DNA Methylation Patterns In Cells Research Articles

PERIPHERAL BLOOD DNA METHYLATION-BASED MACHINE LEARNING MODELS ARE PREDICTIVE OF FUTURE KNEE OA PROGRESSION: BIOSPECIMENS AND DATA FROM THE OSTEOARTHRITIS INITIATIVE AND JOHNSTON COUNTY OSTEOARTHRITIS PROJECT

Purpose: Knee osteoarthritis (OA) is a heterogeneous disease characterized by a variety of clinical and molecular phenotypes, for which there exist no widely-available biomarkers. We have previously published a pilot analysis of baseline peripheral blood cell DNA methylation patterns from a small group of patients from the Osteoarthritis Initiative (OAI), demonstrating that methylation-based models could distinguish future radiographic progressors from nonprogressors. In the current study, we apply this method to the FNIH OsteoArthritis Biomarkers Consortium (OABC) subcohort of the OAI that included pain-, radiographic-, and dual- (both pain and radiographic) progressors compared to nonprogressors and to similarly constructed groups in the Johnston County Osteoarthritis Project (JoCoOA) and an independent OAI cohort from our prior work.

Assessment of seminal cell-free DNA as a potential contaminate in studies of human sperm DNA methylation.

Human seminal cell-free deoxyribonucleic acid (cfDNA) methylation patterns have not yet been thoroughly explored; however, recent work in mouse has suggested that some cfDNA encountered in the epididymis may contaminate DNA methylation studies assessing the mature spermatozoa. Such contamination could clearly prove to be a significant confounder, for many reasons, in epigenetic studies of male factor infertility. To explore the nature of seminal cfDNA methylation and the likelihood that it would be retained following standard semen sample processing for epigenetic analysis. We assessed 12 semen samples collected at Utah Fertility Center. For each sample, seminal cfDNA was isolated from the sperm pellet. The spermatozoa was split into three aliquots including one exposed to DNase to remove any additional cfDNA (termed "pure spermatozoa"), one not exposed to DNase, and one exposed to DNase but reintroduced to seminal cfDNA. We additionally assessed blood DNA as our benchmark for somatic cell DNA methylation patterns. DNA methylation was measured via Illumina's 850k array and assessed for differential regional methylation. Forty-six thousand three hundred fifty-two differentially methylated regions (FDR>40) were identified between pure spermatozoa and seminal cfDNA. We found at these sites that the average DNA methylation in cfDNA always fell somewhere between the average methylation in spermatozoa and blood. We also assessed each sperm treatment groups at all 46,352 regions of interest and found no significant differences at any of these sites. Our data suggest that seminal cfDNA is a clear mixture of both somatic and germline DNA and that cfDNA is not a contaminating feature in sperm DNA methylation studies following standard protocols in human sperm DNA extraction.

The Mouse Papillomavirus Epigenetic Signature Is Characterised by DNA Hypermethylation after Lesion Regression.

Papillomaviruses (PVs) are double-stranded DNA tumour viruses that can infect cutaneous and mucosal epidermis. Human papillomavirus (HPV) types have been linked to the causality of cutaneous squamous cell carcinoma (cSCC); however, HPV DNA is not always detected in the resultant tumour. DNA methylation is an epigenetic change that can contribute to carcinogenesis. We hypothesise that the DNA methylation pattern in cells is altered following PV infection. We tested if DNA methylation was altered by PV infection in the mouse papillomavirus (MmuPV1) model. Immunosuppressed mice were infected with MmuPV1 on cutaneous tail skin. Immunosuppression was withdrawn for some mice, causing lesions to spontaneously regress. Reduced representation bisulphite sequencing was carried out on DNA from the actively infected lesions, visibly regressed lesions, and mock-infected control mice. DNA methylation libraries were generated and analysed for differentially methylated regions throughout the genome. The presence of MmuPV1 sequences was also assessed. We identified 834 predominantly differentially hypermethylated fragments in regressed lesions, and no methylation differences in actively infected lesions. The promoter regions of genes associated with tumorigenicity, including the tumour suppressor protein DAPK1 and mismatch repair proteins MSH6 and PAPD7, were hypermethylated. Viral DNA was detected in active lesions and in some lesions that had regressed. This is the first description of the genome-wide DNA methylation landscape for active and regressed MmuPV1 lesions. We propose that the DNA hypermethylation in the regressed lesions that we report here may increase the susceptibility of cells to ultraviolet-induced cSCC.

Biobehavioral organization shapes the immune epigenome in infant rhesus Macaques (Macaca mulatta)

How individuals respond to and cope with stress is linked with their health and well-being. It is presumed that early stress responsiveness helps shape the health of the developing organism, but the relationship between stress responsiveness and early immune function during development is not well-known. We hypothesized that stress responsiveness may shape epigenetic regulation of immune genes in infancy. We investigated whether aspects of behavioral responsiveness and hypothalamic-pituitary adrenal stress-response were associated with epigenome-wide immune cell DNA methylation patterns in 154 infant rhesus monkeys (3–4 months old). Infants’ behavioral and physiological responses were collected during a standardized biobehavioral assessment, which included temporary relocation and separation from their mother and social group. Genome-wide DNA methylation was quantified using restricted representation bisulfite sequencing (RRBS) from blood DNA collected 2-hours post-separation. Epigenome-wide analyses were conducted using simple regression, multiple regression controlling for immune cell counts, and permutation regression, all corrected for false discovery rate. Across the variables analyzed, there were 20,368 unique sites (in 9,040 genes) at which methylation was significantly associated with at least one behavioral responsiveness or cortisol measure across the three analyses. There were significant associations in 442 genes in the Immune System Process ontology category, and 94 genes in the Inflammation mediated by chemokine and cytokine signaling gene pathway. Out of 35 candidate genes that were selected for further investigation, there were 13 genes with at least one site at which methylation was significantly associated with behavioral responsiveness or cortisol, including two intron sites in the glucocorticoid receptor gene, at which methylation was negatively correlated with emotional behavior the day following the social separation (Day 2 Emotionality; β = −0.39, q < 0.001) and cortisol response following a relocation stressor (Sample 1; β = −0.33, q < 0.001). We conclude that biobehavioral stress responsiveness may correlate with the developing epigenome, and that DNA methylation of immune cells may be a mechanism by which patterns of stress response affect health and immune functioning.

DNA Methylation Patterning and the Regulation of Beta Cell Homeostasis.

Pancreatic beta cells play a central role in regulating glucose homeostasis by secreting the hormone insulin. Failure of beta cells due to reduced function and mass and the resulting insulin insufficiency can drive the dysregulation of glycemic control, causing diabetes. Epigenetic regulation by DNA methylation is central to shaping the gene expression patterns that define the fully functional beta cell phenotype and regulate beta cell growth. Establishment of stage-specific DNA methylation guides beta cell differentiation during fetal development, while faithful restoration of these signatures during DNA replication ensures the maintenance of beta cell identity and function in postnatal life. Lineage-specific transcription factor networks interact with methylated DNA at specific genomic regions to enhance the regulatory specificity and ensure the stability of gene expression patterns. Recent genome-wide DNA methylation profiling studies comparing islets from diabetic and non-diabetic human subjects demonstrate the perturbation of beta cell DNA methylation patterns, corresponding to the dysregulation of gene expression associated with mature beta cell state in diabetes. This article will discuss the molecular underpinnings of shaping the islet DNA methylation landscape, its mechanistic role in the specification and maintenance of the functional beta cell phenotype, and its dysregulation in diabetes. We will also review recent advances in utilizing beta cell specific DNA methylation patterns for the development of biomarkers for diabetes, and targeting DNA methylation to develop translational approaches for supplementing the functional beta cell mass deficit in diabetes.

Exposure to extracellular vesicles from Pseudomonas aeruginosa result in loss of DNA methylation at enhancer and DNase hypersensitive site regions in lung macrophages

ABSTRACT Various pathogens use differing strategies to evade host immune response including modulating the host’s epigenome. Here, we investigate if EVs secreted from P. aeruginosa alter DNA methylation in human lung macrophages, thereby potentially contributing to a dysfunctional innate immune response. Using a genome-wide DNA methylation approach, we demonstrate that P. aeruginosa EVs alter certain host cell DNA methylation patterns. We identified 1,185 differentially methylated CpGs (FDR < 0.05), which were significantly enriched for distal DNA regulatory elements including enhancer regions and DNase hypersensitive sites. Notably, all but one of the 1,185 differentially methylated CpGs were hypomethylated in association with EV exposure. Significantly hypomethylated CpGs tracked to genes including AXL, CFB and CCL23. Gene expression analysis identified 310 genes exhibiting significantly altered expression 48 hours post P. aeruginosa EV treatment, with 75 different genes upregulated and 235 genes downregulated. Some CpGs associated with cytokines such as CSF3 displayed strong negative correlations between DNA methylation and gene expression. Our infection model illustrates how secreted products (EVs) from bacteria can alter DNA methylation of the host epigenome. Changes in DNA methylation in distal DNA regulatory regions in turn can modulate cellular gene expression and potential downstream cellular processes.

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We thank Xu et al for their interest in our work on the role of EZH2 in lupus. We previously reported changes in T cell DNA methylation patterns that are associated with increased disease activity in lupus patients, and identified a possible role for EZH2 in inducing these pro-inflammatory epigenetic changes (1, 2). We followed this by mechanistic work in vitro that resulted in the identification of junctional adhesion molecule A (JAM-A) as a novel important target gene dysregulated by EZH2 in lupus T cells (3).

Structural Basis of DNMT1 and DNMT3A-Mediated DNA Methylation.

DNA methylation, one of the major epigenetic mechanisms, plays critical roles in regulating gene expression, genomic stability and cell lineage commitment. The establishment and maintenance of DNA methylation in mammals is achieved by two groups of DNA methyltransferases (DNMTs): DNMT3A and DNMT3B, which are responsible for installing DNA methylation patterns during gametogenesis and early embryogenesis, and DNMT1, which is essential for propagating DNA methylation patterns during replication. Both groups of DNMTs are multi-domain proteins, containing a large N-terminal regulatory region in addition to the C-terminal methyltransferase domain. Recent structure-function investigations of the individual domains or large fragments of DNMT1 and DNMT3A have revealed the molecular basis for their substrate recognition and specificity, intramolecular domain-domain interactions, as well as their crosstalk with other epigenetic mechanisms. These studies highlight a multifaceted regulation for both DNMT1 and DNMT3A/3B, which is essential for the precise establishment and maintenance of lineage-specific DNA methylation patterns in cells. This review summarizes current understanding of the structure and mechanism of DNMT1 and DNMT3A-mediated DNA methylation, with emphasis on the functional cooperation between the methyltransferase and regulatory domains.

Genome-wide DNA methylation analysis in permanent atrial fibrillation.

Atrial fibrillation (AF) is a highly heterogeneous genetic disease; however, the pathogenesis of AF cannot be explained by genetic variants alone. DNA methylation is a heritable method of gene expression regulation, and may be a potential regulatory mechanism in AF. Therefore, in the present study, the genome‑wide DNA methylation pattern in cells derived from the left atrium of patients with permanent AF (n=7) was compared with that of healthy heart donors (n=4) with a normal sinus rhythm (SR). Enriched biological functions of the differentially methylated genes were assessed. Integrated analysis of genome‑wide methylation and mRNA expression profiles was performed, and reverse transcription quantitative‑polymerase chain reaction (RT‑qPCR) was used to determine the expression levels of four selected genes. A total of 417 differentially methylated CpG sites were identified in the fibrillating atrium (P<0.05; |β|>0.17); the majority of which were located in gene‑body and intergenic regions outside of CpG islands. Aberrantly methylated genes participated in the activation of inflammation, sodium and potassium ion transport, fibrosis and the reduction of lipid metabolism. Hypermethylation in the AF susceptible loci, paired‑like homeodomain transcription factor2 (chromosome 4q25) and coiled‑coil domain containing 141 (chromosome 2q31), aswellas hypomethylation in the calcium voltage‑gated channel subunitα1C (chromosome 12p13) locus, were identified in all patients with AF. Of the 420 upregulated and 567downregulated genes previously identified in patients with AF relative to those with normal SR (fold‑change >2.0; P≤0.05), 12 genes were hypomethylated and eight genes were hypermethylated in each group, respectively (|β|>0.2: P<0.05). RT‑qPCR analysis of four of these genes supported the modulatory effect of DNA methylation on gene expression. These results suggest that DNA methylation‑mediated regulation of gene expression may serve an important role in AF pathogenesis, and several susceptible AF CpG loci were identified which may be involved in the initiation of AF.

Patho-epigenetics of Infectious Diseases Caused by Intracellular Bacteria.

In multicellular eukaryotes including plants, animals and humans, epigenetic reprogramming may play a role in the pathogenesis of a wide variety of diseases. Recent studies revealed that in addition to viruses, pathogenic bacteria are also capable to dysregulate the epigenetic machinery of their target cells. In this chapter we focus on epigenetic alterations induced by bacteria infecting humans. Most of them are obligate or facultative intracellular bacteria that produce either bacterial toxins and surface proteins targeting the host cell membrane, or synthesise effector proteins entering the host cell nucleus. These bacterial products typically elicit histone modifications, i.e. alter the "histone code". Bacterial pathogens are capable to induce alterations of host cell DNA methylation patterns, too. Such changes in the host cell epigenotype and gene expression pattern may hinder the antibacterial immune response and create favourable conditions for bacterial colonization, growth, or spread. Epigenetic dysregulation mediated by bacterial products may also facilitate the production of inflammatory cytokines and other inflammatory mediators affecting the epigenotype of their target cells. Such indirect epigenetic changes as well as direct interference with the epigenetic machinery of the host cells may contribute to the initiation and progression of malignant tumors associated with distinct bacterial infections.

Dynamic changes in DNA modification states during late gestation male germ line development in the rat.

BackgroundEpigenetic reprogramming of fetal germ cells involves the genome-wide erasure and subsequent re-establishment of DNA methylation. Mouse studies indicate that DNA demethylation may be initiated at embryonic day (e) 8 and completed between e11.5 and e12.5. In the male germline, DNA remethylation begins around e15 and continues for the remainder of gestation whilst this process occurs postnatally in female germ cells. Although 5-methylcytosine (5mC) dynamics have been extensively characterised, a role for the more recently described DNA modifications (5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC)) remains unclear. Moreover, the extent to which the developmental dynamics of 5mC reprogramming is conserved across species remains largely undetermined. Here, we sought to describe this process during late gestation in the male rat.ResultsUsing immunofluorescence, we demonstrate that 5mC is re-established between e18.5 and e21.5 in the rat, subsequent to loss of 5hmC, 5fC and 5caC, which are present in germ cells between e14.5 and e16.5. All of the evaluated DNA methyl forms were expressed in testicular somatic cells throughout late gestation. 5fC and 5caC can potentially be excised through Thymine DNA Glycosylase (TDG) and repaired by the base excision repair (BER) pathway, implicating 5mC oxidation in active DNA demethylation. In support of this potential mechanism, we show that TDG expression is coincident with the presence of 5hmC, 5fC and 5caC in male germ cell development.ConclusionThe developmental dependent changes in germ cell DNA methylation patterns suggest that they are linked with key stages of male rat germline progression.

Parameter estimation for an immortal model of colonic stem cell division using approximate Bayesian computation

In this paper we use approximate Bayesian computation to estimate the parameters in an immortal model of colonic stem cell division. We base the inferences on the observed DNA methylation patterns of cells sampled from the human colon. Utilising DNA methylation patterns as a form of molecular clock is an emerging area of research and has been used in several studies investigating colonic stem cell turnover. There is much debate concerning the two competing models of stem cell turnover: the symmetric (immortal) and asymmetric models. Early simulation studies concluded that the observed methylation data were not consistent with the immortal model. A later modified version of the immortal model that included preferential strand segregation was subsequently shown to be consistent with the same methylation data. Most of this earlier work assumes site independent methylation models that do not take account of the known processivity of methyltransferases whilst other work does not take into account the methylation errors that occur in differentiated cells. This paper addresses both of these issues for the immortal model and demonstrates that approximate Bayesian computation provides accurate estimates of the parameters in this neighbour-dependent model of methylation error rates. The results indicate that if colonic stem cells divide asymmetrically then colon stem cell niches are maintained by more than 8 stem cells. Results also indicate the possibility of preferential strand segregation and provide clear evidence against a site-independent model for methylation errors. In addition, algebraic expressions for some of the summary statistics used in the approximate Bayesian computation (that allow for the additional variation arising from cell division in differentiated cells) are derived and their utility discussed.

Abstract 1053: DNMT3B7 alters E-cadherin expression and cell proliferation in breast cancer cells

Abstract Breast cancer is the second-leading cause of cancer death among women in the United States and the progression from non-invasive to invasive/metastatic disease correlates with an increased risk of death. Changes in DNA methylation have been shown to be important in breast cancers. However, the mechanism by which cancer cells acquire and maintain abnormal methylation is not well understood. DNA methyltransferases (DNMTs) are responsible for monitoring and altering DNA methylation patterns in cells. Interestingly, we have identified many aberrant DNMT3B transcripts in cancer cells compared to normal cells. Therefore, we hypothesize that aberrant DNMT3B isoforms alter gene and protein expression to promote tumor progression in breast cancer cells. Our studies indicate that the breast cancer cell lines MCF-7 and MDA-MB-231 express many aberrant DNMT3B isoforms including DNMT3B7 and ΔDNMT3B6. Interestingly, the invasive MDA-MB-231 cells express increased levels of DNMT3B7 and ΔDNMT3B6 compared to the poorly invasive MCF-7 cells. To further our studies, poorly invasive MCF-7 cells were stably transfected with a DNMT3B7 expression construct to determine the role of this aberrant transcript in tumor progression. Preliminary data indicate that the increased expression of DNMT3B7 in these cells correlates with a significant increase in growth. Specifically, cells expressing more DNMT3B7 grow faster than those with lower levels of the aberrant protein. Furthermore, E-cadherin expression is inversely correlated with DNMT3B7 expression indicating that the expression of this aberrant protein may lead to a more aggressive phenotype. Finally, soft agar assays have been performed to determine the functional significance of these findings. The novel results presented in this study suggest the need for additional studies to determine the role of aberrant DNMT3B transcripts in cancer. Ultimately, this work may lead to a better understanding of breast cancer progression and the development of novel therapeutics. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1053. doi:1538-7445.AM2012-1053

Age-dependent decreases in DNA methyltransferase levels and low transmethylation micronutrient levels synergize to promote overexpression of genes implicated in autoimmunity and acute coronary syndromes

T cell DNA methylation levels decline with age, activating genes such as KIR and TNFSF7 (CD70), implicated in lupus-like autoimmunity and acute coronary syndromes. The mechanisms causing age-dependent DNA demethylation are unclear. Maintenance of DNA methylation depends on DNA methyltransferase 1 (Dnmt1) and intracellular S-adenosylmethionine (SAM) levels, and is inhibited by S-adenosylhomocysteine (SAH). SAM levels depend on dietary micronutrients including folate and methionine. SAH levels depend on serum homocysteine concentrations. T cell Dnmt1 levels also decline with age. We hypothesized that age-dependent Dnmt1 decreases synergize with low folate, low methionine or high homocysteine levels to demethylate and activate methylation-sensitive genes. T cells from healthy adults ages 22–81, stimulated and cultured with low folate, low methionine, or high homocysteine concentrations showed demethylation and overexpression of KIR and CD70 beginning at age ∼50 and increased further with age. The effects were reproduced by Dnmt1 knockdowns in T cells from young subjects. These results indicate that maintenance of T cell DNA methylation patterns is more sensitive to low folate and methionine levels in older than younger individuals, due to low Dnmt1 levels, and that homocysteine further increases aberrant gene expression. Thus, attention to proper nutrition may be particularly important in the elderly.

Synthesis of DNA Dumbbell Based Inhibitors for the Human DNA Methyltransferase Dnmt1

Dumbbells that block: Dnmt1 is a crucial enzyme in maintaining the methylation pattern of genes and as such is a critical element of the epigenetic programming process. DNA dumbbell constructs have been developed that inhibit Dnmt1 and have potential in the regulation of DNA methylation patterns in cells (see scheme; SAM=S-adenosylmethionine, Fl=Cy3 fluorescence label, CN=5-azadC, C-Me=5-methyldC).

The establishment and maintenance of DNA methylation patterns in mouse somatic cells

Somatic cell DNA methylation patterns in mammals are established during embryonic development and are then maintained somewhat faithfully for the remainder of the individual's lifetime. Pattern formation can be divided into a series of linked steps that include demethylation, de novo methylation, methylation spreading, methylation blocking, and maintenance methylation. In this review, these steps will be combined to present a model for the formation and maintenance of a methylation pattern in the 5′ region of the mouse Aprt gene. This model suggests that an apparently ‘stable’ methylation pattern results from a dynamic equilibrium between forces that promote and inhibit methylation spreading.

Developmental exposure of male germ cells to 5-azacytidine results in abnormal preimplantation development in rats.

Methylation of cytosine residues in mammalian DNA is established during gametogenesis and embryogenesis; it plays an important role in gene regulation and normal embryonic development and has also been implicated in genomic imprinting. In the present study, we evaluated whether paternal administration of 5-azacytidine, a drug that is incorporated into DNA and blocks DNA methylation, could alter male germ cell development and function. A drug that does not block methylation, 6-azacytidine, served as a control. Adult male Sprague-Dawley rats (n = 4-8 per group) were treated i.p., three times per week for 4 and 11 wk, with saline or 2.5 (low dosage) or 5.0 (high dosage) mg/kg of 5-azacytidine and 6-azacytidine. After each of the treatment periods, males were mated to determine effects on fertility and embryo development. Although neither 6-azacytidine nor 4 wk of 5-azacytidine treatment affected male reproductive organ weights or sperm counts, 11 wk of 5-azacytidine resulted in dose-dependent reductions in testis and epididymal weights and sperm counts. Both dosages of 5-azacytidine resulted in significant increases in preimplantation loss, and the high dosage of 5-azacytidine caused a decrease in fertility. Examination of embryos on Day 2 of gestation revealed a striking dose-dependent increase in the average number of abnormal embryos per litter sired by the males treated with 5-azacytidine (saline, 0.33 +/- 0.24; low dosage, 2.64 +/- 0.92; high dosage, 10.09 +/- 0.95). In summary, paternal administration of 5-azacytidine interfered with normal male germ cell development and resulted in alterations in fertilization and early embryo development. We suggest that 5-azacytidine-induced alterations in germ cell DNA methylation patterns may be one of the underlying mechanisms, since similar dosages of the analogue 6-azacytidine had no effect on male reproduction and progeny outcome.