Eucaryotic DNA Methylation and Gene Mutation

5-methylcytosine (m5C) as a rare base exists in eucaryotic genomes, it is a normal constituent of many eucaryotic DNA, whose existence is a character of eucaryotic DNA. In the regular physiological conditions, cytosine residue of eucaryotic DNA is methylated to be popular. Up to the present, many people consider that the m5C may be mutation hotspots by the m5C deamination leading to gene mutation. Our theoretical investigations indicated that the spontaneous mutation caused by the transition of G - C-A - T, in eukaryotic DNA, may be a result caused by the tautomer changing base pairs and may also be caused by other factor actions, however it could not be caused by the deamination of m5C.

Characterization of Two Groups of Low_copy and Specific DNA Sequences Isolated from Chromosome 7B of Common Wheat

Recent work revealed that, in the genomes of polyploid wheat, there exists a class of low_copy and chromosome_specific sequences that are labile upon polyploid formation. This class of sequences was proposed to play a critical role in the stabilization and establishment of nascent plant polyploids as new species. To further study this issue, five wheat chromosome 7B_specific sequences, isolated from common wheat (Triticum aestivum L.) by chromosome microdissection, were characterized. The sequences were studied by genomic Southern hybridizations on a collection of polyploid wheats and their diploid progenitors. Four sequences hybridized to all polyploid species, but at the diploid level to only species closely related to the B_genome of polyploid wheat. This indicates that these sequences originated with the divergence of the diploid species, and was then vertically transmitted to polyploids. One sequence hybridized to all species at both the diploid and polyploid levels, suggesting its elimination after the polyploid wheat formation. The hybridization of this sequence to two synthetic polyploid wheats indicated that sequence elimination is a rapid event and probably related to methylation status of the sequence. Based on the above results, we suggest that selective changes of low_copy sequences occur rapidly after polyploid formation, which may contribute to the differentiation of chromosomes in newly formed allopolyploid wheats.

Effects of histone acetylation and DNA methylation on p21^(WAF1)regulation

Cell cycle progression is regulated by interactions between cyclins and cyclin-dependent kinases (CDKs). p21(WAF1) is one of the CIP/KIP family which inhibits CDKs activity. Increased expression of p21(WAF1) may play an important role in the growth arrest induced in transformed cells. Although the stability of the p21( WAF1) mRNA could be altered by different signals, cell differentiation and numerous influencing factors. However, recent studies suggest that two known mechanisms of epigenesis, i.e.gene inactivation by methylation in promoter region and changes to an inactive chromatin by histone deacetylation, seem to be the best candidate mechanisms for inactivation of p21( WAF1). To date, almost no coding region p21(WAF1) mutations have been found in tumor cells, despite extensive screening of hundreds of various tumors. Hypermethylation of the p21(WAF1) promoter region may represent an alternative mechanism by which the p21(WAF1/CIP1) gene can be inactivated. The reduction of cellular DNMT protein levels also induces a corresponding rapid increase in the cell cycle regulator p21(WAF1) protein demonstrating a regulatory link between DNMT and p21(WAF1) which is independent of methylation of DNA. Both histone hyperacetylation and hypoacetylation appear to be important in the carcinoma process, and induction of the p21(WAF1) gene by histone hyperacetylation may be a mechanism by which dietary fiber prevents carcinogenesis. Here, we review the influence of histone acetylation and DNA methylation on p21(WAF1) transcription, and affection of pathways or factors associated such as p 53, E2A, Sp1 as well as several histone deacetylation inhibitors.

Characterization of MyDNMT1 and Changes of Global DNA Methylation during the Embryonic Development in Mizuhopecten yessoensis

DNA methylation is a critical epigenetic mechanism that influences gene transcription, genomic stability, X-chromosome inactivation and other factors, and appropriate DNA methylation is crucial in development. DNA methyltransferase 1 （DNMT1） plays an important role in maintaining the established methylation pattern during DNA replication. Although the effect of DNA methylation on embryonic development has been well known in vertebrates, little research has been carried out in invertebrates, especially in marine bivalves. In this study, the DNMT1 gene （MyDNMT1） was firstly identified from Mizuhopecten yessoensis. The full-length cDNA of MyDNMT1 was 5 039 bp, consisted of a 5＇ untranslated region （5＇-UTR） of 79 bp, a 3＇ untranslated region （3＇-UTR） of 199 bp, and a 4 761 bp open reading frame （ORF） encoding a peptide of 1 586 amino acids without a putative signal peptide. The relative mRNA expression level of MyDNMT1 was measured during the embryonic development of M. ydssoensis using real-time PCR, which revealed that the level at stage zygote and trochophore were significantly higher than that at other stages. We further examined the global DNA methylation during development by colorimetric method. The results showed that the methylation level was increased and reached the peak at blastula stage, then dramatically decreased, and fluctuated at early D-shaped larva stage. This study provided greater insight into the DNA methylation of embryonic development, which obtained a better understanding of the relationship between the DNA methylation and the embryonic development in bivalve mollusks.

Stress Treatments and DNA Methylation Affected the Somatic Embryogenesis of Citrus Callus

The evaluation on the callus embryogenesis capacity of 15 genotypes of citrus showed that stress treatments were conducive to somatic embryogenesis and could enhance the recovery of the missed capacity of embryogenesis for some genotypes. Randomly amplified polymorphic DNA (RAPD) and methylation sensitive amplified polymorphism (MSAP) analysis indicated that there existed significant differences in DNA methylation status between the callus capable of producing somatic embryoids and that which missed the embryogenesis capacity of the same genotype Newhall navel orange ( Citrus sinensis Osb. cv. Newhall). The DNA methylation level of the former was lower than that of the latter. However, RAPD profiles did not show any difference between these two kinds of callus.

mRNA expression and DNA methylation in three key genes involved in caste differentiation in female honeybees(Apis mellifera)

In honeybee （Apis mellifera） colonies, queens and workers are altemative forms of the adult female honeybee that develop from genetically identical zygotes but that depend on differential nourishment. Queens and workers display distinct morphologies, anatomies and behavior, better known as caste differentiation. Despite some basic insights, the exact mechanism responsible for this phenomenon, especially at the molecular level, remains unclear although some progress has been achieved. In this study, we examined mRNA levels of the TOR （target of rapamycin） and Dnmt3 （DNA methyltransferase 3） genes, closely related to caste differentiation in honeybees. We also investigated mRNA expression of the S6K （similar to RPS6-p70-protein kinase） gene linked closely to organismal growth and development in queen and worker larvae （1-day and 3-day old）. Last, we investigated the methylation status of these three genes in corresponding castes. We found no difference in mRNA expression for the three genes between 1st instar queen and worker larvae; however, 3rd instar queen larvae had a higher level of TOR mRNA than worker larvae. Methylation levels of all three genes were lower in queen larvae than worker larvae but the differences were not statistically significant. These findings provide basic data for broadening our understanding of caste differentiation in female honeybees.

Genome-wide Identification and Analysis of DNA Methyltransferases in Grape

DNA methylation is an important epigenetic regulation mechanism, which is catalyzed by DNA methyltransferases. In this study, eight DNA methyltransferase genes were identified in grape genome to analyze the selective pressure, gene expression and codon usage bias. The results showed grape DNA methyltransferase MET subfamily underwent relatively strong purifying selection during evolution, while chromomethylase CMT subfamily underwent positive selection during evolution. Under different abiotic(heat, drought or cold) stresses, the expression level of many grape DNA methyltransferase genes changed significantly. The expression level of these genes might be related with cis-regulatory elements of their promoters. The results of codon usage bias analysis showed that synonymous codon bias existed in grape DNA methyltransferase gene family, which might be affected by mutation pressure. These results laid a solid foundation for in-depth study of DNA methyltransferases in grape.

DNA Methylation Analysis of Exogenous Genes Transformed into Sheep by Different Methods

[Objective] This study aimed to investigate the methylation levels of exogenous genes and promoters and the differences of protein expression in transgenic sheep obtained by different transgenic technologies. [Method] Exogenous genes eGFP （enhanced green fluorescent protein） and FGF5 （fibroblast growth factor 5） were separately transformed into sheep by somatic cell cloning, stem cell cloning and perivitelline injection to obtain transgenic sheep, with CMV as the promoter. Bisulfite sequencing method was adopted to detect the methylation status of the promoter region and coding region of exogenous genes in tail tissues of transgenic sheep. Western blot was adopted to detect the expression level of exogenous genes. [Result] The methylation level of the promoter region with stem cell cloning was the highest, followed by somatic cell cloning, while that with perivitelline injection was the lowest; the methylation level of the eGFP coding region with perivitelline injection was the highest, followed by stem cell cloning; the methylation level of the FGF5 coding region with somatic cell cloning was higher than that with perivitelline injection. The exogenous protein expression level was negatively correlated with the methylation level of the promoter region. [Conclusion] This study indicates that different transgenic methods may influence the methylation level of exogenous genes, thus affecting exogenous gene expression.

The altered DNA methylation pattern and its implications in liver cancer

DNA methylation is the most intensively studied epigenetic phenomenon, disturbances of which result in changes ingene transcription, thus exerting drastic imparts onto biological behaviors of cancer. Both the global demethylation andthe local hypermethylation have been widely reported in all types of tumors, providing both challenges and opportunitiesfor a better understanding and eventually controlling of the malignance. However, we are still in the very early stage ofinformation accumulation concerning the tumor associated changes in DNA methylation pattern. A number of excellentrecent reviews have covered this issue in depth. Therefore, this review will summarize our recent data on DNA methy-lation profiling in cancers. Perspectives for the future direction in this dynamic and exciting field will also be given.

DNA methylation in hepatocellular carcinoma

As for many other tumors,development of hepatocellular carcinoma(HCC)must be understood as a multistep process with accumulation of genetic and epigenetic alterations in regulatory genes,leading to activation of oncogenes and inactivation or loss of tumor suppressor genes(TSG).In the last decades,in addition to genetic alterations,epigenetic inactivation of(tumor suppressor) genes by promoter hypermet hylation has been recognized as an important and alternative mechanism in tumorigenesis.In HCC,aberrant methylation of promoter sequences occurs not only in advanced tumors, it has been also observed in premalignant conditions just as chronic viral hepatitis B or C and cirrhotic liver. This review discusses the epigenetic alterations in hepatocellular carcinoma focusing DNA methylation.

Effects of temperature and salinity on survival, growth and DNA methylation of juvenile Pacific abalone, Haliotis discus hannai Ino

Temperature and salinity are two of the most potent abiotic factors influencing marine mollusks. In this study, we investigated the individual and combined effects of temperature and salinity on the survival and growth of juvenile Pacific abalone, Haliotis discus hannai Ino, and also examined the DNA methylation alteration that may underpin the phenotypic variation of abalone exposed to different rearing conditions. The single-factor data showed that the suitable ranges of temperature and salinity were 16-28℃ at a constant salinity of 32, and 24-40 at a constant temperature of 20℃, respectively. The two-factor data indicated that both survival and growth were significantly affected by temperature, salinity and their interaction. The optimal temperature-salinity combination for juveniles was 23-25℃ and 30-36. To explore environment-induced DNA methylation alteration, the methylation-sensitive amplified polymorphism （MSAP） technique was used to analyze the genomic methylation profiles of abalone reared in optimal and adverse conditions. Neither temperature nor salinity induced evident changes in the global methylation level, but 67 and 63 differentially methylated loci were identified in temperature and salinity treatments, respectively. The between-group eigen analysis also showed that both temperature and salinity could induce epigenetic differentiation in H. discus hannai Ino. The results of our study provide optimal rearing conditions for juvenile tt. discus hannai Ino, and represent the first step toward revealing the epigenetic regulatory mechanism of abalone in response to thermal and salt stresses.

BRCA1 affects global DNA methylation through regulation of DNMT1

Global DNA hypomethylation at CpG islands coupled with local hypermethylation is a hallmark for breast cancer, yet the mechanism underlying this change remains elusive. In this study, we showed that DNMT1, which encodes a methylation maintenance enzyme, is a transcriptional target of BRCA1. BRCA1 binds to the promoter of the DNMT1 gene through a potential OCT1 site and the binding is required for maintaining a transcriptional active configuration of the promoter in both mouse and human cells. We further demonstrated that impaired function of BRCA1 leads to global DNA hypomethylation, loss of genomic imprinting, and an open chromatin configuration in several types of tissues examined in a BRCA1 mutant mouse model at premaligant stages. BRCA1 deficiency is also associated with significantly increased expression levels of several protooncogenes, including c-Fos, Ha-Ras, and c-Myc, with a higher expression in tumors, while premalignant mammary epithelial cells displayed an intermediate state between tumors and controls. In human clinical samples, reduced expression of BRCA1 correlates with decreased levels of DNMT1, and reduced methylation of CpG islands. Thus, BRCA1 prevents global DNA hypomethylation through positively regulating DNMT1 expression, and this provides one of mechanisms for BRCAl-associated breast cancer formation.

Changes in Global DNA Methylation Intensity and DNMT1 Transcription During the Aging Process of Scallop Chlamys farreri

DNA methylation is an important epigenetic regulatory mechanism that influences genomic stability, gene activation, X-chromosome inactivation and other factors. A change in DNA methylation is usually associated with aging and cellular senescence. DNA methyltransferase 1(DNMT1) is the most abundant DNA methyltransferase, and it plays an important role in maintaining the established methylation pattern during DNA replication in vertebrates. Although the effect of aging on DNA methylation has been well studied in vertebrates, little research has been conducted in invertebrates, especially in marine bivalves. In this study, we examined global DNA methylation levels in four groups of adult Zhikong scallop Chlamys farreri at different ages. The results showed that both the age and tissue type had a strong effect on the DNA methylation. In addition, a significant decrease in DNA methylation with aging(1–4 years) can be detected in mantle, kidney and hepatopancreas. We further measured the change in DNMT1 transcript abundance using quantitative reverse transcription PCR(q RT-PCR), which revealed that DNMT1 transcription significantly decreased with aging in mantle and hepatopancreas and strongly correlated with DNA methylation(R = 0.72). Our data provided greater insight into the aging-related decline of DNA methylation, which could aid in gaining a better understanding of the relationship between DNA methylation and the aging process in bivalve mollusks.

Cryopreservation by Vitrification of Vitis vinifera cv. ＂Red Globe＂ Zygotic Embryos and Effect on the Expression of DNA Methyltransferase Genes

Mexico is a large producer of table grape （Vitis vinifera L.） and therefore it is important to develop protocols to store the grape varieties germplasm. The objective of the present work was to design a protocol for the cryopreservation by vitrification of zygotic embryos of V. vinifera cv. ＂Red Globe＂ and evaluate possible epigenetics changes. The plant vitrification solution 2 （PVS2） was utilized before the utilization of liquid nitrogen （LN）. The effect of this protocol on embryo viability was tested by the triphenyl-tetrazolium chloride solution, as well as by the in vitro development of grape embryos into plantlet. A cDNA expression library of grape zygotic embryos was created to isolate expressed sequence tags of several DNA methyltrasferases. Gene expression of domains rearranged methyltransferase type 1 （DMR1） and DNA （cytosine-5）-methyltransferase 1 （MET1-2） isozymes was analyzed by quantitative reverse transcriptase PCR. The optimal conditions for vitrification were 10 min in 50% PVS2, followed by I0 min in 100% PVS2. Under these conditions, about 30% of plantlet was obtained from embryos after cryopreservation. It was recorded a reduction in the MET1-2 gene expression, which plays a role in the maintenance of DNA methylation. It is possible to cryopreserve viable grape zygotic embryos, although the treatment seems to induce alterations in the normal DNA methylation pattern of the zygotic embryo genome.

Molecular modeling study of the effect of base methylation on internal interactions and motions in DNA and implication to B-Z conformation change

Methylation in the bases of DNA is known to induce B-Z conformation change. In this work, molecular mechanics and normal mode analysis are used to probe how certain methylation affects the internal interactions and thermodynamic motions in the DNA double helixes in both B and Z conformations, and its implication to B-Z conformation change. By molecular modeling with Insight II, two cases involving cytosine C5 and guanine C8 methylation on both B and Z-form DNA duplex d(CGCGCG)2 are studied in comparison with the corresponding unmethylated duplexes. The internal interaction energies computed based on a molecular mechanics force field and the entropies due to internal motions computed according to a normal mode analysis are in fare agreement with respective observed thermodynamic quantities. The analysis on the computed individual energy terms suggests that the observed B-Z conformation change induced by methylation is primarily driven by enthalpic factors. A combination of changes in Van der Waals interaction, electrostatic interaction and hydrogen bonding likely contributes to the change of enthalpy that favors Z-conformation in the methylated states.

Analysis of Up-Regulation of DNA-PKcs and Its Mechanism in Human Gliomas

OBJECTIVE To detect the differences in gene expression of nonhomologous end-joining pathway including Ku70, Ku80, ERCC4, lig4 and DNA-PKcs between human primary gliomas and normal brain tissues, and furthermore, to explore the underlying mechanism for the expression alteration.METHODS The expression levels of Ku70, Ku80, ERCC4, lig4 and DNA-PKcs in 36 specimens of glioma and 12 specimens of normal brain tissue were measured using SYBR green-based real- time quantitative PCR. Methylation of DNA-PKcs was detected through methylation-specific PCR （MSP）.RESULTS There was no significant difference in expression of Ku70, Ku80, ERCC4 and lig4 between human primary gliomas and normal brain tissues （P 〈 0.05）, while DNA-PKcs were significantly up-regulated （P = 0.002）. The expression of DNA- PKcs was significantly higher in patients with grade III and IV diseases compared to patients with grade II disease or in normal brain tissues （P 〈 0.05）. Moreover, glioma tissue showed weaker methvlation than normal brain tissue.CONCLUSION The up-regulation of the DNA-PKcs may be associated with pathogenesis of glioma. Demethylation of DNA- PKcs promoter is an important reason for its up-regulation.

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.

Aberrantly Methylated MGMT,hMLH1 and hMSH2 in Tumor and Serum DNA of Gliomas Patients

OBJECTIVE This study is to investigate the prevalence ofpromoter CpG island methylation of O^6-methylguananine-DNAmethyltransferase (MGMT), mismatch repair genes (hMLH1 andhMSH2) in both tumor and serum samples of gliomas.METHODS Methylation-specific PCR (MSP) was employed todetect promoter CpG island methylation of the MGMT, hMLH1and hMSH2 genes in 39 samples taken from surgery and 32samples of pretreatment serum all from the patients with gliomas.RESULTS Promoter CpG island methylation of MGMT, hMLH1and hMSH2 was detected and the results were 46.2%, 10.3% and20.5%, respectively in tumor DNA of the cases with gliomas,and 40.6%, 9.4% and 18.8%, respectively in serum DNA of thecases. The methylation pattern in primary tumor and serum wasfound to be concordant in matched tissue and serum samplesof 21 patients. In the cases with positive result of methylationfor MGMT, hMLH1 and hMSH2 in tumor tissues, the results ofdetection for those in the paired serum sample were 77.8% (7/9),66.7% (2/3) and 75.0 % (3/4), respectively. False positive resultswere not obtained in any of the patients who did not exhibitmethylation. No association was found between the promotermethylation of MGMT, hMLH1, and hMSH2 genes in primarygliomas and gender, age, localization, grade of malignant or tumorstage.CONCLUSION Promoter CpG island methylation is a frequentevent in gliomagenesis. Methylation analysis appears to bea promising predictive factor of the prognosis for the gliomapatients treated with alkylating drugs and a noninvasive tumormarker in serum DNA.

Plasma DNA methylation of Wnt antagonists predicts recurrence of esophageal squamous cell carcinoma

AIM:To detect the effects of plasma DNA methylation of Wnt antagonists/inhibitors on recurrence of esophageal squamous cell carcinoma(ESCC).METHODS:We used methylation-specific polymerase chain reaction to detect hypermethylation of the promoter of four Wnt antagonists/inhibitors(SFRP-1,WIF-1,DKK-3 and RUNX3) using DNA from the plasma of ESCC patients(n = 81) and analyzed the association between promoter hypermethylation of Wnt pathway modulator genes and the two-year recurrence of ESCC.RESULTS:Hypermethylation of SFRP-1,DKK-3 and RUNX-3 was significantly associated with an increased risk of ESCC recurrence(P = 0.001,0.003 and 0.001 for SFRP-1,DKK-3 and RUNX3,respectively).Patients carrying two to three methylated genes had a significantly elevated risk of recurrence compared with those not carrying methylated genes(odds ratio = 15.69,95% confidential interval:2.97-83).The area under the receiver operating characteristic curve(AUC) was 77.1 for ESCC recurrence prediction(sensitivity = 66.67 and specificity = 83.3).When combining methylated genes and the clinical stage,the AUC was 83.69,with a sensitivity of 76.19 and a specificity of 83.3.CONCLUSION:The status of promoter hypermethylation of Wnt antagonists/inhibitors in plasma may serve as a non-invasive prognostic biomarker for ESCC.

DNA Methylation Analysis of Cassava （Manihotesculenta Crantz） SC8 and Its Autotetraploid in Response to Cold Stress

DNA methylation plays a vital role in the regulation of gene expression in response to environmental stress. However, little is known about the effect of DNA methylation on the cassava polyploidy. In the present study, methylation-sensitive amplified polymorphisms （MSAP） were used to investigate DNA methylation profiles of cassava polyploidy following cold treatment to identify candidate genes involved in response to cold stress. The result showed that the genome-wide DNA methylation polymorphisms accounted for 34.02%-42.56% in SC8 and its autotetraploid exposed to 5 ~C for 2, 8, 24 and 48 h, respectively. The methylation levels of SC8 at 2 h-cold stress were the highest during 48h under cold treatments. With the time extension within 48 h under cold stress, the methylation levels gradually decreased to the same level as the control but DNA methylation levels of cassava autotetraploid were stable within 48 h. For future analysis of the methylation extent, the cold stress induced more DNA methylation than demethylation in SC8, where DNA methylation was consistent with demethylation in its autotetraploid. The expression analysis demonstrated increase in the transcription of one methylated gene and decrease in the transcription of two demethylated genes. The results revealed that gene methylations in specific sites would be a rapidly epigenetic response to cold stress, further elucidating the methylation functions in its autotetraploid.