Stage-specific differential DNA methylation data analysis during human erythropoiesis in chromosome 16

AbstractPrevious studies have generated controversial findings regarding the correlation between DNA methylation in the human genome and gene expression. Some reports have indicated that promoter methylation is negatively correlated with gene expression levels; however, in some cases, a poor or positive correlation was reported. Most previous findings were based on general trends observed with whole-genome data analysis. Here, we present a novel chromosome-specific statistical analysis design of empirical Bayes differential tests for five phases of erythroid development. To better understand the common methylation patterns of differentially methylated regions (DMRs) during specific stages, we defined differential phases for each CpG locus, based on a maximum log2 fold change. Analyzing hypermethylated and hypomethylated CpG loci separately showed variations in methylation patterns during erythropoiesis in the gene body, promoter and enhancer regions. Hypomethylated DMRs showed stronger associations with erythroid-specific enhancers at the differentiation start phase and with exons in the intermediate phase. To investigate the hypomethylated DMRs further, transcription factor binding site-enrichment analysis was conducted. This analysis highlighted novel transcription factors during each differentiation stage that were not detected by previous differential methylation data analysis. In contrast, hypermethylated DMRs showed a consistent methylation pattern over the different genomic regions. Thus, a closer examination of DNA methylation patterns in a single chromosome during each developmental stage can contribute to verify the association nature between gene expression and DNA methylation.

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5 Gene expression analysis and DNA methylation patterns of porcine somatic cell nuclear transfer blastocysts with high and low incidence of apoptosis

Reproduction Fertility and Development ◽

10.1071/rdv31n1ab5 ◽

2019 ◽

Vol 31 (1) ◽

pp. 128

Author(s):

L. Moley ◽

R. Jones ◽

R. Kaundal ◽

A. Thomas ◽

A. Benninghoff ◽

...

Keyword(s):

Gene Expression ◽

Dna Methylation ◽

Enrichment Analysis ◽

Epigenetic Reprogramming ◽

Reduced Representation ◽

False Discovery ◽

Reduced Representation Bisulfite Sequencing ◽

Methylation Patterns ◽

Dna Methylation Analysis ◽

Go Terms

Somatic cell NT (SCNT) efficiency remains poor, preventing the technology from being regularly used in the agricultural industry. It is believed that faulty epigenetic reprogramming of SCNT embryos leads to the low overall success. A clear apoptotic signature is associated with inappropriate gene expression and epigenomic aberrancies in many experimental cell culture systems, and we hypothesised that an apoptosis biomarker could be used to effectively separate properly reprogrammed porcine SCNT embryos from those that are destined to fail due to incomplete reprogramming. Therefore, our objective was to evaluate global gene expression and DNA methylation patterns in high- and low-apoptosis individual embryos in an effort to characterise the extent of genomic reprogramming that had taken place. Porcine SCNT blastocysts on Day 6 of development were stained with a nontoxic, noninvasive caspase activity reporter, and the top and bottom 20% of detected caspase activity were classified as high and low apoptosis, respectively (3 replicate cloning sessions; n=13 embryos per group). Genomic DNA and total RNA were isolated from each individual blastocyst. The RNA sequencing libraries were prepared using the Ovation SoLo RNA-Seq system (NuGen, San Carlos, CA, USA). Reduced representation bisulfite sequencing libraries were prepared for DNA methylation analysis using a modification of the single-cell reduced representation bisulfite sequencing global DNA methylation analysis approach detailed by Guo et al. (2015 Nat. Protoc. 10, 645-59). The RNA sequencing analysis using EdgeR (https://bioconductor.org/packages/release/bioc/html/edgeR.html) revealed 175 total differentially expressed genes (fold change ≥1.5; false discovery rate ≤0.05) between the high- and low-apoptosis SCNT embryos. This list of differentially expressed genes was used to perform enrichment analysis to identify overrepresented Gene Ontology (GO) terms or Kyoto Encyclopedia of Genes and Genomes pathways (DAVID Ease version 6.8 (https://david.ncifcrf.gov/) against the Sus scrofa background genome). However, no significantly enriched GO terms or pathways were identified (false discovery rate P>0.05). Analysis of global DNA methylation patterns between high- and low-apoptosis SCNT embryos using MethylKit (Akalin et al. 2012Genome Biol. 13, R87) revealed 335 differentially methylated 100-bp regions with at least 25% difference in methylation (adjusted P ≤ 0.01). Gene transcription start sites associated with these regions were used for enrichment analysis; again, no significant enrichment of GO terms or Kyoto Encyclopedia of Genes and Genomes pathways was identified. Principal component analysis of CpG methylation showed the low-apoptosis embryos clustering more tightly than the high-apoptosis embryos, which were highly scattered. Ongoing comparisons of high- and low-apoptosis cloned embryos with naturally fertilized embryos produced invivo may provide more information about which embryos were properly reprogrammed. Although we are still pursuing a link between reprogramming and gene expression in high- and low-apoptosis embryos, we conclude that these data support a model of stochastic epigenetic reprogramming following SCNT and reinforce the necessity of identifying embryos most likely to be successful due to proper epigenetic reprogramming in order to increase SCNT efficiency.

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DNA Methylation in Multiple Myeloma Is Weakly Associated with Gene Transcription

Blood ◽

10.1182/blood.v120.21.1285.1285 ◽

2012 ◽

Vol 120 (21) ◽

pp. 1285-1285

Author(s):

Sungwon Jung ◽

Kim Seungchan ◽

Molly Gale ◽

Rafael Fonseca ◽

John Carpten ◽

...

Keyword(s):

Gene Expression ◽

Dna Methylation ◽

Multiple Myeloma ◽

Pearson Correlation ◽

Enrichment Analysis ◽

Epigenetic Mechanism ◽

Methylation Data ◽

Rt Pcr ◽

Number Of Genes ◽

Unique Genes

Abstract Abstract 1285 Introduction: Recent studies have now demonstrated that both genic and global hypomethylation characterizes the multiple myeloma (MM) epigenome. We previously conducted a study to measure changes in DNA methylation at approximately 1500 CpG (GoldenGate Methylation BeadArray, Illumina) loci in 193 MM samples used in the Multiple Myeloma Research Consortium Genomics Initiative. The purpose of this study was to determine the extent to which DNA methylation changes in MM correlate to changes at the gene expression level. Methods: To evaluate this correlation we performed three independent analyses. In the first approach we analyzed matching gene expression and DNA methylation data using a Pearson correlation to assess the linear relationship between the datasets. In the second approach methylation and gene expression data from 193 matching samples were quantile-normalized and standardized across genes and samples. Methylation data was then discretized into three groups (methylated, unchanged, unmethylated) according to the mean and standard deviation of the values for each probe on the array. We then computed a t-test to compare the gene expression differences for each gene represented on the methylation array between samples in the methylated and unmethylated groups. We also performed a class enrichment analysis to assess DNA methylation/gene expression correlations that might be associated with hyperdiploidy or molecular subtype. In the third approach we performed RT-PCR and methylation pyrosequencing to validate results in a subset of samples used for the analysis and in an independent cohort comprised of 50 MM samples. Results: By Pearson correlation, only 31 (2.1%) CpG loci (corresponding to 24 unique genes) had a gene expression probe with a statistically significant correlation. Using the discretization approach, we identified 382 loci (25.3%, 309 unique genes) with methylation-expression correlations. Although we identified more correlations by discretizing the data, methylated and unmethylated samples were represented by an average of only 30 and 27 samples per group. We identified correlations where the directionality of methylation and expression values were either in the opposite (negative) or same direction (positive). Among the genes identified, the tumor suppressor genes CDKN2A and DLC1 interestingly demonstrated a positive correlation (methylated and expressed) and the IGF1R and IL17RB genes were negatively correlated (unmethylated and expressed). A class enrichment analysis revealed that samples with DLC1, CDKN2A, IGF1R or IL17RB methylation were associated with hyperdiploid MM. Conversely, samples that were unmethylation for these genes were generally non-hyperdiploid. These findings were validated by RT-PCR and methylation pyrosequencing. Conclusion: Overall, our findings demonstrated, albeit in a limited number of genes, that DNA methylation changes are weakly associated to gene expression. These data suggest that CpG methylation may have other functional consequences such as predisposing the genome to global gene transcriptional changes or chromosomal instability. While future studies are needed to determine the exact role of DNA methylation, we identified a number of genes regulated by an epigenetic mechanism with important clinical and biological implications to MM and warrant further study. Disclosures: No relevant conflicts of interest to declare.

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Examination of the dynamics of global DNA methylation pattern establishment during spermatogenesiss

Clinical & Investigative Medicine ◽

10.25011/cim.v30i4.2868 ◽

2007 ◽

Vol 30 (4) ◽

pp. 90

Author(s):

Kirsten Niles ◽

Sophie La Salle ◽

Christopher Oakes ◽

Jacquetta Trasler

Keyword(s):

Dna Methylation ◽

Germ Cell ◽

Germ Cells ◽

Epigenetic Modification ◽

Methylation Pattern ◽

Chromosome 9 ◽

Specific Gene ◽

Global Methylation ◽

Dna Methylation Pattern ◽

Methylation Patterns

Background: DNA methylation is an epigenetic modification involved in gene expression, genome stability, and genomic imprinting. In the male, methylation patterns are initially erased in primordial germ cells (PGCs) as they enter the gonadal ridge; methylation patterns are then acquired on CpG dinucleotides during gametogenesis. Correct pattern establishment is essential for normal spermatogenesis. To date, the characterization and timing of methylation pattern acquisition in PGCs has been described using a limited number of specific gene loci. This study aimed to describe DNA methylation pattern establishment dynamics during male gametogenesis through global methylation profiling techniques in a mouse model. Methods: Using a chromosome based approach, primers were designed for 24 regions spanning chromosome 9; intergenic, non-repeat, non-CpG island sequences were chosen for study based on previous evidence that these types of sequences are targets for testis-specific methylation events. The percent methylation was determined in each region by quantitative analysis of DNA methylation using real-time PCR (qAMP). The germ cell-specific pattern was determined by comparing methylation between spermatozoa and liver. To examine methylation in developing germ cells, spermatogonia from 2 day- and 6 day-old Oct4-GFP (green fluorescent protein) mice were isolated using fluorescence activated cell sorting. Results: As compared to liver, four loci were hypomethylated and five loci were hypermethylated in spermatozoa, supporting previous results indicating a unique methylation pattern in male germ cells. Only one region was hypomethylated and no regions were hypermethylated in day 6 spermatogonia as compared to mature spermatozoa, signifying that the bulk of DNA methylation is established prior to type A spermatogonia. The methylation in day 2 spermatogonia, germ cells that are just commencing mitosis, revealed differences of 15-20% compared to day 6 spermatogonia at five regions indicating that the most crucial phase of DNA methylation acquisition occurs prenatally. Conclusion: Together, these studies provide further evidence that germ cell methylation patterns differ from those in somatic tissues and suggest that much of methylation at intergenic sites is acquired during prenatal germ cell development. (Supported by CIHR)

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Abstract 40: Disturbed Flow Alters Genomewide DNA Methylation Patterns, Regulating Endothelial Gene Expression and Atherosclerosis

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvb.34.suppl_1.40 ◽

2014 ◽

Vol 34 (suppl_1) ◽

Author(s):

Jessilyn Dunn ◽

Haiwei Qiu ◽

Soyeon Kim ◽

Daudi Jjingo ◽

Ryan Hoffman ◽

...

Keyword(s):

Gene Expression ◽

Dna Methylation ◽

Dna Methyltransferase ◽

Methylation Status ◽

Western Diet ◽

Dependent Manner ◽

Gene Promoters ◽

Genome Wide ◽

Methylation Patterns ◽

Endothelial Gene Expression

Atherosclerosis preferentially occurs in arterial regions of disturbed blood flow (d-flow), which alters gene expression, endothelial function, and atherosclerosis. Here, we show that d-flow regulates genome-wide DNA methylation patterns in a DNA methyltransferase (DNMT)-dependent manner. We found that d-flow induced expression of DNMT1, but not DNMT3a or DNMT3b, in mouse arterial endothelium in vivo and in cultured endothelial cells by oscillatory shear (OS) compared to unidirectional laminar shear in vitro. The DNMT inhibitor 5-Aza-2’deoxycytidine (5Aza) or DNMT1 siRNA significantly reduced OS-induced endothelial inflammation. Moreover, 5Aza reduced lesion formation in two atherosclerosis models using ApoE-/- mice (western diet for 3 months and the partial carotid ligation model with western diet for 3 weeks). To identify the 5Aza mechanisms, we conducted two genome-wide studies: reduced representation bisulfite sequencing (RRBS) and transcript microarray using endothelial-enriched gDNA and RNA, respectively, obtained from the partially-ligated left common carotid artery (LCA exposed to d-flow) and the right contralateral control (RCA exposed to s-flow) of mice treated with 5Aza or vehicle. D-flow induced DNA hypermethylation in 421 gene promoters, which was significantly prevented by 5Aza in 335 genes. Systems biological analyses using the RRBS and the transcriptome data revealed 11 mechanosensitive genes whose promoters were hypermethylated by d-flow but rescued by 5Aza treatment. Of those, five genes contain hypermethylated cAMP-response-elements in their promoters, including the transcription factors HoxA5 and Klf3. Their methylation status could serve as a mechanosensitive master switch in endothelial gene expression. Our results demonstrate that d-flow controls epigenomic DNA methylation patterns in a DNMT-dependent manner, which in turn alters endothelial gene expression and induces atherosclerosis.

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TET2 coactivates gene expression through demethylation of enhancers

Science Advances ◽

10.1126/sciadv.aau6986 ◽

2018 ◽

Vol 4 (11) ◽

pp. eaau6986 ◽

Cited By ~ 35

Author(s):

Lu Wang ◽

Patrick A. Ozark ◽

Edwin R. Smith ◽

Zibo Zhao ◽

Stacy A. Marshall ◽

...

Keyword(s):

Gene Expression ◽

Dna Methylation ◽

Regulation Of Gene Expression ◽

Estrogen Receptor Α ◽

Dna Demethylation ◽

Estrogen Response ◽

Dna Base ◽

Modified Dna ◽

Global Increase ◽

Methylation Patterns

The tet methylcytosine dioxygenase 2 (TET2) enzyme catalyzes the conversion of the modified DNA base 5-methylcytosine to 5-hydroxymethylcytosine. TET2 is frequently mutated or dysregulated in multiple human cancers, and loss of TET2 is associated with changes in DNA methylation patterns. Here, using newly developed TET2-specific antibodies and the estrogen response as a model system for studying the regulation of gene expression, we demonstrate that endogenous TET2 occupies active enhancers and facilitates the proper recruitment of estrogen receptor α (ERα). Knockout of TET2 by CRISPR-CAS9 leads to a global increase of DNA methylation at enhancers, resulting in attenuation of the estrogen response. We further identified a positive feedback loop between TET2 and ERα, which further requires MLL3 COMPASS at these enhancers. Together, this study reveals an epigenetic axis coordinating a transcriptional program through enhancer activation via DNA demethylation.

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Nurse cell-derived small RNAs define paternal epigenetic inheritance in Arabidopsis

10.1101/2021.01.25.428150 ◽

2021 ◽

Author(s):

Jincheng Long ◽

James Walker ◽

Wenjing She ◽

Billy Aldridge ◽

Hongbo Gao ◽

...

Keyword(s):

Gene Expression ◽

Dna Methylation ◽

Small Rnas ◽

Nurse Cell ◽

De Novo ◽

Epigenetic Inheritance ◽

Genome Integrity ◽

Nurse Cells ◽

Male Germline ◽

Methylation Patterns

AbstractThe plant male germline undergoes DNA methylation reprogramming, which methylates genes de novo and thereby alters gene expression and facilitates meiosis. Why reprogramming is limited to the germline and how specific genes are chosen is unknown. Here, we demonstrate that genic methylation in the male germline, from meiocytes to sperm, is established by germline-specific siRNAs transcribed from transposons with imperfect sequence homology. These siRNAs are synthesized by meiocyte nurse cells (tapetum) via activity of the tapetum-specific chromatin remodeler CLASSY3. Remarkably, tapetal siRNAs govern germline methylation throughout the genome, including the inherited methylation patterns in sperm. Finally, we demonstrate that these nurse cell-derived siRNAs (niRNAs) silence germline transposons, thereby safeguarding genome integrity. Our results reveal that tapetal niRNAs are sufficient to reconstitute germline methylation patterns and drive extensive, functional methylation reprogramming analogous to piRNA-mediated reprogramming in animal germlines.

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DNA methylation is not a driver of gene expression reprogramming in young honey bee workers

10.1101/2021.03.12.435154 ◽

2021 ◽

Author(s):

Carlos A. M. Cardoso-Junior ◽

Boris Yagound ◽

Isobel Ronai ◽

Emily J. Remnant ◽

Klaus Hartfelder ◽

...

Keyword(s):

Gene Expression ◽

Dna Methylation ◽

Honey Bee ◽

Differential Gene Expression ◽

Social Contexts ◽

Gene Body ◽

Gene Body Methylation ◽

Differential Gene ◽

Honey Bee Workers ◽

Methylation Patterns

AbstractIntragenic DNA methylation, also called gene body methylation, is an evolutionarily-conserved epigenetic mechanism in animals and plants. In social insects, gene body methylation is thought to contribute to behavioral plasticity, for example between foragers and nurse workers, by modulating gene expression. However, recent studies have suggested that the majority of DNA methylation is sequence-specific, and therefore cannot act as a flexible mediator between environmental cues and gene expression. To address this paradox, we examined whole-genome methylation patterns in the brains and ovaries of young honey bee workers that had been subjected to divergent social contexts: the presence or absence of the queen. Although these social contexts are known to bring about extreme changes in behavioral and reproductive traits through differential gene expression, we found no significant differences between the methylomes of workers from queenright and queenless colonies. In contrast, thousands of regions were differentially methylated between colonies, and these differences were not associated with differential gene expression in a subset of genes examined. Methylation patterns were highly similar between brain and ovary tissues and only differed in nine regions. These results strongly indicate that DNA methylation is not a driver of differential gene expression between tissues or behavioral morphs. Finally, despite the lack of difference in methylation patterns, queen presence affected the expression of all four DNA methyltransferase genes, suggesting that these enzymes have roles beyond DNA methylation. Therefore, the functional role of DNA methylation in social insect genomes remains an open question.

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Methylation pattern polymorphism of cyp19a in Nile tilapia and hybrids

Open Life Sciences ◽

10.1515/biol-2018-0040 ◽

2018 ◽

Vol 13 (1) ◽

pp. 327-334 ◽

Cited By ~ 1

Author(s):

Xiaowu Chen ◽

Yonghua Zhao ◽

Yudong He ◽

Jinliang Zhao

Keyword(s):

Gene Expression ◽

Dna Methylation ◽

Methylation Level ◽

Molecular Mechanisms ◽

Methylation Pattern ◽

Male Ratio ◽

Sex Development ◽

Pattern Polymorphism ◽

Fish Hybrids

AbstractSkewed sex development is prevalent in fish hybrids. However, the histological observation and molecular mechanisms remain elusive. In this study, we showed that the interspecific hybrids of the two fish species, Oreochromis niloticus and Oreochromis aureus, had a male ratio of 98.02%. Microscopic examination revealed that the gonads of both male and female hybrids were developmentally retarded. Compared with the ovaries, the testes of both O. niloticus and hybrids showed higher DNA methylation level in two selected regions in the promoter of cyp19a, the gonadal aromatase gene that converts androgens into estrogens, cyp19a showed higher level gene expression in the ovary than in the testis in both O. niloticus and hybrid tilapia. Methylation and gene expression level of cyp19a were negative correlation between the testis and ovary. Gene transcription was suppressed by the methylation of the cyp19a promoter in vitro. While there is no obvious difference of the methylation level in testis or ovary between O. niloticus and hybrids. Thus, the DNA methylation of the promoter of cyp19a may be an essential component of the sex maintenance, but not a determinant of high male ratio and developmental retardation of gonads in tilapia hybrids.

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Abstract P098: An Epigenome-wide Study of Obesity in African American Youth and Young Adults: Novel Findings, Replication in Neutrophils and Relationship With Gene Expression

Circulation ◽

10.1161/circ.135.suppl_1.p098 ◽

2017 ◽

Vol 135 (suppl_1) ◽

Author(s):

Xiaoling Wang ◽

Yue Pan ◽

Haidong Zhu ◽

Guang Hao ◽

Xin Wang ◽

...

Keyword(s):

Gene Expression ◽

Older Adults ◽

Dna Methylation ◽

Young Adults ◽

Methylation Data ◽

Middle Aged ◽

Cpg Sites ◽

Genome Wide ◽

Obesity Status ◽

Youth And Young Adults

Background: Several large-scale epigenome wide association studies on obesity-related DNA methylation changes have been published and in total identified 46 CpG sites. These studies were conducted in middle-aged and older adults of Caucasians and African Americans (AAs) using leukocytes. To what extend these signals are independent of cell compositions as well as to what extend they may influence gene expression have not been systematically investigated. Furthermore, the high prevalence of obesity comorbidities in middle-aged or older population may hide or bias obesity itself related DNA methylation changes. Methods: In this study of healthy AA youth and young adults, genome wide DNA methylation data from leukocytes were obtained from three independent studies: EpiGO study (96 obese cases vs. 92 lean controls, aged 14-21, 50% females, test of interest is obesity status), LACHY study (284 participants from general population, aged 14-18, 50% females, test of interest is BMI), and Georgia Stress and Heart study (298 participants from general population, aged 18-38, 52% females, test of interest is BMI) using the Infinium HumanMethylation450 BeadChip. Genome wide DNA methylation data from purified neutrophils as well as genome wide gene expression data from leukocytes using Illumina HT12 V4 array were also obtained for the EpiGO samples. Results: The meta-analysis on the 3 cohorts identified 76 obesity related CpG sites in leukocytes with p<1х10 -7 . Out of the 46 previously identified CpG sites, 36 can be replicated in this AA youth and young adult sample with same direction and p<0.05. Out of the 107 CpG sites including the 36 replicated ones and the 71 newly identified ones, 71 CpG sites (66%) had their relationship with obesity replicated in purified neutrophils (p<0.05). The analysis on the cis regulation of the 107 CpG sites on gene expression showed that 59 CpG sites had at least one gene within 250kb having expression difference between obese cases and lean controls. Furthermore, out of the 59 CpG sites, 6 showed significantly negative correlations and 1 showed significantly positive correlation with the differentially expressed genes. These CpG sites located in SOCS3, CISH, ABCG1, PIM3 and PTGDS genes. Conclusion: In this study of AA youth and young adults, we identified novel CpG sites associated with obesity and replicated majority of the CpG sites previously identified in middle-aged and older adults. For the first time, we showed that majority of the obesity related CpG sites identified from leukocytes are not driven by cell compositions and provided the direct link between DNA methylation-gene expression-obesity status for 7 CpG sites in 5 genes.

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DNA methylation versus gene expression

Development ◽

10.1242/dev.83.supplement.31 ◽

1984 ◽

Vol 83 (Supplement) ◽

pp. 31-40

Author(s):

Adrian P. Bird

Keyword(s):

Gene Expression ◽

Dna Methylation ◽

Cell Division ◽

Methylation Pattern ◽

Current Evidence ◽

Evolutionary Perspective ◽

Cell Type ◽

Versus Gene ◽

Daughter Cells ◽

The Relationship

Vertebrate DNA is methylated at a high proportion of cytosine residues in the sequence CpG, and it has been suggested that the distribution of methylated and non-methylated CpGs in a given cell type influences the pattern of gene expression in those cells. Since a DNA methylation pattern is normally transmitted faithfully to daughter cells via cell division, this idea suggests an origin for stable, clonally inherited patterns of gene expression. This article discusses some of the current evidence for a relationship between DNA methylation and gene expression. Although the evidence is incomplete, it appears already that the relationship is variable: transcription of some genes is repressed by the presence of 5-methylcytosine at certain CpGs, and may be controlled by methylation, while transcription of other genes is indifferent to methylation. In attempting to explain this variability it is helpful to adopt an evolutionary perspective.

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