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Results for “differential methylation”

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PubMed1983

Differential methylation of chloroplast DNA regulates maternal inheritance in a methylated mutant of Chlamydomonas.

In Chlamydomonas, the maternal inheritance of chloroplast genes correlates with the differential methylation of chloroplast DNA (chlDNA) in females (mt(+)) but not in males (mt(-)). Our previous studies have supported our methylation-restriction model in which the maternal transmission is accounted for by the differential methylation in gametes which protects female but not male chlDNA from degradation during zygote formation. In the mutant me-1 [Bolen, P. L., Grant, D. M., Swinton, D., Boynton, J. E. & Gillham, N. W. (1982) Cell 28, 335-343], chlDNA of vegetative cells of both mating types is heavily methylated even before gametogenesis; nonetheless, maternal inheritance occurs in mutants as in wild type. To investigate the mechanism of maternal inheritance in the me-1 mutant, we have compared restriction fragment patterns after agarose gel electrophoresis of chlDNAs from mutant vegetative cells and gametes with those from wild type, by using a set of 32 restriction enzymes of which 17 were methylation-sensitive in this system. We find that additional methylation occurs during gametogenesis in the mutant female (mt(+)) but not in the corresponding male (mt(-)). Thus, gamete-specific, mating-type-specific methylation occurs in the me-1 mutant as in the wild type, consistent with our methylation-restriction model. In the me-1 mutant, gametic methylation occurs on a background of vegetative cell methylation not present in wild-type cells and irrelevant to the regulation of chloroplast inheritance. Comparison of the me-1 mutation with the mat-1 mutation [Sager, R., Grabowy, C. & Sano, H. (1981) Cell 24, 41-47] provides evidence for the existence of two different chlDNA methylation control systems: mat-1, linked to the mating type locus and regulating the mating-type-specific methylation that correlates with maternal inheritance, and me-1, unlinked to the mating type locus and unrelated to the regulation of maternal inheritance.

Journal Article↗

PubMed2001

DNA methylation and ovarian cancer. I. Analysis of CpG island hypermethylation in human ovarian cancer using differential methylation hybridization.

OBJECTIVE: The aim of this study was to examine CpG island methylation patterns in ovarian cancer and determine whether epigenetic information can be related to clinical data of patients. CpG island (CpGI) hypermethylation is commonly associated with cancer progression, but little is currently known about the role of methylation in ovarian cancer. METHODS: Differential methylation hybridization (DMH) analysis at 742 loci was performed to determine methylation signatures for 20 primary epithelial ovarian carcinomas (Stages II, III, and IV adenocarcinomas, serous papillary), 6 ovarian cancer cell lines, and normal ovarian surface epithelial cells. RESULTS: Between 23 and 108 methylated CpGIs were seen in the ovarian carcinomas. Fewer (P < 0.05) methylated CpGIs were observed in the ovarian cancer cell lines; however, a number of CpGIs were commonly hypermethylated in both the cell lines and the tumor samples. A methylation signature, consisting of frequently (P < 0.05) methylated CpGIs, was determined for the samples. The observed pattern of methylation in ovarian cancers included several (11) CpGI tags that were previously reported to be hypermethylated in human breast cancer. CONCLUSIONS: Epigenetic signatures in ovarian cancer were determined using DMH. This proof-of-concept study lays the foundation for genome-wide screening of methylation to examine epigenotype-phenotype relationships in ovarian cancer.

Adult↗

PubMed2005

[Isolation and analysis of isolation of differentially-methylated fragment CIDM7 in rice induced by cold stress].

By using restriction enzyme isoschizomers that differ in their sensitivity to methylation of their recognition sites, AFLP analysis is carried out to analyse differences of the methylation state of anonymous CCGG sequences between cold-treated(5 degrees for 2 d) and control 9311(Oryza sativa L.) leaves DNA. Demethylation or De novo methylation induced by cold stress are found in some CCGG sites. Some differentially-methylated encoding sequences are isolated, of which a fragment named CIDM7 (cold-inducing differential methylation) is sequenced and executed BLAST against Nipponbare cDNA data, thus we obtain its full length cDNA of 1422 bp encoding a hypothetical F-box protein(425 aa).CIDM7 is demethylated induced by cold stress. Northern blot analysis confirms that CIDM7 gene expression is up-regulated by cold stress. CIDM7 is single copy and lacated on the Nipponbare chromosome 10 (12.56 Mb-12.57 Mb).

Amino Acid Sequence↗

PubMed2006

Developmental profile of H19 differentially methylated domain (DMD) deletion alleles reveals multiple roles of the DMD in regulating allelic expression and DNA methylation at the imprinted H19/Igf2 locus.

The differentially methylated domain (DMD) of the mouse H19 gene is a methylation-sensitive insulator that blocks access of the Igf2 gene to shared enhancers on the maternal allele and inactivates H19 expression on the methylated paternal allele. By analyzing H19 DMD deletion alleles H19DeltaDMD and H19Delta3.8kb-5'H19 in pre- and postimplantation embryos, we show that the DMD exhibits positive transcriptional activity and is required for H19 expression in blastocysts and full activation of H19 during subsequent development. We also show that the DMD is required to establish Igf2 imprinting by blocking access to shared enhancers when Igf2 monoallelic expression is initiated in postimplantation embryos and that the single remaining CTCF site of the H19DeltaDMD allele is unable to provide this function. Furthermore, our data demonstrate that sequence outside of the DMD can attract some paternal-allele-specific CpG methylation 5' of H19 in preimplantation embryos, although this methylation is not maintained during postimplantation in the absence of the DMD. Finally, we report a conditional allele floxing the 1.6-kb sequence deleted from the H19DeltaDMD allele and demonstrate that the DMD is required to maintain repression of the maternal Igf2 allele and the full activity of the paternal Igf2 allele in neonatal liver.

Alleles↗

PubMed2026

MultiDMPcaller: a one-stop software for detection and visualization of differentially methylated positions and regions.

MOTIVATION: Whole-genome bisulfite sequencing (WGBS/BS-Seq) is the gold standard for single-base resolution DNA methylome profiling. However, the diverse statistical models of existing computational methods lead to limited overlap between their results, highlighting the need for novel methods to detect differentially methylated positions (DMPs) and differentially methylated regions (DMRs). RESULTS: We developed MultiDMPcaller, an automated downstream methylome analysis software. It processes upstream outputs to profile DMPs, non-DMPs, DMRs, and context-specific (CpG/CHG/CHH) methylation status, alongside visualizing their chromosomal distribution and enrichment. The software features two key innovations: (i) an adaptive two-step P-value adjustment strategy based on organism-specific methylation patterns, with raw P-value ≤0.05 pre-filtering followed by false discovery rate (FDR) correction, to recover potential DMPs usually missed by standard FDR correction in plant CHG/CHH and animal CpG contexts; and (ii) a multiple pairwise comparison approach, which performs m × n pairwise comparisons for m control and n experimental replicates, followed by a voting system supporting both user-defined majority thresholds and model-based adaptive thresholds, to identify robust and reliable DMPs (with a stricter voting threshold exclusively for loci with low methylation differences) and DMRs. On real datasets from Arabidopsis, apple, and mouse, as well as simulated human datasets, MultiDMPcaller's results showed good agreement with those of other software, exhibiting high conservativeness and superior precision, which suggested a low false discovery proportion. AVAILABILITY AND IMPLEMENTATION: MultiDMPcaller is available at GitHub (https://github.com/jiantaoyuNWAFU/MultiDMPcaller) and via a web server (https://ciebioinfo.nwafu.edu.cn).

Software↗

PubMed2002

Shared role for differentially methylated domains of imprinted genes.

For most imprinted genes, a difference in expression between the maternal and paternal alleles is associated with a corresponding difference in DNA methylation that is localized to a differentially methylated domain (DMD). Removal of a gene's DMD leads to a loss of imprinting. These observations suggest that DMDs have a determinative role in genomic imprinting. To examine this possibility, we introduced sequences from the DMDs of the imprinted Igf2r, H19, and Snrpn genes into a nonimprinted derivative of the normally imprinted RSVIgmyc transgene, created by excising its own DMD. Hybrid transgenes with sequences from the Igf2r DMD2 were consistently imprinted, with the maternal allele being more methylated than the paternal allele. Only the repeated sequences within DMD2 were required for imprinting these transgenes. Hybrid transgenes containing H19 and Snrpn DMD sequences and ones containing sequences from the long terminal repeat of a murine intracisternal A particle retrotransposon were not imprinted. The Igf2r hybrid transgenes are comprised entirely of mouse genomic DNA and behave as endogenous imprinted genes in inbred wild-type and mutant mouse strains. These types of hybrid transgenes can be used to elucidate the functions of DMD sequences in genomic imprinting.

Alleles↗

PubMed1997

Identification and characterization of differentially methylated regions of genomic DNA by methylation-sensitive arbitrarily primed PCR.

We have developed a simple and reproducible fingerprinting method for screening the genome for regions of DNA that have altered patterns of DNA methylation associated with oncogenic transformation. Restriction enzymes with different sensitivities to cytosine methylation in their recognition sites were used to digest genomic DNAs from primary tumors, cell lines, and normal tissues prior to arbitrarily primed PCR amplification. Fragments that showed differential methylation were cloned and sequenced after resolving the PCR products on high-resolution polyacrylamide gels. The cloned fragments were then used as probes for Southern analysis to confirm differential methylation of these regions in colon tissues and cell lines. Forty-four DNA fragments associated with a total of five different regions of genomic DNA containing methylation sites were detected in 10 matched sets of normal and tumor colon DNAs and 7 colon cancer cell lines. A novel CpG island was also isolated that was found to be frequently hypermethylated in bladder and colon tumors. We have demonstrated that this technique is a rapid and efficient method that can be used to screen for altered methylation patterns in genomic DNA and to isolate specific sequences associated with these changes.

Blotting, Southern↗

PubMed2004

Interaction between differentially methylated regions partitions the imprinted genes Igf2 and H19 into parent-specific chromatin loops.

Imprinted genes are expressed from only one of the parental alleles and are marked epigenetically by DNA methylation and histone modifications. The paternally expressed gene insulin-like growth-factor 2 (Igf2) is separated by approximately 100 kb from the maternally expressed noncoding gene H19 on mouse distal chromosome 7. Differentially methylated regions in Igf2 and H19 contain chromatin boundaries, silencers and activators and regulate the reciprocal expression of the two genes in a methylation-sensitive manner by allowing them exclusive access to a shared set of enhancers. Various chromatin models have been proposed that separate Igf2 and H19 into active and silent domains. Here we used a GAL4 knock-in approach as well as the chromosome conformation capture technique to show that the differentially methylated regions in the imprinted genes Igf2 and H19 interact in mice. These interactions are epigenetically regulated and partition maternal and paternal chromatin into distinct loops. This generates a simple epigenetic switch for Igf2 through which it moves between an active and a silent chromatin domain.

Animals↗

PubMed2001

Maternal and paternal chromosomes 7 show differential methylation of many genes in lymphoblast DNA.

Genomic imprinting, the differential expression of paternal and maternal alleles, involves many chromosomal regions and plays a role in development and growth. Differential methylation of maternal and paternal alleles is a hallmark of imprinted genes, and thus methylation assays are widely used to support the identification of novel imprinted genes. Either blood or lymphoblast DNAs are most often used in these assays, even though methylation levels may change in cell culture. We undertook a systematic survey of parent-of-origin-specific methylation of chromosome 7 genes and ESTs by comparing DNA samples from cases of maternal and paternal uniparental disomy for chromosome 7 using DNA from fresh blood and lymphoblast cell lines. Our results revealed that up to 41% of genes and ESTs show parent-of-origin-specific methylation differences in lymphoblast DNA after only a short time in culture, whereas methylation differences were not seen in blood DNA. The methylation changes occurred most commonly on paternal chromosome 7, whereas alterations on maternal chromosome 7 were more infrequent and weaker. These findings indicate that methylation patterns may change significantly during cell culture in a parent-of-origin-dependent manner and suggest that methylation is maintained differently on maternal and paternal chromosomes 7.

Adolescent↗

PubMed1993

DNA regions flanking the mouse Ig 3' alpha enhancer are differentially methylated and DNAase I hypersensitive during B cell differentiation.

Two B cell-specific enhancer elements are associated with the IgH gene cluster. One enhancer is located within the J-C mu intron (E mu), whereas a second enhancer (3' alpha E) is approximately 12.5 kb 3' of the C alpha membrane exon. In an attempt to understand the function of 3' alpha E, we have characterized its surrounding structural milieu during various stages of B cell differentiation through analysis of methylation patterns and the identification of DNAse I-hypersensitive sites. We observed a correlation between the chromatin structure of this region and the differentiation state of the cell. Compared to liver and brain, the region 3' of alpha was hypermethylated in pre-B and T cell lines and became progressively demethylated as B cell differentiation continued. A DNAse I-hypersensitive site was present in pre-B cell lines about 17 kb 3' of 3' alpha E. In fully differentiated myeloma cell lines, a second cluster of DNAse I-hypersensitive sites was present immediately 5' of 3' alpha E. Our data indicate that the 3' alpha enhancer is relatively sequestered during early stages of B cell differentiation and becomes increasingly accessible at later stages.

Animals↗

PubMed1993

Carrier detection in X-linked immunodeficiencies. II: An X inactivation assay based on differential methylation of a line-1 repeat at the DXS255 locus.

The differential methylation of a CpG island 2.5 kb distant from a hypervariable region at the DXS255 locus provides the basis for a Southern blotting X chromosome inactivation analysis system. The technique enables carrier detection in about 90% of females at risk from pedigrees with Wiskott-Aldrich syndrome, X-linked severe combined immunodeficiency or X-linked agammaglobulinemia.

Agammaglobulinemia↗

PubMed2003

Differential methylation pattern of the X-linked lymphoproliferative (XLP) disease gene SH2D1A correlates with the cell lineage-specific transcription.

SH2D1A, the X-linked lymphoproliferative disease (XLP) gene, encodes a cytoplasmic protein that plays an essential role in controlling Epstein-Barr virus infection. It is expressed in T and NK cells, but not in B cells or in granulocytes. The promoter, the regulatory regions, as well as the mechanisms controlling its tissue-specific expression, are still unknown. We tested the hypothesis that DNA methylation might contribute to tissue-specific SH2D1A gene expression and analyzed the methylation status of 2,300 bp upstream of the ATG starting codon, the coding region and part of intron 1. By bisulfite sequencing and methylation-sensitive restriction enzyme digestion, we show that a differential methylation pattern of CpG-rich regions in the 5' region and the adjacent exon 1 of the SH2D1A gene indeed correlates with the tissue-specific gene transcription.

Animals↗

PubMed1992

An X chromosome inactivation assay based on differential methylation of a CpG island coupled to a VNTR polymorphism at the 5' end of the monoamine oxidase A gene.

A CpG island has been identified just upstream of the first exon of the human monoamine oxidase A (MAOA) gene, localized to Xp11.4-Xp11.23. Southern blotting following digestion with the methylation sensitive restriction endonucleases SmaI, HpaII and HhaI, indicated that CpG dinucleotides within the CpG island were unmethylated on the active X chromosome and extensively methylated on the inactive X chromosome. These sites of differential methylation were close to a polymorphic GT-dinucleotide/VNTR region, which is located 1 kb 3' of the first exon and has a heterozygosity value of 75%. PCR primers were designed for amplification of 1.2-1.3 kb DNA fragments, encompassing both the hypervariable region and a cluster of six HpaII sites within the CpG-rich region. Cleavage of HpaII sites was found to be restricted to active X chromosomes. Therefore, following HpaII digestion, DNA fragments were exclusively amplified from inactive X chromosomes. The resulting PCR products were digested with SacI, which reduced the size of the DNA fragments containing the hypervariable region to 230-330 bp, and were subsequently analyzed on denaturating polyacrylamide gels. Because amplified fragments were exclusively derived from the inactive X chromosome, the relative densities of the two allelic fragments should reflect the proportions of cells that have either of the two X chromosome inactivated. The results of this PCR-based X chromosome inactivation assay were fully concordant with Southern blotting methylation analyses at the PGK locus. It therefore provides a rapid and informative method in tumour clonality analysis and carrier detection in X-linked diseases.

Base Sequence↗

PubMed2006

A differentially methylated region of the DAZ1 gene in spermatic and somatic cells.

AIM: To investigate the methylation status of the deleted in azoospermia 1 (DAZ1) gene promoter region in different cell types. METHODS: Using CpG island Searcher software, a CpG island was found in the promoter region of the DAZ1 gene. The methylation status of this region was analyzed in sperm and leukocytes by bisulfited sequencing. RESULTS: The methylation status of the CpG island in the DAZ1 gene promoter region differed in leukocytes and sperm: it was methylated in leukocytes, but unmethylated in sperm. CONCLUSION: A differentially methylated region of the DAZ1 gene exists in spermatic and somatic cells, suggesting that methylation of this region may regulate DAZ1 gene expression in different tissues.

Base Sequence↗

PubMed2006

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.

Aged↗

PubMed2005

TWIST2 demonstrates differential methylation in immunoglobulin variable heavy chain mutated and unmutated chronic lymphocytic leukemia.

PURPOSE: Chronic lymphocytic leukemia (CLL) is a clinically heterogeneous disease for which natural history can be predicted based on the presence or absence of immunoglobulin (Ig) variable heavy chain (V(H)) gene mutations. Herein we report selective epigenetic silencing of the transcription factor TWIST2 (DERMO1) in Ig V(H) mutated CLL and describe a semiquantitative assay to study promoter methylation of this gene in primary tumor cells. MATERIALS AND METHODS: TWIST2 promoter methylation was identified by restriction landmark genome scanning. Southern blot (SB), bisulfite sequencing, and combined bisulfite restriction analysis (COBRA), and quantitative SB-COBRA was performed to study methylation of the TWIST2 promoter. Reverse transcription polymerase chain reaction assays were used to study TWIST2 expression in CLL cells. RESULTS: Following identification and confirmation of TWIST2 methylation in CLL patients, we demonstrated that expression of this transcription factor is related to the degree of promoter methylation. Expression of TWIST2 in a CLL cell line in which the promoter is methylated was increased following decitabine treatment. We next studied 53 patients by COBRA and demonstrated that 72% of patient samples with mutated Ig V(H) show TWIST2 methylation, while only 16% of patient samples with unmutated Ig V(H) were methylated (P < .001). In a subset of patients, methylation of TWIST2 correlated with mRNA expression. CONCLUSION: TWIST2 is differentially methylated in CLL cells relative to Ig V(H) mutational status and can be quantitatively monitored by SB-COBRA. Based on the known role of TWIST2 in silencing p53 function in other malignancies, future studies should focus on the role of TWIST2 in CLL and related lymphoproliferative diseases.

Antimetabolites, Antineoplastic↗

PubMedDate not supplied

Differential methylation in steroid 5 alpha-reductase isozyme genes in epididymis, testis, and liver of the adult rat.

DNA methylation has been largely involved in the regulation of tissue-specific gene expression. The aim of the study was to determine the methylation pattern of steroid 5 alpha-reductase genes 1 and 2 in two reproductive tissues (testis and epididymis) and a nonreproductive tissue (liver) that exhibit different contents of steroid 5 alpha-reductase isozymes. These isozymes induce the bioconversion of testosterone to dihydrotestosterone that in mammals is a key molecule for external genitalia development. Genomic DNA from the testis, the epididymis, and the liver from normal adult rats was used to determine cytosine and adenine methylation pattern of steroid 5 alpha-reductase genes 1 and 2 by restriction fragment length polymorphism (RFLP) analysis using restriction enzymes sensitive to adenine (Mbo I and Sau3A I) and cytosine (Hpa II and MSP I) methylation. We also evaluated the expression of both steroid 5 alpha-reductase genes by northern blot. When genomic DNA was digested with Hpa II or Msp I, we found that steroid 5 alpha-reductase gene 2 was less cytosine methylated in the epididymis and in the testis than in the liver. In contrast, when genomic DNA was digested with Mbo I or Sau3A I, we observed that gene 2 was more adenine methylated in the epididymis and in the testis than in the liver. 5 alpha-Reductase gene 1 presented the same adenine- and cytosine-methylation pattern in the studied tissues. We also found a differential expression of steroid 5 alpha-reductase genes. Gene 2 was expressed both in the testis and the epididymis but not in the liver; whereas gene 1 was only expressed in the latter. Our results suggest that the differential methylation pattern in 5 alpha-reductase gene 2 in reproductive and nonreproductive tissues should be involved in the regulation of its expression.

3-Oxo-5-alpha-Steroid 4-Dehydrogenase↗

PubMed2002

A differentially methylated imprinting control region within the Kcnq1 locus harbors a methylation-sensitive chromatin insulator.

The mechanisms underlying the phenomenon of genomic imprinting remain poorly understood. In one instance, a differentially methylated imprinting control region (ICR) at the H19 locus has been shown to involve a methylation-sensitive chromatin insulator function that apparently partitions the neighboring Igf2 and H19 genes in different expression domains in a parent of origin-dependent manner. It is not known, however, if this mechanism is unique to the Igf2/H19 locus or if insulator function is a common feature in the regulation of imprinted genes. To address this question, we have studied an ICR in the Kcnq1 locus that regulates long range repression on the paternally derived p57Kip2 and Kcnq1 alleles in an imprinting domain that includes Igf2 and H19. We show that this ICR appears to possess a unidirectional chromatin insulator function in somatic cells of both mesodermal and endodermal origins. Moreover, we document that CpG methylation regulates this insulator function suggesting that a methylation-sensitive chromatin insulator is a common theme in the phenomenon of genomic imprinting.

Alleles↗