The multiple promoter methylation profile of PR gene and ERalpha gene in tumor cell lines

The changes of methylation status of various gene promoters are a common feature of malignant cells and these changes can occur early in the progression process. Therefore, abnormal methylation can be used as cancer marker. Such studies will first require the development of a panel of methylated markers that are methylated in cancer tissues but unmethylated in normal tissues or methylated status is different between cancer tissues and normal tissues. By using methylation-specific PCR (MSP) assay method, we observed alterations in DNA methylation at the double promoter regions of the progesterone receptor (PR) gene and estrogen receptor (ERalpha) gene in various tumor cell lines. Compared with normal white blood cell, the methylation status of PRA promoter in various cancer cell lines changed from unmethylation pattern to methylation pattern. That of PRB promoter changed from both unmethylated and methylated alleles to only methylated allele. The methylation status of ERalpha-A and ERalpha-B promoter in various cancer cell lines are cell -specific. This study indicates that PR promoter methylation may be a molecular marker in various cancer detections. And the methylation status of ERalpha-A and ERalpha-B is cell-specific.     

Bisulfite Genomic Sequencing-Derived Methylation Profile of theXistGene throughout Early Mouse Development

Differential epigenetic modification by methylation of CpG dinucleotides is a candidate mechanism that may identify the alleles of imprinted genes and result in monoallelic expression of either the maternal or the paternal allele. Determination of the allelic methylation status of imprinted genes in the gametes and during early development is constrained by the limiting quantities of genomic DNA available from these early developmental stages. To circumvent this problem we have used bisulfite genomic sequencing to determine the allelic methylation status of the minimal promoter and a 1-kb region within theXistgene during preimplantation development. We find that the parentalXistalleles are not differentially methylated in these regions. Our findings are discussed in the context of previous conflicting data obtained using methylation-sensitive restriction enzyme digestion followed by PCR amplification to assay for methylation.     

Linkage disequilibrium analysis of allelic heterogeneity in DNA methylation

Heterogeneity of DNA methylation status among alleles is observed in various cell types and is involved in epigenetic gene regulation and cancer biology. However, the individual methylation profile within each allele has not yet been examined at the whole-genome level. In the present study, we applied linkage disequilibrium analysis to the DNA methylation data obtained from whole-genome bisulfite sequencing studies in mouse germline and other types of cells. We found that the methylation status of 2 consecutive CpG sites showed deviation from equilibrium frequency toward concordant linkage (both methylated or both unmethylated) in germline cells. In the imprinting loci where methylation of constituent alleles is known, our analysis detected the deviation toward the concordant linkage as expected. In addition, we applied this analysis to the transitional zone between methylated and unmethylated regions and to the cells undergoing epigenetic reprogramming. In both cases, deviation to the concordant-linked alleles was conspicuous, indicating that the methylation pattern is not random but rather concordant within each allele. These results will provide the key to understanding the mechanism underlying allelic heterogeneity.     

Genome-wide profiling of DNA methylation reveals a class of normally methylated CpG island promoters

The role of CpG island methylation in normal development and cell differentiation is of keen interest, but remains poorly understood. We performed comprehensive DNA methylation profiling of promoter regions in normal peripheral blood by methylated CpG island amplification in combination with microarrays. This technique allowed us to simultaneously determine the methylation status of 6,177 genes, 92% of which include dense CpG islands. Among these 5,549 autosomal genes with dense CpG island promoters, we have identified 4.0% genes that are nearly completely methylated in normal blood, providing another exception to the general rule that CpG island methylation in normal tissue is limited to X inactivation and imprinted genes. We examined seven genes in detail, including ANKRD30A, FLJ40201, INSL6, SOHLH2, FTMT, C12orf12, and DPPA5. Dense promoter CpG island methylation and gene silencing were found in normal tissues studied except testis and sperm. In both tissues, bisulfite cloning and sequencing identified cells carrying unmethylated alleles. Interestingly, hypomethylation of several genes was associated with gene activation in cancer. Furthermore, reactivation of silenced genes could be induced after treatment with a DNA demethylating agent or in a cell line lacking DNMT1 and/or DNMT3b. Sequence analysis identified five motifs significantly enriched in this class of genes, suggesting that cis-regulatory elements may facilitate preferential methylation at these promoter CpG islands. We have identified a group of non-X-linked bona fide promoter CpG islands that are densely methylated in normal somatic tissues, escape methylation in germline cells, and for which DNA methylation is a primary mechanism of tissue-specific gene silencing.     

DNA Methylation Analysis of Chromosome 21 Gene Promoters at Single Base Pair and Single Allele Resolution

Differential DNA methylation is an essential epigenetic signal for gene regulation, development, and disease processes. We mapped DNA methylation patterns of 190 gene promoter regions on chromosome 21 using bisulfite conversion and subclone sequencing in five human cell types. A total of 28,626 subclones were sequenced at high accuracy using (long-read) Sanger sequencing resulting in the measurement of the DNA methylation state of 580427 CpG sites. Our results show that average DNA methylation levels are distributed bimodally with enrichment of highly methylated and unmethylated sequences, both for amplicons and individual subclones, which represent single alleles from individual cells. Within CpG-rich sequences, DNA methylation was found to be anti-correlated with CpG dinucleotide density and GC content, and methylated CpGs are more likely to be flanked by AT-rich sequences. We observed over-representation of CpG sites in distances of 9, 18, and 27 bps in highly methylated amplicons. However, DNA sequence alone is not sufficient to predict an amplicon's DNA methylation status, since 43% of all amplicons are differentially methylated between the cell types studied here. DNA methylation in promoter regions is strongly correlated with the absence of gene expression and low levels of activating epigenetic marks like H3K4 methylation and H3K9 and K14 acetylation. Utilizing the single base pair and single allele resolution of our data, we found that i) amplicons from different parts of a CpG island frequently differ in their DNA methylation level, ii) methylation levels of individual cells in one tissue are very similar, and iii) methylation patterns follow a relaxed site-specific distribution. Furthermore, iv) we identified three cases of allele-specific DNA methylation on chromosome 21. Our data shed new light on the nature of methylation patterns in human cells, the sequence dependence of DNA methylation, and its function as epigenetic signal in gene regulation. Further, we illustrate genotype–epigenotype interactions by showing novel examples of allele-specific methylation.     

Relationship between DNA methylation, histone H4 acetylation and gene expression in the mouse imprinted Igf2-H19 domain

DNA methylation and histone H4 acetylation play a role in gene regulation by modulating the structure of the chromatin. Recently, these two epigenetic modifications have dynamically and physically been linked. Evidence suggests that both modifications are involved in regulating imprinted genes - a subset of genes whose expression depends on their parental origin. Using immunoprecipitation assays, we investigate the relationship between DNA methylation, histone H4 acetylation and gene expression in the well-characterised imprinted Igf2-H19 domain on mouse chromosome 7. A systematic regional analysis of the acetylation status of the domain shows that parental-specific differences in acetylation of the core histone H4 are present in the promoter regions of both Igf2 and H19 genes, with the expressed alleles being more acetylated than the silent alleles. A correlation between DNA methylation, histone hypoacetylation and gene repression is evident only at the promoter region of the H19 gene. Treatment with trichostatin A, a specific inhibitor of histone deacetylase, reduces the expression of the active maternal H19 allele and this can be correlated with regional changes in acetylation within the upstream regulatory domain. The data suggest that histone H4 acetylation and DNA methylation have distinct functions on the maternal and paternal Igf2-H19 domains.     

Intronic Parent-of-Origin Dependent Differential Methylation at the Actn1 Gene Is Conserved in Rodents but Is Not Associated with Imprinted Expression

Parent-of-origin differential DNA methylation has been associated with regulation of the preferential expression of paternal or maternal alleles of imprinted genes. Based on this association, recent studies have searched for parent-of-origin dependent differentially methylated regions in order to identify new imprinted genes in their vicinity. In a previous genome-wide analysis of mouse brain DNA methylation, we found a novel differentially methylated region in a CpG island located in the last intron of the alpha 1 Actinin (Actn1) gene. In this region, preferential methylation of the maternal allele was observed; however, there were no reports of imprinted expression of Actn1. Therefore, we have tested if differential methylation of this region is common to other tissues and species and affects the expression of Actn1. We have found that Actn1 differential methylation occurs in diverse mouse tissues. Moreover, it is also present in other murine rodents (rat), but not in the orthologous human region. In contrast, we have found no indication of an imprinted effect on gene expression of Actn1 in mice: expression is always biallelic regardless of sex, tissue type, developmental stage or isoform. Therefore, we have identified a novel parent-of-origin dependent differentially methylated region that has no apparent association with imprinted expression of the closest genes. Our findings sound a cautionary note to genome-wide searches on the use of differentially methylated regions for the identification of imprinted genes and suggest that parent-of-origin dependent differential methylation might be conserved for functions other that the control of imprinted expression.     

Analysis of tissue-specific differentially methylated regions (TDMs) in humans

Alterations in DNA methylation have been implicated in mammalian development. Hence, the identification of tissue-specific differentially methylated regions (TDMs) is indispensable for understanding its role. Using restriction landmark genomic scanning of six mouse tissues, 150 putative TDMs were identified and 14 were further analyzed. The DNA sequences of the 14 mouse TDMs are analyzed in this study. Six of the human homologous regions show TDMs to both mouse and human and genes in five of these regions have conserved tissue-specific expression: preferential expression in testis. A TDM, DDX4, is further analyzed in nine testis tissues. An increase in methylation of the promoter region is significantly associated with a marked reduction of the gene expression and defects in spermatogenesis, suggesting that hypomethylation of the DDX4 promoter region regulates DDX4 gene expression in spermatogenic cells. Our results indicate that some genomic regions with tissue-specific methylation and expression are conserved between mouse and human and suggest that DNA methylation may have an important role in regulating differentiation and tissue-/cell-specific gene expression of some genes.     

Methylation status and chromatin structure of the myostatin gene promoter region in the sea perch Lateolabrax japonicus (Perciformes)

Myostatin is a negative regulator of the growth and development of skeletal muscle mass. In fish, myostatin is expressed in several organs in addition to skeletal muscle. To understand the mechanisms regulating myostatin gene expression in the sea perch, Lateolabrax japonicus, we examined the methylation status of the myostatin gene promoter region in several tissues (liver, eye, kidney, brain, and heart) isolated from adult specimens. The frequency of methylated cytosines was very low in all tissues, regardless of the level of myostatin expression, suggesting that DNA methylation is not involved in the tissue-specific regulation of myostatin expression. Southern blot analysis of genomic DNA obtained from micrococcal nuclease-treated nuclei showed that chromatin digestion occurs in tissues where the myostatin gene is actively transcribed and that the myostatin gene is protected from micrococcal nuclease in tissues where myostatin is not expressed. The chromatin structure in the myostatin gene region appears to regulate its expression without DNA methylation.     

DNA methylation-dependent epigenetic regulation of dimethylarginine dimethylaminohydrolase 2 gene in trophoblast cell lineage

Trophoblast cell lineage is established through the first cellular differentiation in mammalian embryogenesis, and its developmental potential is restricted to the extraembryonic tissues contributing solely to the placenta. Several lines of evidence suggest a relative lack of importance of DNA methylation in gene regulation in the extraembryonic tissues when compared with embryonic ones. Here we analyzed the dynamics of epigenetic status in the upstream region of mouse Ddah2 gene, which was found to be specifically repressed in a stem cell population of trophoblast cell lineage. We found a tissue-dependent differentially methylated region in the regulatory region of the Ddah2 gene. This region was hypermethylated in trophoblast stem cells and was hypomethylated in differentiated cells both in vivo and in vitro. This change was well correlated with Ddah2 expression. In addition, in vitro methylation confined to the differentially methylated region was sufficient to repress promoter activity in the reporter assay. Furthermore, a repressive pattern of histone modifications was formed around the differentially methylated region in undifferentiated trophoblast stem cells with repressed Ddah2. Our data suggest that DNA methylation-mediated chromatin remodeling is involved in the regulation of the Ddah2 gene expression and thus is important even in trophoblast cell lineage.     

Differential methylation persists at the mouse Rasgrf1 DMR in tissues displaying monoallelic and biallelic expression

A subset of mammalian genes exhibits genomic imprinting, whereby one parental allele is preferentially expressed. Differential DNA methylation at imprinted loci serves both to mark the parental origin of the alleles and to regulate their expression. In mouse, the imprinted gene Rasgrf1 is associated with a paternally methylated imprinting control region which functions as an enhancer blocker in its unmethylated state. Because Rasgrf1 is imprinted in a tissue-specific manner, we investigated the methylation pattern in monoallelic and biallelic tissues to determine if methylation of this region is required for both imprinted and non-imprinted expression. Our analysis indicates that DNA methylation is restricted to the paternal allele in both monoallelic and biallelic tissues of somatic and extraembryonic lineages. Therefore, methylation serves to mark the paternal Rasgrf1 allele throughout development, but additional factors are required for appropriate tissue-specific regulation of expression at this locus.