Deconvolution of haematological cancer methylation patterns reveals a predominantly non-disease related proliferation signal and uncovers true disease associated methylation changes.

Retinal degeneration is a major cause of irreversible blindness. Stimulation with controlled low-level electrical fields, such as transcorneal electrical stimulation (TES), has recently been postulated as a therapeutic strategy. With promising results, there is a need for detailed molecular characterization of the therapeutic effects of TES. Controlled, non-invasive TES was delivered using a custom contact lens electrode to the retinas of Royal College of Surgeons (RCS) rats, a model of retinal degeneration. DNA methylation in the retina, brain and cell-free DNA in plasma was assessed by reduced representation bisulfite sequencing (RRBS) and gene expression by RNA sequencing. TES induced DNA methylation and gene expression changes implicated in neuroprotection in the retina of RCS rats. We devised an epigenomic-based retinal health score, derived from DNA methylation changes observed with disease progression in RCS rats, and showed that TES improved the epigenomic health of the retina. TES also induced DNA methylation changes in the superior colliculus: the brain which is involved in integrating visual signaling. Lastly, we demonstrated that TES-induced retinal DNA methylation changes were detectable in cell-free DNA derived from plasma. TES induced DNA methylation changes with therapeutic effects, which can be measured in circulation. Based on these changes, we were able to devise a liquid biopsy biomarker for retinal health. These findings shed light on the therapeutic potential and molecular underpinnings of TES, and provide a foundation for the further development of TES to improve the retinal health of patients with degenerative eye diseases.

Changes in first trimester fetal CYP1A1 and AHRR DNA methylation and mRNA expression in response to exposure to maternal cigarette smoking

The effects of ionizing radiation on DNA methylation are of importance due to the role that DNA methylation plays in maintaining genome stability, and the presence of aberrant DNA methylation in many cancers. There is limited evidence that radiation-sensitivity may influence the modulation of DNA methylation by ionizing radiation, resulting in a loss of methylation. The BALB/c, CBA and C57Bl/6 strains are the most commonly utilized mouse strains in radiation research and are classified as radiation sensitive (BALB/c and CBA) or radiation resistant (C57Bl/6). We present here the first direct comparison of changes in repeat element DNA methylation (L1, B1 and Intracisternal A Particle; IAP) over time in these three mouse strains after high-dose radiation exposure. Using a high-resolution melt assay, methylation of the spleen repeat elements was investigated between 1 and 14 days after whole-body irradiation with 1 Gy X rays. Our study demonstrated that rather than a loss of methylation at the elements, all strains exhibited an early increase in L1 methylation one day after irradiation. In the most radiosensitive strain (BALB/c) the increase was also detected at 6 days postirradiation. The radioresistant C57Bl/6 strain exhibited a loss of L1 methylation at 14 days postirradiation. Less extensive changes to the B1 and IAP elements were detected at various time points, and pyrosequencing revealed that the responses of the strains were influenced by sex, with the male BALB/c and CBA mice exhibiting a greater response to the irradiation. The results of our study do not support the hypothesis that the most radiosensitive strains exhibit the greatest loss of repeat element DNA methylation after exposure to high-dose radiation. While the exact mechanism and biological outcome of the changes in DNA methylation observed here are still to be elucidated, this study provides the first evidence that radiation exposure elicits time-dependent changes in the methylation of repeat elements that are influenced by the genetic background, gender and the type of repeat element investigated. Furthermore, it suggest that any induced changes may not be persistent.

Fenofibrate lowers triglycerides (TG) and raises high density lipoprotein cholesterol (HDLc) in dyslipidemic individuals. Several studies have shown genetic variability in lipid responses to fenofibrate treatment. It is, however, not known whether epigenetic patterns are also correlated with the changes in lipids due to fenofibrate treatment. The present study was therefore undertaken to examine the changes in DNA methylation among the participants of Genetics of Lipid Lowering Drugs and Diet Network (GOLDN) study. A total of 443 individuals were studied for epigenome-wide changes in DNA methylation, assessed using the Illumina Infinium HumanMethylation450 array, before and after a 3-week daily treatment with 160 mg of fenofibrate. The association between the change in DNA methylation and changes in TG, HDLc, and low-density lipoprotein cholesterol (LDLc) were assessed using linear mixed models adjusted for age, sex, baseline lipids, and study center as fixed effects and family as a random effect. Changes in DNA methylation were not significantly associated with changes in TG, HDLc, or LDLc after 3 weeks of fenofibrate for any CpG. CpG changes in genes known to be involved in fenofibrate response, e.g., PPAR-α, APOA1, LPL, APOA5, APOC3, CETP, and APOB, also did not show evidence of association. In conclusion, changes in lipids in response to 3-week treatment with fenofibrate were not associated with changes in DNA methylation. Studies of longer duration may be required to detect treatment-induced changes in methylation.

Rye regeneration in anther cultures is problematic and affected by albino plants. DNA methylation changes linked to Cu2+ ions in the induction medium affect reprogramming microspores from gametophytic to sporophytic path. Alternations in S-adenosyl-L-methionine (SAM), glutathione (GSH), or β-glucans and changes in DNA methylation in regenerants obtained under different in vitro culture conditions suggest a crucial role of biochemical pathways. Thus, understanding epigenetic and biochemical changes arising from the action of Cu2+ and Zn2+ that participate in enzymatic complexes may stimulate progress in rye doubled haploid plant regeneration. The Methylation-Sensitive Amplified Fragment Length Polymorphism approach was implemented to identify markers related to DNA methylation and sequence changes following the quantification of variation types, including symmetric and asymmetric sequence contexts. Reverse-Phase High-Pressure Liquid Chromatography (RP-HPLC) connected with mass spectrometry was utilized to determine SAM, GSH, and glutathione disulfide, as well as phytohormones, and RP-HPLC with a fluorescence detector to study polyamines changes originating in rye regenerants due to Cu2+ or Zn2+ presence in the induction medium. Multivariate and regression analysis revealed that regenerants derived from two lines treated with Cu2+ and those treated with Zn2+ formed distinct groups based on DNA sequence and methylation markers. Zn2+ treated and control samples formed separate groups. Also, Cu2+ discriminated between controls and treated samples, but the separation was less apparent. Principal coordinate analysis explained 85% of the total variance based on sequence variation and 69% of the variance based on DNA methylation changes. Significant differences in DNA methylation characteristics were confirmed, with demethylation in the CG context explaining up to 89% of the variance across genotypes. Biochemical profiles also demonstrated differences between controls and treated samples. The changes had different effects on green and albino plant regeneration efficiency, with cadaverine (Cad) and SAM affecting regeneration parameters the most. Analyses of the enzymes depend on the Cu2+ or Zn2+ ions and are implemented in the synthesis of Cad, or SAM, which showed that some of them could be candidates for genome editing. Alternatively, manipulating SAM, GSH, and Cad may improve green plant regeneration efficiency in rye.

Changes in the methylation status of inflammatory bowel disease (IBD)-associated genes could significantly alter levels of gene expression, thereby contributing to disease onset and progression. We previously identified seven disease-associated DNA methylation loci from intestinal tissues of IBD patients using the Illumina GoldenGate BeadArray assay. In this study, we extended this approach to identify IBD-associated changes in DNA methylation in B cells from 18 IBD patients [9 Crohn's disease (CD) and 9 ulcerative colitis (UC)]. B cell DNA methylation markers are particularly favorable for diagnosis due to the convenient access to peripheral blood. We examined DNA methylation profiles of B cell lines using the Illumina GoldenGate BeadArray assay. Disease-associated CpGs/genes with changes in DNA methylation were identified by comparison of methylation profiles between B cell lines from IBD patients and their siblings without IBD. BeadArray data were validated using a bisulfite polymerase chain reaction (PCR)-based restriction fragment length polymorphism (RFLP) method. To verify that observed changes in DNA methylation were not due to virus transformation, we compared specific CpG DNA methylation levels of GADD45A and POMC between B cell lines and matching peripheral blood B lymphocytes from five individuals. Using this approach with strict statistical analysis, we identified 11 IBD-associated CpG sites, 14 CD-specific CpG sites, and 24 UC-specific CpG sites with methylation changes in B cells. IBD- and subtype-specific changes in DNA methylation were identified in B cells from IBD patients. Many of these genes have important immune and inflammatory response functions including several loci within the interleukin (IL)-12/IL-23 pathway.

Epigenetic marks are likely to explain variability of response to antigen in granulomatous lung disease. The objective of this study was to identify DNA methylation and gene expression changes associated with chronic beryllium disease (CBD) and sarcoidosis in lung cells obtained by BAL. BAL cells from CBD (n = 8), beryllium-sensitized (n = 8), sarcoidosis (n = 8), and additional progressive sarcoidosis (n = 9) and remitting (n = 15) sarcoidosis were profiled on the Illumina 450k methylation and Affymetrix/Agilent gene expression microarrays. Statistical analyses were performed to identify DNA methylation and gene expression changes associated with CBD, sarcoidosis, and disease progression in sarcoidosis. DNA methylation array findings were validated by pyrosequencing. We identified 52,860 significant (P < 0.005 and q < 0.05) CpGs associated with CBD; 2,726 CpGs near 1,944 unique genes have greater than 25% methylation change. A total of 69% of differentially methylated genes are significantly (q < 0.05) differentially expressed in CBD, with many canonical inverse relationships of methylation and expression in genes critical to T-helper cell type 1 differentiation, chemokines and their receptors, and other genes involved in immunity. Testing of these CBD-associated CpGs in sarcoidosis reveals that methylation changes only approach significance, but are methylated in the same direction, suggesting similarities between the two diseases with more heterogeneity in sarcoidosis that limits power with the current sample size. Analysis of progressive versus remitting sarcoidosis identified 15,215 CpGs (P < 0.005 and q < 0.05), but only 801 of them have greater than 5% methylation change, demonstrating that DNA methylation marks of disease progression changes are more subtle. Our study highlights the significance of epigenetic marks in lung immune response in granulomatous lung disease.

Changes in DNA methylation can increase or suppress the expression of health-relevant genes. We investigated for the first time the relationship between habitual food consumption and changes in DNA methylation. The German KORA FF4 and KORA Fit studies were used to study the change in methylation over a median follow-up of 4 years. Only subjects participating in both surveys and with available dietary and methylation data were included in the analysis (n = 465). DNA methylation was measured using the Infinium MethylationEPIC BeadChip (Illumina), resulting in 735,527 shared CpGs across both studies. Generalized estimating equation models with an interaction term of exposure and time point were used to analyze the association of 34 food groups, folic acid, and two dietary patterns with changes in DNA methylation over time. The results were corrected for genomic inflation. Significant interaction terms indicate different effects between both time points. We observed only a few significant associations between food intake and change in DNA methylation, except for cream and spirit consumption. The annotated genes include CLN3, PROM1, DLEU7, TLL2, and UGT1A10. We identified weak associations between food consumption and DNA methylation change. The differential results for cream and spirits, both consumed in low quantities, require replication in independent studies.

Breast cancer risk factors include age, genetic alterations, weight, diet and others. More recently accumulating evidence suggests that epigenetic alterations are frequent and play important role in breast cancer development and progression. Epigenetic alterations include changes in DNA methylation, histone modifications and miRNA expression in the absence of changes in DNA sequence. The epigenetic changes in cancer include global loss of DNA methylation that might lead to genomic instability and gain of methylation at many promoters of tumor suppressor genes that leads to gene silencing. Interestingly, work from our own laboratory and others have established that in many different tissues, DNA methylation changes due to age have similar patterns as to changes that occur in cancer. Therefore, the goal of this study is to identify and characterize age-dependent DNA methylation changes in normal breast epithelium and their contribution to breast cancer development. To differentiate between age-dependent and independent DNA methylation changes, we utilized DNA extracted from 27 primary human mammary epithelial cells (age range 33-82 years old) derived from adjacent or contralateral normal mammary tissue of breast cancer patients. We measured DNA methylation using a highly sensitive methodology developed in our laboratory called Digital Restriction Enzyme Analysis of Methylation (DREAM). We also studied DNA methylation in six different breast cancer cell lines by DREAM. We also used publically available 450K TCGA data for 685 breast tumor and 98 normal samples. Using bioinformatic approach, we have defined age dependent (Spearman R &amp;gt; |0.38|, p &amp;lt;0.05) and independent (Spearman R &amp;lt; |0.37|, p&amp;gt;0.05) sites that exhibit DNA methylation changes in normal breast epithelium. Interestingly, we found that the age-related genes are more seeded (exhibit low level of methylation) in their promoter CpG islands and are enriched for polycomb group target genes compared to the non-age related genes (p value = 0.032). Furthermore, these age-related genes (for example HOXD9, TDRD10, MYOD1, DPYS, GDA, GIPC2, FAM162B, LAMA1, PKDREJ, etc) have increased DNA methylation in tumors compared to the normal samples in TCGA with a methylation difference of up to 50%. These changes are significantly different compared to not-age related genes or random genes (p&amp;lt;0.0001). The age-related genes that gained DNA methylation in tumors in TCGA dataset, were also hypermethylated in breast cancer cell lines compared to immortalized human mammary epithelial cells. Thirty one percent of the common genes between TCGA data set and breast cancer cell lines, showed decreased mRNA expression in RNA-seq data available through the TCGA data portal. Therefore, in this study we have characterized age-dependent epigenetic changes in normal breast epithelium as potential breast cancer risk associated alterations. Citation Format: Shoghag B. Panjarian, Carolyn Slater, Jozef Madzo, Jaroslav Jelinek, Xiaowei Chen, Jean-Pierre Issa. Age-dependent DNA methylation in normal breast epithelium and breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1071. doi:10.1158/1538-7445.AM2015-1071

Decitabine induces very early in vivo DNA methylation changes in blasts from patients with acute myeloid leukemia

We present here the first high resolution melt (HRM) assay to quantitatively analyze differences in murine DNA methylation levels utilizing CpG methylation of Long Interspersed Elements-1 (LINE1 or L1). By calculating the integral difference in melt temperature between samples and a methylated control, and biasing PCR primers for unmethylated CpGs, the assay demonstrates enhanced sensitivity to detect changes in methylation in a cell line treated with low doses of 5-aza-2’-deoxycytidine (5-aza). The L1 assay was confirmed to be a good marker of changes in DNA methylation of L1 elements at multiple regions across the genome when compared with total 5-methyl-cytosine content, measured by Liquid Chromatography-Mass Spectrometry (LC-MS). The assay design was also used to detect changes in methylation at other murine repeat elements (B1 and Intracisternal-A-particle Long-terminal Repeat elements). Pyrosequencing analysis revealed that L1 methylation changes were non-uniform across the CpGs within the L1-HRM target region, demonstrating that the L1 assay can detect small changes in CpG methylation among a large pool of heterogeneously methylated DNA templates. Application of the assay to various tissues from Balb/c and CBA mice, including previously unreported peripheral blood (PB), revealed a tissue hierarchy (from hypermethylated to hypomethylated) of PB > kidney > liver > prostate > spleen. CBA mice demonstrated overall greater methylation than Balb/c mice, and male mice demonstrated higher tissue methylation compared with female mice in both strains. Changes in DNA methylation have been reported to be an early and fundamental event in the pathogenesis of many human diseases, including cancer. Mouse studies designed to identify modulators of DNA methylation, the critical doses, relevant time points and the tissues affected are limited by the low throughput nature and exorbitant cost of many DNA methylation assays. The L1 assay provides a high throughput, inexpensive and sensitive screening tool for identifying and characterizing DNA methylation changes to L1 elements at multiple regions across the genome.

BackgroundLow socioeconomic status neighborhood exposure to stress and violence may be sources of negative stimuli that poses significant health risks for children, adolescents and throughout the life course of an individual. The study aims to investigate if aberrant epigenetic DNA methylation changes may be a potential mechanism for regulating neighborhood exposures and health outcomes.MethodsExposure to environmental stressors identified in 98 young African American (AA) adults aged 18–25 years old from the Washington D.C., area were used in the study. We correlated the association between stress markers; cortisol, CRP, IgG, IGA, IgM, and self-reported exposure to violence and stress, with quantitative DNA methylation changes in a panel of gene-specific loci using saliva DNA.ResultsIn all participants studied, the exposure to violence was significant and negatively correlated with DNA methylation of MST1R loci (p = 0.032; r = -0.971) and nominally significant with NR3C1 loci (p = 0.053; r = -0.948). In addition, we observed significant and negative correlation of DNA methylation changes of LINE1 (p = 0.044; r = -0.248); NR3C1 (p = 0.017; r = -0.186); MSTR1 (p = 0.022; r = -0.192); and DRD2 (p = 0.056; r = -0.184; albeit nominal significant correlation) with IgA expression. On the other hand, we observed a significant and position correlation of DNA methylation changes in DRD2 (p = 0.037; r = 0.184) with IgG expression. When participants were stratified by sex, we observed in AA young male adults, significant DNA methylation changes of MST1R (p< 0.05) and association with exposure to violence and IgG level. We also observed significant DNA methylation levels of DRD2 (p< 0.05) and association with IgA, IgG, and cortisol level. Furthermore, we observed significant DNA methylation changes of NR3C1 (p< 0.05) with stress, IgA, and IgG in the male participants only. On the other hand, we only observed significant and a positive association of IgG with DNA methylation levels of ESR1 (p = 0.041) in the young AA female participants.ConclusionOur preliminary observation of significant DNA methylation changes in neuronal and immune genes in saliva samples supports our recently published genome-wide DNA methylations changes in blood samples from young AA male adults indicating that saliva offers a non-invasive means for DNA methylation prediction of exposure to environmental stressors in a gender-specific manner.

Low socioeconomic status neighborhood exposure to stress and violence may be sources of negative stimuli that poses significant health risks for children, adolescents and throughout the life course of an individual. The study aims to investigate if aberrant epigenetic DNA methylation changes may be a potential mechanism for regulating neighborhood exposures and health outcomes. Exposure to environmental stressors identified in 98 young African American (AA) adults aged 18-25 years old from the Washington D.C., area were used in the study. We correlated the association between stress markers; cortisol, CRP, IgG, IGA, IgM, and self-reported exposure to violence and stress, with quantitative DNA methylation changes in a panel of gene-specific loci using saliva DNA. In all participants studied, the exposure to violence was significant and negatively correlated with DNA methylation of MST1R loci (p = 0.032; r = -0.971) and nominally significant with NR3C1 loci (p = 0.053; r = -0.948). In addition, we observed significant and negative correlation of DNA methylation changes of LINE1 (p = 0.044; r = -0.248); NR3C1 (p = 0.017; r = -0.186); MSTR1 (p = 0.022; r = -0.192); and DRD2 (p = 0.056; r = -0.184; albeit nominal significant correlation) with IgA expression. On the other hand, we observed a significant and position correlation of DNA methylation changes in DRD2 (p = 0.037; r = 0.184) with IgG expression. When participants were stratified by sex, we observed in AA young male adults, significant DNA methylation changes of MST1R (p< 0.05) and association with exposure to violence and IgG level. We also observed significant DNA methylation levels of DRD2 (p< 0.05) and association with IgA, IgG, and cortisol level. Furthermore, we observed significant DNA methylation changes of NR3C1 (p< 0.05) with stress, IgA, and IgG in the male participants only. On the other hand, we only observed significant and a positive association of IgG with DNA methylation levels of ESR1 (p = 0.041) in the young AA female participants. Our preliminary observation of significant DNA methylation changes in neuronal and immune genes in saliva samples supports our recently published genome-wide DNA methylations changes in blood samples from young AA male adults indicating that saliva offers a non-invasive means for DNA methylation prediction of exposure to environmental stressors in a gender-specific manner.

Trophoblast cell lineage is established through the first cellular differentiation in mammalian embryogenesis, and its developmental potential is restricted to the extraembryonic tissues contributing solely to the placenta. Several lines of evidence suggest a relative lack of importance of DNA methylation in gene regulation in the extraembryonic tissues when compared with embryonic ones. Here we analyzed the dynamics of epigenetic status in the upstream region of mouse Ddah2 gene, which was found to be specifically repressed in a stem cell population of trophoblast cell lineage. We found a tissue-dependent differentially methylated region in the regulatory region of the Ddah2 gene. This region was hypermethylated in trophoblast stem cells and was hypomethylated in differentiated cells both in vivo and in vitro. This change was well correlated with Ddah2 expression. In addition, in vitro methylation confined to the differentially methylated region was sufficient to repress promoter activity in the reporter assay. Furthermore, a repressive pattern of histone modifications was formed around the differentially methylated region in undifferentiated trophoblast stem cells with repressed Ddah2. Our data suggest that DNA methylation-mediated chromatin remodeling is involved in the regulation of the Ddah2 gene expression and thus is important even in trophoblast cell lineage.

Hibernating mammals conserve energy in the winter by undergoing prolonged bouts of torpor, interspersed with brief arousals back to euthermia. These bouts are accompanied by a suite of reversible physiological and biochemical changes; however, much remains to be discovered about the molecular mechanisms involved. Given the seasonal nature of hibernation, it stands to reason that underlying plastic epigenetic mechanisms should exist. One such form of epigenomic regulation involves the reversible modification of cytosine bases in DNA by methylation. DNA methylation is well known to be a mechanism that confers upon DNA its cellular identity during differentiation in response to innate developmental cues. However, it has recently been hypothesized that DNA methylation also acts as a mechanism for adapting genome function to changing external environmental and experiential signals over different time scales, including during adulthood. Here, we tested the hypothesis that DNA methylation is altered during hibernation in adult wild animals. This study evaluated global changes in DNA methylation in response to hibernation in the liver and skeletal muscle of thirteen-lined ground squirrels along with changes in expression of DNA methyltransferases (DNMT1/3B) and methyl binding domain proteins (MBDs). A reduction in global DNA methylation occurred in muscle during torpor phases whereas significant changes in DNMTs and MBDs were seen in both tissues. We also report dynamic changes in DNA methylation in the promoter of the myocyte enhancer factor 2C (mef2c) gene, a candidate regulator of metabolism in skeletal muscle. Taken together, these data show that genomic DNA methylation is dynamic across torpor-arousal bouts during winter hibernation, consistent with a role for this regulatory mechanism in contributing to the hibernation phenotype.