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Purpose: Renal cell carcinoma (RCC) is a common malignancy, with patients frequently diagnosed at an advanced stage due to the absence of sufficiently sensitive detection technologies, significantly compromising patient survival and quality of life. Advances in cell-free DNA (cfDNA) methylation profiling using liquid biopsies offer a promising non-invasive diagnostic option, but robust biomarkers for early detection are current not available. This study aimed to identify methylation biomarkers for RCC and establish a DNA methylation signature-based prognostic model for this disease. Methods: High-throughput methylation sequencing was performed on peripheral blood samples obtained from 49 primarily Stage I RCC patients and 44 healthy controls. Comparative analysis and Least Absolute Shrinkage and Selection Operator (LASSO) regression methods were employed to identify RCC methylation signatures.Subsequently, methylation markers-based diagnostic and prognostic models for RCC were independently trained and validated using random forest and Cox regression methodologies, respectively. Results: Comparative analysis revealed 864 differentially methylated CpG islands (DMCGIs), 96.3% of which were hypermethylated. Using a training set from The Cancer Genome Atlas (TCGA) dataset of 443 early-stage RCC tumors and matched normal tissues, we applied LASSO regression and identified 23 methylation signatures. We then constructed a random forest-based diagnostic model for early-stage RCC and validated the model using two independent datasets: a TCGA set of 460 RCC tumors and controls, and a blood sample set from our study of 15 RCC cases and 29 healthy controls. For Stage I RCC tissue, the model showed excellent discrimination (AUC-ROC: 0.999, sensitivity: 98.5%, specificity: 100%). Blood sample validation also yielded commendable results (AUC-ROC: 0.852, sensitivity: 73.9%, specificity: 89.7%). Further analysis using Cox regression identified 7 of the 23 DMCGIs as prognostic markers for RCC, allowing the development of a prognostic model with strong predictive power for 1-, 3-, and 5-year survival (AUC-ROC > 0.7). Conclusions: Our findings highlight the critical role of hypermethylation in RCC etiology and progression, and present these identified biomarkers as promising candidates for diagnostic and prognostic applications. [ABSTRACT FROM AUTHOR]

This study aimed to investigate whether genes with different modes of inheritance differ in the presence of promoter-enriched CGI loci. For each autosomal chromosome, the author searched for variations in the total number of diseases' phenotypes with autosomal dominant (AD) and recessive (AR) inheritance for a list of promoter-poor CGI (CGI−) and promoter-enriched CGI (CGI+) genes using the OMIM database. Then, the CGI− and CGI+ genes displaying random allelic or bi-allelic expression were examined. The author evaluated whether there was a distinct distribution of AD and AR diseases in the groups of chromosomes based on their SNP hotspot density. The same analysis was conducted for the X chromosome. The SPSS statistical package was utilized. The distribution of AD and AR diseases between CGI− and CGI+ bi-allelic genes significantly differed in autosomal chromosomes 6 and 17, which show intermediate SNP hotspot density. Additionally, a statistically significant difference was observed in AD and AR diseases in the remaining autosomal chromosomes with low SNP hotspots between their randomly allelic expressed CGI− and CGI+ genes. Specifically, AD diseases were related to CGI− genes, while AR diseases were associated with CGI+ genes. In the X chromosome, X-linked dominant (XLD) diseases were mainly found in CGI+ genes, and X-linked recessive (XLR) diseases were found in CGI− genes, regardless of the X-inactivation process. It is essential to study inheritance and classify genetic variants in a more stochastic way than the terms "Dominant" and "Recessive," and their derivatives, such as "Codominant" and "Incomplete Dominant," are applied in Mendelian and non-Mendelian inheritance. This concept may further explain the "Reduced Penetrance" and "Variable Expressivity" in certain human diseases. All the above suggests a need to reassess how genetic and epigenetic data are studied and utilized for genetic counseling or precision medicine. [ABSTRACT FROM AUTHOR]

Background: Methylation plays a crucial role in genome regulation, serving as an essential epigenetic mechanism. CpG islands, which are regions of DNA rich in CpG dinucleotides, are ubiquitous epigenetic regulatory features that can modulate the functional dynamics of genes, thereby influencing the pathophysiology of pathogenic organisms within a host. Objectives: In this study, we aimed to investigate the distribution of CpG islands on Papillomavirus genomes and assess their potential impact on the neoplastic progression of different Alphapapillomavirus genera within host cells. Methods: We conducted an analysis of dinucleotide frequencies in DNA sequences from various Alphapapillomavirus genera. The sequences for our analysis were sourced from the specialized HPV (human papillomavirus) database (pave.niaid.nih.gov). Following a comprehensive comparative examination of entire genomes from different HPV species, we specifically focused on 63 genomes belonging to the Alphapapillomavirus genus to identify internal CpG island profiles. These investigations were conducted using online bioinformatics software (DBCAT), and statistical analysis was performed using GraphPad Prism v. 8.0.1. Results: Our preliminary findings revealed that 76.1% of the viruses in our study had fewer than 3 CpG islands but multiple CpG sites, while fewer than 25% of the viruses possessed at least 3 CpG islands. High-risk viruses were identified in 68.42% of genotypes with fewer than 3 CpG islands, whereas low-risk genotypes were observed in 15.7% of cases. Notably, some oncogenic viruses lacked CpG islands entirely, and this pattern was also observed in viruses with a single CpG island, occurring in 50% of cases. Based on the absence of CpG islands, genotypes such as HPV 67, HPV 97, and HPV 34 could potentially be classified as carcinogenic. Additionally, the distribution of CpG islands across HPV genes did not appear to be random. Genes like L2 and E2 contained a CpG island in 50% of cases, whereas oncogenes E6 and E7 consistently had a CpG island in 100% of cases in high-risk genotypes. Conclusions: Our findings suggest that the loss of 1 or more CpG islands could potentially transform an HPV genotype into a high-risk genotype. This process may be accelerated in the presence of host-related risk factors. Further research is required to validate whether CpG island distribution can aid in identifying oncogenic viruses. [ABSTRACT FROM AUTHOR]

Initially described as a triad of immunodeficiency, congenital heart defects and hypoparathyroidism, 22q11.2 deletion syndrome (22q11.2DS) now encompasses a great amount of abnormalities involving different systems. Approximately 85% of patients share a 3 Mb 22q11.2 region of hemizygous deletion in which 46 protein-coding genes are included. However, the hemizygosity of the genes of this region cannot fully explain the clinical phenotype and the phenotypic variability observed among patients. Additional mutations in genes located outside the deleted region, leading to "dual diagnosis", have been described in 1% of patients. In some cases, the hemizygosity of the 22q11.2 region unmasks autosomal recessive conditions due to additional mutations on the non-deleted allele. Some of the deleted genes play a crucial role in gene expression regulation pathways, involving the whole genome. Typical miRNA expression patterns have been identified in 22q11.2DS, due to an alteration in miRNA biogenesis, affecting the expression of several target genes. Also, a methylation epi-signature in CpG islands differentiating patients from controls has been defined. Herein, we summarize the evidence on the genetic and epigenetic mechanisms implicated in the pathogenesis of the clinical manifestations of 22q11.2 DS. The review of the literature confirms the hypothesis that the 22q11.2DS phenotype results from a network of interactions between deleted protein-coding genes and altered epigenetic regulation. [ABSTRACT FROM AUTHOR]

Recombination is responsible for breaking up haplotypes, influencing genetic variability, and the efficacy of selection. Bird genomes lack the protein PR domain-containing protein 9, a key determinant of recombination dynamics in most metazoans. Historical recombination maps in birds show an apparent stasis in positioning recombination events. This highly conserved recombination pattern over long timescales may constrain the evolution of recombination in birds. At the same time, extensive variation in recombination rate is observed across the genome and between different species of birds. Here, we characterize the fine-scale historical recombination map of an iconic migratory songbird, the Eurasian blackcap (Sylvia atricapilla), using a linkage disequilibrium–based approach that accounts for population demography. Our results reveal variable recombination rates among and within chromosomes, which associate positively with nucleotide diversity and GC content and negatively with chromosome size. Recombination rates increased significantly at regulatory regions but not necessarily at gene bodies. CpG islands are associated strongly with recombination rates, though their specific position and local DNA methylation patterns likely influence this relationship. The association with retrotransposons varied according to specific family and location. Our results also provide evidence of heterogeneous intrachromosomal conservation of recombination maps between the blackcap and its closest sister taxon, the garden warbler. These findings highlight the considerable variability of recombination rates at different scales and the role of specific genomic features in shaping this variation. This study opens the possibility of further investigating the impact of recombination on specific population-genomic features. [ABSTRACT FROM AUTHOR]

As an important epigenetic modification, DNA methylation is involved in many biological processes such as animal cell differentiation, embryonic development, genomic imprinting and sex chromosome inactivation. As DNA methylation sequencing becomes more sophisticated, it becomes possible to use it to solve more zoological problems. This paper reviews the characteristics of DNA methylation, with emphasis on the research and application of DNA methylation in poultry. [ABSTRACT FROM AUTHOR]

Sogatella furcifera is an important rice pest with wing dimorphism. We previously demonstrated that differentially methylated DNA fragments obtained in macropterous and brachypterous adults of S. furcifera were homologous to the 28 S ribosomal RNA (rRNA) gene, we hypothesized that the methylation levels of 28 S rRNA gene between two wing phenotypes may be different. To test this, we cloned 28 S rRNA gene, which contained 3579 bp and 6 CpG islands. Then methylation-specific primers were designed in the four CpG islands. We found that the four CpG islands were all methylated, and the methylation level remained above 40%, suggesting that the ribosomal gene was the high frequency region for the methylation in the rice pest. We also found that the methylation levels and patterns of 28 S rRNA gene were not consistent between two wing phenotypes individuals of S. furcifera. The findings imply that DNA methylation of 28 S rRNA gene may be related to wing phenotype variations in rice planthoppers, and will facilitate the epigenetic mechanism researches of insect wing dimorphism. [ABSTRACT FROM AUTHOR]

The status of DNA methylation in the human genome changes during the pathogenesis of common diseases and acts as a predictor of life expectancy. Therefore, it is of interest to investigate the methylation level of regulatory regions of genes responsible for general biological processes that are potentially significant for the development of age-associated diseases. Among them there are genes encoding proteins of DNA repair system, which are characterized by pleiotropic effects. Here, results of the targeted methylation analysis of two regions of the human genome (the promoter of the MLH1 gene and the enhancer near the ATM gene) in different tissues of patients with carotid atherosclerosis are present. Analysis of the methylation profiles of studied genes in various tissues of the same individuals demonstrated marked differences between leukocytes and tissues of the vascular wall. Differences in methylation levels between normal and atherosclerotic tissues of the carotid arteries were revealed only for two studied CpG sites (chr11:108089866 and chr11:108090020, GRCh37/hg19 assembly) in the ATM gene. Based on this, we can assume the involvement of ATM in the development of atherosclerosis. "Overload" of the studied regions with transcription factor binding sites (according to ReMapp2022 data) indicate that the tissue-specific nature of methylation of the regulatory regions of the MLH1 and ATM may be associated with expression levels of these genes in a particular tissue. It has been shown that inter-individual differences in the methylation levels of CpG sites are associated with sufficiently distant nucleotide substitutions. [ABSTRACT FROM AUTHOR]

Background: Gene duplication is thought to be a central process in evolution to gain new functions. The factors that dictate gene retention following duplication as well paralog gene divergence in sequence, expression and function have been extensively studied. However, relatively little is known about the evolution of promoter regions of gene duplicates and how they influence gene duplicate divergence. Here, we focus on promoters of paralog genes, comparing their similarity in sequence, in the sets of transcription factors (TFs) that bind them, and in their overall promoter architecture. Results: We observe that promoters of recent duplications display higher sequence similarity between them and that sequence similarity rapidly declines between promoters of more ancient paralogs. In contrast, similarity in cis-regulation, as measured by the set of TFs that bind promoters of both paralogs, does not simply decrease with time from duplication and is instead related to promoter architecture—paralogs with CpG Islands (CGIs) in their promoters share a greater fraction of TFs, while CGI-less paralogs are more divergent in their TF binding set. Focusing on recent duplication events and partitioning them by their duplication mechanism enables us to uncover promoter properties associated with gene retention, as well as to characterize the evolution of promoters of newly born genes: In recent retrotransposition-mediated duplications, we observe asymmetry in cis-regulation of paralog pairs: Retrocopy genes are lowly expressed and their promoters are bound by fewer TFs and are depleted of CGIs, in comparison with the original gene copy. Furthermore, looking at recent segmental duplication regions in primates enable us to compare successful retentions versus loss of duplicates, showing that duplicate retention is associated with fewer TFs and with CGI-less promoter architecture. Conclusions: In this work, we profiled promoters of gene duplicates and their inter-paralog divergence. We also studied how their characteristics are associated with duplication time and duplication mechanism, as well as with the fate of these duplicates. These results underline the importance of cis-regulatory mechanisms in shaping the evolution of new genes and their fate following duplication. [ABSTRACT FROM AUTHOR]

The regulation of glutamate dehydrogenase, an enzyme that is involved in both nitrogen and carbon metabolism, and also links between the tricarboxylic acid cycle and the γ-aminobutyric acid shunt, has been studied. It was found that oxygen deficiency-induced changes in glutamate dehydrogenase activity in maize leaves (Zea mays L.) are to increase its catalytic activity by more than twice. Differential expression of genes was studied by real-time PCR in GDH1 and GDH2, which encode the β- and α-subunits of glutamate dehydrogenase, respectively, in the maize genome. Decreased relative level of gene transcripts GDH2 was accompanied by an increase in the expression activity of the gene GDH1. This, in turn, presumably promoted the amination reaction of 2-oxoglutarate. In the promoter of the gene GDH2, the presence of two CpG islands 404 and 383 bp in size was found. Gene promoter GDH1 does not contain a single CpG island; however, 38% of the CpNpG and CpNpN sites of the total number of studied dinucleotides in its composition were found. To assess the influence of the degree of methylation of individual CpG dinucleotides that are part of the promoter regions of genes GDH1 and GDH2 on their expression under hypoxic conditions, a comparative analysis of the dynamics of the transcriptional activity of the genes of β- and α-subunits of glutamate dehydrogenase from the methyl status of their promoters was carried out. Inversely proportional superposition of changes in the methylation profile of gene promoters GDH1 and GDH2 and transformation of the level of expression of these genes shows their correlation. The data obtained as a result of methyl-specific PCR indicate that an increase in the proportion of methylated CpG dinucleotides leads to a decrease in the amount of mRNA of the gene GDH2, while a decrease in this value for the gene GDH1 causes the induction of its functioning. Methylation of promoter regions of glutamate dehydrogenase genes regulates their transcriptional activity in maize leaves in vivo under conditions of oxygen deficiency. Thus, the little data on the molecular mechanisms of regulation of the synthesis of glutamate dehydrogenase isoenzymes were supplemented by new results on the role of the degree of methylation of gene promoters GDH1 and GDH2 glutamate dehydrogenases in their differential expression during maize's adaptation to hypoxia. [ABSTRACT FROM AUTHOR]

Background & Aims: Gastric cancer is the fifth most common cancer and the fourth leading cause of cancer death in the world. This genetically heterogeneous condition can be sporadic or inherited as an autosomal trait. One of the most important causes of sporadic gastric cancer is environmental factors such as Helicobacter pylori infection. In recent decades, proton-pump inhibitors (PPIs) have been widely used to treat gastrointestinal problems. New researches raise concerns about the link between PPIs and gastric cancer. The CpG island methylator phenotype (CIMP) seen in various malignancies is a set of epigenetic changes associated with promoter methylation and down regulation of the tumor suppressor genes. The present study investigated the methylation profile of MINT2, MINT1, MINT25 and MINT31 promoter regions in patients with gastric cancer. The MINT1 gene, also known as Amyloid-beta A4 precursor protein-binding family A member 1 (APBA1), is located on 9q and encodes a protein which is involved in signal transduction processes. Studies have shown a significant increase in MINT1 hypermethylation in gastric cancer tissues significantly associated with Helicobacter pylori infection. The MINT2 gene, or Amyloid-beta A4 precursor protein-binding, family A, member 2 (APBA2), has been mapped to the 15q region. Abnormal methylation of MINT2 has been identified in the blood and peritoneal fluid of patients with gastric cancer suggesting that it can be used as a predictor of peritoneal micrometastasis. The next two gene regions, MINT25 and MINT31, are functionally different. In fact, MINT25 is the Matrix metalloproteinase 24 (MMP24) gene, located at 20q. Studies have shown that MINT25 hypermethylation is a specific molecular marker for gastric cancer screening. The MINT31 marker is located at 17q in the non-coding region upstream of the CACNA1G gene, which is a T-Type Calcium Channel. MINT31 plays an important role in regulating CACNA1G expression. Studies have shown that hypermethylation of the MINT31 region can be used as a predictor of gastric cancer progression. Based on these findings, the present study investigates the methylation profile of MINT2, MINT1, MINT25 and MINT31 promoter regions in patients with gastric cancer. Another goal of this project was to investigate the correlation between this methylation profile and patients' clinical characteristics. Methods: In this descriptive-analytical study, patients with adenocarcinoma gastric cancer between the ages of 50 and 85 years old were studied. The age matched control group included non-cancerous gastrointestinal patients who referred for endoscopy. Tumor and control tissue samples were collected by endoscopy. Collected tissue samples were extracted using the GENTBIO kit and DNA samples were kept at -20. Methylation-specific PCR (MSP) was used to determine the methylation pattern of selected gene regions. Bisulfite treatment was performed by Bisulfite Conversion Kit (ZYMO RESEARCH). Briefly, bisulfite solutions were prepared according to the kit instructions and were added to the samples. The mixtures were incubated for 5 hours then centrifugation was performed. In the next step a combination of BL buffer and carrier RNA were added to the products. The samples were then thoroughly mixed with ethanol and transferred to Epitect columns. BW wash buffer and BD desulfurizing buffer were used in later stages to improve DNA quality. For each reaction of PCR we used 2X PCR-master mix, 0.5µl of each forward and reverse primers with a concentration of 10μM and 50ng DNA. The following temperature program was applied: 15 minutes at 95°C, 35 cycles including 20 seconds at 95 °C, 30 seconds annealing with variable and specific temperature for each pair of primers, extension for 25 seconds at 72°C and final extension for 5 minutes at 72°C. The annealing temperature of the primer pairs was standardized between 51-60°C. PCR products were evaluated by 8% polyacrylamide gel stained with silver nitrate. After collecting and extracting the results, the data were analyzed using SPSS software version 19. P value less than 0.05 was considered significant. Results: Methylation profile of MINT2, MINT1, MINT25 and MINT31 promoter regions was assessed on polyacrylamide gel after treatment with bisulfite and MS-PCR. Each gene was amplified by a pair of primers for methylated (M) and a pair of primers for non-methylated (U) regions. In case of positive methylation of one or two genes, the situation was considered CIMP-Low phenotype and in case of positive methylation of 3 or 4 genes, phenotype was recorded as a CIMP-High. If the methylation of all four genes was negative, the CIMP-Negative phenotype was recorded. The results showed that CIMP was positive in 52.9% of gastric adenocarcinoma tissues, which included 41.2% CIMP-Low and 11.7% CIMP-High. MINT25 with 76% positive had the highest methylation rate. Interestingly, no positive cases of MINT25 were observed in the control group which could confirm the specificity of this marker. MINT31 was next with 53% positive cases. In control samples for three promoter regions, methylation results were negative and only two positive cases were observed regarding MINT31. MINT1 and MINT2 genes were evaluated positive in 17.7% and 11.8% of cases, respectively. According to these results, there was a statistically significant correlation between PPIs and methylation of MINT25 and MINT31 promoter regions. Considering all 4 gene regions as CIMP phenotype, the significance of the results was reduced, which could be due to the low sample size of positive cases in MINT1 and MINT2. Contrary to expectations, no significant link was observed between the Helicobacter pylori infection and methylation of each gene alone. However, the CIMP-Positive phenotype showed a stronger association with Helicobacter pylori infection than individual genes, although this association was not reached the significant level. Conclusion: Gastric cancer is a multifactorial disease that despite the discovery of several predisposing genes, scientific evidence still indicates the important role of environmental factors in its development. Studies have shown the effective role of epigenetic changes in gastric carcinogenesis. Methylation of many genes has been studied in gastrointestinal cancers, including gastric cancer. Epigenetic instability, characterized by hypermethylation of multiple CpG islands, is known as the CIMP phenotype. CIMP-positive tumors indicate that hypermethylation has occurred simultaneously in the promoter regions of several tumor suppressor genes. This leads to inactivation of gene transcription and consequent loss of their function. So far, different gene sets have been selected to study CIMP in a variety of cancers, due to the heterogeneity of histological features in tumors and patient populations. In the present study, the promoter regions of MINT1, MINT2, MINT25 and MINT31 in patients with gastric cancer were examined and the results confirmed the presence of CIMP-Low and CIMP-High phenotypes in these samples. In our study, the correlation between methylation pattern and clinical characteristics of patients was also investigated. Statistical analyzes showed a significant link between methylation of MINT25 and MINT31 promoter regions with the PPIs. Evidence from several studies suggests that long-term use of PPIs is associated with a higher risk of gastric cancer. However, some studies have suggested that this risk may be limited to people with a history of Helicobacter pylori infection or precancerous lesions of the stomach. Because PPIs induce hypergastrinemia and cellular hyperplasia, they could increase the risk of gastric cancer. Overall, the results of our study show that the CIMP phenotype can be introduced as a biomarker in the diagnosis of gastric cancer, although the methylation profile selection should be standardized based on the characteristics of each population. The results of our study showed that among the 4 proposed gene regions, MINT25 has a statistically significant correlation with gastric cancer and also with a history of PPIs, which indicates the importance of studying this marker in Iranian patients with gastric cancer. [ABSTRACT FROM AUTHOR]

The effect of salt stress on the activity of decarboxylating malate dehydrogenases in maize leaves during the adaptive response of cellular metabolism to salt stress was studied. It was shown that excess NaCl concentrations cause an increase in the enzymatic activity of both NAD+-ME and NADP+-ME. The greatest contribution to the increase in the activity of NAD+-ME is made by the intensification of the expression of the nad-me2 gene. At the same time, the expression of nad-me1 under salinity increases to a lesser extent. Methyl-specific PCR showed an inverse relationship between the expression of nad-me2 and the degree of methylation of the promoter of this gene, while nad-me1 and nadf-me, apparently, are regulated by different mechanisms. A different functional role of each isoform of the studied malic enzymes in the adaptive response of cellular metabolism is assumed. [ABSTRACT FROM AUTHOR]

Methylation of the CpG islands of gene promoter regions is the most common epigenetic modification involved in the regulation of gene expression. A number of studies have shown that ionizing radiation can cause both hyper- and hypomethylation of DNA. Aberrant methylation affects cellular processes and can lead to the development of various pathological states. In the literature, there are few studies on the methylation status of human DNA a long time after radiation exposure. Here, the methylation level of CpG islands of the promoter regions of apoptosis genes (BCL2, ATM, MDM2, CDKN1A, STAT3, and NFKB1), and also its influence on apoptosis of peripheral blood lymphocytes in chronically exposed persons were studied. Residents of the South Ural region who were chronically exposed to radiation (after discharges of radioactive wastes into the Techa river by the "Mayak Production Association" in 1949‒1956) were included in the study. It was established that the proportion of individuals with hypermethylated BCL2 gene promoter among the exposed people was statistically significantly higher than in the control group. The percentage of methylation of the ATM gene promoter weakly positively correlated with dose and age characteristics. Differences in the frequency of lymphocyte apoptosis in exposed individuals with a hypo- or hypermethylated ATM gene promoter were also established. The data indicate that, in the long-term, after chronic low intensity radiation exposure at low and medium doses, epigenetic modifications of the genome occur, which are manifested as changes in methylation of promoter regions of BCL2 and ATM genes. [ABSTRACT FROM AUTHOR]

Background: Imprinting Control Regions (ICRs) are CpG-rich sequences acquiring differential methylation in the female and male germline and maintaining it in a parental origin-specific manner in somatic cells. Despite their expected high mutation rate due to spontaneous deamination of methylated cytosines, ICRs show conservation of CpG-richness and CpG-containing transcription factor binding sites in mammalian species. However, little is known about the mechanisms contributing to the maintenance of a high density of methyl CpGs at these loci. Results: To gain functional insights into the mechanisms for maintaining CpG methylation, we sought to identify the proteins binding the methylated allele of the ICRs by determining the interactors of ZFP57 that recognizes a methylated hexanucleotide motif of these DNA regions in mouse ESCs. By using a tagged approach coupled to LC–MS/MS analysis, we identified several proteins, including factors involved in mRNA processing/splicing, chromosome organization, transcription and DNA repair processes. The presence of the post-replicative mismatch-repair (MMR) complex components MSH2 and MSH6 among the identified ZFP57 interactors prompted us to investigate their DNA binding profile by chromatin immunoprecipitation and sequencing. We demonstrated that MSH2 was enriched at gene promoters overlapping unmethylated CpG islands and at repeats. We also found that both MSH2 and MSH6 interacted with the methylated allele of the ICRs, where their binding to DNA was mediated by the ZFP57/KAP1 complex. Conclusions: Our findings show that the MMR complex is concentrated on gene promoters and repeats in mouse ESCs, suggesting that maintaining the integrity of these regions is a primary function of highly proliferating cells. Furthermore, the demonstration that MSH2/MSH6 are recruited to the methylated allele of the ICRs through interaction with ZFP57/KAP1 suggests a role of the MMR complex in the maintenance of the integrity of these regulatory regions and evolution of genomic imprinting in mammalian species. [ABSTRACT FROM AUTHOR]

Cytosine methylation within the 5′-C-phosphate-G-3′ sequence of nucleotides (called CpG methylation) is a well-known epigenetic modification of genomic DNA that plays an important role in gene expression and development. CpG methylation is likely to be altered in the CpG islands. CpG islands are rich in cytosine, forming a structure called the i-motif via cytosine-cytosine hydrogen bonding. However, little is known about the effect of CpG methylation on the i-motif. In this study, The CpG methylation-induced structural changes on the i-motif was examined by thermal stability, circular dichroism (CD) spectroscopy, and native-polyacrylamide gel electrophoresis (Native-PAGE) evaluation of five i-motif-forming DNAs from four cancer-related genes (VEGF, C-KIT, BCL2, and HRAS). This research shows that CpG methylation increased the transitional pH of several i-motif-forming DNAs and their thermal stability. When examining the effect of CpG methylation on the i-motif in the presence of opposite G4-forming DNAs, CpG methylation influenced the proportion of G4 and i-motif formation. This study showed that CpG methylation altered the stability and structure of the i-motif in CpG islands. [ABSTRACT FROM AUTHOR]

Simple Summary: Hepatocellular carcinoma (HCC) belongs to the most common cancer types and is the third leading cause of cancer-related deaths. To gain insight into the molecular mechanisms of liver cancer cells, we assessed the role of the CpG island regulator SAMD1, which is highly expressed in liver cancer tissues and associated with poor prognosis. We demonstrate that the deletion of SAMD1 in HepG2 cells leads to aberrant gene regulation and to a gene signature linked to a better prognosis. These results establish SAMD1 as a potentially important player in HCC. The unmethylated CpG island-binding protein SAMD1 is upregulated in many human cancer types, but its cancer-related role has not yet been investigated. Here, we used the hepatocellular carcinoma cell line HepG2 as a cancer model and investigated the cellular and transcriptional roles of SAMD1 using ChIP-Seq and RNA-Seq. SAMD1 targets several thousand gene promoters, where it acts predominantly as a transcriptional repressor. HepG2 cells with SAMD1 deletion showed slightly reduced proliferation, but strongly impaired clonogenicity. This phenotype was accompanied by the decreased expression of pro-proliferative genes, including MYC target genes. Consistently, we observed a decrease in the active H3K4me2 histone mark at most promoters, irrespective of SAMD1 binding. Conversely, we noticed an increase in interferon response pathways and a gain of H3K4me2 at a subset of enhancers that were enriched for IFN-stimulated response elements (ISREs). We identified key transcription factor genes, such as IRF1, STAT2, and FOSL2, that were directly repressed by SAMD1. Moreover, SAMD1 deletion also led to the derepression of the PI3K-inhibitor PIK3IP1, contributing to diminished mTOR signaling and ribosome biogenesis pathways. Our work suggests that SAMD1 is involved in establishing a pro-proliferative setting in hepatocellular carcinoma cells. Inhibiting SAMD1's function in liver cancer cells may therefore lead to a more favorable gene signature. [ABSTRACT FROM AUTHOR]

Background: Melanoma Antigen Genes (MAGEs) are a family of genes that have piqued the interest of scientists for their unique expression pattern. The MAGE genes can be classified into type I MAGEs that expressed in testis and other reproductive tissues while type II MAGEs that have broad expression in many tissues. Several MAGE gene families are expressed in embryonic tissues in almost all eukaryotes, which is essential for embryo development mainly during germ cell differentiation. The aim of this study was to analyze the promoter regions and regulatory elements (transcription factors and CpG islands) of MAGE genes encoding for embryonic development in cattle. Results: The in silico analysis revealed the highest promoter prediction scores (1.0) for TSS were obtained for two gene sequences (MAGE B4-like and MAGE-L2) while the lowest promoter prediction scores (0.8) was obtained for MAGE B17-like. It also revealed that the best common motif, motif IV, bear a resemblance with three TF families including Zinc-finger family, SMAD family and E2A related factors. From thirteen identified TFs candidates, majority of them (11/13) were clustered to Zinc-finger family serving as transcriptionally activator role whereas three (SP1, SP3 and Znf423) of them as activator or repressor in response to physiological and pathological stimuli. On the other hand we revealed slightly rich CpG islands in the gene body and promoter regions of MAGE genes encoding for embryonic development in cattle. Conclusion: This in silico analysis of gene promoter regions and regulatory elements in MAGE genes could be useful for understanding regulatory networks and gene expression patterns during embryo development in bovine. [ABSTRACT FROM AUTHOR]

The Polycomb repressive complex 2 (PRC2) is an essential chromatin regulatory complex involved in repressing the transcription of diverse developmental genes. PRC2 consists of a core complex; possessing H3K27 methyltransferase activity and various associated factors that are important to modulate its function. During evolution, the composition of PRC2 and the functionality of PRC2 components have changed considerably. Here, we compare the PRC2 complex members of Drosophila and mammals and describe their adaptation to altered biological needs. We also highlight how the PRC2.1 subcomplex has gained multiple novel functions and discuss the implications of these changes for the function of PRC2 in chromatin regulation. [ABSTRACT FROM AUTHOR]

Background and purpose: Alcohol-induced osteonecrosis of the femoral head (ONFH) is a complex and heterogeneous disease. Genetic factors and epigenetic modifications are one of the pathogenesis of the disease. However, the influence of epigenetic factors on the disease has not been systematically studied. Our research aims to determine the methylation changes of alcohol-induced ONFH. Methods: An analytical cross-sectional study of a Chinese male population (50 alcohol-induced ONFH patients and 50 controls). The EpiTYPER of the Sequenom MassARRAY platform was used to detect the DNA methylation status of 132 cytosine-phosphate-guanine (CpG) sites in the OPG/RANKL/RANK gene promoter region. Results: In the whole study group, the chi-square test was used to analyze the methylation rate between the two groups, and six CpG sites were found to be different, among which OPG1_CpG_2, OPG3_CpG_4, RANK1_CpG_6, RANK3_CpG_10, RANKL2_CpG_21, and RANKL2_CpG_46 in the case group were higher than those in the control group, while OPG4_CpG_2 was lower than that in the control group. The results showed that in patients with alcohol-induced ONFH, 146 CpG sites were examined for differences in methylation levels compared with healthy controls, 32 of which were not detected, and 23 of the remaining 114 sites showed differences in methylation levels compared with alcohol-induced ONFH patients. Receiver operator characteristic (ROC) curve analysis demonstrated the methylation levels of OPG/RANKL/RANK could efficiently predict the existence of alcohol-induced ONFH. Conclusion: Our study of Chinese men suggests that several CpG sites in the OPG/RANKL/RANK gene in peripheral blood leukocytes of patients with alcohol-induced ONFH are in an abnormal methylation state (hypermethylation tended to be more frequent). [ABSTRACT FROM AUTHOR]

• DNA methylation techniques unravel underlying molecular mechanisms related to disease. • Epigenetics has been applied to study cancer, obesity and aging. • Epigenetics is an emerging key field to understand the complexity of biology. • DNA methylation methods present different advantages and certain limitations. • Researchers may choose the most appropriate approach for their analysis. Methylation of CpG dinucleotides plays a crucial role in the regulation of gene expression and therefore in the development of different pathologies. Aberrant methylation has been associated to the majority of the diseases, including cancer, neurodegenerative, cardiovascular and autoimmune disorders. Analysis of DNA methylation patterns is crucial to understand the underlying molecular mechanism of these diseases. Moreover, DNA methylation patterns could be used as biomarker for clinical management, such as diagnosis, prognosis and treatment response. Nowadays, a variety of high throughput methods for DNA methylation have been developed to analyze the methylation status of a high number of CpGs at once or even the whole genome. However, identification of specific methylation patterns at specific loci is essential for validation and also as a tool for diagnosis. In this review, we describe the most commonly used approaches to evaluate specific DNA methylation. There are three main groups of techniques that allow the identification of specific regions that are differentially methylated: bisulfite conversion-based methods, restriction enzyme-based approaches, and affinity enrichment-based assays. In the first group, specific restriction enzymes recognize and cleave unmethylated DNA, leaving methylated sequences intact. Bisulfite conversion methods are the most popular approach to distinguish methylated and unmethylated DNA. Unmethylated cytosines are deaminated to uracil by sodium bisulfite treatment, while the methyl cytosines remain unconverted. In the last group, proteins with methylation binding domains or antibodies against methyl cytosines are used to recognize methylated DNA. In this review, we provide the theoretical basis and the framework of each technique as well as the analysis of their strength and the weaknesses. [ABSTRACT FROM AUTHOR]

Background: X-chromosome inactivation (XCI) in eutherian mammals is the epigenetic inactivation of one of the two X chromosomes in XX females in order to compensate for dosage differences with XY males. Not all genes are inactivated, and the proportion escaping from inactivation varies between human and mouse (the two species that have been extensively studied). Results: We used DNA methylation to predict the XCI status of X-linked genes with CpG islands across 12 different species: human, chimp, bonobo, gorilla, orangutan, mouse, cow, sheep, goat, pig, horse and dog. We determined the XCI status of 342 CpG islands on average per species, with most species having 80–90% of genes subject to XCI. Mouse was an outlier, with a higher proportion of genes subject to XCI than found in other species. Sixteen genes were found to have discordant X-chromosome inactivation statuses across multiple species, with five of these showing primate-specific escape from XCI. These discordant genes tended to cluster together within the X chromosome, along with genes with similar patterns of escape from XCI. CTCF-binding, ATAC-seq signal and LTR repeats were enriched at genes escaping XCI when compared to genes subject to XCI; however, enrichment was only observed in three or four of the species tested. LINE and DNA repeats showed enrichment around subject genes, but again not in a consistent subset of species. Conclusions: In this study, we determined XCI status across 12 species, showing mouse to be an outlier with few genes that escape inactivation. Inactivation status is largely conserved across species. The clustering of genes that change XCI status across species implicates a domain-level control. In contrast, the relatively consistent, but not universal correlation of inactivation status with enrichment of repetitive elements or CTCF binding at promoters demonstrates gene-based influences on inactivation state. This study broadens enrichment analysis of regulatory elements to species beyond human and mouse. [ABSTRACT FROM AUTHOR]

Platelet‐derived growth factor signalling pathways play a fundamental role in inducing and sustaining the proliferative and prosurvival stimuli in canine osteosarcomas (cOSAs). The increased expression of platelet‐derived growth factor receptors (PDGFRs) α and β, and their cognate ligands, were almost invariably observed in cOSAs and OSA‐derived cell lines. In particular, overexpression of PDGFRβ‐mediated signalling pathways was found in both the tumour microenvironment, where it drives stromal cell recruitment, and in neoangiogenesis, such as in tumour cells where it triggers aberrant proliferation, migration and local invasion. The majority of the pathological consequences of PDGFRβ signalling are because of aberrant expression. In fact, epigenetic dysregulation of oncogenes throughout demethylation of their promoter has emerged as a pivotal mechanism driving oncogenesis. The aim of this study was to assess the methylation status of the PDGFRβ promoter and to clarify its role in modulating the expression of the tyrosine kinase receptor in canine osteosarcoma. The CpG island of the PDGFRβ promoter was identified using a mixed in silico and experimental approach, and a method based upon the methylation‐sensitive high‐resolution melting assay for quantitatively and precisely assessing the methylation status of the promoter was then set up. The method herein described was then exploited to assess the methylation status of the promoter in a case series of cOSAa. COSAs consistently but variably expressed PDGFRβ. However, the promoter was almost completely demethylated, and its methylation status did not correlate with the expression levels. This finding supported the hypothesis that post‐transcriptional regulatory mechanisms may act in cOSAs. [ABSTRACT FROM AUTHOR]

The aggregation and joint analysis of large numbers of exome sequences has recently made it possible to derive estimates of intolerance to loss-of-function (LoF) variation for human genes. Here, we demonstrate strong and widespread coupling between genic LoF intolerance and promoter CpG density across the human genome. Genes downstream of the most CpG-rich promoters (top 10% CpG density) have a 67.2% probability of being highly LoF intolerant, using the LOEUF metric from gnomAD. This is in contrast to 7.4% of genes downstream of the most CpG-poor (bottom 10% CpG density) promoters. Combining promoter CpG density with exonic and promoter conservation explains 33.4% of the variation in LOEUF, and the contribution of CpG density exceeds the individual contributions of exonic and promoter conservation. We leverage this to train a simple and easily interpretable predictive model that outperforms other existing predictors and allows us to classify 1,760 genes—which are currently unascertained in gnomAD—as highly LoF intolerant or not. These predictions have the potential to aid in the interpretation of novel variants in the clinical setting. Moreover, our results reveal that high CpG density is not merely a generic feature of human promoters but is preferentially encountered at the promoters of the most selectively constrained genes, calling into question the prevailing view that CpG islands are not subject to selection. [ABSTRACT FROM AUTHOR]

RNA helicase DHX33 was found to regulate the transcription of multiple genes involved in cancer development. But the underlying molecular mechanism remains unclear. Here, we found DHX33 associated extensively with gene promoters at CG-rich region. Its deficiency reduced the loading of active RNA polymerase II at gene promoters. Furthermore, we observed a functional interaction between DHX33, AP-2, and DNA demethylation protein Gadd45a (growth arrest and DNA damage inductile protein 45a) at specific gene promoters. DHX33 is required to recruit GADD45a, thereby causing local DNA demethylation through further recruiting teneleven- translocation (Tet) methylcytosine dioxygenase enzyme, as manifested by reduced 5-hydroxymethyl cytosine levels for a subset of genes after DHX33 deficiency. This process might involve R-loop formation in GC skew as a guidance signal at promoter sites. Our report provides for the first time, to our knowledge, original evidence that DHX33 alters epigenetic marks and regulates specific gene transcription through interaction with Gadd45a. [ABSTRACT FROM AUTHOR]

Background: Identifying molecular differences between primary and metastatic colorectal cancers-now possible with the aid of omics technologies-can improve our understanding of the biological mechanisms of cancer progression and facilitate the discovery of novel treatments for late-stage cancer. We compared the DNA methylomes of primary colorectal cancers (CRCs) and CRC metastases to the liver. Laser microdissection was used to obtain epithelial tissue (10 to 25 × 106 μm2) from sections of fresh-frozen samples of primary CRCs (n = 6), CRC liver metastases (n = 12), and normal colon mucosa (n = 3). DNA extracted from tissues was enriched for methylated sequences with a methylCpG binding domain (MBD) polypeptide-based protocol and subjected to deep sequencing. The performance of this protocol was compared with that of targeted enrichment for bisulfite sequencing used in a previous study of ours.Results: MBD enrichment captured a total of 322,551 genomic regions (249.5 Mb or ~ 7.8% of the human genome), which included over seven million CpG sites. A few of these regions were differentially methylated at an expected false discovery rate (FDR) of 5% in neoplastic tissues (primaries: 0.67%, i.e., 2155 regions containing 279,441 CpG sites; liver metastases: 1%, i.e., 3223 regions containing 312,723 CpG sites) as compared with normal mucosa samples. Most of the differentially methylated regions (DMRs; 94% in primaries; 70% in metastases) were hypermethylated, and almost 80% of these (1882 of 2396) were present in both lesion types. At 5% FDR, no DMRs were detected in liver metastases vs. primary CRC. However, short regions of low-magnitude hypomethylation were frequent in metastases but rare in primaries. Hypermethylated DMRs were far more abundant in sequences classified as intragenic, gene-regulatory, or CpG shelves-shores-island segments, whereas hypomethylated DMRs were equally represented in extragenic (mainly, open-sea) and intragenic (mainly, gene bodies) sequences of the genome. Compared with targeted enrichment, MBD capture provided a better picture of the extension of CRC-associated DNA hypermethylation but was less powerful for identifying hypomethylation.Conclusions: Our findings demonstrate that the hypermethylation phenotype in CRC liver metastases remains similar to that of the primary tumor, whereas CRC-associated DNA hypomethylation probably undergoes further progression after the cancer cells have migrated to the liver. [ABSTRACT FROM AUTHOR]

Somatic embryogenesis (SE) is a more rapid and controllable method for plant propagation than traditional breeding methods. However, it often suffers from limited efficiency. SERK1 promotes SE in several plants, including pineapple (Ananas comosus L.). We investigate the embryonic cell-specific transcriptional regulation of AcSERK1 by methylation analysis of CpG islands in AcSERK1 regulatory sequences. This revealed differences in the methylation status of CpG islands between embryonic callus and non-embryonic callus; the methylation inhibitor 5-azaC increased AcSERK1 expression and also accelerated SE. These findings indicate that the expression of AcSERK1 is regulated epigenetically. This study lays the foundation for further analysis of epigenetic regulatory mechanisms that may enhance the efficiency of SE in pineapple and other plants. [ABSTRACT FROM AUTHOR]

Background: In vertebrate genomes, CpG sites can be clustered into CpG islands, and the amount of methylation in a CpG island can change due to gene regulation processes. Thus, single regulatory events can simultaneously change the methylation states of many CpG sites within a CpG island. This should be taken into account when quantifying the amount of change in methylation, for example in form of a branch length in a phylogeny of cell types. Results: We propose a probabilistic model (the IWE-SSE model) of methylation dynamics that accounts for simultaneous methylation changes in multiple CpG sites belonging to the same CpG island. We further propose a Markov-chain Monte-Carlo (MCMC) method to fit this model to methylation data from cell type phylogenies and apply this method to available data from murine haematopoietic cells and from human cell lines. Combined with simulation studies, these analyses show that accounting for CpG island wide methylation changes has a strong effect on the inferred branch lengths and leads to a significantly better model fit for the methylation data from murine haematopoietic cells and human cell lines. Conclusion: The MCMC based parameter estimation method for the IWE-SSE model in combination with our MCMC based inference method allows to quantify the amount of methylation changes at single CpG sites as well as on entire CpG islands. Accounting for changes affecting entire islands can lead to more accurate branch length estimation in the presence of simultaneous methylation change. [ABSTRACT FROM AUTHOR]

Reproductive decline in older female mice can be attributed to a failure of the uterus to decidualise in response to steroid hormones. Here, we show that normal decidualisation is associated with significant epigenetic changes. Notably, we identify a cohort of differentially methylated regions (DMRs), most of which gain DNA methylation between the early and late stages of decidualisation. These DMRs are enriched at progesterone-responsive gene loci that are essential for reproductive function. In female mice nearing the end of their reproductive lifespan, DNA methylation fidelity is lost at a number of CpG islands (CGIs) resulting in CGI hypermethylation at key decidualisation genes. Importantly, this hypermethylated state correlates with the failure of the corresponding genes to become transcriptionally upregulated during the implantationwindow. Thus, ageassociated DNA methylation changes may underlie the decidualisation defects that are a common occurrence in older females. Alterations to the epigenome of uterine cells may therefore contribute significantly to the reproductive decline associated with advanced maternal age. [ABSTRACT FROM AUTHOR]

Racial/ethnic disparities have a significant impact on bladder cancer outcomes with African American patients demonstrating inferior survival over European-American patients. We hypothesized that epigenetic difference in methylation of tumor DNA is an underlying cause of this survival health disparity. We analyzed bladder tumors from African American and European-American patients using reduced representation bisulfite sequencing (RRBS) to annotate differentially methylated DNA regions. Liquid chromatography–mass spectrometry (LC-MS/MS) based metabolomics and flux studies were performed to examine metabolic pathways that showed significant association to the discovered DNA methylation patterns. RRBS analysis showed frequent hypermethylated CpG islands in African American patients. Further analysis showed that these hypermethylated CpG islands in patients are commonly located in the promoter regions of xenobiotic enzymes that are involved in bladder cancer progression. On follow-up, LC-MS/MS revealed accumulation of glucuronic acid, S-adenosylhomocysteine, and a decrease in S-adenosylmethionine, corroborating findings from the RRBS and mRNA expression analysis indicating increased glucuronidation and methylation capacities in African American patients. Flux analysis experiments with 13C-labeled glucose in cultured African American bladder cancer cells confirmed these findings. Collectively, our studies revealed robust differences in methylation-related metabolism and expression of enzymes regulating xenobiotic metabolism in African American patients indicate that race/ethnic differences in tumor biology may exist in bladder cancer. [ABSTRACT FROM AUTHOR]

Objective To investigate the methylation status in promoter region of estrogen receptor alpha (ERа)-A gene and its relation with plasma homocysteine (Hcy) level and cognitive impairment in white matter hyperintensity (WMH) patients. Patients and Methods 210 patients aged 65 and older were selected. The methylation status of CpG islands in ERа-A gene promoter was analyzed by nested methylation-specific PCR. Serum Hcy and estradiol levels were measured by enzyme-linked immunosorbent assay. Cognitive function were evaluated using minimum mental state examination, the montreal cognitive assessment, Stroop color-word test, symbol digit modalities, trail making test B and instrumental activities of daily living (IADL). The severity of WMH was evaluated with the Fazekas scale by brain magnetic resonance imaging. Results We found a significant association between the severity of WMH and CpG island methylation of ERа-A gene (P < 0.05). Multiple regression analysis showed that serum Hcy level, methylation of ERа-A gene and WMH severity were significant determining factors for cognitive impairment (P < 0.05). The spearman rank correlation analysis showed a significant correlation of methylation of ERа-A gene with serum Hcy level, WMH severity, cognitive function and IADL status (P < 0.05). Conclusion Methylation of ERа-A gene promoter has a high frequency in WMH patients with cognitive impairment and is correlated with high plasma Hcy level. [ABSTRACT FROM AUTHOR]

Highlights • Acute DNMT inhibition improves short-term object pattern separation memory. • Acute DNMT inhibition does not affect long-term object pattern separation memory. • DNMT inhibition increases Bdnf1 expression in dorsal hippocampi of mice. • DNMT inhibition increases methylation in three CpG islands at Bdnf1 promoter. Abstract Enhancement of synaptic plasticity through changes in neuronal gene expression is a prerequisite for improved cognitive performance. Moreover, several studies have shown that DNA methylation is able to affect the expression of (e.g. plasticity) genes that are important for several cognitive functions. In this study, the effect of the DNA methyltransferase (DNMT) inhibitor RG108 was assessed on object pattern separation (OPS) task in mice. In addition, its effect on the expression of target genes was monitored. Administration of RG108 before the test led to a short-lasting, dose-dependent increase in pattern separation memory that was not present anymore after 48 h. Furthermore, treatment with RG108 did not enhance long-term memory of the animals when tested after a 24 h inter-trial interval in the same task. At the transcriptomic level, acute treatment with RG108 was accompanied by increased expression of Bdnf1 , while expression of Bdnf4 , Bdnf9 , Gria1 and Hdac2 was not altered within 1 h after treatment. Methylation analysis of 14 loci in the promoter region of Bdnf1 revealed a counterintuitive increase in the levels of DNA methylation at three CpG sites. Taken together, these results indicate that acute administration of RG108 has a short-lasting pro-cognitive effect on object pattern separation that could be explained by increased Bdnf1 expression. The observed increase in Bdnf1 methylation suggests a complex interplay between Bdnf methylation-demethylation that promotes Bdnf1 expression and associated cognitive performance. Considering that impaired pattern separation could constitute the underlying problem of a wide range of mental and cognitive disorders, pharmacological agents including DNA methylation inhibitors that improve pattern separation could be compelling targets for the treatment of these disorders. In that respect, future studies are needed in order to determine the effect of chronic administration of such agents. [ABSTRACT FROM AUTHOR]

Abstract Bone marrow (BM)-derived endothelial progenitor cells (EPCs) are the key players in angiogenesis and vascular function. Cystathionine-β-synthase (CBS), an H 2 S-generating enzyme in methionine metabolism, regulates the function of these EPCs. This study aims to examine whether CBS hyper-methylation contributes to the bone marrow endothelial progenitor cell (BM-EPCs) function and subsequent bone blood flow in mice fed with a high methionine diet (HMD). Bone marrow (BM) cells were collected from HMD and control mice, differentiated into BM-EPCs, and were characterized by acLDL-DiI labeling. CBS mRNA expression was analyzed by real-time PCR, and the global methylation status and methylation of the CBS promoter were detected by nuclear 5-mC assay and methylation-specific PCR (qMSP) respectively. The result reveals that CBS promoter in BM-EPCs from HMD mice was hyper-methylated and the methylation level was, indeed, negatively correlated with CBS mRNA and angiogenic function of BM-EPCs. In addition, global methylation (5-mC) and DNA methyltransferase-1 (DNMT1) expression were increased in HMD condition. In vitro study also shows that HMD induced hyperhomocysteinemia (HHcy) impaired both adhesion and angiogenesis properties of BM-EPCs, accompanied by higher methylation level of CBS promoter that compared to control. Furthermore, bone blood flow was found to be decreased in HMD mice as compared to wild-type mice. To dissect the epigenetic mechanism, we also administrated DNMT inhibitor, 5-azacytidine (5-Aza) to HMD mice. The administration of 5-Aza in HMD mice restored the CBS expression, EPC mediated angiogenesis and blood flow by reducing abnormal DNA hyper-methylation. In conclusion, HHcy dismantles BM-EPCs function and bone blood flow through the hyper-methylation of the CBS promoter in HMD fed mice. Highlights • High methionine diet (HMD) fed mice develop the hyperhomocysteinemia phenotype. • CBS gene promoter is Hyper-methylated in HMD fed mice. • Hypermethylation of CBS promoter causes BM-EPCs dysfunction and bone blood flow in HMD mice. • Administration of 5-Aza (DNMT inhibitor) in HMD mice restores the BM-EPCs function and bone blood flow. [ABSTRACT FROM AUTHOR]

Abstract DNA vaccines require a vector to replicate genes and express encoding antigens. Antibiotic resistance genes are often used as selection markers, which must not be released to the environment upon final product commercialization. For this reason, generation of antibiotic resistance-free vectors is imperative. The pPAL vector contains the cytomegalovirus enhancer and promoter for expression in mammalian cells and the E. coli fabI chromosomal gene as a selectable marker. The fabI gene encodes the enoyl-ACP reductase (FabI). The bacteriostatic compound triclosan is an inhibitor of this enzyme. Therefore, the selection of positive clones depends on the enzyme:inhibitor molar ratio. According to western blot analysis, the pPAL vector is functional for expression of the Leishmania infantum (Kinetoplastid: Trypanosomatidae) gene encoding for the protein kinase C receptor analog (LACK/p36) in the HEK293T human cell line transfected with pPAL-LACK. The fabI gene sequence contains a 210 bp CpG island, suggesting a potential role as an adjuvant of the antibiotic resistance-free pPAL vector. In fact, Th1 response induction levels against canine leishmaniasis only using pPAL-LACK was shown to be as strong as in previous strategies using a recombinant vaccinia virus in combination with standard mammalian expression plasmid vectors. In summary, the pPAL plasmid contains the essential elements for manipulation and expression of any cloned DNA sequence in prokaryotic and mammalian cells using an E. coli endogenous gene as a selectable marker, which also provides a long CpG island. This element enhances Th1 immune response against L. infantum infection in dogs using the gene encoding for the LACK antigen. Therefore, this antibiotic resistance-free plasmid is a vaccine vector actively participating in protection against canine leishmaniasis and may be potentially tested as a vaccine vector with other antigens against different pathogens. Graphical abstract Unlabelled Image Highlights • pPAL is an antibiotic resistance-free mammalian expression plasmid vector based on the E. coli fabI gene selection system. • The Leishmania infantum LACK gene is successfully over-expressed in the HEK293T human cell line. • Th1 cytokine levels are similar using pPAL-LACK and previous strategies which include recombinant vaccinia viruses. [ABSTRACT FROM AUTHOR]

Abstract Normal development involves the interplay of genetic and epigenetic regulatory mechanisms. Pax6 is an eye-selector factor responsible for initiating the regulatory cascade for the development of the eyes. For the olive ridley sea turtle (Lepidochelys olivacea), a threatened species, eye malformations have been reported. In order to study the DNA methylation status of the putative promoter of the Pax6 gene in embryos with ocular malformations, an exploratory study was carried out in which DNA was isolated from embryos with anophthalmia, microphthalmia, and cyclopia, as well as from their normal counterparts. The 5′-flanking region from the Pax6 gene was isolated, showing two CpG islands (CGIs). The methylation status of CGIs in malformed embryos was compared with that of normal embryos by bisulfite sequencing. Putative transcription factor binding sites and regulatory features were identified. Methylation patterns were observed in both CpG and non-CpG contexts, and were unique for each malformed embryo; in the CpG context, an embryo with cyclopia showed a methylated cytosine upstream the CGI-1 not present in other embryos, an embryo with left anophthalmia presented two methylated cytosines in the CGI-1, whereas an embryo with left anophthalmia and right microphthalmia showed two methylated cytosines in the CGI-2. Normal embryos did not show methylated cytosines in the CGI-1, but one of them showed one methylcytosine in the CGI-2. Methylated transcription factor-binding sites may affect Pax6 expression associated to the cellular response to environmental compounds and hypoxia, signal transduction, cell cycle, lens physiology and development, as well as the transcription rate. Although preliminary, these results suggest that embryos with ocular malformations present unique DNA methylation patterns in the putative promoter of the Pax6 gene in L. olivacea , and probably those subtle, random changes in the methylation status can cause (at least in part) the aberrant phenotypes observed in these embryos. Graphical abstract Unlabelled Image Highlights • The olve ridley Pax6 putative promoter region was isolated and characterized. • The methylation status was analyzed in embryos with eye malformations. • Malformed embryos showed different methylcytosines in specific TFBS. • The methylation status was apparently random and individual. • Subtle changes in methylation may be associated to aberrant phenotypes. [ABSTRACT FROM AUTHOR]

Abstract As an important epigenetic modification DNA methylation is catalyzed by DNA methylation transferases (DNMTs) and occurs mainly in CpG islands. DNA methylation plays an important role in regulates gene expression, cell differentiation, genetic imprinting and tumor therapy. Retinoic acid-binding proteins (RAC) is vital for the absorption, transport, metabolism and maintenance of homeostasis of retinoic acid, which in turn regulates the differentiation and proliferation of cells by regulating the transcription of many target genes, therefore, these proteins influence differentiation and proliferation of adipocytes and muscle fibroblasts. Thus, cellular retinoic acid binding protein 2 (CRABP2) may be a candidate gene which affects beef quality, yield and fat deposition. The aim of this study was to evaluate the expression and the methylation pattern on the differentially methylated region (DMR) of the promoter of CRABP2. The DNA methylation pattern was tested by bisulfite sequencing polymerase chain reaction (BSP), the quantitative real-time PCR (qPCR) was used to analysis the expression of CRABP2 gene. The results showed that the DNA methylation level was higher in purebred cattle breed than that in hybrid cattle breeds which was negative correlation with the expression of the gen. These results indicate that the methylation status of the CRABP2 DMR can regulate mRNA expression. What's more, there are different methylation and expression patterns in different breeds and tissues which may influence the phenotype, and the results may be a useful parameter to investigate the function of CRABP2 in muscle and fat developmental in Chinese cattle. Highlights • The CRABP2 plays an important role in beef quality, yield and fat deposition. • The appropriate DNA methylation is essential for normal gene function. • The Jianxian Red cattle exhibited a higher mRNA level and DNA methylation level than the hybrid cattle during in muscle and fat tissues. • A useful molecular marker for muscle developmental and as a model for studies in other species. [ABSTRACT FROM AUTHOR]

Objective: The pathogenesis of endometriosis is still mysterious, being retrograde menstruation and coelomic metaplasia the most accepted hypotheses. Recently, it has been proposed that endometriosis is caused by fine-tuning alterations of the female genital system development during the foetal life and that in utero exposition to endocrine disruptors can be one of the factors causing the disease, possibly acting on the methylation status of the genome. In this study, we have evaluated the methylation status of HOXA10 gene regulation regions in a cohort of 22 endometriosis patients respect to a control group of 6 healthy women. Results: The methylation study was carried out on three CpG islands, previously described hypermethylated in the endometrium of endometriosis patients and include 22 CpG sites, 21 CpG sites and 10 CpG sites, respectively identified through the online platform MethPrimer. The analysis did not find significant differences between patients with endometriosis and healthy control individuals. These results confirm previous studies on genome wide methylation analysis in endometriosis patients. Therefore, other epigenetically altered genes should be considered more related to the pathogenesis of endometriosis. [ABSTRACT FROM AUTHOR]

The article looks at a study which evaluates impact of azacytidine treatment on aberrant DNA methylation in Juvenile Myelomonocytic Leukaemia (JMML) patients. Topics discussed include membrane elements such as adhesion molecules, developmental processes and cell cycle-related genes, assessment and genomic DNA extraction and sequence analysis and scenario of pathogenetic epigenetic regulation and specific levels of hypermethylation.

Rbpms (RNA-binding protein with multiple splicing) is a member of the RRM (RNA Recognition Motif) family of RNA-binding proteins, which is expressed as multiple alternatively spliced transcripts encoding different protein isoforms. We have shown earlier that Rbpms expression in the retina is restricted to retinal ganglion cells (RGCs), and have characterized this gene as a marker for RGCs. The aim of this study was to identify isoforms representing Rbpms in human retinas and to analyze its transcriptional regulation. We found that Rbpms is expressed as transcription variants 1 and 3 encoding isoforms A and C, respectively. These isoforms are encoded by the same first 6 exons but have different C-terminal ends encoded by exon 8 in variant 1 and exon 7 in variant 3. Computational analysis of the Rbpms 5′ untranslated and flanking regions reveals the presence of three CpG islands and four predicted promoter regions (PPRs). The effect of PPR 1 (− 1672/− 1420) and PPR2 (− 330/− 79) on transcriptional activation was minimal, whereas PPR 3 (− 73/+ 177) and PPR4 (+ 274/+ 524) induced the expression by ~ 7 and ninefold compared to control, respectively. The maximum activity, a 30-fold increase above the control level, was obtained from the construct containing both PPRs 3 and 4. Site-directed mutagenesis of several cis-elements within PPR3 and PPR4 including five for Sp1, one for AP1, and two for NF-kB showed that mutation of the first three and especially the first GC box resulted in a threefold downregulation of gene expression. AP1, NF-kB, and two downstream Sp1 sites had no significant effect on expression level. The possible involvement of the GC box 1 at position − 54 in transcriptional regulation of Rbpms was corroborated by EMSA, which showed formation of a DNA-protein complex in the presence of the oligonucleotide corresponding to this Sp1-binding site. [ABSTRACT FROM AUTHOR]

CpG islands (CGIs) are associated with ∼60% of mammalian promoters. Most unmethylated CGIs exhibit transcriptional activity, which has led to their co-option as promoters by retrogenes. CGIs may also serve as alternative promoters for downstream genes with methylated promoters, with implications on aberrant activation of oncogenes in cancer phenotypes. [ABSTRACT FROM AUTHOR]

We present MethyMer, a Python-based tool aimed at selecting primers for amplification of complete CpG islands. These regions are difficult in terms of selecting appropriate primers because of their low-complexity, high GC content. Moreover, bisulfite treatment, in fact, leads to the reduction of the 4-letter alphabet (ATGC) to 3-letter one (ATG, except for methylated cytosines), and this also reduces region complexity and increases mispriming potential. MethyMer has a flexible scoring system, which optimizes the balance between various characteristics such as nucleotide composition, thermodynamic features (melting temperature, dimers G, etc.), the presence of CpG sites and polyN tracts, and primer specificity, which is assessed with aligning primers to the bisulfite-treated genome using bowtie (up to three mismatches are allowed). Users are able to customize desired or limit ranges of various parameters as well as penalties for non-desired values. Moreover, MethyMer allows picking up the optimal combination of PCR primer pairs to perform the amplification of a large genomic locus, e.g. CpG island or other hard-to-study region, with minimal overlap of the individual amplicons. MethyMer incorporates ENCODE genome annotation records (promoter/enhancer/insulator), The Cancer Genome Atlas (TCGA) CpG methylation data derived with Illumina Infinium 450K microarrays, and records on correlations between TCGA RNA-Seq and CpG methylation data for 20 cancer types. These databases are included in the MethyMer release. Our tool is available at . [ABSTRACT FROM AUTHOR]

Complexity in tissues affected by cancer arises from somatic mutations and epigenetic modifications in the genome. The mutation susceptible hotspots present within the genome indicate a non-random nature and/or a position specific selection of mutation. An association exists between the occurrence of mutations and epigenetic DNA methylation. This study is primarily aimed at determining mutation status, and identifying a signature for predicting mutation prone zones of tumor suppressor (TS) genes. Nearby sequences from the top five positions having a higher mutation frequency in each gene of 42 TS genes were selected from a cosmic database and were considered as mutation prone zones. The conserved motifs present in the mutation prone DNA fragments were identified. Molecular docking studies were done to determine putative interactions between the identified conserved motifs and enzyme methyltransferase DNMT1. Collective analysis of 42 TS genes found GC as the most commonly replaced and AT as the most commonly formed residues after mutation. Analysis of the top 5 mutated positions of each gene (210 DNA segments for 42 TS genes) identified that CG nucleotides of the amino acid codons (e.g., Arginine) are most susceptible to mutation, and found a consensus DNA “T/AGC/GAGGA/TG” sequence present in these mutation prone DNA segments. Similar to TS genes, analysis of 54 oncogenes not only found CG nucleotides of the amino acid Arg as the most susceptible to mutation, but also identified the presence of similar consensus DNA motifs in the mutation prone DNA fragments (270 DNA segments for 54 oncogenes) of oncogenes. Docking studies depicted that, upon binding of DNMT1 methylates to this consensus DNA motif (C residues of CpG islands), mutation was likely to occur. Thus, this study proposes that DNMT1 mediated methylation in chromosomal DNA may decrease if a foreign DNA segment containing this consensus sequence along with CG nucleotides is exogenously introduced to dividing cancer cells. [ABSTRACT FROM AUTHOR]

MicroRNAs (miRNAs) are single-stranded RNAs of 18-25 nucleotides that regulate gene expression at the post-transcriptional level. They are involved in many physiological and pathological processes, including cell proliferation, apoptosis, development and carcinogenesis. Because of the central role of miRNAs in the regulation of gene expression, their expression needs to be tightly controlled. Here, we summarize the different mechanisms of epigenetic regulation of miRNAs, with a particular focus on DNA methylation and histone modification. [ABSTRACT FROM AUTHOR]

DNA methylation is a prevalent epigenetic modification involved in transcriptional regulation and essential for mammalian development. While the genome-wide distribution of this mark has been studied to great detail, the mechanisms responsible for its correct deposition, as well as the cause for its aberrant localization in cancers, have not been fully elucidated. Here, we have compared the activity of individual DNMT3A isoforms in mouse embryonic stem and neuronal progenitor cells and report that these isoforms differ in their genomic binding and DNA methylation activity at regulatory sites. We identify that the longer isoform DNMT3A1 preferentially localizes to the methylated shores of bivalent CpG island promoters in a tissue-specific manner. The isoform-specific targeting of DNMT3A1 coincides with elevated hydroxymethylcytosine (5-hmC) deposition, suggesting an involvement of this isoform in mediating turnover of DNA methylation at these sites. Through genetic deletion and rescue experiments, we demonstrate that this isoform-specific recruitment plays a role in de novo DNA methylation at CpG island shores, with potential implications on H3K27me3-mediated regulation of developmental genes. [ABSTRACT FROM AUTHOR]

Background: DNA methylation is an epigenetic modification that is studied at a single-base resolution with bisulfite treatment followed by high-throughput sequencing. After alignment of the sequence reads to a reference genome, methylation counts are analyzed to determine genomic regions that are differentially methylated between two or more biological conditions. Even though a variety of software packages is available for different aspects of the bioinformatics analysis, they often produce results that are biased or require excessive computational requirements. Results: DMRfinder is a novel computational pipeline that identifies differentially methylated regions efficiently. Following alignment, DMRfinder extracts methylation counts and performs a modified single-linkage clustering of methylation sites into genomic regions. It then compares methylation levels using beta-binomial hierarchical modeling and Wald tests. Among its innovative attributes are the analyses of novel methylation sites and methylation linkage, as well as the simultaneous statistical analysis of multiple sample groups. To demonstrate its efficiency, DMRfinder is benchmarked against other computational approaches using a large published dataset. Contrasting two replicates of the same sample yielded minimal genomic regions with DMRfinder, whereas two alternative software packages reported a substantial number of false positives. Further analyses of biological samples revealed fundamental differences between DMRfinder and another software package, despite the fact that they utilize the same underlying statistical basis. For each step, DMRfinder completed the analysis in a fraction of the time required by other software. Conclusions: Among the computational approaches for identifying differentially methylated regions from high-throughput bisulfite sequencing datasets, DMRfinder is the first that integrates all the post-alignment steps in a single package. Compared to other software, DMRfinder is extremely efficient and unbiased in this process. DMRfinder is free and open-source software, available on GitHub (github.com/jsh58/DMRfinder); it is written in Python and R, and is supported on Linux. [ABSTRACT FROM AUTHOR]

Objective: As a frequent hypertensive disorder in pregnancy, preeclampsia causes 15% of all preterm deliveries. This study aims to understand its mechanism via analyzing gene expression and methylation data.Methods: Human gene expression microarray data (GSE73374) and corresponding DNA methylation microarray data (GSE73375) of preeclamptic placental tissues (n = 19) and normotensive placental tissues (n = 17) were obtained from the Gene Expression Omnibus. Differentially expressed genes (DEGs), differentially methylated regions (DMRs) and CpG islands (DMIs) were identified and compared. Pathway enrichment analysis was performed, and the functional interactions were investigated.Results: A total of 392 DEGs, 2667 DMRs, and 464 DMIs were found between the two groups, and a functional interaction network of DEGs was established. Then, 28 potential key genes were identified, which were oppositely regulated in preeclampsia regarding expression and methylation. Eight of the potential key genes had DMIs, among which ITGA5 interacted with FN1 and FOS. The 3 DEGs were up-regulated, and their DMIs were hypomethylated.Conclusion: FN1, FOS, and ITGA5 might promote the development of preeclampsia via modulating differentiation/apoptosis/invasion of human extravillous trophoblasts, as well as focal adhesion, VEGFR3 signaling, β1 integrin cell surface interactions, α9β1/α4β1 integrin signaling, AP-1/ATF-2/HIF-1α transcription factor networks, and Fc-epsilon receptor I signaling in mast cells. [ABSTRACT FROM AUTHOR]

Evolutionary studies of DNA methylation offer insights into the mechanisms governing the variation of genomic DNA methylation across different species. Comparisons of gross levels of DNA methylation between distantly related species indicate that the size of the genome and the level of genomic DNA methylation are positively correlated. In plant genomes, this can be reliably explained by the genomic contents of repetitive sequences. In animal genomes, the role of repetitivfe sequences on genoimc DNAmethylation is less clear. On a shorter timescale, population-level comparisons demonstrate that genetic variation can explain the observed variabilityofDNAmethylation tosomedegree. The amount ofDNAmethylation variationthat hasbeenattributedtogenetic variationin the human population studies so far is substantially lower than that from Arabidopsis population studies, but this disparity might reflect the differences in the computational and experimental techniques used. The effect of genetic variation on DNAmethylation has been directly examined inmammalian systems, revealing several causative factors that govern DNA methylation. On the other hand, studies from Arabidopsis have furthered our understanding of spontaneous mutations of DNA methylation, termed "epimutations." Arabidopsis has an extremely high rate of spontaneous epimutations, which may play a major role in shaping the global DNA methylation landscape in this genome. Key missing information includes the frequencies of spontaneous epimutations in other lineages, in particular animal genomes, and howpopulation-level variation ofDNAmethylation leads to species-level differences. [ABSTRACT FROM AUTHOR]

A novel sensitive method for detection of DNA methylation was developed with thioglycollic acid (TGA)-capped CdTe quantum dots (QDs) as fluorescence probes. Recognition of methylated DNA sites would be useful strategy due to the important roles of methylation in disease occurrence and developmental processes. DNA methylation occurs most often at cytosine-guanine sites (CpG dinucleotides) of gene promoters. The QDs significantly interacted with hybridized unmethylated and methylated DNA. The interaction of CpG rich methylated and unmethylated DNA hybrid with quantum dots as an optical probe has been investigated by fluorescence spectroscopy and electrophoresis assay. The fluorescence intensity of QDs was highly dependent to unmethylated and methylated DNA. Specific site of CpG islands of Adenomatous polyposis coli (APC), a well-studied tumor suppressor gene, was used as the detection target. Under optimum conditions, upon the addition of unmethylated dsDNA, the fluorescence intensity increased in linear range from 1.0 × 10 to 1.0 × 10M with detection limit of 6.2 × 10 M and on the other hand, the intensity of QDs showed no changes with addition of methylated dsDNA. We also demonstrated that the unmethylated and methylated DNA and QDs complexes showed different mobility in electrophoresis assay. This easy and reliable method could distinguish between methylated and unmethylated DNA sequences. [ABSTRACT FROM AUTHOR]

Background: DNA methylation is an important tissue-specific epigenetic event that influences transcriptional regulation of gene expression. Differentially methylated CpG sites may act as mediators between genetic variation and gene expression, and this relationship can be exploited while mapping multi-tissue expression quantitative trait loci (eQTL). Current multi-tissue eQTL mapping techniques are limited to only exploiting gene expression patterns across multiple tissues either in a joint tissue or tissue-by-tissue frameworks. We present a new statistical approach that enables us to model the effect of germ-line variation on tissue-specific gene expression in the presence of effects due to DNA methylation. Results: Our method efficiently models genetic and epigenetic variation to identify genomic regions of interest containing combinations of mRNA transcripts, CpG sites, and SNPs by jointly testing for genotypic effect and higher order interaction effects between genotype, methylation and tissues. We demonstrate using Monte Carlo simulations that our approach, in the presence of both genetic and DNA methylation effects, gives an improved performance (in terms of statistical power) to detect eQTLs over the current eQTL mapping approaches. When applied to an array-based dataset from 150 neuropathologically normal adult human brains, our method identifies eQTLs that were undetected using standard tissue-by-tissue or joint tissue eQTL mapping techniques. As an example, our method identifies eQTLs by leveraging methylated CpG sites in a LIM homeobox member gene (LHX9), which may have a role in the neural development. Conclusions: Our score test-based approach does not need parameter estimation under the alternative hypothesis. As a result, our model parameters are estimated only once for each mRNA - CpG pair. Our model specifically studies the effects of non-coding regions of DNA (in this case, CpG sites) on mapping eQTLs. However, we can easily model micro-RNAs instead of CpG sites to study the effects of post-transcriptional events in mapping eQTL. Our model's flexible framework also allows us to investigate other genomic events such as alternative gene splicing by extending our model to include gene isoform-specific data. [ABSTRACT FROM AUTHOR]

Vertebrate ALDH18A 1 genes encode a bifunctional mitochondrial enzyme, catalyzing a 2-step conversion of glutamate to glutamyl semialdehyde, subsequently converted into proline, ornithine and arginine. Bioinformatic analyses of vertebrate and invertebrate genomes were undertaken using known ALDH18A1 amino acid sequences. G5K (glutamyl kinase) and GPR (glutamyl phosphate reductase) domain sequences were identified for all vertebrate and invertebrate genomes examined, whereas bacterial sequences encoded separate enzymes. Vertebrate ALDH18A1 (also called P5CS) sequences were highly conserved throughout vertebrate evolution. A mechanism for generating two major vertebrate ALDH18A1 isoforms is proposed with ‘a’ isoform containing Asn239-Val240 with wide tissue expression, whereas the ‘b’ isoform lacking the dipeptide has been reported in gut tissues. Phylogenetic analyses describe the relationships and potential origins of the ALDH18A1 gene during vertebrate and invertebrate evolution and a proposal for generating the bifunctional vertebrate and invertebrate ALDH18A1 gene from a bacterial operon ( proBA ) encoding G5K and GPR. A more recent Aldh18a1 gene duplication event has apparently occurred with a primordial rat genome. [ABSTRACT FROM AUTHOR]

Prostate cancer (PCa), a multifocal clinically heterogeneous disease, is the most commonly diagnosed non-cutaneous neoplasm in men worldwide. The epigenome of PCa is a typical representation of catastrophic model of epigenetic alterations during tumorigenesis and its progression. Alterations in methylation patterns in tumor suppressors, cell cycle, oncogenes and metabolism-related genes are the most commonly observed epigenetic alterations in PCa. In this study, we have developed a computational strategy to identify methylated biomarker signature panels as potential targets of PCa by screening >160 genes reported to be epigenetically dysregulated, and shortlisted 26 differentially methylated genes (DMGs) that significantly contribute to oncogenesis. The gene ontology and functional enrichment analysis were performed, which showed that identified DMGs contribute to cellular and metabolic processes such as cell communication, cell cycle, response to drugs, apoptosis and p53 signaling. The top hub genes AR, CDH13, CDKN2A, DAPK1, GSTP1, CD44 and RASSF1 identified from protein-protein interaction network construction using Search Tool for the Retrieval of Interacting Genes contributed to hormonal response, inflammatory response, cell cycle, reactive oxygen species activity and receptor kinase activity, which are related to hallmarks of cancer as revealed by their functional enrichment analysis by Cytoscape. These genes were further scrutinized for CpG islands, transcription start sites and positions of methylated cytosines to study their methylation profiles. Our analysis revealed high negative correlation values between methylation frequencies and gene expressions of the hub genes, namely, AR, CDH13, CDKN2A, DAPK1, CD44, GSTP1 and RASSF1, which can be used as potential diagnostic biomarkers for PCa. [ABSTRACT FROM AUTHOR]