A gene-encoded FRET fluorescent sensor designed for detecting asymmetric dimethylation levels in vitro and in living cells.

Arginine methylation is involved in many important biological processes. PRMT1 is a major arginine methyltransferase in mammalian cells and is highly conserved in eukaryotes. It catalyzes the methylation of various of substrates, including histones, and PRMT1 has been reported to be overexpressed in many cancers, indicating that it is a potential therapeutic target. No tool for efficient methylation level detection in living cells has been available to date. In this work, we designed and constructed a gene-encoded fluorescence resonance energy transfer (FRET) fluorescent sensor for detecting dimethylation levels in living cells and evaluated its functional efficiency both in vitro and in living cells. Both site-directed mutagenesis and PRMT1 inhibition experiments verified that the fluorescent sensor responded to changes in PRMT1 activity and to different PRMT1-induced methylation levels in vitro. Finally, we verified that this optimized methyl sensor responded sensitively to changes in methylation levels in living cells by overexpressing and inhibiting PRMT1, which makes it a useful tool for real-time imaging of arginine methylation. As a new tool for detecting arginine dimethylation levels in living cells, the designed FRET sensor is very important for posttranslational studies and may show a wide range of applications.

m6Aexpress-Reader: Prediction of m6A regulated expression genes by integrating m6A sites and reader binding information in specific- context.

N6-methyladenosine (m6A) is the most abundant form of mRNA modification and plays an important role in regulating gene expression. However, the mechanisms of m6A regulated gene expression in cell or condition specific, are still poorly understood. Even though, some methods are able to predict m6A regulated expression (m6A-reg-exp) genes in specific context, they don't introduce the m6A reader binding information, while this information can help to predict m6A-reg-exp genes and more clearly to explain the mechanisms of m6A-mediated gene expression process. Thus, by integrating m6A sites and reader binding information, we proposed a novel method (called m6Aexpress-Reader) to predict m6A-reg-exp genes from limited MeRIP-seq data in specific context. m6Aexpress-Reader adopts the reader binding signal strength to weight the posterior distribution of the estimated regulatory coefficients for enhancing the prediction power. By using m6Aexpress-Reader, we found the complex characteristic of m6A on gene expression regulation and the distinct regulated pattern of m6A-reg-exp genes with different reader binding. m6A readers, YTHDF2 or IGF2BP1/3 all play an important role in various cancers and the key cancer pathways. In addition, m6Aexpress-Reader reveals the distinct m6A regulated mode of reader targeted genes in cancer. m6Aexpress-Reader could be a useful tool for studying the m6A regulation on reader target genes in specific context and it can be freely accessible at: https://github.com/NWPU-903PR/m6AexpressReader.

Abnormal TNS3 gene methylation in patients with congenital scoliosis.

BACKGROUND: Congenital scoliosis (CS) is a congenital deformity of the spine resulting from abnormal and asymmetrical development of vertebral bodies during pregnancy. However, the etiology and mechanism of CS remain unclear. Epigenetics is the study of heritable variations in gene expression outside of changes in nucleotide sequence. Among these, DNA methylation was described first and is the most characteristic and most stable epigenetic mechanism. Therefore, in this study, we aim to explore the association between genome methylation and CS which are not been studied before. METHODS: Two pairs of monozygotic twins were included, with each pair involving one individual with and one without CS. Agilent SureSelect XT Human Methyl-Sequencing was used for genome methylation sequencing. MethylTarget was used to detect methylation levels in target regions. Immunohistochemistry was performed to visualize expression of associated genes in candidate regions. RESULTS: A total of 75 differentially methylated regions were identified, including 24 with an increased methylation level and 51 with a decreased methylation level in the CS group. Nine of the differentially methylated regions were selected (TNS3, SEMAC3, GPR124, MEST, DLK1, SNTG1, PPIB, DEF8, and GRHL2). The results showed that the methylation level of the promoter region of TNS3 was 0.72 ± 0.08 in the CS group and 0.43 ± 0.06 in the control group (p = 0.00070 < 0.01). There was no significant difference in the degree of methylation of SEMAC3, GPR124, MEST, DLK1, SNTG1, PPIB, DEF8, or GRHL2 between the two groups. Immunohistochemistry showed significantly decreased TNS3 expression in the cartilage of the articular process in CS (CS: 0.011 ± 0.002; control: 0.018 ± 0.006, P = 0.003 < 0.01). CONCLUSION: Compared with the control group, high-level methylation of the TNS3 promoter region and low TNS3 expression in the cartilage layer of the articular process characterize CS. Thus, DNA methylation and TNS3 may play important roles in the pathogenesis of CS.

Sequence, Expression, and Anti-GCRV Function of the Ferritin from the Grass Carp, Ctenopharyngodon idellus.

Ferritin possesses an immune function to defend against pathogen infection. To elucidate the immunity-protecting roles of ferritin from Ctenopharyngodon idellus (Ciferritin) against virus infection, the cDNA and promoter sequences of Ciferritin were determined, and the correlations between Ciferrtin expressions and promoter methylation levels were analyzed. In addition, the functional role of Ciferrtin on GCRV (grass carp reovirus) infection was assessed. The full-length cDNA of Ciferritin is 1053 bp, consists of a 531 bp open-reading frame, and encodes 176 amino acids. Ciferritin showed the highest sequence identity with the ferritin middle subunit of Mylopharyngodon piceus (93.56%), followed by the subunits of Megalobrama amblycephala and Sinocyclocheilus rhinocerous. Ciferritin contains a conserved ferritin domain (interval: 10−94 aa), and the caspase recruitment domain (CARD) and Rubrerythrin domain were also predicted. In the spleen and kidney, significantly higher Ciferritin expressions were observed at 6, 12, 24, or 168 h post GCRV infection than those in the PBS injection group (p < 0.05). The Ciferrtin expression level in the progeny of maternal-immunized grass carp was significantly higher than that in the progeny of common grass carp (p < 0.05). Ciferritin promoter methylation level in the progeny from common grass carp was 1.27 ± 0.15, and in the progeny of the maternal-immunized group was 1.00 ± 0.14. In addition, methylation levels of "CpG9" and "CpG10" loci were significantly lower in the progeny of maternal-immunized fish than those in the common group. Except for the "CpG5", methylation levels of all other detected "CpG" loci negatively correlated with Ciferritin expression levels. Furthermore, the total methylation level of "CpG1−10" negatively correlated with the Ciferritin expressions. The Ciferritin expression level was significantly up-regulated, and the VP7 protein levels were significantly reduced, at 24 h post GCRV infection in the Ciferritin over-expression cells (p < 0.05). The results from the present study provide sequence, epigenetic modification and expression, and anti-GCRV functional information of Ciferritin, which provide a basis for achieving resistance to GCRV in grass carp breeding.

Influence of Benzo(a)pyrene on Different Epigenetic Processes.

Epigenetic changes constitute one of the processes that is involved in the mechanisms of carcinogenicity. They include dysregulation of DNA methylation processes, disruption of post-translational patterns of histone modifications, and changes in the composition and/or organization of chromatin. Benzo(a)pyrene (BaP) influences DNA methylation and, depending on its concentrations, as well as the type of cell, tissue and organism it causes hypomethylation or hypermethylation. Moreover, the exposure to polyaromatic hydrocarbons (PAHs), including BaP in tobacco smoke results in an altered methylation status of the offsprings. Researches have indicated a potential relationship between toxicity of BaP and deregulation of the biotin homeostasis pathway that plays an important role in the process of carcinogenesis. Animal studies have shown that parental-induced BaP toxicity can be passed on to the F1 generation as studied on marine medaka (Oryzias melastigma), and the underlying mechanism is likely related to a disturbance in the circadian rhythm. In addition, ancestral exposure of fish to BaP may cause intergenerational osteotoxicity in non-exposed F3 offsprings. Epidemiological studies of lung cancer have indicated that exposure to BaP is associated with changes in methylation levels at 15 CpG; therefore, changes in DNA methylation may be considered as potential mediators of BaP-induced lung cancer. The mechanism of epigenetic changes induced by BaP are mainly due to the formation of CpG-BPDE adducts, between metabolite of BaP-BPDE and CpG, which leads to changes in the level of 5-methylcytosine. BaP also acts through inhibition of DNA methyltransferases activity, as well as by increasing histone deacetylases HDACs, i.e., HDAC2 and HDAC3 activity. The aim of this review is to discuss the mechanism of the epigenetic action of BaP on the basis of the latest publications.

Uncovering the mechanistic basis for specific recognition of monomethylated H3K4 by the CW domain of Arabidopsis histone methyltransferase SDG8.

Chromatin consists of DNA and histones, and specific histone modifications that determine chromatin structure and activity are regulated by three types of proteins, called writer, reader, and eraser. Histone reader proteins from vertebrates, vertebrate-infecting parasites, and higher plants possess a CW domain, which has been reported to read histone H3 lysine 4 (H3K4). The CW domain of Arabidopsis SDG8 (also called ASHH2), a histone H3 lysine 36 methyltransferase, preferentially binds monomethylated H3K4 (H3K4me1), unlike the mammalian CW domain protein, which binds trimethylated H3K4 (H3K4me3). However, the molecular basis of the selective binding by the CW domain of SDG8 (SDG8-CW) remains unclear. Here, we solved the 1.6-Å-resolution structure of SDG8-CW in complex with H3K4me1, which revealed that residues in the C-terminal α-helix of SDG8-CW determine binding specificity for low methylation levels at H3K4. Moreover, substitutions of key residues, specifically Ile-915 and Asn-916, converted SDG8-CW binding preference from H3K4me1 to H3K4me3. Sequence alignment and mutagenesis studies revealed that the CW domain of SDG725, the homolog of SDG8 in rice, shares the same binding preference with SDG8-CW, indicating that preference for low methylated H3K4 by the CW domain of ASHH2 homologs is conserved among higher-order plants. Our findings provide first structural insights into the molecular basis for specific recognition of monomethylated H3K4 by the H3K4me1 reader protein SDG8 from Arabidopsis.

Defining a prognostic score based on O6-methylguanine-DNA methyltransferase cut-off methylation level determined by pyrosequencing in patients with glioblastoma multiforme.

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.

Individualized analysis reveals CpG sites with methylation aberrations in almost all lung adenocarcinoma tissues.

BACKGROUND: Due to the heterogeneity of cancer, identifying differentially methylated (DM) CpG sites between a set of cancer samples and a set of normal samples cannot tell us which patients have methylation aberrations in a particular DM CpG site. METHODS: We firstly showed that the relative methylation-level orderings (RMOs) of CpG sites within individual normal lung tissues are highly stable but widely disrupted in lung adenocarcinoma tissues. This finding provides the basis of using the RankComp algorithm, previously developed for differential gene expression analysis at the individual level, to identify DM CpG sites in each cancer tissue compared with its own normal state. Briefly, through comparing with the highly stable normal RMOs predetermined in a large collection of samples for normal lung tissues, the algorithm finds those CpG sites whose hyper- or hypo-methylations may lead to the disrupted RMOs of CpG site pairs within a disease sample based on Fisher's exact test. RESULTS: Evaluated in 59 lung adenocarcinoma tissues with paired adjacent normal tissues, RankComp reached an average precision of 94.26% for individual-level DM CpG sites. Then, after identifying DM CpG sites in each of the 539 lung adenocarcinoma samples from TCGA, we found five and 44 CpG sites hypermethylated and hypomethylated in above 90% of the disease samples, respectively. These findings were validated in 140 publicly available and eight additionally measured paired cancer-normal samples. Gene expression analysis revealed that four of the five genes, HOXA9, TAL1, ATP8A2, ENG and SPARCL1, each harboring one of the five frequently hypermethylated CpG sites within its promoters, were also frequently down-regulated in lung adenocarcinoma. CONCLUSIONS: The common DNA methylation aberrations in lung adenocarcinoma tissues may be important for lung adenocarcinoma diagnosis and therapy.

CpG methylation of dmrt1 and cyp19a promoters in relation to their sexual dimorphic expression in the Japanese flounder Paralichthys olivaceus.

To better understand the effects of DNA methylation on the expression patterns of dmrt1 (Doublesex and Mab-3-related transcription factor 1) and cyp19a (Cytochrome P450 19a) in the Japanese flounder Paralichthys olivaceus, quantitative expressions, cellular distributions and cytosine-p-guanine (CpG) methylation patterns of these two genes in the gonads were analysed. The results showed that P. olivaceus dmrt1 expression was 70 times higher in the testis than in the ovary (P < 0·05). Its mRNA was detected clearly in spermatocytes and Sertoli cells of the testis, but weakly in the ovary. Paralichthys olivaceus cyp19a expression was 40 times higher in the ovary than in the testis (P < 0·01). Its mRNA was detected clearly in follicular cells of the ovary, but weakly in spermatocytes of the testis. The dmrt1 promoter CpGs were not methylated in the testis, whereas 57·69% were methylated in the ovary. For the cyp19a promoter CpGs, 97·5% were methylated in the testis and 73·33% were methylated in the ovary. These findings demonstrate that P. olivaceus dmrt1 and cyp19a are sex-related genes with sexual dimorphic expression, CpG methylation levels of the two genes are consistent with their expression quantities, and this epigenetic modification can influence the differential expression of genes in the gonads of P. olivaceus.

Identification of clinical characteristics biomarkers for rheumatoid arthritis through targeted DNA methylation sequencing.

OBJECTIVES: Rheumatoid arthritis (RA) is a complex disease with a challenging diagnosis, especially in seronegative patients. The aim of this study is to investigate whether the methylation sites associated with the overall immune response in RA can assist in clinical diagnosis, using targeted methylation sequencing technology on peripheral venous blood samples. METHODS: The study enrolled 241 RA patients, 30 osteoarthritis patients (OA), and 30 healthy volunteers control (HC). Fifty significant cytosine guanine (CG) sites between undifferentiated arthritis and RA were selected and analyzed using targeted DNA methylation sequencing. Logistic regression models were used to establish diagnostic models for different clinical features of RA, and six machine learning methods (logit model, random forest, support vector machine, adaboost, naive bayes, and learning vector quantization) were used to construct clinical diagnostic models for different subtypes of RA. Least absolute shrinkage and selection operator regression and detrended correspondence analysis were utilized to screen for important CGs. Spearman correlation was used to calculate the correlation coefficient. RESULTS: The study identified 16 important CG sites, including tumor necrosis factort receptor associated factor 5 (TRAF5) (chr1:211500151), mothers against decapentaplegic homolog 3 (SMAD3) (chr15:67357339), tumor endothelial marker 1 (CD248) (chr11:66083766), lysosomal trafficking regulator (LYST) (chr1:235998714), PR domain zinc finger protein 16 (PRDM16) (chr1:3307069), A-kinase anchoring protein 10 (AKAP10) (chr17:19850460), G protein subunit gamma 7 (GNG7) (chr19:2546620), yes1 associated transcriptional regulator (YAP1) (chr11:101980632), PRDM16 (chr1:3163969), histone deacetylase complex subunit sin3a (SIN3A) (chr15:75747445), prenylated rab acceptor protein 2 (ARL6IP5) (chr3:69134502), mitogen-activated protein kinase kinase kinase 4 (MAP3K4) (chr6:161412392), wnt family member 7A (WNT7A) (chr3:13895991), inhibin subunit beta B (INHBB) (chr2:121107018), deoxyribonucleic acid replication helicase/nuclease 2 (DNA2) (chr10:70231628) and chromosome 14 open reading frame 180 (C14orf180) (chr14:105055171). Seven CG sites showed abnormal changes between the three groups (P < 0.05), and 16 CG sites were significantly correlated with common clinical indicators (P < 0.05). Diagnostic models constructed using different CG sites had an area under the receiver operating characteristic curve (AUC) range of 0.64-0.78 for high-level clinical indicators of high clinical value, with specificity ranging from 0.42 to 0.77 and sensitivity ranging from 0.57 to 0.88. The AUC range for low-level clinical indicators of high clinical value was 0.63-0.72, with specificity ranging from 0.48 to 0.74 and sensitivity ranging from 0.72 to 0.88. Diagnostic models constructed using different CG sites showed good overall diagnostic accuracy for the four subtypes of RA, with an accuracy range of 0.61-0.96, a balanced accuracy range of 0.46-0.94, and an AUC range of 0.46-0.94. CONCLUSIONS: This study identified potential clinical diagnostic biomarkers for RA and provided novel insights into the diagnosis and subtyping of RA. The use of targeted deoxyribonucleic acid (DNA) methylation sequencing and machine learning methods for establishing diagnostic models for different clinical features and subtypes of RA is innovative and can improve the accuracy and efficiency of RA diagnosis.

Tissue-specific RNA methylation prediction from gene expression data using sparse regression models.

N6-methyladenosine (m6A) is a highly prevalent and conserved post-transcriptional modification observed in mRNA and long non-coding RNA (lncRNA). Identifying potential m6A sites within RNA sequences is crucial for unraveling the potential influence of the epitranscriptome on biological processes. In this study, we introduce Exp2RM, a novel approach that formulates single-site-based tissue-specific elastic net models for predicting tissue-specific methylation levels utilizing gene expression data. The resulting ensemble model demonstrates robust predictive performance for tissue-specific methylation levels, with an average R-squared value of 0.496 and a median R-squared value of 0.482 across all 22 human tissues. Since methylation distribution varies among tissues, we trained the model to incorporate similar patterns, significantly improves accuracy with the median R-squared value increasing to 0.728. Additonally, functional analysis reveals Exp2RM's ability to capture coefficient genes in relevant biological processes. This study emphasizes the importance of tissue-specific methylation distribution in enhancing prediction accuracy and provides insights into the functional implications of methylation sites.

Quantitative Investigation of Methylation Heterogeneity by Digital Melting Curve Analysis on a SlipChip for Atrial Fibrillation.

Methylation is an essential epigenetic modification involved in regulating gene expression and maintaining genome stability. Methylation patterns can be heterogeneous, exhibiting variations in both level and density. However, current methods of methylation analysis, including sequencing, methylation-specific PCR, and high-resolution melting curve analysis (HRM), face limitations of high cost, time-consuming workflows, and the difficulty of both accurate heterogeneity analysis and precise quantification. Here, a droplet array SlipChip-based (da-SlipChip-based) digital melting curve analysis (MCA) method was developed for the accurate quantification of both methylation level (ratio of methylated molecules to total molecules) and methylation density (ratio of methylated CpG sites to total CpG sites). The SlipChip-based digital MCA system supplements an in situ thermal cycler with a fluorescence imaging module for real-time MCA. The da-SlipChip can generate 10,656 droplets of 1 nL each, which can be separated into four lanes, enabling the simultaneous analysis of four samples. This method's clinical application was demonstrated by analyzing samples from ten healthy individuals and twenty patients with atrial fibrillation (AF), the most common arrhythmia. This method can distinguish healthy individuals from those with AF of both the paroxysmal and persistent types. It also holds potential for broader application in various research and clinical settings requiring methylation analysis.

5-Methylcytosine profiles in mouse transcriptomes suggest the randomness of m5C formation catalyzed by RNA methyltransferase.

OBJECTIVE: 5-Methylcytosine (m5C) is a type of chemical modification on the nucleotides and is widespread in both DNA and RNA. Although the DNA m5C has been extensively studied over the past years, the distribution and biological function of RNA m5C still remain to be elucidated. Here, I explored the profiles of RNA m5C in four mouse tissues by applying a RNA cytosine methylation data analysis tool to public mouse RNA m5C data. RESULTS: I found that the methylation rates of cytosine were the same with the averages of methylation level at single-nucleotide level. Furthermore, I gave a mathematical formula to describe the observed relationship and analyzed it deeply. The sufficient necessary condition for the given formula suggests that the methylation levels at most m5C sites are the same in four mouse tissues. Therefore, I proposed a hypothesis that the m5C formation catalyzed by RNA methyltransferase is random and with the same probability at most m5C sites, which is the methylation rate of cytosine. My hypothesis can be used to explain the observed profiles of RNA m5C in four mouse tissues and will be benefit to future studies of the distribution and biological function of RNA m5C in mammals.

Methylation level of potato gene OMT30376 regulates tuber anthocyanin transformations.

After anthocyanin synthesis, a variety of anthocyanin compounds are produced through further methylation, glycosylation, and acylation. However, the effect of the potato methylase gene on anthocyanin biosynthesis has not been reported. Red and purple mutation types appear in tubers of the potato cultivar 'Purple Viking' with chimeric skin phenotypes. In this study, transcriptome and anthocyanin metabolome analyses were performed on skin of Purple Viking tubers and associated mutants. According to the metabolome analysis, the transformation of delphinidin into malvidin-3-O-glucoside and petunidin 3-O-glucoside and that of cyanidin into rosinidin O-hexoside and peonidin-3-O-glucoside were hindered in red tubers. Expression of methyltransferase gene OMT30376 was significantly lower in red tubers than in purple ones, whereas the methylation level of OMT30376 was significantly higher in red tubers. In addition, red skin appeared in tubers from purple tuber plants treated with S-adenosylmethionine (SAM), indicating the difference between purple and red was caused by the methylation degree of the gene OMT30376. Thus, the results of the study suggest that the OMT30376 gene is involved in the transformation of anthocyanins in potato tubers. The results also provide an important reference to reveal the regulatory mechanisms of anthocyanin biosynthesis and transformation.

Circulating methylation level of HTR2A is associated with inflammation and disease activity in rheumatoid arthritis.

Objectives: HTR2A is previously identified as a susceptibility gene for rheumatoid arthritis (RA). In this study, we performed the association analysis between DNA methylation of HTR2A with RA within peripheral blood samples. Methods: We enrolled peripheral blood samples from 235 patients with RA, 30 osteoarthritis (OA) patients, and 30 healthy controls. The DNA methylation levels of about 218 bp from chr13: 46898190 to chr13: 46897973 (GRCh38/hg38) around HTR2A cg15692052 from patients were analyzed by targeted methylation sequencing. Results: We measured methylation status for 7 CpGs in the promoter region of HTR2A and obseved overall methylation status are signficantly increased in RA compared with normal inviduals (FDR= 9.05 x 10-5). The average cg15692052 methylation levels (methylation score) showed a positive correlation with CRP (r=0.15, P=0.023). Compared with the OA group or HC group, the proportion of haplotypes CCCCCCC (FDR=0.02 and 2.81 x 10-6) is signficantly increased while TTTTTCC (FDR =0.01) and TTTTTTT(FDR =6.92 x 10-3) are significantly decreased in RA. We find methylation haplotypes combining with RF and CCP could signficantly enhance the performance of the diagnosing RA and its comorbidities (hypertension, interstitial lung disease, and osteoporosis), especially in interstitial lung disease. Conclusions: In our study, we found signficant hypermethylation of promoter region of HTR2A which indicates the potential clinical diagnostic role in rheumatoid arthritis.

Quantification Methods for Methylation Levels in Illumina Arrays.

The genome-wide patterns of methylation levels and identifications of differentially methylated genetic loci or regions of CpG sites are being important, because the methylome has the potential for large effects in disease etiology. There are several quantification methods of methylation level at each CpG site for a given sample, which include ß-value, M-value, and N-value. The performance of three quantification methods of methylation levels has been examined with simulation study and 27K Illumina array data for obesity.

Association of FTO gene methylation with incident type 2 diabetes mellitus: A nested case-control study.

OBJECTIVES: This study aimed to investigate the association of FTO methylation level with type 2 diabetes mellitus (T2DM) in a nested case-control study. METHODS: This nested case-control study included 287 pairs of T2DM cases and controls identified from a rural Chinese cohort study with a 6-year follow-up. Controls were matched to the cases on a 1:1 basis by age, sex, ethnicity, marital status, and residence. Conditional multivariate logistic regression models were used to calculate odds ratios (ORs) and 95% confidence intervals (CIs) for the association of cytosine guanine (CpG) locus and tag-single nucleotide polymorphisms (Tag-SNPs) with T2DM. Spearman correlation analysis was used to evaluate the association between FTO methylation and possible risk factors for T2DM in the control group. RESULTS: The methylation level on the CpG9 site significantly differs between cases and controls, with a significant association between the CpG9 site methylation and probability of T2DM: OR 2.19 (95%CI: 1.31-3.65) after adjusting for potential confounders. The Tag-SNPs (rs72803657, rs1558902, rs17817449, rs11076023) were not associated with T2DM. Further, FTO methylation was associated with some risk factors for T2DM. CONCLUSIONS: A CpG locus of FTO was positively associated with T2DM, but SNPs were not. FTO methylation were also associated with some T2DM risk factors. Further study with a large sample size and data on metabolic product are needed to confirm the association.

Effect of LHX2 gene methylation level and its function on radiotherapy of cervical cancer.

BACKGROUND: Cervical cancer is the most common malignancy of the female reproductive system, for which radiotherapy is one of the main treatments. Gene methylation in cells is an important factor in tumorigenesis, and radiotherapy can change DNA methylation in cells. At the same time, combined with the clinical effect of radiotherapy, key genes of LIM homeobox 2 (LHX2) significantly related to cervical cancer. The LHX2 are LIM-homeobox genes that play important roles in signal transduction, cell differentiation, tissue-specific differentiation, and body formation. METHODS: In this study, bisulfite genomic sequencing (BSP-Seq) technology was used to analyze the methylation level of LHX2 in patients with cervical cancer before and after radiotherapy. In addition, combined with the clinical effect of radiotherapy, the function of LHX2 in siHA and C33A cells were analyzed with the help of overexpression, small interfering RNA (siRNA), cell invasion, and migration ability. The expression level of the migration- and apoptosis-related genes which were affected by LHX2 were tested with quantitative real time polymerase chain reaction (qRT-PCR). RESULTS: Combined with clinical treatment, methylation level difference, and correlation enrichment analysis, it was found that LHX2 genes were closely related to the occurrence and development of cervical cancer. After 5-aza-2'-deoxycytidine (5-Aza-dC) and radiotherapy, the methylation of LHX2 genes in siHA and C33A squamous cell carcinoma cells was decreased, and the messenger RNA (mRNA) and protein expression levels were relatively increased; meanwhile, the LHX2 could accelerate the ability for cell invasion and migration and inhibited the apoptosis of the cell after treatment with radiotherapy. CONCLUSIONS: The methylation and expression levels of LHX2 genes are closely related to cervical cancer. The methylation level of LHX2 was reduced after radiation therapy. The LHX2 gene has a positive effect on cervical cancer through acceleration of the cell invasion and migration ability and inhibition of cell apoptosis after radiotherapy treatment.

Detection of average methylation level of specific genes by binary-probe hybridization.

We developed a simple and highly-selective method for 5-methylcytosine detection of specific gene sequence based on binary-probe DNA hybridization. The sequence complementary to the target was designed into two probes, and each fragment of binary probes bound to a relatively short sequence of the target, which made it sensitive to the base mismatches introduced by bisulfite treatment. The advantages of a low detection limit of methylation abundance of 0.1% for the fully methylated target and high sensitivity of 10 pM have been proved by the successful design of binary-probe hybridization. The successful design of the binary probes makes it possible to quantify the average methylation levels of five CpG sites. Thirty-two DNA strands containing 5, 4, 3, 2, 1 and 0 CpG sites were successfully analyzed with the same pair of binary probes. The higher the average methylation level of the target was, the higher the degree of the hybridization reaction. Based on the simple construction of the binary-probe hybridization, the developed biosensor exhibited signals proportional to the average methylation level of the vimentin gene and could evaluate the average methylation level of artificial mixtures. Furthermore, the method has been used to detect vimentin methylation in a genomic context with good specificity, which indicated its potential in the pre-diagnosis of methylation related disease.

Interaction between early in ovo stimulation of the gut microbiota and chicken host - splenic changes in gene expression and methylation.

BACKGROUND: Epigenetic regulation of the gene expression results from interaction between the external environment and transcription of the genetic information encoded in DNA. Methylated CpG regions within the gene promoters lead to silencing of the gene expression in most cases. Factors contributing to epigenetic regulation include intestinal microbiota, which in chicken can be potently modified by in ovo stimulation. The main aim of this study was to determine global and specific methylation patterns of the spleen under the influence of host-microbiome interaction. RESULTS: Fertilized eggs of two genotypes: Ross 308 and Green-legged Partridgelike were in ovo stimulated on d 12 of incubation. The injected compounds were as follows: probiotic - Lactococcus lactis subsp. cremoris IBB477, prebiotic - galactooligosaccharides, and synbiotic - combination of both. Chickens were sacrificed on d 42 post-hatching. Spleen was collected, RNA and DNA were isolated and intended to gene expression, gene methylation and global methylation analysis. We have proved that negative regulation of gene expression after administration of bioactive substances in ovo might have epigenetic character. Epigenetic changes depend on the genotype and the substance administered in ovo. CONCLUSION: Epigenetic nature of microbial reprogramming in poultry and extension of issues related to host-microbiome interaction is a new direction of this research.