223 ABERRENT TIMP-2 GENE EXPRESSION IN CLONED BOVINE NEONATAL PLACENTA IS DUE TO ABNORMAL EPIGENETIC MODIFICATIONS

A global proteomics approach by 2-D gel electrophoresis and mass spectrometry was used to analyze the differential protein patterns of three placentae obtained after postnatal death of fetuses from SCNT of Korean Native cattle and three normal placentae obtained after birth of AI fetuses, as previously reported (Kim et al. 2004 Reprod. Fertil. Develop. 16, 145). One differentially up-regulated proteins in SCNT placenta was identified as TIMP-2 protein that is related to extracellular matrix degradation and tissue remodeling processes. To ensure that the identified protein was truly up-regulated in SCNT placenta, a small portion of the protein lysates were subjected to Western blotting with the antibodies against TIMP-2 and β-actin protein. Band images were scanned with a GT-6000 Scanner (Epson-Seiko, Suwa, Nagano, Japan) and densitometric analyses were performed using NIH Image (version 1.56). Proteins were normalized against the levels of β-actin protein. Indeed, Western blot analysis revealed a significant increase in TIMP-2 protein level (about 5-fold) in SCNT placenta compared with normal. Epigenetic programming of the genome by DNA methylation and histone modification ensures proper gene expression during development. To examine how the TIMP-2 gene was programmed to be active in SCNT placenta, we first investigated the status of DNA methylation in the promoter region of the TIMP-2 gene. Sodium bisulfite mapping revealed that most CpG sites in SCNT placenta cells were unmethylated in agreement with expression of TIMP-2 compared with those in normal placenta cells, suggesting that TIMP-2 gene programming by hypomethylation of CpG sites in the promoter region was responsible for the expression in SCNT placenta. We then examined histone modification of the nucleosome in the coding region of the TIMP-2 gene of SCNT placenta cells and normal placenta cells. Chromatin immunoprecipitation (ChIP) assay revealed that H3-K9/K14 acetylation of the TIMP-2 locus in SCNT placenta cells was about 2-fold higher than in normal placenta cells. Therefore, it is postulated that the SCNT placenta at the end of gestation seems to be unusual in tissue remodeling via improper expression of TIMP-2 by abnormal epigenetic modification, and that may affect placental abnormality such as enlarged and dysfunctional placentae as reported in cloned bovine and mouse.

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Association of Aberrant DNA Methylation Level in the CD4 and JAK-STAT-Pathway-Related Genes with Mastitis Indicator Traits in Chinese Holstein Dairy Cattle

Animals ◽

10.3390/ani12010065 ◽

2021 ◽

Vol 12 (1) ◽

pp. 65

Author(s):

Tahir Usman ◽

Nawab Ali ◽

Yachun Wang ◽

Ying Yu

Keyword(s):

Gene Expression ◽

Dna Methylation ◽

Dairy Cattle ◽

Methylation Level ◽

Promoter Region ◽

Promoter Regions ◽

Cpg Sites ◽

Cell Counts ◽

Aberrant Dna Methylation ◽

Stat Pathway

The present study was designed to evaluate the gene expression and DNA methylation level in the promoter region of the CD4 and the JAK-STAT-pathway-related genes. A total of 24 samples were deployed in the gene expression and 118 samples were used in the DNA methylation study. Student’s t-tests were used to analyze the gene expression and DNA methylation. The evaluation of DNA methylation in promoter regions of JAK2 and STAT5A revealed hypo-methylation levels of CpG sites and higher gene expression in cows diagnosed with mastitis as compared to the healthy control, and vice versa in those with CD4. DNA methylation was negatively correlated with gene expression in JAK2, STAT5A, and CD4 genes. Six, two, and four active transcription factors were identified on the CpG sites in the promoter regions of JAK2, STAT5A, and CD4 genes, respectively. Regarding correlation analysis, the DNA methylation levels of CD4 showed significantly higher positive correlations with somatic cell counts (p < 0.05). Findings of the current study inferred that aberrant DNA methylation in the CpG sites at the 1 kb promoter region in JAK2, STAT5A, and CD4 genes due to mastitis in cows can be used as potential epigenetic markers to estimate bovine mastitis susceptibility in dairy cattle.

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Correlation Patterns Between DNA Methylation and Gene Expression in The Cancer Genome Atlas

Cancer Informatics ◽

10.1177/1176935119828776 ◽

2019 ◽

Vol 18 ◽

pp. 117693511982877 ◽

Cited By ~ 28

Author(s):

John CG Spainhour ◽

Hong Seo Lim ◽

Soojin V Yi ◽

Peng Qiu

Keyword(s):

Gene Expression ◽

Dna Methylation ◽

Promoter Region ◽

The Cancer Genome Atlas ◽

Gene Body ◽

Cpg Sites ◽

Cpg Site ◽

Cancer Genome Atlas ◽

Genome Atlas

Background: DNA methylation is a form of epigenetic modification that has been shown to play a significant role in gene regulation. In cancer, DNA methylation plays an important role by regulating the expression of oncogenes. The role of DNA methylation in the onset and progression of various cancer types is now being elucidated as more large-scale data become available. The Cancer Genome Atlas (TCGA) provides a wealth of information for the analysis of various molecular aspects of cancer genetics. Gene expression data and DNA methylation data from TCGA have been used for a variety of studies. A traditional understanding of the effects of DNA methylation on gene expression has linked methylation of CpG sites in the gene promoter region with the decrease in gene expression. Recent studies have begun to expand this traditional role of DNA methylation. Results: Here we present a pan-cancer analysis of correlation patterns between CpG methylation and gene expression. Using matching patient data from TCGA, 33 cancer-specific correlations were calculated for each CpG site and the expression level of its corresponding gene. These correlations were used to identify patterns on a per-site basis as well as patterns of methylation across the gene body. Using these identified patterns, we found genes that contain conflicting methylation signals beyond the commonly accepted association between the promoter region methylation and silencing of gene expression. Beyond gene body methylation in whole, we examined individual CpG sites and show that, even in the same gene body, some sites can have a contradictory effect on gene expression in cancers. Conclusions: We observed that within promoter regions there was a substantial amount of positive correlation between methylation and gene expression, which contradicts the commonly accepted association. We observed that the correlation between CpG methylation and gene expression does not exhibit in a tissue-specific manner, suggesting that the effects of methylation on gene expression are largely tissue independent. The analysis of correlation associated with the location of the CpG site in the gene body has led to the identification of several different methylation patterns that affect gene expression, and several examples of methylation activating gene expression were observed. Distinctly opposing or conflicting effects were seen in close proximity on the gene body, where negative and positive correlations were seen at the neighboring CpG sites.

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EPIGENETIC ALTERATIONS ASSOCIATED WITH PREMATURE OVARIAN FAILURE

Clinical & Investigative Medicine ◽

10.25011/cim.v31i4.4804 ◽

2008 ◽

Vol 31 (4) ◽

pp. 11

Author(s):

Manda Ghahremani ◽

Courtney W Hannah ◽

Maria Peneherrera ◽

Karla L Bretherick ◽

Margo R Fluker ◽

...

Keyword(s):

Dna Methylation ◽

Premature Ovarian Failure ◽

Promoter Region ◽

Gene Promoter ◽

Ovarian Failure ◽

P Value ◽

Epigenetic Alterations ◽

Methylation Array ◽

Cpg Sites ◽

Deficient Mice

Background/Purpose: Premature ovarian failure (POF) affects 1% of women with a largely idiopathic and poorly understood etiology. The objective of this study was to identify specific epigenetic alterations by measuring DNA methylation of gene regulatory regions in women with POF vs. controls. Methods: Blood samples were collected from idiopathic POFpatients (Amenorrhea for at least 3 months and 2 serum FSH levels of > 40mIU/ml obtained > 1 month apart prior to age 40) and control women (CW) (healthy pregnancy after age 37 with out a pregnancy loss). Genomic DNA was extracted from EDTA anticoagulated blood and bisulfite converted for analysis using the Illumina Golden Gate Methylation Panel which measures DNA methylation at 1506 CpG sites in the promoter regions of 807 genes in 10 POF and 12 CW. Candidate genes with altered epigenetic marks between POF and CW at a nominal P-value < 0.05 were identified using a t-testcomparison within the Illumina bead studio software. Genes of interest were further analyzed for quantitative methylation at specific CpG sites using pyrosequencing in 30 POF and 30 CW. Results: Comparison of DNA methylation profiles of our initial POF and CW groups identified several genes with statistically significanthyper- or hypo- methylation in the POF group (P < 0.05), including the Androgen Receptor (AR)promoter region, which was significantly hypermethylated. To further validate these results, DNA methylation of the AR gene promoter was quantified bypryosequencing in a larger group of POF and CW. Pyrosequencing further confirmed a significantly higher DNA methylation of the AR promoter region inPOF vs. CW (P=0.007). Conclusions: This is a novel study identifying epigenetic alterations in POF. The hypermethylation of the AR gene in POF patients may cause decreased level of the AR in these women. This is especially interesting given a recent report of induced POF in AR deficient mice^1. Specific epigenetic markers, as identified by DNA methylation array profiling in blood, may serve as useful biomarkers for POF and other fertility disorders. However, it will need to be determined if these methylation changes are present prior to diagnosis, or are a consequence of menopause itself. Reference: 1.Hiroko S. et al. Premature ovarian failure in androgenreceptor deficient mice. PNAS;103:224-9

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Age-related differences in monocyte DNA methylation and immune function in healthy Kenyan adults and children

Immunity & Ageing ◽

10.1186/s12979-021-00223-2 ◽

2021 ◽

Vol 18 (1) ◽

Author(s):

Katherine R. Dobbs ◽

Paula Embury ◽

Emmily Koech ◽

Sidney Ogolla ◽

Stephen Munga ◽

...

Keyword(s):

Gene Expression ◽

Dna Methylation ◽

Young Adults ◽

Expression Profiles ◽

Gene Expression Profiles ◽

Innate Immune ◽

Inflammatory Gene Expression ◽

Cpg Sites ◽

Age Related ◽

Adults And Children

Abstract Background Age-related changes in adaptive and innate immune cells have been associated with a decline in effective immunity and chronic, low-grade inflammation. Epigenetic, transcriptional, and functional changes in monocytes occur with aging, though most studies to date have focused on differences between young adults and the elderly in populations with European ancestry; few data exist regarding changes that occur in circulating monocytes during the first few decades of life or in African populations. We analyzed DNA methylation profiles, cytokine production, and inflammatory gene expression profiles in monocytes from young adults and children from western Kenya. Results We identified several hypo- and hyper-methylated CpG sites in monocytes from Kenyan young adults vs. children that replicated findings in the current literature of differential DNA methylation in monocytes from elderly persons vs. young adults across diverse populations. Differentially methylated CpG sites were also noted in gene regions important to inflammation and innate immune responses. Monocytes from Kenyan young adults vs. children displayed increased production of IL-8, IL-10, and IL-12p70 in response to TLR4 and TLR2/1 stimulation as well as distinct inflammatory gene expression profiles. Conclusions These findings complement previous reports of age-related methylation changes in isolated monocytes and provide novel insights into the role of age-associated changes in innate immune functions.

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Unraveling the functional role of DNA methylation using targeted DNA demethylation by steric blockage of DNA methyltransferase with CRISPR/dCas9

10.1101/2020.03.28.012518 ◽

2020 ◽

Cited By ~ 1

Author(s):

Daniel M. Sapozhnikov ◽

Moshe Szyf

Keyword(s):

Gene Expression ◽

Dna Methylation ◽

Promoter Region ◽

Dna Methyltransferase ◽

Dna Demethylation ◽

New Method ◽

Inducible Promoters ◽

Specific Targeting ◽

Main Effect

AbstractAlthough associations between DNA methylation and gene expression were established four decades ago, the causal role of DNA methylation in gene expression remains unresolved. Different strategies to address this question were developed; however, all are confounded and fail to disentangle cause and effect. We developed here a highly effective new method using only deltaCas9(dCas9):gRNA site-specific targeting to physically block DNA methylation at specific targets in the absence of a confounding flexibly-tethered enzymatic activity, enabling examination of the role of DNA methylation per se in living cells. We show that the extensive induction of gene expression achieved by TET/dCas9-based targeting vectors is confounded by DNA methylation-independent activities, inflating the role of DNA methylation in the promoter region. Using our new method, we show that in several inducible promoters, the main effect of DNA methylation is silencing basal promoter activity. Thus, the effect of demethylation of the promoter region in these genes is small, while induction of gene expression by different inducers is large and DNA methylation independent. In contrast, targeting demethylation to the pathologically silenced FMR1 gene targets robust induction of gene expression. We also found that standard CRISPR/Cas9 knockout generates a broad unmethylated region around the deletion, which might confound interpretation of CRISPR/Cas9 gene depletion studies. In summary, this new method could be used to reveal the true extent, nature, and diverse contribution to gene regulation of DNA methylation at different regions.

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DNA Methylation Landscape of 16 Canine Somatic Tissues by Methylation-Sensitive Restriction Enzyme-Based Next Generation Sequencing

10.21203/rs.3.rs-146319/v1 ◽

2021 ◽

Author(s):

Jumpei Yamazaki ◽

Yuki Matsumoto ◽

Jaroslav Jelinek ◽

Teita Ishizaki ◽

Shingo Maeda ◽

...

Keyword(s):

Gene Expression ◽

Dna Methylation ◽

Next Generation Sequencing ◽

Restriction Enzyme ◽

Companion Animals ◽

Next Generation ◽

Cpg Sites ◽

Somatic Tissues ◽

Methylation Sensitive Restriction Enzyme ◽

Generation Sequencing

Abstract Background: DNA methylation plays important functions in gene expression regulation that is involved in individual development and various diseases. DNA methylation has been well studied in human and model organisms, but only limited data exist in companion animals like dog. Results: Using methylation-sensitive restriction enzyme-based next generation sequencing (Canine DREAM), we obtained canine DNA methylation maps from 16 somatic tissues. In total, we evaluated 130,861 CpG sites. The majority of CpG sites were either highly methylated (>70%, 52.5%-64.6% of all CpG sites analyzed) or unmethylated (<30%, 22.5%-28.0% of all CpG sites analyzed) which are methylation patterns similar to other species. The overall methylation status of CpG sites across the 32 methylomes were remarkably similar. However, the tissue types were clearly defined by principle component analysis and hierarchical clustering analysis with DNA methylome. We found 6416 CpG sites located closely at promoter region of genes and inverse correlation between DNA methylation and gene expression of these genes. Conclusions: Our study provides basic dataset for DNA methylation profiles in dogs.

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EGFR DNA Methylation Correlates With EGFR Expression, Immune Cell Infiltration, and Overall Survival in Lung Adenocarcinoma

Frontiers in Oncology ◽

10.3389/fonc.2021.691915 ◽

2021 ◽

Vol 11 ◽

Author(s):

Zhanyu Xu ◽

Fanglu Qin ◽

Liqiang Yuan ◽

Jiangbo Wei ◽

Yu Sun ◽

...

Keyword(s):

Dna Methylation ◽

Protein Expression ◽

Lung Adenocarcinoma ◽

Methylation Level ◽

Promoter Region ◽

Body Region ◽

Gene Body ◽

Immune Infiltration ◽

Cpg Sites ◽

Gene Body Region

BackgroundThe epidermal growth factor receptor (EGFR) is a primary target of molecular targeted therapy for lung adenocarcinoma (LUAD). The mechanisms that lead to epigenetic abnormalities of EGFR in LUAD are still unclear. The purpose of our study was to evaluate the abnormal methylation of EGFR CpG sites as potential biomarkers for LUAD.MethodsTo assess the differentially methylation CpG sites of EGFR in LUAD, we used an integrative study of Illumina HumanMethylation450K and RNA-seq data from The Cancer Genome Atlas (TCGA). We evaluated and compared EGFR multiple-omics data to explore the role of CpG sites located in EGFR promoter regions and gene body regions and the association with transcripts, protein expression levels, mutations, and somatic copy number variation. We calculated the correlation coefficients between CpG sites of EGFR and immune infiltration fraction (by MCPcounter and ESTIMATE) and immune-related pathways in LUAD. Finally, we validated the differential methylation of clinically and prognostically relevant CpG sites using quantitative methylation-specific PCR (qMSP).ResultsWe found that the methylation level of many EGFR CpGs in the promoter region was negatively correlated with the transcription level, protein expression, and SCNV, while the methylation at the gene body region was positively correlated with these features. The methylation level of EGFR CpGs in the promoter region was positively correlated with the level of immune infiltration and IFN-γ signature, while the opposite was found for methylation of the gene body region. The qMSP results showed that cg02316066 had a high methylation level, while cg02166842 had a low methylation level in LUAD. There was a high degree of co-methylation between cg02316066 and cg03046247.ConclusionOur data indicate that EGFR is an epigenetic regulator in LUAD acting through DNA methylation. Our research provides a theoretical basis for the further detection of EGFR DNA methylation as a predictive biomarker for LUAD survival and immunotherapy.

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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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Changes in DNA Methylation and Gene Expression of Insulin and Obesity-Related Gene PIK3R1 after Roux-en-Y Gastric Bypass

International Journal of Molecular Sciences ◽

10.3390/ijms21124476 ◽

2020 ◽

Vol 21 (12) ◽

pp. 4476

Author(s):

Marcela A S Pinhel ◽

Natália Y Noronha ◽

Carolina F Nicoletti ◽

Vanessa AB Pereira ◽

Bruno AP de Oliveira ◽

...

Keyword(s):

Gene Expression ◽

Dna Methylation ◽

Weight Loss ◽

Gastric Bypass ◽

Related Gene ◽

Cpg Sites ◽

Genome Wide ◽

Genetically Determined ◽

Methylation Patterns ◽

Weight Loss Interventions

Weight regulation and the magnitude of weight loss after a Roux-en-Y gastric bypass (RYGB) can be genetically determined. DNA methylation patterns and the expression of some genes can be altered after weight loss interventions, including RYGB. The present study aimed to evaluate how the gene expression and DNA methylation of PIK3R1, an obesity and insulin-related gene, change after RYGB. Blood samples were obtained from 13 women (35.9 ± 9.2 years) with severe obesity before and six months after surgical procedure. Whole blood transcriptome and epigenomic patterns were assessed by microarray-based, genome-wide technologies. A total of 1966 differentially expressed genes were identified in the pre- and postoperative periods of RYGB. From these, we observed that genes involved in obesity and insulin pathways were upregulated after surgery. Then, the PIK3R1 gene was selected for further RT-qPCR analysis and cytosine-guanine nucleotide (CpG) sites methylation evaluation. We observed that the PI3KR1 gene was upregulated, and six DNA methylation CpG sites were differently methylated after bariatric surgery. In conclusion, we found that RYGB upregulates genes involved in obesity and insulin pathways.

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