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Meta-analysis of human methylomes reveals stably methylated sequences surrounding CpG islands associated with high gene expression.

ABSTRACT: DNA methylation is thought to play an important role in the regulation of mammalian gene expression, partly based on the observation that a lack of CpG island methylation in gene promoters is associated with high transcriptional activity. However, the CpG island methylation level only accounts for a fraction of the variance in gene expression, and methylation in other domains is hypothesized to play a role. We hypothesized that regions of very high stability in methylation would exist and provide biological insight into the role of methylation both within and outside CpG islands.We set out to identify highly stable regions in the human methylome, based on the subset of CpGs assayed with an Illumina Infinium 450 K array. Using 1,737 samples from 30 publically available studies, we identified 15,224 CpGs that are 'ultrastable' in their state across tissues and developmental stages (974 always methylated; 14,250 always unmethylated). Further analysis of ultrastable CpGs led us to identify a novel subset of CpG islands, 'ravines', which exhibit a markedly consistent pattern of low methylation with highly methylated flanking shores and shelves. We distinguish ravines from other CpG islands characterized by a broader flanking region of low methylation. Interestingly, ravines are associated with higher gene expression compared to typical unmethylated CpG islands, and are more often found near housekeeping genes.The identification of ultrastable sites in the human methylome led us to identify a subclass of CpG islands characterized by a very stable pattern of methylation encompassing the island and flanking regions, established early in development and maintained through differentiation. This pattern is associated with particularly high levels of gene expression, providing new evidence that methylation beyond the CpG island could play a role in gene expression.

SUBMITTER: Edgar R

PROVIDER: S-EPMC4260796 | biostudies-other | 2014

REPOSITORIES: biostudies-other

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Meta-analysis of human methylomes reveals stably methylated sequences surrounding CpG islands associated with high gene expression.

Edgar Rachel R Tan Powell Patrick Cheng PP Portales-Casamar Elodie E Pavlidis Paul P

Epigenetics & chromatin 20141023 1

<h4>Background</h4>DNA methylation is thought to play an important role in the regulation of mammalian gene expression, partly based on the observation that a lack of CpG island methylation in gene promoters is associated with high transcriptional activity. However, the CpG island methylation level only accounts for a fraction of the variance in gene expression, and methylation in other domains is hypothesized to play a role. We hypothesized that regions of very high stability in methylation wou ...[more]

PMID: 25493099

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Project description:BackgroundMammalian CpG islands (CGIs) normally escape DNA methylation in all adult tissues and developmental stages. However, in our previous study we unexpectedly identified many methylated CGIs in human peripheral blood leukocytes. Methylated CpG dinucleotides convert to TpG dinucleotides through deaminization of their cytosine bases more frequently than hypomethylated CpG dinucleotides. Therefore, we wondered how methylated CGIs in germline or non-germline cells maintain their CpG-rich sequences. It is known that events such as germline hypomethylation, CpG selection, biased gene conversion (BGC), and frequent CpG fixation can contribute to the maintenance of CpG-rich sequences in methylated CGIs in germline or non-germline cells. However, it has not been investigated which of the processes maintain CpG-rich sequences of methylated CGIs in each genomic position.ResultsIn this study, we comprehensively examined the contribution of the processes described above to the maintenance of CpG-rich sequences in methylated CGIs in germline and non-germline cells which were classified by genomic positions. Approximately 60-80% of CGIs with high methylation in H1 cell line (H1-HM) in all the genomic positions showed a low average CpG→TpG/CpA substitution rate. In contrast, fewer than half the numbers of CGIs with H1-HM in all the genomic positions showed a low average CpG→TpG/CpA substitution rate and low levels of methylation in sperm cells (SPM-LM). Furthermore, a small fraction of CGIs with a low average CpG→TpG/CpA substitution rate and high levels of methylation in sperm cells (SPM-HM) showed CpG selection. On the other hand, independent of the positions in genes, most CGIs with SPM-HM showed a slightly higher average TpG/CpA→CpG substitution rate compared with those with SPM-LM.ConclusionsRelatively high numbers (approximately 60-80%) of CGIs with H1-HM in all the genomic positions preserve their CpG-rich sequences by a low CpG→TpG/CpA substitution rate caused mainly by their SPM-LM, and for those with SPM-HM partly by CpG selection and TpG/CpA→CpG fixation. BGC has little contribution to the maintenance of CpG-rich sequences of CGIs with SPM-HM which were classified by genomic positions.

Project description:Hypermethylation of promoter CpG islands is strongly correlated to transcriptional gene silencing and epigenetic maintenance of the silenced state. As well as its role in tumor development, CpG island methylation contributes to the acquisition of resistance to chemotherapy. Differential Methylation Hybridisation (DMH) is one technique used for genome-wide DNA methylation analysis. The study of such microarray data sets should ideally account for the specific biological features of DNA methylation and the non-symmetrical distribution of the ratios of unmethylated and methylated sequences hybridised on the array. We have therefore developed a novel algorithm tailored to this type of data, Methylation Linear Discriminant Analysis (MLDA).MLDA was programmed in R (version 2.7.0) and the package is available at CRAN 1. This approach utilizes linear regression models of non-normalised hybridisation data to define methylation status. Log-transformed signal intensities of unmethylated controls on the microarray are used as a reference. The signal intensities of DNA samples digested with methylation sensitive restriction enzymes and mock digested are then transformed to the likelihood of a locus being methylated using this reference. We tested the ability of MLDA to identify loci differentially methylated as analysed by DMH between cisplatin sensitive and resistant ovarian cancer cell lines. MLDA identified 115 differentially methylated loci and 23 out of 26 of these loci have been independently validated by Methylation Specific PCR and/or bisulphite pyrosequencing.MLDA has advantages for analyzing methylation data from CpG island microarrays, since there is a clear rational for the definition of methylation status, it uses DMH data without between-group normalisation and is less influenced by cross-hybridisation of loci. The MLDA algorithm successfully identified differentially methylated loci between two classes of samples analysed by DMH using CpG island microarrays.

Project description:DNA methylation is a covalent modification of the nucleotide cytosine that is stably inherited at the dinucleotide CpG by somatic cells, and 70% of CpG dinucleotides in the genome are methylated. The exception to this pattern of methylation are CpG islands, CpG-rich sequences that are protected from methylation, and generally are thought to be methylated only on the inactive X-chromosome and in tumors, as well as differentially methylated regions (DMRs) in the vicinity of imprinted genes. To identify chromosomal regions that might harbor imprinted genes, we devised a strategy for isolating a library of normally methylated CpG islands. Most of the methylated CpG islands represented high copy number dispersed repeats. However, 62 unique clones in the library were characterized, all of which were methylated and GC-rich, with a GC content >50%. Of these, 43 clones also showed a CpG(obs)/CpG(exp) >0.6, of which 30 were studied in detail. These unique methylated CpG islands mapped to 23 chromosomal regions, and 12 were differentially methylated regions in uniparental tissues of germline origin, i.e., hydatidiform moles (paternal origin) and complete ovarian teratomas (maternal origin), even though many apparently were methylated in somatic tissues. We term these sequences gDMRs, for germline differentially methylated regions. At least two gDMRs mapped near imprinted genes, HYMA1 and a novel homolog of Elongin A and Elongin A2, which we term Elongin A3. Surprisingly, 18 of the methylated CpG islands were methylated in germline tissues of both parental origins, representing a previously uncharacterized class of normally methylated CpG islands in the genome, and which we term similarly methylated regions (SMRs). These SMRs, in contrast to the gDMRs, were significantly associated with telomeric band locations (P =.0008), suggesting a potential role for SMRs in chromosome organization. At least 10 of the methylated CpG islands were on average 85% conserved between mouse and human. These sequences will provide a valuable resource in the search for novel imprinted genes, for defining the molecular substrates of the normal methylome, and for identifying novel targets for mammalian chromatin formation.

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Meta-analysis of human methylomes reveals stably methylated sequences surrounding CpG islands associated with high gene expression.

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Meta-analysis of human methylomes reveals stably methylated sequences surrounding CpG islands associated with high gene expression.

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