The Effect of the Cyp19a1 Gene Methylation Modification on Temperature-dependent Sex Determination of Reeves' Turtle(Mauremys reevesii)

Temperature-dependent sex determination（TSD） is a type of environmental sex determination in which the sex of the embryos depends on the ambient temperature; however,the molecular mechanisms governing this process remain unknown.Aromatase,encoded by the cyp19a1 gene,which converts androgens into estrogens in animals,was considered to be the key gene for TSD.In this study,the 5＇-flanking region of the cyp19a1 gene in Reeves＇ turtle（Mauremys reevesii） was cloned,and the promoter region was identified using the luciferase reporter assay.Then the eggs of Reeves＇ turtle were incubated at different temperatures（26°C： male-biased temperature; 29°C： non-sex-biased temperature and 32°C： female-biased temperature）.During the thermosensitive period,the adrenal kidney gonad complexes（AKG） were sampled.DNA methylation analysis of the AKG samples showed that the promoter region of the cyp19a1 gene was significantly de-methylated in the female-biased temperature regime（P＆lt;0.01）.Quantitative analysis of the cyp19a1 gene and estrogen by q PCR and Elisa assay showed that the expression level of the cyp19a1 gene and estrogen content were both upregulated significantly at the female-biased temperature（P＆lt;0.01）.These results indicated that the de-methylation response of the cyp19a1 gene to incubation temperature,especially at the female-biased temperature,could lead to temperature-specific expression of aromatase and increased estrogen levels,which may further determine gonadal development in Reeves＇ turtle.These findings provide insights into the genetic mechanisms underlying TSD.

DNA methylation in poultry:a review

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.

Silence of HIN-1 expression through methylation of its gene promoter in gastric cancer

AIM: To clarify the role of high in normal-1 (HIN-1) gene promoter methylation during gastric cancer development. METHODS: Gastric cancer cell lines and tissue specimens were analyzed for expression of HIN-1 mRNA and protein using the semi-quantitative reverse transcription polymerase chain reaction and immunohistochemistry. The methylation of the HIN-1 gene promoter was detected in gastric carcinoma cells and tissues using methylation-specific polymerase chain reaction. The 3-(4,5-dimethylthiazol-2yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium cell viability assay and flow cytometry were used to assess the changes in behaviors of gastric cancer cells with or without 5-aza-2’-deoxycytidine treatment. RESULTS: HIN-1 was not expressed in 4 of 5 gastric cancer cell lines. The demethylation reagent 5-aza-2’-deoxycytidine was able to induce or upregulate HIN-1 expression in gastric cancer cell lines, which is associated with reduction of tumor cell viability. Furthermore, methylation of the HIN-1 gene promoter was shown in 57.8% (26/45) of the primary gastric cancer and 42.1% (17/38) of adjacent tissue samples, but was not shown in normal gastric mucosa (0/10). From the clinicopathological data of the patients, methylation of the HIN-1 gene promoter was found to be associated with tumor differentiation (P = 0.000). CONCLUSION: High methylation of HIN-1 gene promoter results in silence of HIN-1 expression in gastric cancer. 5-aza-2’-deoxycytidine reverses HIN-1 methylation and reduces viability of gastric cancer cells.

Methylation Characteristics of Perivisceral Fat Gene in Obese Rats with Phlegm-dampness Syndrome and the Effect of Wen Dan Decoction

Objective To observe the characteristics of gene methylation in obese rats with phlegm-dampness syndrome induced by the high-fat diet, and to study the effect of Wen Dan Decoction on gene methylation after the intervention. Methods Methylation sites of genes were detected by the MeDIP-seq method. Bioinformatics method was used to analyze the gene methylation characteristics of obesity with phlegmdampness syndrome and the effect of Wen Dan Decoction. Results (1) There were 3 242 methylation differential loci in dietinduced obesity with phlegm-dampness syndrome, of which 1 243 were down-regulated and 1 999 were up-regulated, involving 1 579 differential genes. GO analysis showed that "offactory receptor activity" and others were enriched. The possible signal pathways involved were "Olfactory transduction""Tuberculosis""Systemic lupus erythematosus" and "Ribosome".(2) After the intervention of Wen Dan Decoction in obesity with phlegmdampness syndrome, 4 046 different methylation loci were obtained, including 1 067 down-regulated loci and 2 979 up-regulated loci, involving 2 068 genes. GO analysis showed that "offactory receptor activity" and others were enriched. These genes involved seven signaling pathways, such as "Metabolic pathways".(3) Between diet-induced obesity with phlegm-dampness syndrome and Wen Dan Decoction intervening obesity with the phlegm-dampness syndrome, 582 common genes of methylation differential genes were obtained. After the intervention of Wen Dan Decoction, the number of GO enrichment items was more than that of obesity with phlegm-dampness syndrome, and even the same GO enrichment items involved more genes. Conclusions The phlegm-dampness syndrome of obesityinduced by diet had the characteristics of gene methylation changes, and the intervention of Wen Dan Decoction could also affect the status of gene methylation. The genes affected by Wen Dan Decoction were closely related to the methylation gene of phlegm-dampness syndrome of obesity-induced by diet but covered a wider range.

Methylation of the PCDH8 (Protocadherin-8) gene in gastric cancer

Objective: To investigate the methylation status of the PCDH8 (Protocadherin-8) gene in gastric cancer tissues and find out the relationship between methylation status of the PCDH8 and clinicopathological features in gastric cancer patients. Methods: We first investigated the methylation status of the PCDH8 (Protocadherin-8) gene in 65 gastric cancer and detected aberrant promoter methylation in gastric cancers; and then analyzed he relationship between methylation status of the PCDH8 and clinicopathological status with SPSS 13.0 software. Results: We first investigated the methylation status of the PCDH8 (Protocadherin-8) gene in 65 gastric cancer and detected aberrant promoter methylation in 36 of 65 (55.4%) gastric cancers. There was no significant difference in the distribution of patients with methylation or unmethylation of PCDH8 in terms of age, sex, tumor size, distant metastasis, or TNM stage. Methylation of PCDH8 was significantly correlated to negative pathological lymph node metastasis (P=0.038) and tumor differentiation (P=0.01). These two factors were proved to be of prognostic importance. Conclusion: Methylated PCDH8 seems to have a trend for worse prognosis in gastric cancer. However, a further large series of tumor samples and a longer follow-up period are required to elucidate its potential role.

DNA methylation and microRNAs in cancer

DNA methylation is a type of epigenetic modification in the human genome,which means that gene expression is regulated without altering the DNA sequence.Methylation and the relationship between methylation and cancer have been the focus of molecular biology researches.Methylation represses gene expression and can influence embryogenesis and tumorigenesis.In different tissues and at different stages of life,the level of methylation of DNA varies,implying a fundamental but distinct role for methylation.When genes are repressed by abnormal methylation,the resulting effects can include instability of that gene and inactivation of a tumor suppressor gene.MicroRNAs have some aspects in common with this regulation of gene expression.Here we reviewed the influence of gene methylation on cancer and analyzed the methods used to profile methylation.We also assessed the correlation between methylation and other epigenetic modifications and microRNAs.About 55 845 research papers have been published about methylation,and one-fifth of these are about the appearance of methylation in cancer.We conclude that methylation does play a role in some cancer types.

Aberrant methylation of SPARC in human hepatocellular carcinoma and its clinical implication

AIM:To investigate the methylation status of secreted protein acidic and rich in cysteine(SPARC) in human hepatocellular carcinoma(HCC) and evaluate its clinical implication.METHODS:The methylation status of SPARC was analyzed in one HCC cell line(SMMC-7721) and 60 pairs of HCC and corresponding nontumorous tissues by methylation-specific polymerase chain reaction and bisulfite sequencing.The expression of SPARC mRNA and protein were examined by reverse transcription polymerase chain reaction and immunohistochemistry,respectively.The correlations between the methylation status and the gene expression,the clinicopathological parameters,as well as the prognosis after surgery were analyzed.RESULTS:In the SMMC-7721 cell line,the loss of SPARC expression was correlated with the aberrant methylation and could be reactivated by the demethylating agent 5-aza-2'-deoxycytidine.Methylation frequency of SPARC in HCC was significantly higher than that in the corresponding nontumorous tissues(45/60 vs 7/60,P < 0.001),and it was correlated with the pathological classification(P = 0.019).The downregulation of the SPARC mRNA expression in HCC was correlated with the SPARC methylation(P = 0.040).The patients with methylated SPARC had a poorer overall survival than those without methylated SPARC(28.0 mo vs 41.0 mo,P = 0.043).CONCLUSION:Aberrant methylation is an important mechanism for SPARC inactivation in HCC and SPARC methylation may be a promising biomarker for the diagnosis and prognosis of HCC.

Genic C-Methylation in Soybean Is Associated with Gene Paralogs Relocated to Transposable Element-Rich Pericentromeres

Most plants are polyploid due to whole-genome duplications （WGD） and can thus have duplicated genes. Following a WGD, paralogs are often fractionated （lost） and few duplicate pairs remain. Little attention has been paid to the role of DNA methylation in the functional divergence of paralogous genes. Using high- resolution methylation maps of accessions of domesticated and wild soybean, we show that in soybean, a recent paleopolyploid with many paralogs, DNA methylation likely contributed to the elimination of ge- netic redundancy of polyploidy-derived gene paralogs. Transcriptionally silenced paralogs exhibit partic- ular genomic features as they are often associated with proximal transposable elements （TEs） and are pref- erentially located in pericentromeres, likely due to gene movement during evolution. Additionally, we provide evidence that gene methylation associated with proximal TEs is implicated in the divergence of expression profiles between orthologous genes of wild and domesticated soybean, and within populations.

5-aza-2'-deoxycitydine induces demethylation and up-regulates transcription of p16^(INK4A) gene in human gastric cancer cell lines

Background To investigate the effects of DNA methylation on the expression of tumor-associated genes and the cell cycle in human gastric cancer cells. Methods Two gastric cancer cell lines (MKN-45 and HGC-27) were treated with DNA methyltransferase (DNMT) inhibitor,5-aza-2’-deoxycytidine (5-aza-dC). The expressions of p16 INK4A,p21 WAF1,p53 , p73 ,c-Ha-ras and c-myc genes mRNA were detected by using reverse transcription PCR (RT-PCR). DNA methylation status of p16 INK4A gene promoter was assayed by bisulfite modification and sequencing. The cell cycle was analyzed by using flow cytometry (FCM). Results 5-aza-dC induced the demethylation of p16 INK4A gene promoter. The expression of p16 INK4A mRNA was obviously up-regulated by treatment with 10 μmol/L (MKN-45 cells) or 5 μmol/L (HGC-27 cells) of 5-aza-dC for 24 hours. However,5-aza-dC treatment failed to regulate the expressions of p21 WAF1,p53 , p73 ,c-Ha-ras and c-myc genes in MKN-45 and HGC-27 cells. Furthermore,5-aza-dC induced the cell cycle arrest in G1 phase in HGC-27 cell,but not in MKN-45 cell. Conclusions DNA methylation regulates the transcription of p16 INK4A but not p21 WAF1 and proto-oncogenes in human gastric cancer cell lines MKN-45 and HGC-27.

The exploration of the correlation between the risk of obesity and the promoter methylation of PRDM16 gene

Objective To explore the association between the Cp G methylation level of positive regulatory domain containing 16(PRDM16)gene promoter and obesity or body mass index(BMI).Methods A total of 116 patients(91female adults and 25 male adults)with abdominal operation in a municipal hospital of Henan province were enrolled in this study and they were divided into

Efficacy of Yisui granule(益髓颗粒)on myelodysplastic syndromes in SKM-1 mouse xenograft model through suppressing Wnt/β-catenin signaling pathway

OBJECTIVE:To unmask the underlying mechanisms of Yisui granule(益髓颗粒,YSG)for the treatment of Myelodysplastic syndromes(MDS).METHODS:Our study used an SKM-1 mouse xenograft model of MDS to explore the anti-tumor potential of YSG and its safety,assess its effect on overall survival(OS),and evaluate whether its mechanism is associated with the demethylation of the secreted frizzled related protein 5(s FRP5)gene and suppressing Wnt/β-catenin pathway.Bisulfite amplicon sequencing was applied to detect the level of methylation of the s FRP5 gene;western blotting,immunofluorescence staining,and real-time Polymerase Chain Reaction were performed to detect DNA methyltransferase 1(DNMT1),s FRP5,and other Wnt/β-catenin pathway-related m RNA and protein expression.RESULTS:The results showed that high-dosage YSG exerted an anti-tumor effect similar to that of decitabine,improved OS,and reduced long-term adverse effects in the long term.Mechanically,YSG reduced the expression of DNMT1 methyltransferase,decreased the methylation,and increased the expression of the Wnt/β-catenin pathway antagonist-s FRP5.Furthermore,components of the Wnt/β-catenin pathway,including Wnt3a,β-catenin,c-Myc,and cyclin D1,were down-regulated in response to YSG,suggesting that YSG could treat MDS by demethylating the s FRP5 gene and suppressing the Wnt/β-catenin pathway.CONCLUSIONS:Our findings demonstrated that YSG could be used alone or in combination with decitabine to improve outcomes in the MDS animal model,providing an alternative solution for treating MDS.

Genome-wide analysis of differential DNA methylation in Silver-Russell syndrome

Silver-Russell Syndrome(SRS) is clinically heterogeneous disorder characterized by low birth weight, postnatal growth restriction, and variable dysmorphic features. Current evidence strongly implicates imprinted genes as an important etiology of SRS. Although almost half of the patients showed DNA hypomethylation at the H19/IGF2 imprinted domain, and approximately7%–10% of SRS patients have maternal uniparental disomy of chromosome 7(UPD(7) mat); the rest of the SRS patients shows unknown etiology. In this study, we investigate whether there are further DNA methylation defects in SRS patients. We measured DNA methylation in seven SRS patients and five controls at more than 485,000 CpG sites using DNA methylation microarrays. We analyzed methylation changes genome-wide and identified the differentially methylated regions(DMRs) using bisulfite sequencing and digital PCR. Our analysis identifies epimutations at the previously characterized domains of H19/IGF2,providing proof of principle that our methodology can detect the changes in DNA methylation at imprinted loci. In addition,our results showed a novel SRS associated imprinted gene OSBPL5 located on chromosome 11p14 with the probe cg25963939,which is hypomethylated in 4/7 patients(P=0.023, β=.0.243). We also report DMRs in other genes including TGFβ3, HSF1,GAP43, NOTCH4 and MYH14. These DMRs were found to be associated with SRS using GO pathway analysis. In this study,we identified the probe cg25963939, located at the 5′UTR of imprinted gene OSBPL5, as a novel DMR that is associated with SRS. This finding provides new insights into the mechanism of SRS etiology and aid the further stratification of SRS patients by molecular phenotypes.

Specifications of Targeting Heterochromatin Modifications in Plants

Plants encode a diverse repertoire of DNA methyltransferases that have specialized to target cytosines for methylation in specific sequence contexts. These include the de novo methyltransferase, DOMAINS REARRANGED METHYLTRANSFERASE 2 （DRM2）, which methylates cytosines in all sequence contexts through an RNA-guided process, the CHROMOMETHYLASES （CMTs）, which methylate CHH and CHG cytosines （where H is A, T, or C）, and METHYLTRANSFERASE 1 （MET1）, which maintains methylation of symmetrical CG contexts. In this review, we discuss the sequence specificities and targeting of each of these pathways. In particular, we highlight recent studies that indicate CMTs preferentially target CWG or CWA/CAW motifs （where W is A or T）, and discuss how self-reinforcing feedback loops between DNA methyltransferases and histone modifications characteristic of heterochromatin specify targeting. Finally, the initiating events that lead to gene body methylation are discussed as a model illustrating how interde- pendent targeting of different silencing pathways can potentiate the establishment of off-target epialleles.

EARLY FLOWERING IN SHORT DAYS(EFS) regulates the seed size in Arabidopsis

Post-transcriptional modifications,including histone modifications and DNA methylation,alter the chromatin landscape to regulate gene expression,thus control various cellular processes in plants.EARLY FLOWERING IN SHORT DAYS(EFS)is the major contributor for H3K36 methylation in Arabidopsis and is important for plant development.Here,we find that EFS is expressed in different stages of embryo morphogenesis,and the efs mutant produces larger embryo that results in enlarged seeds.Further analysis reveals that an imprinted gene MOP9.5 is hypomethylated at the promoter region and its expression is derepressed in efs mutant.MOP9.5 promoter is marked by various epigenetic modifications,and we find that following the increase of H3K36me3,H3K27me3 and H3K9me2 levels are reduced in efs mutant.This data indicates an antagonistic regulation between H3K36me3 and DNA methylation,and/or H3K27me3 at MOP9.5.Our results further show that both maternal and paternal EFS alleles are responsible for the seed size regulation,which unraveled a novel function of EFS in plant development.