DNA Methylation Profiles of Vegans and Non-Vegetarians in the Adventist Health Study-2 Cohort

We sought to determine if DNA methylation patterns differed between vegans and non-vegetarians in the Adventist Health Study-2 cohort. Genome-wide DNA methylation derived from buffy coat was profiled in 62 vegans and 142 non-vegetarians. Using linear regression, methylation of CpG sites and genes was categorized or summarized according to various genic/intergenic regions and CpG island-related regions, as well as the promoter. Methylation of genes was measured as the average methylation of available CpG’s annotated to the nominated region of the respective gene. A permutation method defining the null distribution adapted from Storey et al. was used to adjust for false discovery. Differences in methylation of several CpG sites and genes were detected at a false discovery rate < 0.05 in region-specific and overall analyses. A vegan diet was associated predominantly with hypomethylation of genes, most notably methyltransferase-like 1 (METTL1). Although a limited number of differentially methylated features were detected in the current study, the false discovery method revealed that a much larger proportion of differentially methylated genes and sites exist, and could be detected with a larger sample size. Our findings suggest modest differences in DNA methylation in vegans and non-vegetarians, with a much greater number of detectable significant differences expected with a larger sample.

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Exploring Differentially Methylated Genes in Vulvar Squamous Cell Carcinoma

Cancers ◽

10.3390/cancers13143580 ◽

2021 ◽

Vol 13 (14) ◽

pp. 3580

Author(s):

Shatavisha Dasgupta ◽

Patricia C. Ewing-Graham ◽

Sigrid M. A. Swagemakers ◽

Thierry P. P. van den Bosch ◽

Peggy N. Atmodimedjo ◽

...

Keyword(s):

Dna Methylation ◽

Squamous Cell Carcinoma ◽

Cell Carcinoma ◽

Squamous Cell ◽

Dna Sequences ◽

Cpg Island ◽

Epigenetic Modification ◽

Vulvar Squamous Cell Carcinoma ◽

Differentially Methylated Genes ◽

Methylation Profiling

DNA methylation is the most widely studied mechanism of epigenetic modification, which can influence gene expression without alterations in DNA sequences. Aberrations in DNA methylation are known to play a role in carcinogenesis, and methylation profiling has enabled the identification of biomarkers of potential clinical interest for several cancers. For vulvar squamous cell carcinoma (VSCC), however, methylation profiling remains an under-studied area. We sought to identify differentially methylated genes (DMGs) in VSCC, by performing Infinium MethylationEPIC BeadChip (Illumina) array sequencing, on a set of primary VSCC (n = 18), and normal vulvar tissue from women with no history of vulvar (pre)malignancies (n = 6). Using a false-discovery rate of 0.05, beta-difference (Δβ) of ± 0.5, and CpG-island probes as cut-offs, 199 DMGs (195 hyper-methylated, 4 hypo-methylated) were identified for VSCC. Most of the hyper-methylated genes were found to be involved in transcription regulator activity, indicating that disruption of this process plays a vital role in VSCC development. The majority of VSCCs harbored amplifications of chromosomes 3, 8, and 9. We identified a set of DMGs in this exploratory, hypothesis-generating study, which we hope will facilitate epigenetic profiling of VSCCs. Prognostic relevance of these DMGs deserves further exploration in larger cohorts of VSCC and its precursor lesions.

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Stage-differentiated modelling of DNA methylation landscapes uncovers salient biomarkers and prognostic signatures in colorectal cancer progression

10.21203/rs.3.rs-244816/v1 ◽

2021 ◽

Author(s):

Sangeetha Muthamilselvan ◽

Abirami Raghavendran ◽

Ashok Palaniappan

Keyword(s):

Colorectal Cancer ◽

Dna Methylation ◽

Cpg Island ◽

Early Stage ◽

P Value ◽

Consensus Analysis ◽

One Stage ◽

Differentially Methylated Genes ◽

Methylation Patterns ◽

The Impact

Abstract Background: Aberrant DNA methylation acts epigenetically to skew the gene transcription rate up or down, with causative roles in the etiology of cancers. However research on the role of DNA methylation in driving the progression of cancers is limited. In this study, we have developed a comprehensive computational framework for the stage-differentiated modelling of DNA methylation landscapes in colorectal cancer (CRC), and unravelled significant stagewise signposts of CRC progression. Methods: The methylation β - matrix was derived from the public-domain TCGA data, converted into M-value matrix, annotated with AJCC stages, and analysed for stage-salient genes using multiple approaches involving stage-differentiated linear modelling of methylation patterns and/or expression patterns. Differentially methylated genes (DMGs) were identified using a contrast against controls (adjusted p-value <0.001 and |log fold-change of M-value| >2). These results were filtered using a series of all possible pairwise stage contrasts (p-value <0.05) to obtain stage-salient DMGs. These were then subjected to a consensus analysis, followed by Kaplan–Meier survival analysis to evaluate the impact of methylation patterns of consensus stage-salient biomarkers on disease prognosis.Results: We found significant genome-wide changes in methylation patterns in cancer cases relative to controls agnostic of stage. Our stage-differentiated analysis yielded the following stage-salient genes: one stage-I gene (FBN1), one stage-II gene (FOXG1), one stage-III gene (HCN1) and four stage-IV genes (NELL1, ZNF135, FAM123A, LAMA1). All the biomarkers were hypermethylated, indicating down-regulation and signifying a CpG island Methylator Phenotype (CIMP) manifestation. A significant prognostic signature consisting of FBN1 and FOXG1 survived all the steps of our analysis pipeline, and represents a novel early-stage biomarker. Conclusions: We have designed a workflow for stage-differentiated consensus analysis, and identified stage-salient diagnostic biomarkers and an early-stage prognostic biomarker panel. Our studies further yield a novel CIMP-like signature of potential clinical import underlying CRC progression.

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RPS19 and JAK2 Are Not Silenced by DNA Methylation in Diamond-Blackfan Anemia.

Blood ◽

10.1182/blood.v108.11.1312.1312 ◽

2006 ◽

Vol 108 (11) ◽

pp. 1312-1312

Author(s):

Jaroslav Jelinek ◽

Jean-Pierre J. Issa ◽

Rong He ◽

Radek Cmejla ◽

Jana Cmejlova ◽

...

Keyword(s):

Dna Methylation ◽

Tyrosine Kinase ◽

Ribosomal Proteins ◽

Cpg Island ◽

Methylation Frequency ◽

Diamond Blackfan Anemia ◽

Cpg Sites ◽

Causal Therapy ◽

Epigenetic Suppression ◽

Control Samples

Abstract Diamond Blackfan Anemia (DBA) is a congenital disorder characterized by decreased red blood cell production accompanied by developmental abnormalities in 30% patients. Twenty-five percent of DBA patients display heterozygous mutations of ribosomal protein S19 (RPS19) on chromosome 19q13.2. No mutations were found in genes for other ribosomal proteins of the translation initiation complex. Although a second DBA locus has been proposed on in the region 8p23.3–8p22, the precise molecular defect is not known in 75% of DBA patients. The exact mechanism of how RPS19 mutations affect erythropoiesis remains unclear. Haploinsufficency of RPS19 may hamper translation machinery important for rapid erythroid differentiation. Reduced gene expression of a cluster of ribosomal proteins including RPS19 in DBA patients was recently reported. No causal therapy for DBA is available, with the exception of bone marrow transplantation. Some DBA patients benefit therapeutically from corticosteroids, cyclosporine A, or metoclopramide. Recently, a long-lasting remission was described in a DBA patient treated with valproic acid, a histone deacetylase inhibitor, suggesting epigenetic suppression of genes critical for erythropoiesis may be involved in the pathogenesis of DBA. DNA methylation of promoter-associated CpG islands is an epigenetic modification resulting in transcriptional silencing functionally equivalent to a loss-of-function mutation. Constitutive activation of JAK2 tyrosine kinase by a somatic V617F mutation leads to excessive erythropoiesis in polycythemia vera, an antonym of DBA. We hypothesized that silencing by DNA methylation of promoter-associated CpG island of the RPS19 or JAK2 genes may play a role in DBA. To test the hypothesis, we analyzed DNA methylation of RPS19 and JAK2 genes in 14 patients from the Czech DBA Registry. Genomic DNA isolated from blood cells of 3 DBA patients carrying heterozygous RPS19 mutations, 11 DBA patients without RPS19 mutation and 4 control samples was treated with bisulfite to convert all unmethylated cytosines to uracils while methylated cytosines were spared from the conversion. A region spanning 13 CpG sites positioned from 1–160 bases downstream from transcription start site (TSS) of RPS19 gene was PCR amplified and cloned in a sequencing vector. Individual bacterial clones were isolated and PCR inserts were sequenced in 8–12 clones per sample. Bisulfite cloning and sequencing revealed that more than 99% of CpG sites were converted to TpG and thus not methylated either in DBA samples (only 4/1466 CpG sites were methylated, methylation frequency was 0.3%) or control samples (2/555 CpG sites methylated, methylation frequency 0.4%). To explore a possibility of epigenetic suppression of erythropoietin signaling in DBA we analyzed DNA methylation of the CpG island of JAK2 tyrosine kinase gene in the same set of samples. Bisulfite-treated DNA was PCR amplified and T/C polymorphisms corresponding to unmethylated or methylated CpG sites were quantified by pyrosequencing. All DBA and control samples showed the absence of DNA methylation at four CpG sites located 12 to 25 bases downstream of TSS. We conclude that epigenetic silencing by DNA methylation is not involved in the expression of ribosomal structural protein RPS19; neither it affects the expression of a transducer of erythropoietin signaling JAK2 tyrosine kinase.

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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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Epigenome-Wide Association Study of Prostate Cancer in African Americans Identifies DNA Methylation Biomarkers for Aggressive Disease

Biomolecules ◽

10.3390/biom11121826 ◽

2021 ◽

Vol 11 (12) ◽

pp. 1826

Author(s):

Yifan Xu ◽

Chia-Wen Tsai ◽

Wen-Shin Chang ◽

Yuyan Han ◽

Maosheng Huang ◽

...

Keyword(s):

Prostate Cancer ◽

Dna Methylation ◽

African American ◽

African Americans ◽

Zinc Finger Protein ◽

African American Men ◽

Pathway Enrichment Analysis ◽

Cpg Sites ◽

Differentially Methylated Genes ◽

Incidence And Mortality

DNA methylation plays important roles in prostate cancer (PCa) development and progression. African American men have higher incidence and mortality rates of PCa than other racial groups in U.S. The goal of this study was to identify differentially methylated CpG sites and genes between clinically defined aggressive and nonaggressive PCa in African Americans. We performed genome-wide DNA methylation profiling in leukocyte DNA from 280 African American PCa patients using Illumina MethylationEPIC array that contains about 860K CpG sties. There was a slight increase of overall methylation level (mean β value) with the increasing Gleason scores (GS = 6, GS = 7, GS ≥ 8, P for trend = 0.002). There were 78 differentially methylated CpG sites with P < 10−4 and 9 sites with P < 10−5 in the trend test. We also found 77 differentially methylated regions/genes (DMRs), including 10 homeobox genes and six zinc finger protein genes. A gene ontology (GO) molecular pathway enrichment analysis of these 77 DMRs found that the main enriched pathway was DNA-binding transcriptional factor activity. A few representative DMRs include HOXD8, SOX11, ZNF-471, and ZNF-577. Our study suggests that leukocyte DNA methylation may be valuable biomarkers for aggressive PCa and the identified differentially methylated genes provide biological insights into the modulation of immune response by aggressive PCa.

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Neonatal Lead (Pb) Exposure and DNA Methylation Profiles in Dried Bloodspots

International Journal of Environmental Research and Public Health ◽

10.3390/ijerph17186775 ◽

2020 ◽

Vol 17 (18) ◽

pp. 6775

Author(s):

Luke Montrose ◽

Jaclyn M. Goodrich ◽

Masako Morishita ◽

Joseph Kochmanski ◽

Zachary Klaver ◽

...

Keyword(s):

Dna Methylation ◽

Inductively Coupled Plasma ◽

Housing Stock ◽

The United States ◽

P Value ◽

Inductively Coupled ◽

Cpg Sites ◽

Differentially Methylated Genes ◽

The World ◽

Pb Exposure

Lead (Pb) exposure remains a major concern in the United States (US) and around the world, even following the removal of Pb from gasoline and other products. Environmental Pb exposures from aging infrastructure and housing stock are of particular concern to pregnant women, children, and other vulnerable populations. Exposures during sensitive periods of development are known to influence epigenetic modifications which are thought to be one mechanism of the Developmental Origins of Health and Disease (DOHaD) paradigm. To gain insights into early life Pb exposure-induced health risks, we leveraged neonatal dried bloodspots in a cohort of children from Michigan, US to examine associations between blood Pb levels and concomitant DNA methylation profiles (n = 96). DNA methylation analysis was conducted via the Infinium MethylationEPIC array and Pb levels were assessed via high resolution inductively coupled plasma mass spectrometry (HR-ICP-MS). While at-birth Pb exposure levels were relatively low (average 0.78 µg/dL, maximum of 5.27 ug/dL), we identified associations between DNA methylation and Pb at 33 CpG sites, with the majority (82%) exhibiting reduced methylation with increasing Pb exposure (q < 0.2). Biological pathways related to development and neurological function were enriched amongst top differentially methylated genes by p-value. In addition to increases/decreases in methylation, we also demonstrate that Pb exposure is related to increased variability in DNA methylation at 16 CpG sites. More work is needed to assess the accuracy and precision of metals assessment using bloodspots, but this study highlights the utility of this unique resource to enhance environmental epigenetics research around the world.

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Genome-Wide Analysis of DNA Methylation in Human Amnion

The Scientific World JOURNAL ◽

10.1155/2013/678156 ◽

2013 ◽

Vol 2013 ◽

pp. 1-11 ◽

Cited By ~ 8

Author(s):

Jinsil Kim ◽

Mitchell M. Pitlick ◽

Paul J. Christine ◽

Amanda R. Schaefer ◽

Cesar Saleme ◽

...

Keyword(s):

Dna Methylation ◽

Preterm Birth ◽

Cpg Island ◽

Biological Data ◽

Valuable Insight ◽

Sequencing Analysis ◽

Human Amnion ◽

Cpg Dinucleotides ◽

Genome Wide ◽

Differentially Methylated Genes

The amnion is a specialized tissue in contact with the amniotic fluid, which is in a constantly changing state. To investigate the importance of epigenetic events in this tissue in the physiology and pathophysiology of pregnancy, we performed genome-wide DNA methylation profiling of human amnion from term (with and without labor) and preterm deliveries. Using the Illumina Infinium HumanMethylation27 BeadChip, we identified genes exhibiting differential methylation associated with normal labor and preterm birth. Functional analysis of the differentially methylated genes revealed biologically relevant enriched gene sets. Bisulfite sequencing analysis of the promoter region of the oxytocin receptor (OXTR) gene detected two CpG dinucleotides showing significant methylation differences among the three groups of samples. Hypermethylation of the CpG island of the solute carrier family 30 member 3 (SLC30A3) gene in preterm amnion was confirmed by methylation-specific PCR. This work provides preliminary evidence that DNA methylation changes in the amnion may be at least partially involved in the physiological process of labor and the etiology of preterm birth and suggests that DNA methylation profiles, in combination with other biological data, may provide valuable insight into the mechanisms underlying normal and pathological pregnancies.

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DNA Methylation Analysis of 807 Genes in Chronic Myeloid Leukemia and Acute Promyelocytic Leukemia.

Blood ◽

10.1182/blood.v110.11.2122.2122 ◽

2007 ◽

Vol 110 (11) ◽

pp. 2122-2122 ◽

Cited By ~ 1

Author(s):

Hyang-Min Byun ◽

Shahrooz Eshaghian ◽

Jia Yi Jiang ◽

Si Ho Choi ◽

John Soussa ◽

...

Keyword(s):

Dna Methylation ◽

Cpg Island ◽

Blast Crisis ◽

Bimodal Distribution ◽

Epigenetic Therapy ◽

Dna Hypermethylation ◽

Cpg Sites ◽

Genetic Translocation ◽

Methylator Phenotype ◽

Hypermethylated Genes

Abstract DNA methylation changes are a common finding in leukemia, and hypermethylation of CpG island promoters is associated with aberrant gene silencing. Some abnormal cancer related methylation changes have been associated with clinical phenotype including pathologic features, prognosis, and treatment response. However, other DNA methylation changes do not appear to have phenotypic consequences and may reflect a stochastic event or a downstream event of tumorogenesis, such as the CpG island methylator phenotype (CIMP). In order to obtain a better understanding of the DNA methylation changes found in leukemia we analyzed 18 acute promyelocytic leukemia (APL) and 36 chronic myeloid leukemia (CML) patients. We specifically chose to study APL and CML as these leukemia are initiated by specific genetic translocation events, t(15:17) and t(9:22) respectively. To measure the DNA methylation status, we used the GoldenGate Assay for Methylation and BeadArray technology from Illumina, Inc. The Standard Methylation Cancer Panel I from Illumina interrogates 1505 CpG sites, selected from 807 genes (231 genes contain one CpG site per gene, 463 genes contain two CpG sites and 114 genes have three or more CpG sites). In our study we found 142 and 269 genes that were hypermethylated in CML and APL. 31 genes were uniquely hypermethylated in CML, 158 genes were hypermethylated only in APL, and 111 genes were hypermethylated in both leukemias. There was a unique pattern of hypermethylated genes in each cancer; such there was a high concordance of hypermethylated genes within each leukemia type. These data suggest that the epigenetic events were a result of the genetic translocation BCR/ABL or PML/RARα (associated with chromosomal aberrations t(9:22) or t(15:17)) that initiates these leukemias. Analysis of the number of hypermethylated genes in these two leukemias showed a bimodal distribution suggestive of CIMP, however, closer examination showed that this bimodal distribution could be attributed to the two different types of leukemia. APL patients had mean of 280 genes hypermethylated while CML patients only had a mean of 193 genes hypermethylated. APL had a stronger methylator phenotype than CML for the loci studied, which underscores the possible relationship of CIMP to a genetic phenotype. Subset analysis of our CML samples by chronic phase (23 patients), accelerated phase (5 patients), and blast crisis (8 patients) revealed 42 genes that became hypermethylated with progression of CML. It is possible that hypermethylation of these genes are clinically important in the leukemia phenotype, and maybe targets for epigenetic therapy. We examined the DNA methylation changes induced by the DNA methylation inhibitor, azacitidine, in a patient with blast crisis CML and refractory to imatinib mesylate therapy. Azacitidine could reverse the aberrant hypermethylation associated with progression of CML to blast crisis and supports the use of this drug as an epigenetic therapy. Our data show that the majority of DNA hypermethylation events in leukemia are dependent on genetic events, but there is a subset of DNA hypermethylation events that are involved in the progression of leukemia and may be therapeutically reversed by DNA methylation inhibitors.

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Epigenome-450K-wide methylation signatures of active cigarette smoking: The Young Finns Study

Bioscience Reports ◽

10.1042/bsr20200596 ◽

2020 ◽

Vol 40 (7) ◽

Author(s):

Pashupati P. Mishra ◽

Ismo Hänninen ◽

Emma Raitoharju ◽

Saara Marttila ◽

Binisha H. Mishra ◽

...

Keyword(s):

Dna Methylation ◽

Olfactory Receptor ◽

Cpg Island ◽

Receptor Activity ◽

Gene Set Analysis ◽

Gene Set ◽

Cpg Sites ◽

Gene Sets ◽

The Impact ◽

Young Finns Study

Abstract Smoking as a major risk factor for morbidity affects numerous regulatory systems of the human body including DNA methylation. Most of the previous studies with genome-wide methylation data are based on conventional association analysis and earliest threshold-based gene set analysis that lacks sensitivity to be able to reveal all the relevant effects of smoking. The aim of the present study was to investigate the impact of active smoking on DNA methylation at three biological levels: 5′-C-phosphate-G-3′ (CpG) sites, genes and functionally related genes (gene sets). Gene set analysis was done with mGSZ, a modern threshold-free method previously developed by us that utilizes all the genes in the experiment and their differential methylation scores. Application of such method in DNA methylation study is novel. Epigenome-wide methylation levels were profiled from Young Finns Study (YFS) participants’ whole blood from 2011 follow-up using Illumina Infinium HumanMethylation450 BeadChips. We identified three novel smoking related CpG sites and replicated 57 of the previously identified ones. We found that smoking is associated with hypomethylation in shore (genomic regions 0–2 kilobases from CpG island). We identified smoking related methylation changes in 13 gene sets with false discovery rate (FDR) ≤ 0.05, among which is olfactory receptor activity, the flagship novel finding of the present study. Overall, we extended the current knowledge by identifying: (i) three novel smoking related CpG sites, (ii) similar effects as aging on average methylation in shore, and (iii) a novel finding that olfactory receptor activity pathway responds to tobacco smoke and toxin exposure through epigenetic mechanisms.

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Homocysteine Inhibits Cyclin A Promoter Methylation Via DNMT3 Inactivation in Human Endothelial Cells.

Blood ◽

10.1182/blood.v108.11.1822.1822 ◽

2006 ◽

Vol 108 (11) ◽

pp. 1822-1822

Author(s):

S. Jamaluddin ◽

Irene Chen ◽

Fan Yang ◽

Xiaoming Liu ◽

Andrew I. Schafer ◽

...

Keyword(s):

Dna Methylation ◽

Cpg Island ◽

Trichostatin A ◽

Umbilical Vein ◽

Gc Content ◽

Pcr Amplification ◽

Cyclin A ◽

Methyl Transferase ◽

Cpg Sites ◽

Transcriptional Inhibition

Abstract Hyperhomocysteinemia is a significant and independent risk factor for cardiovascular disease. We reported previously that homocysteine (Hcy) inhibits endothelial cell (EC) growth by transcriptional inhibition of the cyclin A gene. This is associated with an increase in S-adenosylhomocysteine, a potent inhibitor of methyltransferase. We hypothesized that Hcy inhibits EC growth and cyclin A transcription via hypomethylation and studied the effect of Hcy on epigenetic regulation of the cyclin A gene in EC. We found that the levels of cyclin A mRNA were significantly suppressed by azacytidne (AZC), a potent DNA methyl transferase (DNMT) inhibitor, in human umbilical vein EC (HUVEC). The cyclin A promoter (−518/256) is rich in GC content (59.1%), and has a CpG island spanning a 477 bp region (−277/200). Bisulphite sequencing followed by PCR amplification of the cyclin A core (−267/37) promoter, which contains 23 CpG sites serving as potential methylation sites, showed that Hcy (50 mM) eliminated methylation at two CpG sites, position 1 and 35, in the cyclin A promoter. Hcy selectively inhibited the activity of DNA methyl transferase 1 (DNMT1) by 40%, and had no effect on DNA methyl transferase 3 (DNMT3) activity in HUVEC. Furthermore, chromatin immunoprecipitation (ChIP) assays demonstrated that Hcy reduced the binding of methyl CpG binding protein 2 (MeCP2) and increased the binding of acetylated histone H3 and H4 in the cyclin A promoter in HUVEC but not in human aortic smooth muscle cells. The binding of MeCP2 to the cyclin A promoter was completely suppressed by AZC and trichostatin A, a histone deacetylase (HDAC) inhibitor, indicating that HDAC and DNA methylation mediate MeCP2 binding to cyclin A promoter. Finally, adenovirus tranduced DNMT1 overexpression, but not DNMT3, reversed Hcy-induced growth inhibition. In conclusion, we found that Hcy inhibits EC growth by suppressing cyclin A transcription, and that Hcy exerts this action by inhibiting DNA methylation through the suppression of DNMT1 and HDAC activity.

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