restriction of Dnmt3b expression to neural progenitor cells, as well as the mild cognitive defects observed in ICF patients, suggests that DNMT3B may play an important role in early neurogenesis. We performed RNAi knockdown of DNMT3B in human embryonic stem cells (hESCs) in order to investigate the mechanistic contribution of DNMT3B to DNA methylation and early neuronal differentiation. While DNMT3B was not required for early neuroepithelium specification, DNMT3B deficient neuroepithelium exhibited accelerated maturation with earlier expression, relative to normal hESCs, of mature neuronal markers (such as NEUROD1) and of early neuronal regional specifiers (such as those for the neural crest). Genome-wide analyses of DNA methylation by MethylC-seq identified novel regions of hypomethylation in the DNMT3B knockdowns along the X chromosome as well as pericentromeric regions, rather than changes to promoters of specific dysregulated genes. We observed a loss of H3K27me3 and the polycomb complex protein EZH2 at the promoters of early neural and neural crest specifier genes during differentiation of DNMT3B knockdown but not normal hESCs. Our results indicate that DNMT3B mediates large-scale methylation patterns in hESCs and that DNMT3B deficiency in the cells alters the timing of their neuronal differentiation and maturation.     

Genome-wide and single-base resolution DNA methylomes of the Pacific oyster Crassostrea gigas provide insight into the evolution of invertebrate CpG methylation

Background

Studies of DNA methylomes in a wide range of eukaryotes have revealed both conserved and divergent characteristics of DNA methylation among phylogenetic groups. However, data on invertebrates particularly molluscs are limited, which hinders our understanding of the evolution of DNA methylation in metazoa. The sequencing of the Pacific oyster Crassostrea gigas genome provides an opportunity for genome-wide profiling of DNA methylation in this model mollusc.

Results

Homologous searches against the C. gigas genome identified functional orthologs for key genes involved in DNA methylation: DNMT1, DNMT2, DNMT3, MBD2/3 and UHRF1. Whole-genome bisulfite sequencing (BS-seq) of the oyster’s mantle tissues revealed that more than 99% methylation modification was restricted to cytosines in CpG context and methylated CpGs accumulated in the bodies of genes that were moderately expressed. Young repeat elements were another major targets of CpG methylation in oysters. Comparison with other invertebrate methylomes suggested that the 5’-end bias of gene body methylation and the negative correlation between gene body methylation and gene length were the derived features probably limited to the insect lineage. Interestingly, phylostratigraphic analysis showed that CpG methylation preferentially targeted genes originating in the common ancestor of eukaryotes rather than the oldest genes originating in the common ancestor of cellular organisms.

Conclusions

Comparative analysis of the oyster DNA methylomes and that of other animal species revealed that the characteristics of DNA methylation were generally conserved during invertebrate evolution, while some unique features were derived in the insect lineage. The preference of methylation modification on genes originating in the eukaryotic ancestor rather than the oldest genes is unexpected, probably implying that the emergence of methylation regulation in these ''relatively young’ genes was critical for the origin and radiation of eukaryotes.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-1119) contains supplementary material, which is available to authorized users.     

S-adenosylhomocysteine Hydrolase Participates in DNA Methylation Inheritance

DNA (cytosine-5) methyltransferase 1 (DNMT1) is essential for mammalian development and maintenance of DNA methylation following DNA replication in cells. The DNA methylation process generates S-adenosyl-l-homocysteine, a strong inhibitor of DNMT1. Here we report that S-adenosylhomocysteine hydrolase (SAHH/AHCY), the only mammalian enzyme capable of hydrolyzing S-adenosyl-l-homocysteine binds to DNMT1 during DNA replication. SAHH enhances DNMT1 activity in vitro, and its overexpression in mammalian cells led to hypermethylation of the genome, whereas its inhibition by adenosine periodate or siRNA-mediated knockdown resulted in hypomethylation of the genome. Hypermethylation was consistent in both gene bodies and repetitive DNA elements leading to aberrant gene regulation. Cells overexpressing SAHH specifically up-regulated metabolic pathway genes and down-regulated PPAR and MAPK signaling pathways genes. Therefore, we suggest that alteration of SAHH level affects global DNA methylation levels and gene expression.     

The Role of Dietary Extra Virgin Olive Oil and Corn Oil on the Alteration of Epigenetic Patterns in the Rat DMBA-Induced Breast Cancer Model

Disruption of epigenetic patterns is a major change occurring in all types of cancers. Such alterations are characterized by global DNA hypomethylation, gene-promoter hypermethylation and aberrant histone modifications, and may be modified by environment. Nutritional factors, and especially dietary lipids, have a role in the etiology of breast cancer. Thus, we aimed to analyze the influence of different high fat diets on DNA methylation and histone modifications in the rat dimethylbenz(a)anthracene (DMBA)-induced breast cancer model. Female Sprague-Dawley rats were fed a low-fat, a high corn-oil or a high extra-virgin olive oil (EVOO) diet from weaning or from induction with DMBA. In mammary glands and tumors we analyzed global and gene specific (RASSF1A, TIMP3) DNA methylation by LUMA and bisulfite pyrosequencing assays, respectively. We also determined gene expression and enzymatic activity of DNA methyltransferases (DNMT1, DNMT3a and DNMT3b) and evaluated changes in histone modifications (H3K4me2, H3K27me3, H4K20me3 and H4K16ac) by western-blot. Our results showed variations along time in the global DNA methylation of the mammary gland displaying decreases at puberty and with aging. The olive oil-enriched diet, on the one hand, increased the levels of global DNA methylation in mammary gland and tumor, and on the other, changed histone modifications patterns. The corn oil-enriched diet increased DNA methyltransferase activity in both tissues, resulting in an increase in the promoter methylation of the tumor suppressor genes RASSF1A and TIMP3. These results suggest a differential effect of the high fat diets on epigenetic patterns with a relevant role in the neoplastic transformation, which could be one of the mechanisms of their differential promoter effect, clearly stimulating for the high corn-oil diet and with a weaker influence for the high EVOO diet, on breast cancer progression.     

A systematic comparison of quantitative high-resolution DNA methylation analysis and methylation-specific PCR

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.     

FOXM1 induces a global methylation signature that mimics the cancer epigenome in head and neck squamous cell carcinoma

The oncogene FOXM1 has been implicated in all major types of human cancer. We recently showed that aberrant FOXM1 expression causes stem cell compartment expansion resulting in the initiation of hyperplasia. We have previously shown that FOXM1 regulates HELLS, a SNF2/helicase involved in DNA methylation, implicating FOXM1 in epigenetic regulation. Here, we have demonstrated using primary normal human oral keratinocytes (NOK) that upregulation of FOXM1 suppressed the tumour suppressor gene p16^INK4A (CDKN2A) through promoter hypermethylation. Knockdown of HELLS using siRNA re-activated the mRNA expression of p16^INK4A and concomitant downregulation of two DNA methyltransferases DNMT1 and DNMT3B. The dose-dependent upregulation of endogenous FOXM1 (isoform B) expression during tumour progression across a panel of normal primary NOK strains (n = 8), dysplasias (n = 5) and head and neck squamous cell carcinoma (HNSCC) cell lines (n = 11) correlated positively with endogenous expressions of HELLS, BMI1, DNMT1 and DNMT3B and negatively with p16^INK4A and involucrin. Bisulfite modification and methylation-specific promoter analysis using absolute quantitative PCR (MS-qPCR) showed that upregulation of FOXM1 significantly induced p16^INK4A promoter hypermethylation (10-fold, P<0.05) in primary NOK cells. Using a non-bias genome-wide promoter methylation microarray profiling method, we revealed that aberrant FOXM1 expression in primary NOK induced a global hypomethylation pattern similar to that found in an HNSCC (SCC15) cell line. Following validation experiments using absolute qPCR, we have identified a set of differentially methylated genes, found to be inversely correlated with in vivo mRNA expression levels of clinical HNSCC tumour biopsy samples. This study provided the first evidence, using primary normal human cells and tumour tissues, that aberrant upregulation of FOXM1 orchestrated a DNA methylation signature that mimics the cancer methylome landscape, from which we have identified a unique FOXM1-induced epigenetic signature which may have clinical translational potentials as biomarkers for early cancer screening, diagnostic and/or therapeutic interventions.     

Loss of DNMT1o Disrupts Imprinted X Chromosome Inactivation and Accentuates Placental Defects in Females

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. Dnmt1o^mat−/− mouse embryos born to Dnmt1^Δ1o/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 Dnmt1^Δ1o/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.