Cytosine methylation of the sequence GATC in a mycoplasma

Mycoplasma virus L2 is subject to host-specific restriction and modification in Acholeplasma laidlawii strains JA1 and K2. We have examined the DNAs from both host cells and viruses propagated on these strains with respect to susceptibility to cleavage by restriction endonucleases and for DNA base modifications. We show that, in strain K2 and L2 virus grown on K2 cells, cytosine in the sequence GATC is methylated to 5-methylcytosine and, although strain K2 and L2 viruses grown on K2 contain N6-methyladenine in their DNA, adenine in the sequence GATC is not methylated. In contrast to K2, strain JA1 and L2 virus grown on JA1 cells contain no detectable methylated bases. It is not known which of the methylated bases in K2 is the basis for the K2 restriction-modification system operative on L2 virus.     

Mapping global histone methylation patterns in the coding regions of human genes

Histone methylation patterns in the human genome, especially in euchromatin regions, have not been systematically characterized. In this study, we examined the profile of histone H3 methylation (Me) patterns at different lysines (Ks) in the coding regions of human genes by genome-wide location analyses by using chromatin immunoprecipitation linked to cDNA arrays. Specifically, we compared H3-KMe marks known to be associated with active gene expression, namely, H3-K4Me, H3-K36Me, and H3-K79Me, as well as those associated with gene repression, namely, H3-K9Me, H3-K27Me, and H4-K20Me. We further compared these to histone lysine acetylation (H3-K9/14Ac). Our results demonstrated that: first, close correlations are present between active histone marks except between H3-K36Me2 and H3-K4Me2. Notably, histone H3-K79Me2 is closely associated with H3-K4Me2 and H3-K36Me2 in the coding regions. Second, close correlations are present between histone marks associated with gene silencing such as H3-K9Me3, H3-K27Me2, and H4-K20Me2. Third, a poor correlation is observed between euchromatin marks (H3-K9/K14Ac, H3-K4Me2, H3-K36Me2, and H3-K79Me2) and heterochromatin marks (H3-K9Me2, H3-K9Me3, H3-K27Me2, and H4-K20Me2). Fourth, H3-K9Me2 is neither associated with active nor repressive histone methylations. Finally, histone H3-K4Me2, H3-K4Me3, H3-K36Me2, and H3-K79Me2 are associated with hyperacetylation and active genes, whereas H3-K9Me2, H3-K9Me3, H3-K27Me2, and H4-K20Me2 are associated with hypoacetylation. These data provide novel new information regarding histone KMe distribution patterns in the coding regions of human genes.     

Dynamic stage-specific changes in imprinted differentially methylated regions during early mammalian development and prevalence of non-CpG methylation in oocytes

Mammalian imprinted genes are associated with differentially methylated regions (DMRs) that are CpG methylated on one of the two parental chromosomes. In mice, at least 21 DMRs acquire differential methylation in the germline and many of them act as imprint centres. We previously reported the physical extents of differential methylation at 15 DMRs in mouse embryos at 12.5 days postcoitum. To reveal the ontogeny of differential methylation, we determined and compared methylation patterns of the corresponding regions in sperm and oocytes. We found that the extent of the gametic DMRs differs significantly from that of the embryonic DMRs, especially in the case of paternal gametic DMRs. These results suggest that the gametic DMR sequences should be used to extract the features specifying methylation imprint establishment in the germline: from this analysis, we noted that the maternal gametic DMRs appear as unmethylated islands in male germ cells, which suggests a novel component in the mechanism of gamete-specific marking. Analysis of selected DMRs in blastocysts revealed dynamic changes in allelic methylation in early development, indicating that DMRs are not fully protected from the major epigenetic reprogramming events occurring during preimplantation development. Furthermore, we observed non-CpG methylation in oocytes, but not in sperm, which disappeared by the blastocyst stage. Non-CpG methylation was frequently found at maternally methylated DMRs as well as non-DMR regions, suggesting its prevalence in the oocyte genome. These results provide evidence for a unique methylation profile in oocytes and reveal the surprisingly dynamic nature of DMRs in the early embryo.     

Hypoxia-induced DNA hypermethylation in human pulmonary fibroblasts is associated with Thy-1 promoter methylation and the development of a pro-fibrotic phenotype

Background

Pulmonary fibrosis is a debilitating and lethal disease with no effective treatment options. Understanding the pathological processes at play will direct the application of novel therapeutic avenues. Hypoxia has been implicated in the pathogenesis of pulmonary fibrosis yet the precise mechanism by which it contributes to disease progression remains to be fully elucidated. It has been shown that chronic hypoxia can alter DNA methylation patterns in tumour-derived cell lines. This epigenetic alteration can induce changes in cellular phenotype with promoter methylation being associated with gene silencing. Of particular relevance to idiopathic pulmonary fibrosis (IPF) is the observation that Thy-1 promoter methylation is associated with a myofibroblast phenotype where loss of Thy-1 occurs alongside increased alpha smooth muscle actin (α-SMA) expression. The initial aim of this study was to determine whether hypoxia regulates DNA methylation in normal human lung fibroblasts (CCD19Lu). As it has been reported that hypoxia suppresses Thy-1 expression during lung development we also studied the effect of hypoxia on Thy-1 promoter methylation and gene expression.

Methods

CCD19Lu were grown for up to 8 days in hypoxia and assessed for global changes in DNA methylation using flow cytometry. Real-time PCR was used to quantify expression of Thy-1, α-SMA, collagen I and III. Genomic DNA was bisulphite treated and methylation specific PCR (MSPCR) was used to examine the methylation status of the Thy-1 promoter.

Results

Significant global hypermethylation was detected in hypoxic fibroblasts relative to normoxic controls and was accompanied by increased expression of myofibroblast markers. Thy-1 mRNA expression was suppressed in hypoxic cells, which was restored with the demethylating agent 5-aza-2^′-deoxycytidine. MSPCR revealed that Thy-1 became methylated following fibroblast exposure to 1% O₂.

Conclusion

These data suggest that global and gene-specific changes in DNA methylation may play an important role in fibroblast function in hypoxia.     

Developmental changes in the methylation of the rat albumin and alpha-fetoprotein genes

We have analyzed methylation of the rat albumin and alpha-fetoprotein (AFP) genes by hydridizing labeled cDNA clones to HpaII and MspI digests of DNA from different stages of development. These CCGG-cutting enzymes distinguish 5-methylcystosine in mCCGG (sensitive to HpaII) and CmCGG (sensitive to MspI). In the liver, the albumin gene is heavily methylated at 18 days gestation and uniformly demethylated in the adult. The AFP gene is also heavily methylated at 18 days gestation, and develops demethylated regions at the 3' half of the gene in the adult. These methylation changes are not observed in other embryonic or adult tissues. We also evaluated expression of these genes by measuring their corresponding mRNAs. The albumin gene is actively transcribed in 18-day fetal liver, when it is heavily methylated, as well as in adult liver, when it is unmethylated. In contrast, the AFP gene is transcribed only in fetal liver, even though it is less methylated in adult liver. These findings suggest that specific methylation changes are associated with changes in gene expression, but that this association is not adequately described by the simple hypothesis that methylation turns genes off.     

DHPLC-based method for DNA methylation analysis of differential methylated regions from imprinted genes

The bisulfite genomic sequencing method is one of the most widely used techniques for methylation analysis in heterogeneous unbiased PCR, amplifying for both methylated and unmethylated alleles simultaneously. However, it requires labor-intensive and time-consuming cloning and sequencing steps. In the current study, we used a denaturing high-performance liquid chromatography (DHPLC) procedure in a complementary way with the bisulfite genomic sequencing to analyze the methylation of differentially methylated regions (DMRs) of imprinted genes. We showed reliable and reproducible results in distinguishing overall methylation profiles of DMRs regions of human SNRPN, H19, MEST/PEG1, LIT1, IGF2, TSSC5, WT1 antisense, and mouse H19, Mest/Peg1, Igf2R imprinted genes. These DHPLC profiles were in accordance with bisulfite genomic sequencing data and may serve as a type of "fingerprint," revealing the overall methylation status of DMRs associated with sample heterogeneity. We conclude that DHPLC analysis could be used to increase the throughput efficiency of methylation pattern analysis of imprinted genes after the bisulfite conversion of genomic DNA and unbiased PCR amplification.     

Clustered DNA methylation changes in polycomb target genes in early-stage liver cancer

Polycomb-group proteins mark specific chromatin conformations in embryonic and somatic stem cells that are critical for maintenance of their "stemness". These proteins also mark altered chromatin modifications identified in various cancers. In normal differentiated cells or advanced cancerous cells, these polycomb-associated loci are frequently associated with increased DNA methylation. It has thus been hypothesized that changes in DNA methylation status within polycomb-associated loci may dictate cell fate and that abnormal methylation within these loci may be associated with tumor development. To assess this, we examined the methylation states of four polycomb target loci -Trip10, Casp8AP2, ENSA, and ZNF484 - in liver cancer. These four targets were selected because their methylation levels are increased during mesenchymal stem cell-to-liver differentiation. We found that these four loci were hypomethylated in most early-stage liver cancer specimens. For comparison, two non-polycomb tumor suppressor genes, HIC1 and RassF1A, were also examined. Whereas the methylation level of HIC1 did not differ significantly between normal and tumor samples, RassF1A was significantly hypermethylated in liver tumor samples. Unsupervised clustering analysis classified the methylation changes within polycomb and non-polycomb targets to be independent, indicating independent epigenetic evolution. Thus, pre-deposited polycomb marks within somatic stem cells may contribute to the determination of methylation changes during hepatic tumorigenesis.     

Cytosine methylation can induce local distortions in the structure of duplex DNA.

Methyl groups at the C5 position of pyrimidines located within oligopurine-oligopyrimidine tracts in DNA have been shown previously to modulate curvature generated by those tracts. However, it was not known whether the influence of such methyl groups is consequent to the altered helical structure within the tracts themselves. In the current study, it is demonstrated that methylation of cytosines up to three base pairs away from a (dA)5.(dT)5 tract (A-tract) can still result in alterations of the net curvature of the A-tract-containing DNA, as measured by alterations in electrophoretic mobility. This latter effect depends strongly on both the sequence of the non-A-tract DNA and the positions of the methylated C residues. The current results lend further support to the notion that the biological consequences of cytosine methylation may be effected through local alterations in DNA structure as well as through direct protein-DNA interactions.     

Three enhancer regions regulate gbx2 gene expression in the isthmic region during zebrafish development

In vertebrate embryos, positioning of the boundary between the midbrain and hindbrain (MHB) and subsequent isthmus formation are dependent upon the interaction between the Otx2 and Gbx genes. In zebrafish, sequential expression of gbx1 and gbx2 in the anterior hindbrain contributes to this process, whereas in mouse embryos, a single Gbx gene (Gbx2) is responsible for MHB development. In the present study, to investigate the regulatory mechanism of gbx2 in the MHB/isthmic region of zebrafish embryos, we cloned the gene and showed that its organization is conserved among different vertebrates. Promoter analyses revealed three enhancers that direct reporter gene expression after the end of epiboly in the anterior-most hindbrain, which is a feature of the zebrafish gbx2 gene. One of the enhancers is located upstream of gbx2 (AMH1), while the other two enhancers are located downstream of gbx2 (AMH2 and AMH3). Detailed analysis of the AMH1 enhancer showed that it directs expression in the rhombomere 1 (r1) region and the dorsal thalamus, as has been shown for gbx2, whereas no expression was induced by the AMH1 enhancer in other embryonic regions in which gbx2 is expressed. The AMH1 enhancer is composed of multiple regulatory subregions that share the same spatial specificity. The most active of the regulatory subregions is a 291-bp region that contains at least two Pax2-binding sites, both of which are necessary for the function of the main component (PB1-A region) of the AMH1 enhancer. In accordance with these results, enhancer activity in the PB1-A region, as well as gbx2 expression in r1, was missing in no isthmus mutant embryos that lacked functional pax2a. In addition, we identified an upstream conserved sequence of 227bp that suppresses the enhancer activity of AMH1. Taken together, these findings suggest that gbx2 expression during the somitogenesis stage in zebrafish is regulated by a complex mechanism involving Pax2 as well as activators and suppressors in the regions flanking the gene.     

DNA methylation in the promoter regions of the laminin family genes in normal and breast carcinoma tissues

Molecular Biology - Extracellular glycoproteins of the laminin family are essential components of basement membranes involved in a number of biological processes, including tissue differentiation,...     

Cytosine methylation in the EcoRI site of active and inactive herpesvirus thymidine kinase promoters

The herpesvirus thymidine kinase (tk) gene integrated in the human cell line, 2.1-a, can be inactivated by limited de novo methylation. All these TK- clones show partial EcoRI digestion of the recognition site (cGAATTCg) in the tk promoter in contrast to complete digestion of this site in the original cell line. Studies on well-defined substrates prepared in vitro showed that methylation of one cytosine in the EcoRI recognition sequence resulted in partial and methylation of both cytosines in severe inhibition of digestion by EcoRI. This characteristic was used to determine whether no, one or both cytosines in the EcoRI site of the tk promoter were methylated in various TK- clones derived from 2.1-a and in TK+ clones re-expressing the gene after 5-azacytidine treatment. A high correlation was found between inactivity of the tk gene and methylation of only one of the two cytosines in the EcoRI recognition site. The results also show that the tk promoter can be active despite the presence of a methylated cytosine.     

Tissue specific differentially methylated regions (TDMR): Changes in DNA methylation during development

Tissue specific differentially methylated regions (TDMRs) were identified and localized in the mouse genome using second generation virtual RLGS (vRLGS). Sequenom MassARRAY quantitative methylation analysis was used to confirm and determine the fine structure of tissue specific differences in DNA methylation. TDMRs have a broad distribution of locations to intragenic and intergenic regions including both CpG islands, and non-CpG islands regions. Somewhat surprising, there is a strong bias for TDMR location in non-promoter intragenic regions. Although some TDMRs are within or close to repeat sequences, overall they are less frequently associated with repetitive elements than expected from a random distribution. Many TDMRs are methylated at early developmental stages, but unmethylated later, suggesting active or passive demethylation, or expansions of populations of cells with unmethylated TDMRs. This is notable during postnatal testis differentiation where many testis specific TDMRs become progressively "demethylated". These results suggest that methylation changes during development are dynamic, involve demethylation and methylation, and may occur at late stages of embryonic development or even postnatally.