Spreading of methylation along DNA.
Ontology highlight
ABSTRACT:
SUBMITTER: Lindsay H
PROVIDER: S-EPMC1217954 | biostudies-other | 1996 Dec
REPOSITORIES: biostudies-other
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Project description:TET1 is a 5-methylcytosine dioxygenase and its DNA demethylating activity has been implicated in pluripotency and reprogramming. However, the precise role of TET1 in DNA methylation regulation outside of developmental reprogramming is still unclear. Here, we show that overexpression of the TET1 catalytic domain but not full length TET1 (TET1-FL) induces massive global DNA demethylation in differentiated cells. Genome-wide mapping reveals that 5-hydroxymethylcytosine production by TET1-FL is inhibited as DNA methylation increases, which can be explained by the preferential binding of TET1-FL to unmethylated CpG islands (CGIs) through its CXXC domain. TET1-FL specifically accumulates 5-hydroxymethylcytosine at the edges of hypomethylated CGIs, while knockdown of endogenous TET1 induces methylation spreading from methylated edges into hypomethylated CGIs. We also found that gene expression changes after TET1-FL overexpression are relatively small and independent of its dioxygenase function. Thus, our results identify TET1 as a maintenance DNA demethylase that does not purposely decrease methylation levels, but specifically prevents aberrant methylation spreading into CGIs in differentiated cells.
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Project description:BackgroundDNA methylation is a major epigenetic modification important for regulating gene expression and suppressing spurious transcription. Most methods to scan the genome in different tissues for differentially methylated sites have focused on the methylation of CpGs in CpG islands, which are concentrations of CpGs often associated with gene promoters.ResultsHere, we use a methylation profiling strategy that is predominantly responsive to methylation differences outside of CpG islands. The method compares the yield from two samples of size-selected fragments generated by a methylation-sensitive restriction enzyme. We then profile nine different normal tissues from two human donors relative to spleen using a custom array of genomic clones covering the euchromatic portion of human chromosome 1 and representing 8% of the human genome. We observe gross regional differences in methylation states across chromosome 1 between tissues from the same individual, with the most striking differences detected in the comparison of cerebellum and spleen. Profiles of the same tissue from different donors are strikingly similar, as are the profiles of different lobes of the brain. Comparing our results with published gene expression levels, we find that clones exhibiting extreme ratios reflecting low relative methylation are statistically enriched for genes with high expression ratios, and vice versa, in most pairs of tissues examined.ConclusionThe varied patterns of methylation differences detected between tissues by our methylation profiling method reinforce the potential functional significance of regional differences in methylation levels outside of CpG islands.
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Project description:Transposable elements (TEs) and their relics play major roles in genome evolution. However, mobilization of TEs is usually deleterious and strongly repressed. In plants and mammals, this repression is typically associated with DNA methylation, but the relationship between this epigenetic mark and TE sequences has not been investigated systematically. Here, we present an improved annotation of TE sequences and use it to analyze genome-wide DNA methylation maps obtained at single-nucleotide resolution in Arabidopsis. We show that although the majority of TE sequences are methylated, ∼26% are not. Moreover, a significant fraction of TE sequences densely methylated at CG, CHG and CHH sites (where H = A, T or C) have no or few matching small interfering RNA (siRNAs) and are therefore unlikely to be targeted by the RNA-directed DNA methylation (RdDM) machinery. We provide evidence that these TE sequences acquire DNA methylation through spreading from adjacent siRNA-targeted regions. Further, we show that although both methylated and unmethylated TE sequences located in euchromatin tend to be more abundant closer to genes, this trend is least pronounced for methylated, siRNA-targeted TE sequences located 5' to genes. Based on these and other findings, we propose that spreading of DNA methylation through promoter regions explains at least in part the negative impact of siRNA-targeted TE sequences on neighboring gene expression.
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Project description:We used a transgene system to study spreading of RNA-directed DNA methylation (RdDM) during transcriptional gene silencing in Arabidopsis thaliana. Forward and reverse genetics approaches using this system delineated a stepwise pathway for the biogenesis of secondary siRNAs and unidirectional spreading of methylation from an upstream enhancer element into downstream sequences. Trans-acting, hairpin-derived primary siRNAs induce primary RdDM, independently of an enhancer-associated 'nascent' RNA, at the target enhancer region. Primary RdDM is a key step in the pathway because it attracts the secondary siRNA-generating machinery, including RNA polymerase IV, RNA-dependent RNA polymerase2 and Dicer-like3 (DCL3). These factors act in a turnover pathway involving a nascent RNA, which normally accumulates stably in non-silenced plants, to produce cis-acting secondary siRNAs that induce methylation in the downstream region. The identification of DCL3 in a forward genetic screen for silencing-defective mutants demonstrated a strict requirement for 24-nt siRNAs to direct methylation. A similar stepwise process for spreading of DNA methylation may occur in mammalian genomes, which are extensively transcribed in upstream regulatory regions.
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Project description:Global changes in the state of spatially distributed systems can often be traced back to perturbations that arise locally. Whether such local perturbations grow into global changes depends on the system geometry and the spatial spreading of these perturbations. Here, we investigate how different spreading behaviors of local perturbations determine their global impact in 1-dimensional systems of different size. Specifically, we assessed sliding arrest events in in vitro motility assays where myosins propel actin, and simulated the underlying mechanochemistry of myosins that bind along the actin filament. We observed spontaneous sliding arrest events that occurred more frequently for shorter actin filaments. This observation could be explained by spontaneous local arrest of myosin kinetics that stabilizes once it spreads throughout an entire actin filament. When we introduced intermediate concentrations of the actin cross-linker filamin, longer actin was arrested more frequently. This observation was reproduced by simulations where filamin binding induces persistent local arrest of myosin kinetics, which subsequently spreads throughout the actin filament. A spin chain model with nearest-neighbor coupling reproduced key features of our experiments and simulations, thus extending to other linear systems with nearest-neighbor coupling the following conclusions: 1) perturbations that are persistent only once they spread throughout the system are more effective in smaller systems, and 2) perturbations that are persistent upon their establishment are more effective in larger systems. Beyond these general conclusions, our work also provides a theoretical model of collective myosin kinetics with a finite range of mechanical coupling along the actin filament.
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Project description:(1) Background: Epithelial⁻mesenchymal plasticity (EMP) is a dynamic process whereby epithelial carcinoma cells reversibly acquire morphological and invasive characteristics typical of mesenchymal cells. Identifying the methylation differences between epithelial and mesenchymal states may assist in the identification of optimal DNA methylation biomarkers for the blood-based monitoring of cancer. (2) Methods: Methylation-sensitive high-resolution melting (MS-HRM) was used to examine the promoter methylation status of a panel of established and novel markers in a range of breast cancer cell lines spanning the epithelial⁻mesenchymal spectrum. Pyrosequencing was used to validate the MS-HRM results. (3) Results: VIM, DKK3, and CRABP1 were methylated in the majority of epithelial breast cancer cell lines, while methylation of GRHL2, MIR200C, and CDH1 was restricted to mesenchymal cell lines. Some markers that have been used to assess minimal residual disease such as AKR1B1 and APC methylation proved to be specific for epithelial breast cell lines. However, RASSF1A, RARβ, TWIST1, and SFRP2 methylation was seen in both epithelial and mesenchymal cell lines, supporting their suitability for a multimarker panel. (4) Conclusions: Profiling DNA methylation shows a distinction between epithelial and mesenchymal phenotypes. Understanding how DNA methylation varies between epithelial and mesenchymal phenotypes may lead to more rational selection of methylation-based biomarkers for circulating tumour DNA analysis.
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Project description:While large-scale studies applying various statistical approaches have identified hundreds of mutated driver genes across various cancer types, the contribution of epigenetic changes to cancer remains more enigmatic. This is partly due to the fact that certain regions of the cancer genome, due to their genomic and epigenomic properties, are more prone to dysregulated DNA methylation than others. Thus, it has been difficult to distinguish which promoter methylation changes are really driving carcinogenesis from those that are mostly just a reflection of their genomic location. By developing a novel method that corrects for epigenetic covariates, we reveal a small, concise set of potential epigenetic driver events. Interestingly, those changes suggest different modes of epigenetic carcinogenesis: first, we observe recurrent inactivation of known cancer genes across tumour types suggesting a higher convergence on common tumour suppressor pathways than previously anticipated. Second, in prostate cancer, a cancer type with few recurrently mutated genes, we demonstrate how the epigenome primes tumours towards higher tolerance of other aberrations.
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Project description:Colorectal laterally spreading tumors (LSTs) grow to extremely large size while rarely invade deeply. Also, there is a low tendency to become cancerous. We used the Illumina Human Methylation 450K array to query the main epigenetic difference of LSTs. We built a discovery cohort with 10 matched cases, and a validation cohort with 9 additional matched cases. Our results suggest that LST displays significant decrease in DNA methylation, highlighted by the discovery of 1,018 hypomethylated intergenic regions (IGRs). Comparing to classic differentially methylated probes and regions that overlap transcription starting site and CpG island, IGR-regions were associated more closely with genes involved in functional biological processes and correlated with specific histone modifications. Hypomethylated IGR regions were often annotated as tissue-specific regulatory elements for noncolon tissues and were typically epigenetically silenced in normal colon mucosa. By integration of public data, we defined the commonality and specific epigenetic signatures for adenomas, LSTs and colon adenocarcinomas. Only 435 hypermethylated differentially methylated probes (DMPs) and differentially methylated regions (DMRs) and 517 hypomethylated DMPs and DMRs were shared by the three diseases. However, our pathway-level analysis discovered that genes in four pathways were common target of epimutations in LSTs, adenomas and CRCs. More interestingly, different diseases seem to employ distinct epigenetic insult to disturb specific pathways. Between LST and adenoma, we found eight pathways including Ras signaling and Rap1 signaling pathway were commonly targeted but the epimutation patterns were opposite. Comparison between precancerous conditions and invasive states revealed the key pathways governing the progression to malignancy, including PI3K-Akt pathways.
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Project description:Repression of many tumor suppressor genes in cancer is concurrent with aberrantly increased DNA methylation levels at promoter CpG islands (CGIs). About one-fourth of empirically defined human promoters are surrounded by or contain clustered repetitive elements. It was previously observed that a sharp transition of methylation exists between highly methylated repetitive elements and unmethylated promoter-CGIs in normal tissues. The factors that lead to aberrant CGI hypermethylation in cancer remain poorly understood. Here, we established a site-specific integration system with enforced local transcriptional repression in colorectal cancer cells and monitored the occurrence of initial de novo methylation at specific CG sites adjacent to the CGI of the INSL6 promoter, which could be accelerated by binding a KRAB-containing transcriptional factor. Additional repetitive elements from P16 and RIL (PDLIM4), if situated adjacent to the promoter of INSL6, could confer DNA methylation spreading into the CGI particularly in the setting of KRAB-factor binding. However, a repressive chromatin alone was not sufficient to initiate DNA methylation, which required specific DNA sequences and was integration-site (and/or cell-line) specific. Overall, these results demonstrate a requirement for specific DNA sequences to trigger de novo DNA methylation, and repetitive elements as cis-regulatory factors to cooperate with advanced transcriptional repression in promoting methylation spreading.
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