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In this study, we analyzed the regulatory properties of 26 (twenty-six) genes associated with nonsyndromic male infertility. We applied an in silico analysis in order to determine the number and distribution of promoters and identify relevant promoter consensus sequences and potential transcription factors. Underlining the concept of alternative transcriptional initiation (ATI), we have found that 65.4% of genes associated with nonsyndromic male infertility have 1 (one) to 6 (six) promoters, located in the region 1 kb upstream of the TSS, and 41% of them are located at a position below −500 bp. Although the TATA box consensus sequence TAWAAA, such as W is A or T, appears at a common location in all genes, it is shifted for at least 10 bp in the EFCAB9 gene. The C2H2 zinc finger is found to be the most significant common transcription factor, binding genes' promoters GLIS1, ZSCAN21, GLIS3, GLIS1, ZNF770, ZNF780A, ZNF81, and ZNF264. On the other hand, basic leucine zipper factors (bZIPs) bind the JUNB gene promoter specifically, exhibiting unique regulatory properties of all genes associated with nonsyndromic male infertility. Two genes, NANOS1 and ZMYND15, are expected to be less susceptible to DNA methylation, due to the high density of CpG content found in their promoter regions. [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]

G-quadruplexes (G4s) are secondary structures in DNA that have been shown to be involved in gene regulation. They play a vital role in the cellular processes and several pathogens including bacteria, fungi, and viruses have also been shown to possess G4s that help them in their pathogenesis. Additionally, cross-talk among the CpG islands and G4s has been shown to influence biological processes. The virus-encoded G4s are affected by the mutational landscape leading to the formation/deletion of these G4s. Therefore, understanding and predicting these multivariate effects on traditional and non-traditional quadruplexes forms an important area of research, that is, yet to be investigated. We have designed a user-friendly webserver QUFIND (http://soodlab.com/qufinder/) that can predict traditional as well as non-traditional quadruplexes in a given sequence. QUFIND is connected with ENSEMBL and NCBI so that the sequences can be fetched in a real-time manner. The algorithm is designed in such a way that the user is provided with multiple options to customize the base (A, T, G, or C), size of the stem (2-5), loop length (1-30), number of bulges (1-5) as well as the number of mismatches (0-2) enabling the identification of any of the secondary structure as per their interest. QUFIND is designed to predict both CpG islands as well as G4s in a given sequence. Since G4s are very short as compared to the CpG islands, hence, QUFIND can also predict the overlapping G4s within CpG islands. Therefore, the user has the flexibility to identify either overlapping or non-overlapping G4s along with the CpG islands. Additionally, one section of QUFIND is dedicated to comparing the G4s in two viral sequences. The visualization is designed in such a manner that the user is able to see the unique quadruplexes in both the input sequences. The efficiency of QUFIND is calculated on G4s obtained from G4 high throughput sequencing data (n = 1000) or experimentally validated G4s (n = 329). Our results revealed that QUFIND is able to predict G4-quadruplexes obtained from G4-sequencing data with 90.06% prediction accuracy whereas experimentally validated quadruplexes were predicted with 97.26% prediction accuracy. [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]

The jaagsiekte sheep retrovirus (JSRV), belonging to the betaretrovirus genus of the retroviridae family, includes both exogenous and endogenous jaagsiekte sheep retroviruses (exJSRV and enJSRV, respectively). At the proviral genome level, exJSRV and enJSRV strains have a high degree of similarity with their main variation regions being the LTR, gag, and env genes. In this study, for the first time, we investigated and compared the distribution of CpG islands between these enJSRV and exJSRV strains. Specifically, we analyzed a total of 42 fulllength JSRV genomic sequences obtained from the GenBank® database to identify CpG islands in the exJSRV and enJSRV genomes using the MethPrimer software. Our results showed that the CpG islands in the two JSRV strains were mainly distributed in the LTR, gag, and env genes. In exJSRVs, 66.66% (6/9), 33.33% (3/9), and 100% (9/9) of the sequences presented at least one CpG island in LTR, gag, env genes, respectively, and for enJSRVs, 84.84% (28/33), 57.57% (19/33), and 96.96% (32/33) of the sequences presented at least one CpG island in the LTR, gag, and env genes. These findings suggested that the distribution, length, and genetic traits of CpG islands were different for the exJSRV and enJSRV strains. In future, it would be necessary to demonstrate the biological significance of CpG islands within these genes in exJSRV and enJSRV genomes. This will enhance understanding regarding the potential role of CpG islands in epigenetic regulation. [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]

Phosphorus (P) is the second-most essential macronutrient required for the growth and development of plants. It is involved in a number of cellular processes that contribute to the plant's growth and development. This study investigated Saccharum spp. hybrid and Sorghum bicolor promoter regions of Phosphate transporters (PHT), viz., PHT1, PHT2, PHT3, PHT4, and PHO1, through in silico analysis. The transcription start sites (TSS), conserved motifs, and CpG islands were studied using various computational techniques. The distribution of TSSs indicated the highest promoter prediction scores (1.0). MSh2 and MSb4 were recognized as the common promoter motifs for PHT promoters, found in with 85 to 100% percentage of distribution. The CpG analysis revealed that the promoter regions of most PHT genes had low CpG density, indicating a possible tissue-specific expression. The PHT promoters were investigated for the presence of biotic- and abiotic-stress-associated transcription factor binding sites (TFbs) that revealed the presence of binding motifs for major transcription factors (TFs), namely, AP2/ERF, bHLH, bZIP, MYB, NAC, and WRKY. Therefore, the in-silico analysis of the promoter regions helps us to understand the regulation mechanism of phosphate transporter promoters and gene expression under stress management. The 5′ regulatory region of the EaPHT gene was isolated from Erianthus, a wild relative of the genus Saccharum. The promoter construct was prepared and transformed in tobacco wherein the promoter drove the expression of GUS. Analysis of GUS expression in transgenic tobacco revealed enhanced expression of GUS under salt-stress conditions. This is the first report of the isolation and characterization of a phosphate transporter gene promoter from Erianthus and is expected to be useful for the development of salt-stress transgenic crop plants. [ABSTRACT FROM AUTHOR]

Hepatitis B virus (HBV) infection led to 66% liver deaths world-wide in year 2015. Thirty-seven per cent of these deaths were the result of chronic hepatitis B (CHB)-associated hepatocellular carcinoma (HCC). Although early diagnosis of HCC improves survival, early detection is rare. Methylation of HBV DNA including covalently closed circular DNA (cccDNA) is more often encountered in HCC cases than those in CHB and cirrhosis. Three typical CpG islands within the HBV genome are the common sites for methylation. The HBV cccDNA methylation affects the viral replication and protein expression in the course of infection and may associate with the disease pathogenesis and HCC development. We review the current findings in HBV DNA methylation that provide insights into its role in HCC diagnosis. [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: Gene silence via methylation of the CpG islands is cancer's most common epigenetic modification. Given the highly significant role of NIS in thyroid cancer (TC) differentiation, this cross-sectional study aimed to investigate the DNA methylation pattern in seven CpG islands (CpG1-7 including +846, +918, +929, +947, +953, +955, and +963, respectively) of the NIS promoter in patients diagnosed with papillary (PTC), follicular (FTC), and multinodular goiter (MNG). Additionally, a systematic review of the literature was conducted to compare our results with studies concerning methylation of the NIS gene promoter. Methods: Thyroid specimens from 64 patients met the eligibility criteria, consisting of 28 PTC, 9 FTC, and 27 benign MNG cases. The mRNA of NIS was tested by qRT-PCR. The bisulfite sequencing PCR (BSP) technique was performed to evaluate the promoter methylation pattern of the NIS gene. Sequencing results were received in chromatograph, FASTA, SEQ, and pdf formats and were analyzed using Chromas. The methylation percentage at each position and for each sample was calculated by mC/(mC+C) formula for all examined CpGs; following that, the methylation percentage was also calculated at each CpG site. Besides, a literature search was conducted without restricting publication dates. Nine studies met the eligibility criteria after removing duplicates, unrelated articles, and reviews. Results: NIS mRNA levels decreased in tumoral tissues of PTC (P = 0.04) and FTC (P = 0.03) patients compared to their matched non-tumoral ones. The methylation of NIS promoter was not common in PTC samples, but it was frequent in FTC (P < 0.05). Significant differences were observed in the methylation levels in the 4th(+ 947), 6th(+ 955), and 7th(+ 963) CpGs sites in the forward strand of NIS promoter between FTC and MNG tissues (76.34 ± 3.12 vs 40.43 ± 8.42, P = 0.004, 69.63 ± 3.03 vs 23.29 ± 6.84, P = 0.001 and 50.33 ± 5.65 vs 24 ± 6.89, P = 0.030, respectively). There was no significant correlation between the expression and methylation status of NIS in PTC and FTC tissues. Conclusion: Perturbation in NIS promoter's methylation individually may have a potential utility in differentiating MNG and FTC tissues. The absence of a distinct methylation pattern implies the importance of other epigenetic processes, which may alter the production of NIS mRNA. In addition, according to the reversibility of DNA methylation, it is anticipated that the design of particular targeted demethylation medicines will lead to a novel cancer therapeutic strategy. [ABSTRACT FROM AUTHOR]

Investigation of gene-environment cross talk through epigenetic modifications led to better understanding of the number of complex diseases. Clinical heterogeneity and differential treatment response often contributed by the epigenetic signatures which could be personal. DNA methylation at CpG islands presents a critical nuclear process as a result of gene-environment interactions. These CpG islands are frequently present near the promoter sequence of genes and get differentially methylated under specific environmental conditions. Technical advancements facilitate in high throughput screening of differentially methylated CpG islands. Recent epigenetic studies unraveled several CD susceptibility genes expressed in peripheral blood lymphocytes (PBLs), duodenal mucosa, lamina and epithelial cells that are influenced by differentially methylated CpG islands. Here we highlighted these susceptibility genes; classify these genes based on cellular functions and tissue of expression. We further discussed how these genes interacts with each other to influence critical pathways like NF-κB signaling pathway, IL-17 signaling cascade, RIG-I like receptor signaling pathway, NOD-like receptor pathways among several others. This review also shed light on how gut microbiota may lead to the differential methylation of CpG islands of CD susceptibility genes. Large scale epigenetic studies followed by estimation of heritability of these CpG methylation and polygenic risk score estimation of these genes would prioritize potentially druggable targets for better therapeutics. In vivo studies are warranted to unravel further cellular responses to CpG methylation. [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]

Transcriptional silencing of 35S rDNA loci inherited from one parental species is occurring relatively frequently in allopolyploids. However, molecular mechanisms by which it is selected for transcriptional silencing remain unclear. We applied NGS, silver staining and bisulfite sequencing to study the structure, expression and methylation landscape of 35S rDNA in two allopolyploids of common origin, allotetraploid Anemone multifida (2n = 4x = 32, genome composition BBDD) and allohexaploid A. baldensis (2n = 6x = 48, AABBDD), and their genome donors, A. sylvestris (2n = 16, AA), A. cylindrica (2n = 16, BB) and A. parviflora (2n = 16, DD). The size of the recovered 35S rDNA units varied from 10,489 bp in A. cylindrica to 12,084 bp in A. sylvestris. Anemone showed an organization typical of most ribosomal 35S rDNA composed of NTS, ETS, rRNA genes, TTS and TIS with structural features of plant IGS sequences and all functional elements needed for rRNA gene activity. The NTS was more variable than the ETS and consisted of SRs which are highly variable among Anemone. Five to six CpGrich islands were found within the ETS. CpG island located adjacent to the transcription initiation site (TIS) was highly variable regarding the sequence size and methylation level and exhibited in most of the species lower levels of methylation than CpG islands located adjacent to the 18S rRNA gene. Our results uncover hypomethylation of A. sylvestrisand A. parviflora-derived 35S rDNA units in allopolyploids A. multifida and A. baldensis. Hypomethylation of A. parviflora-derived 35S rDNA was more prominent in A. baldensis than in A. multifida. We showed that A. baldensis underwent coupled A. sylvestrisderived 35S rDNA array expansion and A. parviflora-derived 35S rDNA copy number decrease that was accompanied by lower methylation level of A. sylvestris-derived 35S rDNA units in comparison to A. parviflora-derived 35S rDNA units. These observations suggest that in A. baldensis nucleolar dominance is directed toward A. sylvestris-derived chromosomes. This work broadens our current knowledge of the 35S rDNA organization in Anemone and provides evidence of the progenitor-specific 35S rDNA methylation in nucleolar dominance [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]

Myotonic dystrophy type 1 (DM1) is a progressive, non-treatable, multi-systemic disorder. To investigate the contribution of epigenetics to the complexity of DM1, we compared DNA methylation profiles of four annotated CpG islands (CpGis) in the DMPK locus and neighbouring genes, in distinct DM1 tissues and derived cells, representing six DM1 subtypes, by bisulphite sequencing. In blood, we found no differences in CpGi 74, 43 and 36 in DNA methylation profile. In contrast, a CTCF1 DNA methylation gradient was found with 100% methylation in congenital cases, 50% in childhood cases and 13% in juvenile cases. CTCF1 methylation correlated to disease severity and CTG expansion size. Notably, 50% of CTCF1 methylated cases showed methylation in the CTCF2 regions. Additionally, methylation was associated with maternal transmission. Interestingly, the evaluation of seven families showed that unmethylated mothers passed on an expansion of the CTG repeat, whereas the methylated mothers transmitted a contraction. The analysis of patient-derived cells showed that DNA methylation profiles were highly preserved, validating their use as faithful DM1 cellular models. Importantly, the comparison of DNA methylation levels of distinct DM1 tissues revealed a novel muscle-specific epigenetic signature with methylation of the CTCF1 region accompanied by demethylation of CpGi 43, a region containing an alternative DMPK promoter, which may decrease the canonical promoter activity. Altogether, our results showed a distinct DNA methylation profile across DM1 tissues and uncovered a novel and dual epigenetic signature in DM1 muscle samples, providing novel insights into the epigenetic changes associated with DM1. [ABSTRACT FROM AUTHOR]

Chrysanthemum is a well-known ornamental plant with numerous uses. WRKY is a large family of transcription factors known for a variety of functions ranging from stress resistance to plant growth and development. Due to the limited research on the WRKY family in chrysanthemums, we examined them for the first time in Chrysanthemum lavandulifolium. A total of 138 ClWRKY genes were identified, which were classified into three groups. Group III in C. lavandulifolium contains 53 members, which is larger than group III of Arabidopsis. The number of introns varied from one to nine in the ClWRKY gene family. The "WRKYGQK" motif is conserved in 118 members, while other members showed slight variations. AuR and GRE responsive cis -acting elements were located in the promoter region of WRKY members, which are important for plant development and flowering induction. In addition, the W box was present in most genes; the recognition site for the WRKY gene may play a role in autoregulation and cross-regulation. The expression of the most variable 19 genes in terms of different parameters was observed at different stages. Among them, 10 genes were selected due to the presence of CpG islands, while nine genes were selected based on their close association with important Arabidopsis genes related to floral traits. ClWRKY36 and ClWRKY45 exhibit differential expression at flowering stages in the capitulum, while methylation is detected in three genes, including ClWRKY31, ClWRKY100 , and ClWRKY129. Our results provide a basis for further exploration of WRKY members to find their functions in plant growth and development, especially in flowering traits. [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: DNA methylation at CpG sites is a vital epigenetic modification of the human genome affecting gene expression, and potentially, health outcomes. However, evidence is just budding on the effects of aerobic exercise-induced adaptation on DNA methylation in older mild cognitively impaired (MCI) elderly African American (AAs). Therefore, we examined the effects of a 6-month aerobic exercise-intervention on genome-wide DNA methylation in elderly AA MCI volunteers. Design: Elderly AA volunteers confirmed MCI assigned into a 6-month program of aerobic exercise (eleven participants) underwent a 40-min supervised-training 3-times/week and controls (eight participants) performed stretch training. Participants had maximal oxygen consumption (VO2max) test and Genome-wide methylation levels at CpG sites using the Infinium HumanMethylation450 BeadChip assay at baseline and after a 6-month exercise program. We computed false discovery rates (FDR) using Sidak to account for multiplicity of tests and performed quantitative real-time polymerase chain-reaction (qRT-PCR) to confirm the effects of DNA methylations on expression levels of the top 5 genes among the aerobic participants. CpG sites identified from aerobic-exercise participants were similarly analyzed by the stretch group to quantify the effects of exercise-induced methylation changes among the group of stretch participants. Results: Eleven MCI participants (aerobic: 73% females; mean age 72.3 ± 6.6 years) and eight MCI participants (stretch: 75% female; mean age 70.6 ± 6.7 years) completed the training. Aerobic exercise-training was associated with increases in VO2max and with global hypo- and hypermethylation changes. The most notable finding was CpG hypomethylation within the body of the VPS52 gene (P = 5.4 × 10–26), a Golgi-associated protein, involved in intracellular protein trafficking including amyloid precursor protein. qRT-PCR confirmed a nearly twofold increased expression of VPS52. Other top findings with FDR q -value < 10–5, include hypomethylations of SCARB1 (8.8 × 10–25), ARTN (6.1 × 10–25), NR1H2 (2.1 × 10–18) and PPP2R5D (9.8 × 10–18). Conclusion: We conclude that genome-wide DNA methylation patterns is associated with exercise training-induced methylation changes. Identification of methylation changes around genes previously shown to interact with amyloid biology, intracellular protein trafficking, and lipoprotein regulations provide further support to the likely protective effect of exercise in MCI. Future studies in larger samples are needed to confirm our findings. [ABSTRACT FROM AUTHOR]

The genes for long noncoding RNAs (lncRNAs) are widely represented in mammals, but their functions remain largely unknown. One possible way to study lncRNAs is a detailed comparison of various characteristics of lncRNA and protein-coding genes, since there is extensive information on the functions of the latter. A feature of protein-coding genes in mammals is the high evolutionary conservation of the primary exon sequences. Although the conservation of the primary lncRNA exon sequences is not as pronounced as that of the protein-coding genes, it is nevertheless significantly higher than the intron sequences of the lncRNA genes. We assessed the conservation of the aforementioned traits based on multiple mammal alignments: human, chimpanzee, mouse, and rat. The conservation rate was assessed based on the gene segments, e.g., exons, introns, and promoter segments (300 bp upstream of transcription start site). A study of the relationship between the lncRNA gene conservation rate and the presence/absence of CpG islands (CGIs) found greater conservation of the lncRNA genes, which are located next to CGIs. This trend may be the cause of the previously identified association between conservation and the lncRNA expression level. A separate task was to annotate the types of lncRNA within the samples (sense, antisense, and intergenic lncRNA and pseudogenes). It was found that sense-lncRNAs (which reside preferentially in the coding loci) maintain the highest ratio of promoter CGIs. The second and third lncRNA types in terms of the occurrence of CGIs are pseudogenes and antisense lncRNAs (AS-lncRNAs), respectively. The least CGI-enriched lncRNAs are intergenic RNAs. This indicates that CpG islands are more inherent in the promoters of coding genes than noncoding ones. The overall conservation rate of promoter CGIs across all lncRNA classes has been estimated as 45%. The study highlights the presence of gene-specific signals in noncoding RNAs. For the first time (as far as we know), we have extended the spectrum of the (coding) gene-specific signals with the promoter CGI analysis. [ABSTRACT FROM AUTHOR]

Gene therapy can cure heritage diseases if the transmitted genes achieve long-term therapeutic expression in the specific tissues. The involvement of nucleases and immune response usually makes expression short-lived. DNA methylation is one of the most important mechanisms of gene expression regulation that is called epigenetics. The (pTagRFP-C) is a mammalian expression vector used to study DNA methylation. E. coli HB101 and JM109 used for the propagation unmethylated and methylated vector respectively. Both forms of vector are digested by MscI, un-methylated form of the vector is cut in two locations, but methylated form remain uncut after digestion. The CpG islands prediction shows about 10 islands were detected by meth-prime software. The size of island ranged (120-452) bp with GC% 50% and O/E 0.6. the methylation of RFP gene shows three islands with the size ranged (129-279) bp with 50% of GC and 0.6 O/E. figure (5) shows the CpG motifs. The CMV promoters didn’t contains CpG islands. [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]

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]

The purpose of this study was to investigate the association between physical effort and DNA methylation in the promoter region of the dopamine transporter gene (DAT1). The research group included 100 athletes (mean age = 22.88, SD = 6.35), whereas the control group were 239 healthy male volunteers matched for age (mean age = 21.69, SD = 3.39). Both, the control and the research group, included individuals with Caucasian origin from the same region of Poland. DNA was extracted from peripheral blood leukocytes using a DNA isolation kit (A&A Biotechnology, Gdynia, Poland). Bisulfite modification of 250 ng DNA was performed using the EZ DNA Methylation Kit (Zymo Research, Orange, CA, USA), according to manufacturer's instructions. The methylation-specific PCR assay was carried out in a Mastercycler epgradient S (Eppendorf, Germany). We observed that the level of general methylation of the CpG island was similar for both groups. Further exploration of individual CpG sites allowed to notice that there were significant differences in methylation status in specific positions. Nonetheless, there was no rule that would indicate either higher or lower methylation of individual sites, four of them were methylated at a higher level (positions 1, 4, 5, 7, 8, 9, 10, 11, 12, 13, 16, 17, 18, 23, 25, 26, 27, 29 and 30), while one showed an inverse trend (position 3). More precise analysis with the usage of Bonferroni correction for multiple tests indicated that differences in CpG site methylation were mainly increased in several positions and decreased in position 3. [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]

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]

Increasing evidence suggests that DNA methylation has key roles in the replication of retroviruses, including lentiviruses, and pathogenesis of diseases. However, the precise characteristics of CpG islands are not known for many retroviruses. In this study, we compared the distribution of CpG islands among strains of equine infectious anemia virus (EIAV), a lentivirus in the family Retroviridae and a model for HIV research. We identified CpG islands in 32 full-length EIAV genomic sequences obtained from the GenBank database using MethPrimer. Only one CpG island, from 100 to 120 bp, was identified in the genomes of EIAV strains DV10, DLV3-A, and DLV5-10 from China, V26 and V70 from Japan, and IRE H3, IRE F2, IRE F3, and IRE F4 from Ireland. Importantly, the CpG island was located within the Rev gene, which is required for the expression of viral cis-acting elements and the production of new virions. These results suggest that the distribution, length, and genetic properties of CpG islands differ among EIAV strains. Future research should focus on the biological significance of this CpG island within rev to improve our understanding of the precise roles of CpG islands in epigenetic regulation in the species. [ABSTRACT FROM AUTHOR]

It is well established that 5-methylcytosine (5mC) in genomic DNA of mammalian cells can be oxidized into 5-hydroxymethylcytosine (5hmC) and other derivates by DNA dioxygenase TETs. While conversion of 5mC to 5hmC plays an important role in active DNA demethylation through further oxidation steps, a certain proportion of 5hmCs remain in the genome. Although 5hmCs contribute to the flexibility of chromatin and protect bivalent promoters from hypermethylation, the direct effect of 5hmCs on gene transcription is unknown. In this present study, we have engineered a zinc-finger protein-based P16-specific DNA dioxygenase (P16-TET) to induce P16 hydroxymethylation and demethylation in cancer cells. Our results demonstrate, for the first time, that although the hydroxymethylated P16 alleles retain transcriptionally inactive, hydroxymethylation could increase the susceptibility of reactivation of methylated P16 alleles. [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]

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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]

The silencing of tumor suppressor genes by promoter CpG island (CGI) methylation is an important cause of oncogenesis. Silencing of MLH1 and BRCA1, two examples of oncogenic events, results from promoter CGI methylation. Interestingly, both MLH1 and BRCA1 have a divergent promoter, from which another gene on the opposite strand is also transcribed. Although studies have shown that divergent transcription is an important factor in transcriptional regulation, little is known about its implication in aberrant promoter methylation in cancer. In this study, we analyzed the methylation status of CGI in divergent promoters using a recently enriched transcriptome database. We measured the extent of CGI methylation in 119 colorectal cancer (CRC) clinical samples (65 microsatellite instability high [MSI‐H] CRC with CGI methylator phenotype, 28 MSI‐H CRC without CGI methylator phenotype and 26 microsatellite stable CRC) and 21 normal colorectal tissues using Infinium MethylationEPIC BeadChip. We found that CGI within divergent promoters are less frequently methylated than CGI within unidirectional promoters in normal cells. In the genome of CRC cells, CGI within unidirectional promoters are more vulnerable to aberrant methylation than CGI within divergent promoters. In addition, we identified three DNA sequence motifs that correlate with methylated CGI. We also showed that methylated CGI are associated with genes whose expression is low in normal cells. Thus, we here provide fundamental observations regarding the methylation of divergent promoters that are essential for the understanding of carcinogenesis and development of cancer prevention strategies. In this study, we revealed that the extent of promoter methylation was different depending on the arrangement of genes using DNA methylation data of 119 colorectal cancer specimens. Divergent promoters were hypomethylated compared to unidirectional promoters. The provide data is essential for the understanding of the carcinogenic mechanism. [ABSTRACT FROM AUTHOR]

Purpose: Epigenetic variations in the O6-methylguanine-methyltransferase gene had been widely associated with a favorable impact on survival in patients affected by glioblastoma multiforme (GBM). Aim of this study is to explore a scoring system based on the gene promoter methylation in order to predict patients’ prognosis.Methods: A series of 128 patients with GBM was retrospectively analyzed. A training set and a validations set were then generated. The methylation level of CpGi from 74 to 83 was determined by pyrosequencing. In accordance to previous literature, each island was assigned with 1 point if the corresponding methylation level was higher than 9%. The sum consisted in a score that went from 0 (all CpGi < 9%) to 10 (all CpGi ≥ 9%). A threshold capable to detect a favorable outcome (overall survival, OS > 24 months) was identified by ROC analysis.Results: Median OS and follow-up were 14 and 32.6 months respectively. Among the total population, 35% of the pts had a score of 0, while 29% had a score of 10. A score ≥ 6 was associated with a favorable prognosis also when corrected for age (> 70 vs. ≤ 70 years) and ECOG performance status (0-1 vs. 2-3). Similar results were observed also in terms of PFS. Results were consistent in the training and in the validation set.Conclusions: The present manuscript explored a novel scoring system capable to take into consideration the methylation status of each single CpGi, capable to better predict prognosis in GBM patients. [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]

We address the problem of the annotation of CpG islands (CGIs) clusters in the human genome. Upon analyzing gene content within CGIs clusters, piRNA, tRNA, and miRNA-encoding genes were found as well as CpG-rich homeobox genes reported previously. Chromosome-wide CGI density is positively correlated with replication timing, confirming that CGIs may serve as open chromatin markers. Early embryonic stage expressed KRAB-ZNF genes abundant at chromosome 19 were found to be interlinked with CGI clusters. We detected that a number of long CGIs and CGI clusters are, in fact, tandem copies with multiple annotated macrosatellites and paralogous genes. This finding implies that tandem expansion of CGIs may serve as a substrate for nonhomologous recombination events. [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]