Methylation Level Of Promoter Region Research Articles

N6-methyladenosine (m6A)-circHECA from secondary hair follicle of cashmere goats: identification, regulatory network and expression regulated potentially by methylation of its host gene promoter.

The objective of this study was to identify the m6A-circHECA molecule in secondary hair follicles (SHFs) of cashmere goats, and generate its potential regulatory network, as well as explore the potential relationship between transcriptional pattern of m6A-circHECA and promoter methylation of its host gene (HECA). The validation of circHECA m6A sites was performed using Methylation Immunoprecipitation (Me-RIP) along with reverse transcription-quantitative polymerase chain reaction (RT-qPCR) technique. The nucleus and cytoplasm localizations of m6A-circHECA were performed using SHF stem cells of cashmere goats with RT-qPCR analysis. Based on in-silico analysis, the regulatory networks of m6A-circHECA were generated with related signal pathway enrichment. The methylation level of promoter region of m6A-circHECA host gene (HECA) was assessed by the bisulfite sequencing PCR (BSP-PCR) technique. The m6A-circHECA was confirmed to contain four m6A modification sites including m6A-213, m6A-297, m6A-780, and m6A-927, and it was detected mainly in cytoplasm of the SHF stem cells of cashmere goats. The integrated regulatory network analysis showed directly or indirectly complex regulatory relationships between m6A-circHECA of cashmere goats and its potential target molecules: miRNAs, mRNAs, and proteins. The regulatory network and pathway enrichment indicated that m6A-circHECA might play multiple roles in the SHF physiology process of cashmere goats through directly or indirectly interacting or regulating its potential target molecules. A higher methylation level of promoter region of HECA gene in SHFs of cashmere goats might cause the lower expression of m6A-circHECA. The m6A-circHECA might play multiple roles in SHF physiology process of cashmere goats through miRNA mediated pathways along with directly or indirectly interaction with its target proteins. The promoter methylation of m6A-circHECA host gene (HECA) most likely was implicated in its expression inhibition in SHFs of cashmere goats.

Analysis of Chromatin Accessibility and DNA Methylation to Reveal the Functions of Epigenetic Modifications in Cyprinus carpio Gonads.

Epigenetic modifications are critical in precisely regulating gene expression. The common carp (Cyprinus carpio) is an economically important fish species, and females exhibit faster growth rates than males. However, the studies related to epigenetic modifications in the common carp gonads are limited. In this study, we conducted the Assay for Transposase Accessible Chromatin sequencing (ATAC-seq) and Bisulfite sequencing (BS-seq) to explore the roles of epigenetic modifications in the common carp gonads. We identified 84,207 more accessible regions and 77,922 less accessible regions in ovaries compared to testes, and some sex-biased genes showed differential chromatin accessibility in their promoter regions, such as sox9a and zp3. Motif enrichment analysis showed that transcription factors (TFs) associated with embryonic development and cell proliferation were heavily enriched in ovaries, and the TFs Foxl2 and SF1 were only identified in ovaries. We also analyzed the possible regulations between chromatin accessibility and gene expression. By BS-seq, we identified 2087 promoter differentially methylated genes (promoter-DMGs) and 5264 gene body differentially methylated genes (genebody-DMGs) in CG contexts. These genebody-DMGs were significantly enriched in the Wnt signaling pathway, TGF-beta signaling pathway, and GnRH signaling pathway, indicating that methylation in gene body regions could play an essential role in sex maintenance, just like methylation in promoter regions. Combined with transcriptomes, we revealed that the expression of dmrtb1-like, spag6, and fels was negatively correlated with their methylation levels in promoter regions. Our study on the epigenetic modifications of gonads contributes to elucidating the molecular mechanism of sex differentiation and sex maintenance in the common carp.

A pan-cancer analysis revealing the role of LFNG, MFNG and RFNG in tumor prognosis and microenvironment

BackgroundFringe is a glycosyltransferase involved in tumor occurrence and metastasis. However, a comprehensive analysis of the Fringe family members lunatic fringe (LFNG), manic fringe (MFNG), radical fringe (RFNG) in human cancers is lacking.MethodsIn this study, we performed a pan-cancer analysis of Fringe family members in 33 cancer types with transcriptomic, genomic, methylation data from The Cancer Genome Atlas (TCGA) project. The correlation between Fringe family member expression and patient overall survival, copy number variation, methylation, Gene Ontology enrichment, and tumor-infiltrating lymphocytes (TILs) was investigated by using multiple databases, such as cBioPortal, Human Protein Atlas, GeneCards, STRING, MSigDB, TISIDB, and TIMER2. In vitro experiments and immunohistochemical assays were performed to validate our findings.ResultsHigh expression levels of LFNG, MFNG, RFNG were closely associated with poor overall survival in multiple cancers, particularly in pancreatic adenocarcinoma (PAAD), uveal melanoma (UVM), and brain lower-grade glioma (LGG). Copy number variation analysis revealed that diploid and gain mutations of LFNG was significantly increased in PAAD and stomach adenocarcinoma (STAD), and significantly associated with the methylation levels in promoter regions. Significant differential genes between high and low expression groups of these Fringe family members were found to be consistently enriched in immune response and T cell activation pathway, extracellular matrix adhesion pathway, RNA splicing and ion transport pathways. Correlation between the abundance of tumor-infiltrating lymphocytes (TILs) and LFNG, MFNG, and RFNG expression showed that high LFNG expression was associated with lower TIL levels, particularly in PAAD. In vitro experiment by using pancreatic cancer PANC1 cells showed that LFNG overexpression promoted cell proliferation and invasion. Immunohistochemical assay in 90 PAAD patients verified the expression level of LFNG and its relationship with the prognosis.ConclusionsOur study provides a relatively comprehensive understanding of the expression, mutation, copy number, promoter methylation level changes along with prognosis values of LFNG, MFNG, and RFNG in different tumors. High LFNG expression may serve as a poor prognosis molecular marker for PAAD.

Methylation of eNOS in the rat penile corpus cavernosum under different pathological states and its relationship with erectile function.

It has been shown that methylation in the promoter region of eNOS can downregulate eNOS expression resulting in the endothelial dysfunction. However, it is unclear whether low androgen levels and type 1 diabetes cause ED by methylating the promoter region of eNOS in the penile corpus cavernosum. To clarify the effects of type 1 diabetes and hypo-androgen status on the methylation level of the promoter region of the eNOS gene in penile cavernous tissue and their relationship with the erectile function. Fifty-eight eight-week-old male Sprague-Dawley rats were randomly divided into six groups (n=6): sham operation group, castration group, castration+testosterone (cast+T) group, normoglycemia group, diabetic group, and diabetic+methyltransferase inhibitor (5-aza-dc, 1.5mg/kg) group. The ICPmax/MAP, serum T, the concentration of nitric oxide (NO), the expression of DNMT1, DNMT3a, DNMT3b, and eNOS, and the methylation level of the eNOS promoter region in penile corpus cavernosum of rat were examined 4weeks after surgery in the sham-operated group, the castration group, and the castration + testosterone replacement group. Those tests were examined after 6weeks using of methylation inhibitors in the normoglycemic group, the diabetic group, and the diabetic + methylation inhibitor group. ICPmax/MAP, DNMT1, DNMT3a, DNMT3b, eNOS, and NO levels were significantly lower in castrated rats than in sham and cast+T rats (P<0.05). ICPmax/MAP, eNOS, and NO levels were lower, and DNMT1, DNMT3a, and DNMT3b expression levels were significantly increased in the diabetic group compared with the normoglycemic and diabetic+methyltransferase inhibitor groups (P<0.05). There was no significant difference in the methylation level of the promoter region of eNOS in penile cavernous tissue of castrated rats compared with the sham group or the testosterone replacement group. The methylation level of the promoter region of eNOS in penile cavernous tissue was significantly higher in the diabetic group than in the normoglycemic group and diabetic+methyltransferase inhibitor group (P<0.05). Although low androgen status inhibited the level of methyltransferase in rat penile cavernous tissue, did not affect the level of methylation in the promoter region of eNOS. Hyperglycemia inhibits the NO level in the penile cavernous tissue and the erectile function of rats by upregulating the methyltransferase level in the penile cavernous tissue and the methylation level in the promoter region of eNOS. Methylation inhibitors can partly improve the erectile function in type 1 diabetic rats.

Effects of fecal microbiota transplant on DNA methylation in patients with systemic lupus erythematosus

Systemic lupus erythematosus (SLE) is a highly heterogeneous autoimmune disease characterized by multiple organ damage accompanied by the over-production of autoantibodies. Decreased intestinal flora diversity and disruption of homeostasis have been proven to be associated with pathogenesis of SLE. In previous study, a clinical trial was conducted to verify the safety and effectiveness of fecal microbiota transplantation (FMT) in the treatment of SLE. To explore the mechanism of FMT in the treatment of SLE, we included 14 SLE patients participating in clinical trials, including 8 in responders group (Rs) and 6 in non-responders group (NRs), and collected peripheral blood DNA and serum. We found that the serum of S-adenosylmethionine (SAM), methylation group donor, was upregulated after FMT, accompanied by an increase in genome-wide DNA methylation level in Rs. We further showed that the methylation levels in promoter regions of Interferon-γ (IFN-γ), induced Helicase C Domain Containing Protein 1 (IFIH1), endoplasmic reticulum membrane protein complex 8 (EMC8), and Tripartite motif-containing protein 58 (TRIM58) increased after FMT treatment. On the contrary, there was no significant change in the methylation of IFIH1 promoter region in the NRs after FMT, and the methylation level of IFIH1 in the Rs was significantly higher than that in the NRs at week 0. We included 850 K methylation chip sequencing, combining previous data of metagenomic sequencing, and metabolomic sequencing for multi-omics analysis to discuss the relationship between flora-metabolite-methylation in FMT. Finally, we found that hexanoic acid treatment can up-regulate the global methylation of peripheral blood mononuclear cells in SLE patients. Overall, our results delineate changes in methylation level after FMT treatment of SLE and reveal possible mechanisms of FMT treatment in terms of the recovery of abnormal hypomethylation.

Local diversity of drought resistance and resilience in Populus tomentosacorrelates with the variation of DNA methylation.

Little information is known about DNA methylation variation in shaping environment-specific drought resistance and resilience for tree adaptation. In this study, we leveraged RNA sequencing and whole-genome bisulfite sequencing data to dissect the distinction of epigenetic regulation under drought stress and rewater condition of Populus tomentosa accessions from three geographical regions. We demonstrated low resistance and high resilience for accessions from South. Non-CG methylation levels in promoter regions of Southern accessions were lower than accessions from higher latitudes and negatively regulated gene expression. CHH context methylation was more sensitive to drought stress, and the geographical-specific differentially methylated regions were scarcely changed by environmental fluctuation. We identified 60 conserved hub genes within the co-expression networks that correlate with photosynthetic and stomatal morphological traits. Epigenome-wide association studiesand genome-wide association studiesof these 60 hub genes revealed the interdependency between genetic and epigenetic variation in GATA9 and LECRK-VIII.2, whichwas associated with stomatal morphology and chlorophyll content. The natural epigenetic variation in GATA9 was also faithfully transmitted to progenies in two family-based F1 populations. This study indicates a functional relationship of DNA methylation diversity with drought resistance and resilience which offers new insights into plants' local adaptation to a stressful environment.

Chinese Tuina Protects against Neonatal Hypoxia-Ischemia through Inhibiting the Neuroinflammatory Reaction.

Aim. Neonatal hypoxic-ischemic encephalopathy (HIE) is a significant cause of perinatal morbidity and mortality. Chinese Tuina is an effective treatment for HIE, but the molecular mechanisms are yet unknown. This study investigated the effect and mechanisms of Chinese Tuina on the inflammatory response in neonatal HIE rats. Main Methods. 30 male neonatal rats were divided randomly into 3 groups: sham, HIE, and HIE with Chinese Tuina (CHT) groups. The HIE and CHT groups were subjected to left common carotid occlusion and hypoxia at 3 days postnatal (P3). The pups in the CHT group received Chinese Tuina treatment on the next day for 28 days. The weight was measured at P4, P9, P13, P21, and P31. The behavioral functions were determined at P21. The protein expression and the methylation level in promoter regions of TNF-α and IL-10 were determined by Western blotting, immunohistochemistry, and pyrosequencing, respectively, at P33. Key Findings. The weight gain in the HIE group was slow compared with that of the CHT group. The rats in the CHT group performed better both in the balance beam and hang plate experiment. Chinese Tuina inhibited the expression of TNF-α and upregulated the expression of IL-10. Neonatal hypoxic-ischemic injury downregulated the methylation level in promoter regions of TNF-α at all CpG points but not IL-10. However, Chinese Tuina did not change the methylation level in promoter regions of TNF-α and IL-10. Significance. Chinese Tuina protected against HIE through inhibiting the neuroinflammatory reaction. While HIE markedly downregulated the methylation level of TNF-α, the protective effects of Chinese Tuina were independent of the regulation of the methylation level of TNF-α and IL-10.

DNA methylation in the medial prefrontal cortex regulates alcohol-related behavior in rats.

Alcohol abuse causes tissue and organ damage, and may participate neuropsychiatric diseases. Studies have shown that DNA methylation plays an important role in gene expression and behavioral changes induced by alcohol, however the causative neurobiological mechanisms have not been clarified. In this study, 32 healthy adult male SD rats were randomly divided into a drinking water control group (n=16) and a chronic alcohol exposure group (n=16). The alcohol preference and locomotor activity of rats were evaluated by two-bottle choice test (TBCT) and open-field test (OFT). DNA methylation in the medial prefrontal cortex (mPFC) tissue was detected by the reduced representative bisulfite sequencing (RRBS) technology. The methylation differential genes closely related to alcohol abuse were screened. qRT-PCR was used to verify the mRNA expression patterns of differential genes. qRT-PCR and Western blot were used to detect the expression of DNA methyltransferases (DNMTs) and methyl CpG binding protein 2 (MeCP2). Furthermore, the effect of short-term alcohol exposure (7 days) on DNMTs and MeCP2 in the mPFC of rats was tested (n=8/group). The results indicated that the methylation level of promoter region in the mPFC of rats exposed to chronic alcohol was significantly increased. In addition, the increased methylation levels in the promoter of Ntf3 and Ppm1G were accompanied by down-regulated mRNA levels in the chronic alcohol exposure group. The decreased methylation levels in the promoter of Hap1 and DUSP1 were accompanied by up-regulated mRNA levels. Furthermore, chronic alcohol exposure increased the mRNA and protein levels of DNMT3B and MeCP2. However, short term alcohol exposure did not affect their expression. This present study provides evidence that DNA methylation is associated with the development of alcohol abuse, which may be regulated by DNMT3B and MeCP2. The target genes Ntf3, Ppm1G, Hap1, and DUSP1 related to alcohol abuse were discovered as well, providing new insights into the neurobiological mechanism of alcohol abuse and the potential pharmacological targets for the treatment of alcohol abuse.

Epigenome-wide association study of depression symptomatology in elderly monozygotic twins

Depression is a severe and debilitating mental disorder diagnosed by evaluation of affective, cognitive and physical depression symptoms. Severity of these symptoms strongly impacts individual’s quality of life and is influenced by a combination of genetic and environmental factors. One of the molecular mechanisms allowing for an interplay between these factors is DNA methylation, an epigenetic modification playing a pivotal role in regulation of brain functioning across lifespan. The aim of this study was to investigate if there are DNA methylation signatures associated with depression symptomatology in order to identify molecular mechanisms contributing to pathophysiology of depression. We performed an epigenome-wide association study (EWAS) of continuous depression symptomatology score measured in a cohort of 724 monozygotic Danish twins (346 males, 378 females). Through EWAS analyses adjusted for sex, age, flow-cytometry based blood cell composition, and twin relatedness structure in the data we identified depression symptomatology score to be associated with blood DNA methylation levels in promoter regions of neuropsin (KLK8, p-value = 4.7 × 10−7) and DAZ associated protein 2 (DAZAP2, p-value = 3.13 × 10−8) genes. Other top associated probes were located in gene bodies of MAD1L1 (p-value = 5.16 × 10−6), SLC29A2 (p-value = 6.15 × 10−6) and AKT1 (p-value = 4.47 × 10−6), all genes associated before with development of depression. Additionally, the following three measures (a) DNAmAge (calculated with Horvath and Hannum epigenetic clock estimators) adjusted for chronological age, (b) difference between DNAmAge and chronological age, and (c) DNAmAge acceleration were not associated with depression symptomatology score in our cohort. In conclusion, our data suggests that depression symptomatology score is associated with DNA methylation levels of genes implicated in response to stress, depressive-like behaviors, and recurrent depression in patients, but not with global DNA methylation changes across the genome.

Double‐negative feedback interaction between DNA methyltransferase 3A and microRNA‐145 in the Warburg effect of ovarian cancer cells

Ovarian cancer is the most lethal gynecological malignancy because of its poor prognosis. The Warburg effect is one of the key mechanisms mediating cancer progression. Molecules targeting the Warburg effect are therefore of significant therapeutic value for the treatment of cancers. Many microRNAs (miR) are dysregulated in cancers, and aberrant miR expression patterns have been suggested to correlate with the Warburg effect in cancer cells. In our study, we found that miR‐145 negatively correlated with DNA methyltransferase (DNMT)3A expression at cellular/histological levels. miR‐145 inhibited the Warburg effect by targeting HK2. Luciferase reporter assays confirmed that miR‐145‐mediated downregulation of DNMT3A occurred through direct targeting of its mRNA 3′‐UTRs, whereas methylation‐specific PCR (MSP) assays found that knockdown of DNMT3A increased mRNA level of miR‐145 and decreased methylation levels of promoter regions in the miR‐145 precursor gene, thus suggesting a crucial crosstalk between miR‐145 and DNMT3A by a double‐negative feedback loop. DNMT3A promoted the Warburg effect through miR‐145. Coimmunoprecipitation assays confirmed no direct binding between DNMT3A and HK2. In conclusion, a feedback loop between miR‐145 and DNMT3A is a potent signature for the Warburg effect in ovarian cancer, promising a potential target for improved anticancer treatment.

Bioinformatics Analysis of DNA Methylation in the Promoters of LincRNA in Lung Adenocarcinoma

背景与目的肿瘤相关长链非编码RNA(lincRNA)的异常表达影响癌症的进程, 是肿瘤进展机制研究的热点, 然而其调控机制目前并不清楚。本研究通过生物信息学方法研究肺腺癌lincRNA启动子DNA甲基化水平与基因表达和预后的关系。方法从TCGA网站下载肺腺癌Illumina Methylation 450K芯片的全基因组DNA甲基化数据及转录组数据, 分析DNA甲基化在lincRNA基因附近的分布情况及与基因表达的关系; 比较肿瘤组织和癌旁组织DNA甲基化和表达变化。结果lincRNA启动子附近DNA甲基化水平较低, 且与基因的表达水平呈负相关。在肿瘤组织和癌旁组织间, 15个lincRNA的DNA甲基化水平存在显著差异, 与其表达呈反向变化, 包括与癌症发生发展相关的FINDER基因, 该基因启动子高甲基化的肺腺癌病人预后较差, 与基因表达作用刚好相反。结论肺腺癌中一些lincRNA启动子的DNA甲基化水平能调控lincRNA的表达, 进而与肺腺癌的预后有关。

Transgene-independent heredity of RdDM-mediated transcriptional gene silencing of endogenous genes in rice.

SummaryTo induce transcriptional gene silencing (TGS) of endogenous genes of rice (Oryza sativa L.), we expressed double‐strand RNA of each promoter region and thus induced RNA‐directed DNA methylation (RdDM). We targeted constitutively expressed genes encoding calnexin (CNX), protein disulphide isomerase (PDIL1‐1) and luminal binding protein (BiP1); an endoplasmic reticulum stress‐inducible gene (OsbZIP50); and genes with seed‐specific expression encoding α‐globulin (Glb‐1) and glutelin‐B4 (GluB4). TGS of four genes was obtained with high efficiency (CNX, 66.7% of regenerated plants; OsBiP1, 67.4%; OsbZIP50, 63.4%; GluB4, 66.1%), whereas the efficiency was lower for PDIL1‐1 (33.3%) and Glb‐1 TGS lines (10.5%). The heredity of TGS, methylation levels of promoter regions and specificity of silencing of the target gene were investigated in some of the TGS lines. In progeny of CNX and OsbZIP50 TGS lines, suppression of the target genes was preserved (except in the endosperm) even after the removal of trigger genes (T‐DNA) by segregation. TGS of CNX was reverted by demethylation treatment, and a significant difference in CG and CHG methylation levels in the −1 to −250 bp region of the CNX promoter was detected between the TGS and revertant lines, suggesting that TGS is closely related to the methylation levels of promoter. TGS exhibited specific suppression towards the target gene compared with post‐transcriptional gene silencing when GluB4 gene from glutelin multigene family was targeted. Based on these results, future perspectives and problems to be solved in the application of RdDM to new plant breeding techniques in rice are discussed.

Association between the DNA methylations of POMC, MC4R, and HNF4A and metabolic profiles in the blood of children aged 7\u20139\xa0years

BackgroundProopiomelanocortin (POMC), melanocortin 4 receptor (MC4R), and hepatocyte nuclear factor 4 alpha (HNF4A) are closely associated with weight gain and metabolic traits. In a previous study, we demonstrated associations between the methylations of POMC, MC4R, and HNF4A and metabolic profiles at birth. However, little is known about these associations in obese children. To evaluate the clinical utility of epigenetic biomarkers, we investigated to determine whether an association exists between the methylations of POMC, MC4R, and HNF4A and metabolic profiles in blood of normal weight and overweight and obese children.MethodsWe selected 79 normal weight children and 41 overweight and obese children aged 7–9 years in the Ewha Birth and Growth Cohort study. POMC methylation levels at exon 3, and MC4R and HNF4A methylation levels in promoter regions were measured by pyrosequencing. Serum glucose, total cholesterol (TC), triglyceride, high-density lipoprotein cholesterol (HDL–c), and insulin levels were analyzed using a biochemical analyzer and an immunoradiometric assay. Partial correlation and multiple regression analysis were used to assess relationships between POMC, MC4R, and HNF4A methylation levels and metabolic profiles.ResultsSignificant correlations were found between POMC methylation and HDL–c levels, and between HNF4A methylation and both TC and HDL–c levels. Interestingly, associations were found between POMC methylation status and HDL–c levels, and between HNF4A methylation status and TC levels independent of body mass index.ConclusionsThese findings show that POMC, MC4R, and HNF4A methylation status in the blood of children are associated with metabolic profiles. Therefore, we suggest that the DNA methylation status might serve as a potential epigenetic biomarkers of metabolic syndrome.

Locus- and Site-Specific DNA Methylation of 19 kDa Zein Genes in Maize.

An interesting question in maize development is why only a single zein gene is highly expressed in each of the 19-kDa zein gene clusters (A and B types), z1A2-1 and z1B4, in the immature endosperm. For instance, epigenetic marks could provide a structural difference. Therefore, we investigated the DNA methylation of the arrays of gene copies in both promoter and gene body regions of leaf (non-expressing tissue as a control), normal endosperm, and cultured endosperm. Although we could show that expressed genes have much lower methylation levels in promoter regions than silent ones in both leaf and normal endosperm, there was surprisingly also a difference in the pattern of the z1A and z1B gene clusters. The expression of z1B gene is suppressed by increased DNA methylation and activated with reduced DNA methylation, whereas z1A gene expression is not. DNA methylation in gene coding regions is higher in leaf than in endosperm, whereas no significant difference is observed in gene bodies between expressed and non-expressed gene copies. A median CHG methylation (25–30%) appears to be optimal for gene expression. Moreover, tissue-cultured endosperm can reset the DNA methylation pattern and tissue-specific gene expression. These results reveal that DNA methylation changes of the 19-kDa zein genes is subject to plant development and tissue culture treatment, but varies in different chromosomal locations, indicating that DNA methylation changes do not apply to gene expression in a uniform fashion. Because tissue culture is used to produce transgenic plants, these studies provide new insights into variation of gene expression of integrated sequences.

Thymine DNA Glycosylase Gene Knockdown Can Affect the Differentiation of Pig Preadipocytes

Aims: To study the effect of thymine DNA glycosylase (TDG) gene knockdown on the differentiation of pig preadipocytes. Methods: Preadipocytes were obtained from subcutaneous adipose tissue from the neck of 1- to 7-day-old pigs. The TDG gene was knocked down using siRNA, and cell differentiation was induced. The mRNA expression level was measured using fluorescence quantitative PCR, and the protein expression level was determined using Western blot analysis. The DNA methylation levels in promoter regions of differentiation-related genes were also evaluated. Results: TDG gene knockdown decreased the mRNA expression levels of the peroxisome proliferator-activated receptorγ (PPARγ) and Fatty acid binding proteins 4(FABP4 Also known as aP2) genes (P<0.01), while the mRNA expression level of the CCAAT/enhancer binding protein alpha(C/EBPα) gene did not change significantly (P>0.05). In addition, after induced differentiation, the lipid droplet production significantly decreased, and the percentages of methylation in the promoter regions of C/EBPα, PPARγ, and aP2 genes were 0.9%, 80%, and 76%, respectively. In contrast, the percentages of methylation in the negative control groups were 0.5%, 67.5%, and 58%, respectively. Conclusion: TDG gene knockdown could inhibit the differentiation of pig preadipocytes and affect the DNA methylation levels of some transcription factors.

ABERRANT METHYLATION OF THE PROMOTER OF APC, CDH13 AND MGMT GENES IN COLORECTAL CANCER PATIENTS

Aberrant methylation of gene promoter regions is the main epigenetic change characterizing colorectal cancer. Methylation levels of 42 CpG-sites of promoter regions of the MGMT, APC and CDH13 genes in colorectal cancer were studied in comparison with methylation levels of the adjacent normal tissue in 25 patients. Pyrosequencing showed an increase in methylation levels of promoter regions of the MGMT, APC and CDH13 genes in tumor samples by 3 to 5 times. These tumor samples were screened for activating SNP-mutations in the KRAS (40 %), NRAS (0 %) and BRAF (0 %) oncogenes. SNP-mutations in the KRAS gene were accompanied by hypermethylation of one or more promoters of the studied genes. Association of this epigenetic index with tumor metastasis was proved. The data on an increase in methylation of the promoter regions of oncosupressor genes can be used as sensitive prognostic markers of progression and metastasis of colorectal cancer.

FOXP3 gene promoter methylation in endometrial cancer cells

To determine the methylation level of promoter region of the FOXP3 gene promoter depending on the heterogeneity of intracellular localization of its protein product in endometrial cancer (EC) cells and assess its relation to the clinical and morphological features of tumor. Samples of surgical material of 40 EC patients who have not received any specific treatment before the surgery, were studied. Real time methylation-specific PCR (MSP) as well as morphological and immunohistochemical methods were used in the study. Methylation of promoter region of the FOXP3 gene was determined in all EC cases, but variability of the methylation level in EC cells from 45.0% to 85.0% was observed. With tumor progression and in tumors with deep (≥ 1/2) invasion in myometrium, an increase of the methylation level of the FOXP3 and of cell number with cytoplasmic FOXP3 localization was observed. In EC patients the correlation between of methylation level of the FOXP3 gene and the number of FOXP3(+) tumor cells with cytoplasmic expression (r = 0.41) was determined. The methylation level of FOXP3 gene promoter region and intracellular localization of its protein product are associated with tumor differentiation grade and the depth of myometrial invasion.

DNA methylation level of promoter region of activating transcription factor 5 in glioma.

Transcription factors, which represent an important class of proteins that play key roles in controlling cellular proliferation and cell cycle modulation, are attractive targets for cancer therapy. Previous researches have shown that the expression level of activating transcription factor 5 (ATF5) was frequently increased in glioma and its acetylation level was related to glioma. The purposes of this study were to explore the methylation level of ATF5 in clinical glioma tissues and to explore the effect of ATF5 methylation on the expression of ATF5 in glioma. Methylation of the promoter region of ATF5 was assayed by bisulfite-specific polymerase chain reaction (PCR) sequencing analysis in 35 cases of glioma and 5 normal tissues. Quantitative real-time PCR (qRT-PCR) was also performed to detect ATF5 mRNA expression in 35 cases of glioma and 5 normal tissues. Clinical data were collected from the patients and analyzed. The percentages of methylation of the ATF5 gene in the promoter region in healthy control, patients with well-differentiated glioma, and those with poorly differentiated glioma were 87.78%, 73.89%, and 47.70%, respectively. Analysis of the methylation status of the promoter region of the ATF5 gene showed a gradually decreased methylation level in poorly differentiated glioma, well-differentiated glioma, and normal tissues (P<0.05). There was also a significant difference between well-differentiated glioma and poorly differentiated glioma (P<0.05). ATF5 mRNA expression in glioma was significantly higher than that in the normal tissues (P<0.05). This study provides the first evidence that the methylation level of ATF5 decreased, and its mRNA expression was evidently up-regulated in glioma.

DNA methylation is associated with expression level changes of galectin gene in mantle wound healing process of pearl oyster, Pinctada fucata.

Galectin is one important member of pattern recognition proteins that plays a pivotal role in regulating innate immune response of invertebrates. In this study, we cloned the promoter sequence of a tandem-repeat galectin from the pearl oyster Pinctada fucata (P. fucata). The quantitative real-time PCR analysis revealed that galectin mRNA expression in mantle tissues were firstly up-regulated from time points of 2 h-24 h, and then down-regulated from time points of 24 h-168 h after mantle injury. The genome methylation level of mantle tissue was inversely related to galectin mRNA expression (Pearson correlation: -0.554, P: 0.154). The "CpG4-6" methylation level in promoter region of galectin was significant positive correlated with the mRNA expression (Pearson correlation: 0.313, P: 0.049). The results indicated that galectin gene may be involved in immune response in mantle wound healing process of P. fucata, and DNA methylation may be a regulation factor of gene expression.

Increased methylation of interleukin 6 gene is associated with obesity in Korean women.

Obesity is the fifth leading risk for death globally, and a significant challenge to global health. It is a common, complex, non-malignant disease and develops due to interactions between the genes and the environment. DNA methylation can act as a downstream effector of environmental signals; analysis of this process therefore holds substantial promise for identifying mechanisms through which genetic and environmental factors jointly contribute to disease risk. To assess the effects of excessive weight and obesity on gene-specific methylation levels of promoter regions, we determined the methylation status of four genes involved in inflammation and oxidative stress [interleukin 6 (IL6), tumor necrosis factor α (TNFα), mitochondrial transcription factor A (TFAM), and glucose transport 4 (GLUT4)] in blood cell-derived DNA from healthy women volunteers with a range of body mass indices (BMIs) by methylation-specific PCR. Interestingly, the samples from obese individuals (BMI ≥ 30 kg/m2) showed significantly increased hypermethylation for IL6 gene compared to normal weight (BMI < 23 kg/m2) and overweight samples (23 kg/m2 ≤ BMI < 30 kg/m2) (P = 0.034 and P = 0.026). However, there was no statistically significant difference in promoter methylation of the other 3 genes between each group. These findings suggest that aberrant DNA methylation of IL6 gene promoter may play an important role in the etiology and pathogenesis of obesity and IL6 methylation could be used as molecular biomarker for obesity risk assessment. Further studies are required to elucidate the potential mechanisms underlying this relationship.