Characteristics of DNA methylation and gene expression in regulatory features on the Infinium 450k Beadchip

Understanding the relationship between variations in DNA methylation and gene expression has been challenging. Evidence suggests the function of DNA methylation may vary with genomic context, and few consistent rules linking methylation to expression have been noted. For array-based studies, the content of current DNA methylation array platforms provide broad coverage of the genome but target only a fraction of the potentially methylated CG dinucleotides. A better understanding of the interplay between DNA methylation and gene expression is beneficial for users of these platforms, and may aid with candidate prioritization in epigenome-wide association studies (EWAS). To address this we examined the relationship between DNA methylation levels and gene expression in primary T-lymphocytes at discreet genomic regions around the transcriptional unit (Promoters, gene body, untranslated regions) and at CpG island-associated regions (islands, shores and shelves), stratifying by high and low expressed genes. As anticipated we found evidence that DNA methylation at CpG sites near promoter regions are tightly correlated with gene expression in both the stably expressed and developmentally regulated genes, however this is dependent on CpG density. DNA methylation within the gene body was not consistently associated with changes in gene expression. CpG islands and island shores exhibited strong correlations with gene expression, but this was not true for island shelves. We found these relationships were generally preserved at both dynamic and steady state genes, with some notable exceptions. In combination these insights may be useful for prioritising candidates identified in epigenome-wide association studies for subsequent functional studies.

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Genome-Wide DNA Methylation Analysis Shows Enrichment of Differential Methylation in “Open Seas” and Enhancers and Reveals Hypomethylation in DNMT3A Mutated Cytogenetically Normal AML (CN-AML)

Blood ◽

10.1182/blood.v120.21.653.653 ◽

2012 ◽

Vol 120 (21) ◽

pp. 653-653 ◽

Cited By ~ 2

Author(s):

Ying Qu ◽

Andreas Lennartsson ◽

Verena I. Gaidzik ◽

Stefan Deneberg ◽

Sofia Bengtzén ◽

...

Keyword(s):

Gene Expression ◽

Dna Methylation ◽

Cpg Island ◽

Cpg Islands ◽

Differential Methylation ◽

Methylation Analysis ◽

Cpg Sites ◽

Genome Wide ◽

Genomic Regions ◽

Methylation Patterns

Abstract Abstract 653 DNA methylation is involved in multiple biologic processes including normal cell differentiation and tumorigenesis. In AML, methylation patterns have been shown to differ significantly from normal hematopoietic cells. Most studies of DNA methylation in AML have previously focused on CpG islands within the promoter of genes, representing only a very small proportion of the DNA methylome. In this study, we performed genome-wide methylation analysis of 62 AML patients with CN-AML and CD34 positive cells from healthy controls by Illumina HumanMethylation450K Array covering 450.000 CpG sites in CpG islands as well as genomic regions far from CpG islands. Differentially methylated CpG sites (DMS) between CN-AML and normal hematopoietic cells were calculated and the most significant enrichment of DMS was found in regions more than 4kb from CpG Islands, in the so called open sea where hypomethylation was the dominant form of aberrant methylation. In contrast, CpG islands were not enriched for DMS and DMS in CpG islands were dominated by hypermethylation. DMS successively further away from CpG islands in CpG island shores (up to 2kb from CpG Island) and shelves (from 2kb to 4kb from Island) showed increasing degree of hypomethylation in AML cells. Among regions defined by their relation to gene structures, CpG dinucleotide located in theoretic enhancers were found to be the most enriched for DMS (Chi χ2<0.0001) with the majority of DMS showing decreased methylation compared to CD34 normal controls. To address the relation to gene expression, GEP (gene expression profiling) by microarray was carried out on 32 of the CN-AML patients. Totally, 339723 CpG sites covering 18879 genes were addressed on both platforms. CpG methylation in CpG islands showed the most pronounced anti-correlation (spearman ρ =-0.4145) with gene expression level, followed by CpG island shores (mean spearman rho for both sides' shore ρ=-0.2350). As transcription factors (TFs) have shown to be crucial for AML development, we especially studied differential methylation of an unbiased selection of 1638 TFs. The most enriched differential methylation between CN-AML and normal CD34 positive cells were found in TFs known to be involved in hematopoiesis and with Wilms tumor protein-1 (WT1), activator protein 1 (AP-1) and runt-related transcription factor 1 (RUNX1) being the most differentially methylated TFs. The differential methylation in WT 1 and RUNX1 was located in intragenic regions which were confirmed by pyro-sequencing. AML cases were characterized with respect to mutations in FLT3, NPM1, IDH1, IDH2 and DNMT3A. Correlation analysis between genome wide methylation patterns and mutational status showed statistically significant hypomethylation of CpG Island (p<0.0001) and to a lesser extent CpG island shores (p<0.001) and the presence of DNMT3A mutations. This links DNMT3A mutations for the first time to a hypomethylated phenotype. Further analyses correlating methylation patterns to other clinical data such as clinical outcome are ongoing. In conclusion, our study revealed that non-CpG island regions and in particular enhancers are the most aberrantly methylated genomic regions in AML and that WT 1 and RUNX1 are the most differentially methylated TFs. Furthermore, our data suggests a hypomethylated phenotype in DNMT3A mutated AML. Disclosures: No relevant conflicts of interest to declare.

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Identification of blood autosomal cis-expression quantitative trait methylation (cis-eQTMs) in children

10.1101/2020.11.05.368076 ◽

2020 ◽

Author(s):

Carlos Ruiz-Arenas ◽

Carles Hernandez-Ferrer ◽

Marta Vives-Usano ◽

Sergi Marí ◽

Inés Quintela ◽

...

Keyword(s):

Gene Expression ◽

Dna Methylation ◽

Quantitative Trait ◽

Association Studies ◽

Cell Type ◽

Cell Type Specificity ◽

Biological Interpretation ◽

Cell Type Specific ◽

Using Data ◽

The Relationship

AbstractBackgroundThe identification of expression quantitative trait methylation (eQTMs), defined as correlations between gene expression and DNA methylation levels, might help the biological interpretation of epigenome-wide association studies (EWAS). We aimed to identify autosomal cis-eQTMs in child blood, using data from 832 children of the Human Early Life Exposome (HELIX) project.MethodsBlood DNA methylation and gene expression were measured with the Illumina 450K and the Affymetrix HTA v2 arrays, respectively. The relationship between methylation levels and expression of nearby genes (transcription start site (TSS) within a window of 1 Mb) was assessed by fitting 13.6 M linear regressions adjusting for sex, age, and cohort.ResultsWe identified 63,831 autosomal cis-eQTMs, representing 35,228 unique CpGs and 11,071 unique transcript clusters (TCs, genes). 74.3% of these cis-eQTMs were located at <250 kb, 60.0% showed an inverse relationship and 23.9% had at least one genetic variant associated with the methylation and expression levels. They were enriched for active blood regulatory regions. Adjusting for cellular composition decreased the number of cis-eQTMs to 37.7%, suggesting that some of them were cell type-specific. The overlap of child blood cis-eQTMs with those described in adults was small, and child and adult shared cis-eQTMs tended to be proximal to the TSS, enriched for genetic variants and with lower cell type specificity. Only half of the cis-eQTMs could be captured through annotation to the closest gene.ConclusionsThis catalogue of blood autosomal cis-eQTMs in children can help the biological interpretation of EWAS findings, and is publicly available at https://helixomics.isglobal.org/.

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Genome-wide Differential-based Analysis of the Relationship between DNA Methylation and Gene Expression in Cancer

Current Bioinformatics ◽

10.2174/1574893614666190424160046 ◽

2019 ◽

Vol 14 (8) ◽

pp. 783-792 ◽

Cited By ~ 2

Author(s):

Yuanyuan Zhang ◽

Chuanhua Kou ◽

Shudong Wang ◽

Yulin Zhang

Keyword(s):

Gene Expression ◽

Dna Methylation ◽

Epigenetic Modification ◽

Epigenetic Mechanism ◽

Gene Body ◽

Differential Analysis ◽

Promoter Regions ◽

Genome Wide ◽

Significant Difference ◽

The Relationship

Background:: DNA methylation is an epigenetic modification that plays an important role in regulating gene expression. There is evidence that the hypermethylation of promoter regions always causes gene silencing. However, how the methylation patterns of other regions in the genome, such as gene body and 3’UTR, affect gene expression is unknown. Objective:: The study aimed to fully explore the relationship between DNA methylation and expression throughout the genome-wide analysis which is important in understanding the function of DNA methylation essentially. Method:: In this paper, we develop a heuristic framework to analyze the relationship between the methylated change in different regions and that of the corresponding gene expression based on differential analysis. Results:: To understande the methylated function of different genomic regions, a gene is divided into seven functional regions. By applying the method in five cancer datasets from the Synapse database, it was found that methylated regions with a significant difference between cases and controls were almost uniformly distributed in the seven regions of the genome. Also, the effect of DNA methylation in different regions on gene expression was different. For example, there was a higher percentage of positive relationships in 1stExon, gene body and 3’UTR than in TSS1500 and TSS200. The functional analysis of genes with a significant positive and negative correlation between DNA methylation and gene expression demonstrated the epigenetic mechanism of cancerassociated genes. Conclusion:: Differential based analysis helps us to recognize the change in DNA methylation and how this change affects the change in gene expression. It provides a basis for further integrating gene expression and DNA methylation data to identify disease-associated biomarkers.

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Genome Wide Study of DNA Methylation In AML

Blood ◽

10.1182/blood.v116.21.3618.3618 ◽

2010 ◽

Vol 116 (21) ◽

pp. 3618-3618

Author(s):

Marwa Saied ◽

Sabah Khaled ◽

Thomas Down ◽

Jacek Marzec ◽

Paul Smith ◽

...

Keyword(s):

Gene Expression ◽

Dna Methylation ◽

Cpg Island ◽

Cpg Islands ◽

Differential Methylation ◽

Methylation Analysis ◽

Promoter Regions ◽

Gene Body Methylation ◽

Normal Bone ◽

Genome Wide

Abstract Abstract 3618 DNA methylation is the most stable epigenetic modification and has a major role in cancer initiation and progression. The two main aims for this research were, firstly, to use the genome wide analysis of DNA methylation to better understand the development of acute myeloid leukemia (AML). The second aim was to detect differentially methylated genes/regions between certain subtypes of AML and normal bone marrow (NBM). We used the methylated DNA immunoprecipitation technique followed by high-throughput sequencing by Illumina Genome Analyser II (MeDIP -seq) for 9 AML samples for which ethical approval has been obtained. The selected leukemias included three with the t(8; 21), three with the t(15; 17) translocations and three with normal karyotypes (NK). The control samples were 3 normal bone marrows (NBMs) from healthy donors. The number of reads generated from Illumina ranged between 18– 20 million paired-end reads/lane with a good base quality from both ends (base quality > 30 represented 75%-85% of reads). The reads were aligned using 2 algorithms (Maq and Bowtie) and the methylation analysis was performed by Batman software (Bayesian Tool for Methylation Analysis). The creation of this genome-wide methylation map for AML permits the examination of the patterns for key genetic elements. Investigation of the 35,072 promoter regions identified 80 genes, which showed a significant differential methylation levels in leukemic cases in comparison to NBM; consistently high methylation levels in leukaemia were detected in the promoters of 70 genes e.g. DPP6, ID4, DCC, whereas high methylation levels in NBM, lost in leukaemia was observed in 10 genes e.g. ATF4. For each AML subtype, we also identified significant differentially methylated promoter regions e.g. PAX1 for t(8; 21), GRM7 for t(15; 17), NPM2 for NK. An analysis of gene body methylation identified 49 genes with significantly higher methylation in AML in comparison to NBM e.g. MYOD1 and 31 genes with a higher methylation in NBMs than AML e.g. GNG8. A similar analysis of 23,600 CpG islands identified 400 CpG islands with significant differential methylation levels between leukaemia and NBMs (212 CpG islands were found to have significantly increased methylation in leukaemia and 188 CpG islands had significantly higher methylation in NBMs). The pattern of methylation in CpG island “shores” (2 KB from either side of each CpG island) has been investigated and 312 CpG island shores showed a higher methylation in leukaemia and 88 CpG shores had a significant increase methylation levels in NBMs. This genome wide methylation map has been validated by using direct bisulfite sequencing of the regions identified above (Spearman r= 0.8, P <0.0001) and also by using Illumina Infinium assay (Spearman r= 0.7 P <0.0001) which interrogates regions at single representative CpGs. Comparison of previous array based gene expression data with this methylation map revealed a significant negative correlation between promoter methylation and gene expression (Pearson r= -0.9, P< 0.0001) while, gene body methylation showed a small negative correlation with gene expression, that was found in genes of CpG density >3% (Pearson r= -0.3, P< 0.0001). Conclusion: we have established a high-resolution (100bp) map of DNA methylation in AML and thus identified a novel list of genes, which have significantly differential methylation levels in AML. Disclosures: No relevant conflicts of interest to declare.

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DNA Methylation Independent Silencing of the RARα Gene Expression in Acute Myeloid Leukemia.

Blood ◽

10.1182/blood.v108.11.2225.2225 ◽

2006 ◽

Vol 108 (11) ◽

pp. 2225-2225

Author(s):

Annegret Glasow ◽

Angela Barrett ◽

Manuel Boix Chornet ◽

Rajeev Gupta ◽

Da-cheng Zhou ◽

...

Keyword(s):

Gene Expression ◽

Dna Methylation ◽

Retinoic Acid ◽

Cell Lines ◽

Cpg Island ◽

Synergistic Effects ◽

Cpg Islands ◽

Regulatory Region ◽

Histone H3 ◽

Acute Myeloid

Abstract All-trans-retinoic acid (ATRA) and the gene encoding retinoic acid receptor-α (RARα) have been implicated in the pathogenesis and treatment of acute promyelocytic leukemia (APL). Nevertheless, the role of these molecules in the pathogenesis and therapy of non-APL acute myeloid leukemias (AMLs) remains unclear. Previously we have shown that expression of the ATRA-inducible RARα2 isoform is downregulated in a variety of AML cell lines and increases with hematopoietic differentiation along the myelomonocytic lineage. Using quantitative real-time PCR we have now investigated expression of the RARα gene in primary AML cells (n=23) and report that as in AML cell lines the levels of RARα2 mRNAs are markedly reduced (by 48 fold in APL, p≤0.05, and by 52 fold in non-APL AML, p≤0.01) relative to cord blood (CB) derived CD33 (or CD34) positive cell population (n=4). However, in contrast to the AML cell lines and normal hematopoietic progenitors, the expression of the RARα1 isoform was also significantly reduced in primary AML samples (up to 26 fold, p≤0.05). Examination of potential mechanisms underlying the silencing of the RARα gene expression in AML revealed that the RARα2 promoter possesses two small CpG islands that are fully methylated in all AML cell lines examined. Consistent with the expression pattern of RARα1 a single CpG island in the RARα1 promoter region was unmethylated in all these samples. As expected from such results expression of RARα2, but not RARα1, could be stimulated with a DNA demethylating agent 5-aza-2′deoxycytidine and synergistic effects between 5-aza-2′deoxycytidine and ATRA were observed on both RARα2 expression and cellular differentiation of APL and non-APL AML cell lines. Extending this analysis to clinical material we have surprisingly discovered that RARα1 and RARα2 CpG islands are unmethylated in all AML patient samples, including 3 APL cases, suggesting that DNA methylation may not play a significant role in silencing of the RARα gene expression in primary AML cells. Chromatin immunoprecipitation of the RARα2 regulatory region with antibodies to specific histone modifications revealed presence of other negatively acting chromatin states in primary AML samples (relative to normal CD33 positive CB cells), including decreased histone H3 acetylation as well as decreased di- and tri-methylation of histone H3 lysine 4. A possibility that microRNAs targeting sequences in the common 3′-UTR regions of the RARα1 and α2 isoforms may also contribute to silencing of both RARα1 and α2 expression in AML cannot be excluded. These results underscore the complexities of mechanisms that are responsible for silencing of gene expression in AML and support the notion that diminished RARα expression contributes to leukemogenesis.

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The proto CpG island methylator phenotype of sessile serrated adenomas/polyps

10.1101/357178 ◽

2018 ◽

Cited By ~ 1

Author(s):

Hannah R. Parker ◽

Stephany Orjuela ◽

Andreia Martinho Oliveira ◽

Fabrizio Cereatti ◽

Matthias Sauter ◽

...

Keyword(s):

Gene Expression ◽

Dna Methylation ◽

Cpg Island ◽

Cpg Islands ◽

Normal Mucosa ◽

Cpg Island Methylator Phenotype ◽

Molecular Features ◽

Colon Cancers ◽

Methylator Phenotype ◽

Serrated Adenomas

AbstractSessile serrated adenomas/polyps (SSA/Ps) are the putative precursors of the ˜20% of colon cancers with the CpG island methylator phenotype (CIMP), but their molecular features are poorly understood. We used high-throughput analysis of DNA methylation and gene expression to investigate the epigenetic phenotype of SSA/Ps. Fresh-tissue samples of 17 SSA/Ps and (for comparison purposes) 15 conventional adenomas (cADNs)—each with a matched sample of normal mucosa— were prospectively collected during colonoscopy (total no. samples analyzed: 64). DNA and RNA were extracted from each sample. DNA was subjected to bisulfite next-generation sequencing to assess methylation levels at ˜2.7 million CpG sites located predominantly in gene regulatory regions and spanning 80.5Mb (˜2.5% of the genome); RNA was sequenced to define the samples’ transcriptomes. An independent series of 61 archival lesions was used for targeted verification of DNA methylation findings. Compared with normal mucosa samples, SSA/Ps and cADNs exhibited markedly remodeled methylomes. In cADNs, hypomethylated regions were far more numerous (18,417 vs 4288 in SSA/Ps) and rarely affected CpG islands/shores. SSA/Ps seemed to have escaped this wave of demethylation. Cytosine hypermethylation in SSA/Ps was more pervasive (hypermethylated regions: 22,147 vs 15,965 in cADNs; hypermethylated genes: 4938 vs 3443 in cADNs) and more extensive (region for region), and it occurred mainly within CpG islands and shores. Given its resemblance to the CIMP typical of SSA/Ps’ putative descendant colon cancers, we refer to the SSA/P methylation phenotype as proto-CIMP. Verification studies of six hypermethylated regions (3 SSA/P-specific and 3 common) demonstrated the high potential of DNA methylation markers for predicting the diagnosis of SSA/Ps and cADNs. Surprisingly, proto-CIMP in SSA/Ps was associated with upregulated gene expression (n=618 genes vs 349 that were downregulated); downregulation was more common in cADNs (n=712 vs 516 upregulated genes). The epigenetic landscape of SSA/Ps differs markedly from that of cADNs. These differences are a potentially rich source of novel tissue-based and noninvasive biomarkers that can add precision to the clinical management of the two most frequent colon-cancer precursors.

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DNA Methylation of Human Choline Kinase Alpha Promoter-Associated CpG Islands in MCF-7 Cells

Genes ◽

10.3390/genes12060853 ◽

2021 ◽

Vol 12 (6) ◽

pp. 853

Author(s):

Siti Aisyah Faten Mohamed Sa’dom ◽

Sweta Raikundalia ◽

Shaharum Shamsuddin ◽

Wei Cun See Too ◽

Ling Ling Few

Keyword(s):

Dna Methylation ◽

Transcription Factor ◽

Binding Site ◽

Promoter Activity ◽

Cytosine Methylation ◽

Cpg Island ◽

Cpg Islands ◽

Site Directed Mutagenesis ◽

Choline Kinase ◽

Mcf 7

Choline kinase (CK) is the enzyme catalyzing the first reaction in CDP-choline pathway for the biosynthesis of phosphatidylcholine. Higher expression of the α isozyme of CK has been implicated in carcinogenesis, and inhibition or downregulation of CKα (CHKA) is a promising anticancer approach. This study aimed to investigate the regulation of CKα expression by DNA methylation of the CpG islands found on the promoter of this gene in MCF-7 cells. Four CpG islands have been predicted in the 2000 bp promoter region of ckα (chka) gene. Six CpG island deletion mutants were constructed using PCR site-directed mutagenesis method and cloned into pGL4.10 vectors for promoter activity assays. Deletion of CpG4C region located between –225 and –56 significantly increased the promoter activity by 4-fold, indicating the presence of important repressive transcription factor binding site. The promoter activity of methylated full-length promoter was significantly lower than the methylated CpG4C deletion mutant by 16-fold. The results show that DNA methylation of CpG4C promotes the binding of the transcription factor that suppresses the promoter activity. Electrophoretic mobility shift assay analysis showed that cytosine methylation at MZF1 binding site in CpG4C increased the binding of putative MZF1 in nuclear extract. In conclusion, the results suggest that DNA methylation decreased the promoter activity by promoting the binding of putative MZF1 transcription factor at CpG4C region of the ckα gene promoter.

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Temporal changes in DNA methylation and RNA expression in a small song bird: within- and between-tissue comparisons

BMC Genomics ◽

10.1186/s12864-020-07329-9 ◽

2021 ◽

Vol 22 (1) ◽

Author(s):

Melanie Lindner ◽

Irene Verhagen ◽

Heidi M. Viitaniemi ◽

Veronika N. Laine ◽

Marcel E. Visser ◽

...

Keyword(s):

Gene Expression ◽

Dna Methylation ◽

Environmental Conditions ◽

Brain Regions ◽

Temporal Changes ◽

Egg Laying ◽

Gene Body ◽

Cpg Site ◽

Repeated Sampling ◽

Over Time

Abstract Background DNA methylation is likely a key mechanism regulating changes in gene transcription in traits that show temporal fluctuations in response to environmental conditions. To understand the transcriptional role of DNA methylation we need simultaneous within-individual assessment of methylation changes and gene expression changes over time. Within-individual repeated sampling of tissues, which are essential for trait expression is, however, unfeasible (e.g. specific brain regions, liver and ovary for reproductive timing). Here, we explore to what extend between-individual changes in DNA methylation in a tissue accessible for repeated sampling (red blood cells (RBCs)) reflect such patterns in a tissue unavailable for repeated sampling (liver) and how these DNA methylation patterns are associated with gene expression in such inaccessible tissues (hypothalamus, ovary and liver). For this, 18 great tit (Parus major) females were sacrificed at three time points (n = 6 per time point) throughout the pre-laying and egg-laying period and their blood, hypothalamus, ovary and liver were sampled. Results We simultaneously assessed DNA methylation changes (via reduced representation bisulfite sequencing) and changes in gene expression (via RNA-seq and qPCR) over time. In general, we found a positive correlation between changes in CpG site methylation in RBCs and liver across timepoints. For CpG sites in close proximity to the transcription start site, an increase in RBC methylation over time was associated with a decrease in the expression of the associated gene in the ovary. In contrast, no such association with gene expression was found for CpG site methylation within the gene body or the 10 kb up- and downstream regions adjacent to the gene body. Conclusion Temporal changes in DNA methylation are largely tissue-general, indicating that changes in RBC methylation can reflect changes in DNA methylation in other, often less accessible, tissues such as the liver in our case. However, associations between temporal changes in DNA methylation with changes in gene expression are mostly tissue- and genomic location-dependent. The observation that temporal changes in DNA methylation within RBCs can relate to changes in gene expression in less accessible tissues is important for a better understanding of how environmental conditions shape traits that temporally change in expression in wild populations.

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On the origin and evolutionary consequences of gene body DNA methylation

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1604666113 ◽

2016 ◽

Vol 113 (32) ◽

pp. 9111-9116 ◽

Cited By ~ 149

Author(s):

Adam J. Bewick ◽

Lexiang Ji ◽

Chad E. Niederhuth ◽

Eva-Maria Willing ◽

Brigitte T. Hofmeister ◽

...

Keyword(s):

Gene Expression ◽

Dna Methylation ◽

Recombinant Inbred Lines ◽

Dna Methyltransferases ◽

Inbred Lines ◽

Gene Body ◽

Gene Body Methylation ◽

Eutrema Salsugineum ◽

And Function ◽

Evolutionary Consequences

In plants, CG DNA methylation is prevalent in the transcribed regions of many constitutively expressed genes (gene body methylation; gbM), but the origin and function of gbM remain unknown. Here we report the discovery that Eutrema salsugineum has lost gbM from its genome, to our knowledge the first instance for an angiosperm. Of all known DNA methyltransferases, only CHROMOMETHYLASE 3 (CMT3) is missing from E. salsugineum. Identification of an additional angiosperm, Conringia planisiliqua, which independently lost CMT3 and gbM, supports that CMT3 is required for the establishment of gbM. Detailed analyses of gene expression, the histone variant H2A.Z, and various histone modifications in E. salsugineum and in Arabidopsis thaliana epigenetic recombinant inbred lines found no evidence in support of any role for gbM in regulating transcription or affecting the composition and modification of chromatin over evolutionary timescales.

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