Analysis Of DNA Methylation Patterns Research Articles

Long Distance Trans DNA Hypomethylation in Cyclin D1 Deregulated B Cell Malignancies.

Cyclin D1 is a common target gene in B cell malignancies. Cyclin D1 is deregulated by translocation in most patients with mantle cell lymphoma (MCL) and 15–20% of patients with multiple myeloma (MM). Cyclin D1 is not expressed in normal lymphocytes. Gene targeting experiments in cyclin D1 overexpressing MCL and MM were carried out and genetic variants isolated which had lost the translocated or inserted 11:14 chromosomes. These clones no longer expressed cyclin D1 but expressed high levels of cyclin D3. Analysis of DNA methylation patterns in these clones demonstrated that the translocated chromosome exerts a long distance trans DNA hypomethylating effect on the normal chromosome (transvection) at the cyclin D1 locus and at least 100 kilobases upstream. Thus, in the absence of the translocated chromosome, the normal chromosome is densely DNA methylated. This long distance trans hypomethylating effect was also demonstrated in a MM cell line (U266) which contains an insertion rather than a translocation of IgH sequences. Combined FISH/ immunofluorescent antibody labelling studies have shown the presence of both the normal and translocated chromosome 11's at the outer nucleolar membrane, where they are tethered by the proteins CTCF and nucleophosmin, as demonstrated using chromatin immunoprecipitation assays. Tethering of the translocated and nontranslocated chromosomes by CTCF/nucleophosmin provides a mechanism for pairing and long distance DNA transhypomethylation.

In Vivo Analysis of DNA Methylation Patterns Recognized by Specific Proteins: Coupling ChIP and Bisulfite Analysis

The three-way connection between DNA methylation, chromatin configuration, and transcriptional regulation is under increasing attention, but the fine rules governing the epigenetic control are still poorly understood. In several studies, the authors have concluded that the methylation status of CpG sites could be critical for the binding of factors to DNA and, consequently, for chromatin conformation. We tested the possibility that a novel technical approach combining chromatin immunoprecipitation and bisulfite genomic sequencing analysis (ChIP-BA) could provide useful information on the role of specific CpG methylation patterns in driving the association in vivo of proteins to given genomic regions. Our results show that ChIP-BA permits the establishment in vivo of the methylation patterns required for the binding of a methyl-CpG binding protein and, in addition, can potentially identify methylation patterns that do not allow a protein to bind specific genomic regions. Possible fields of application are discussed. We believe that wide use of ChIP-BA could make possible the exploration of a novel aspect of the intricate epigenetic web.

De novo quantitative bisulfite sequencing using the pyrosequencing technology

Current protocols for DNA methylation analysis are either labor intensive or limited to the measurement of only one or two CpG positions. Pyrosequencing is a real-time sequencing technology that can overcome these limitations and be used as an epigenotype-mapping tool. Initial experiments demonstrated reliable quantification of the degree of DNA methylation when 2–6 CpGs were analyzed. We sought to improve the sequencing protocol so as to analyze as many CpGs as possible in a single sequencing run. By using an improved enzyme mix and adding single-stranded DNA-binding protein to the reaction, we obtained reproducible results for as many as 10 successive CpGs in a single sequencing reaction spanning up to 75 nucleotides. A minimum amount of 10 ng of bisulfite-treated DNA is necessary to obtain good reproducibility and avoid preferential amplification. We applied the assay to the analysis of DNA methylation patterns in four CpG islands in the vicinity of IGF2 and H19 genes. This allowed accurate and quantitative de novo sequencing of the methylation state of each CpG, showing reproducible variations of methylation state in contiguous CpGs, and proved to be a useful adjunct to current technologies.

Crucial role of DNA methylation in determination of clonally distributed killer cell Ig-like receptor expression patterns in NK cells.

Human NK cells are characterized by the expression of surface receptors of the killer cell Ig-like receptor (KIR) family, which are involved in the specific recognition of pathogenic target cells. Each NK cell expresses and maintains an individual subset of inhibitory and stimulatory KIR and in this way contributes to a diversified NK cell repertoire. To date, the molecular basis for generation of clonally distributed KIR expression patterns has been elusive. Here, analyses of DNA methylation patterns of KIR genes in NK cell lines as well as in NK cells, freshly isolated from peripheral blood, demonstrated that a small CpG island surrounding the transcriptional start site of each KIR gene is consistently demethylated in expressed KIR and methylated in unexpressed KIR. DNA-demethylating treatment resulted in a rapid and stable induction of transcription and cell surface expression of all formerly unexpressed KIR in NK cell lines, NK cell clones, and freshly isolated NK cells, but not in other cell types. In vitro methylation of KIR CpG islands repressed reporter gene expression in NK cells. We conclude that clonal patterns of KIR expression are mainly epigenetically determined and maintained through DNA methylation.

Down-regulation of BRCA1 in human sporadic breast cancer; analysis of DNA methylation patterns of the putative promoter region.

Germ-line alterations of BRCA1 are responsible for about 50% of familial breast cancers. Although its biological function(s) has not yet been fully determined, it has been suggested that it may act as a tumor suppressor gene in breast and ovarian cancers. In sporadic breast cancers alterations of BRCA1 have not been detected and in vitro experiments have indicated that BRCA1 negatively regulates cellular proliferation. The present study was designed to identify and quantify, the BRCA1 mRNA levels, in normal and neoplasic human breast tissue. BRCA1 mRNA molecules were quantified using competitive reverse transcriptase PCR assays. DNA methylation patterns of this gene have been analysed by Southern blot experiments using methylation sensitive restriction enzymes. We found that BRCA1 mRNA levels were significantly lower in sporadic breast cancers (37 cases analysed, 24 cases of invasive ductal carcinomas not otherwise specified (NOS), two lobular carcinomas in situ two medullary carcinomas, four invasive lobular carcinomas, two invasive mucinous carcinomas and three invasive ductal carcinomas with predominantly in situ component) compared with normal breast tissues (P=0.0003). This down-regulation of BRCA1 is observed in all histologic types analysed. In invasive ductal carcinomas NOS, this down-regulation does not correlate with any of the prognostic factors studied (tumor size, node status, histologic grade, hormone receptor status). In the samples analysed, alterations of DNA methylation patterns were not dectected in the vicinity of the major transcription start site. These data suggest the involvement of BRCA1 in the carcinogenesis of these histologic types.

DNA Methylation Differences Associated with Tumor Tissues Identified by Genome Scanning Analysis

Most investigations on the role of DNA methylation in cancer have focused on epigenetic changes associated with known tumor suppressor genes. This may have led to an underestimation of the number of CpG islands altered by DNA methylation, since it is possible that a subset of unknown genes relevant to cancer development may preferentially be affected by epigenetic rather than genetic means and would not be identified as familial deletions, mutations, or loss of heterozygosity. We used a recently developed screening procedure (methylation-sensitive arbitrarily primed-polymerase chain reaction to scan genomic DNA for CpG islands methylated in white blood cells (WBCs) and in tumor tissues. DNA methylation pattern analysis showed little interindividual differences in the WBCs and normal epithelium (adjacent to colon, bladder, and prostate cancer cells), but with some tissue-specific differences. Cancer cells showed marked methylation changes that varied considerably between different tumors, suggesting variable penetrance of the methylation phenotype in patients. Direct sequencing of 8 of 45 bands altered in these cancers showed that several of them were CpG islands, and 2 of these sequences were identified in GenBank. Surprisingly, three of the bands studied corresponded to transcribed regions of genes. Thus, hypermethylation of CpG islands in cancer cells is not confined to the promoters of growth regulatory genes but is also found in actively transcribed regions.

Desmoid fibromatosis is a clonal process

Desmoid fibromatosis is a locally aggressive proliferative soft tissue lesion of controversial nature. The authors investigated the clonality of this process by molecular genetic analysis of DNA methylation pattern at a polymorphic site at the human androgen-receptor gene (HUMARA) to examine the inactivation pattern of the X chromosome. Twenty desmoid fibromatoses including primary and recurrent lesions from 11 female patients were studied. Sixteen lesions from eight patients showed nonrandom X inactivation, consistent with a clonal origin and, therefore, a true neoplastic nature. Furthermore, multiple recurrent lesions from two patients exhibited the same inactivation pattern as the corresponding primary lesions, suggesting that they were derived from the same cell clone as the primary lesion. One patient was homozygous at the HUMARA locus, and two patients had the same skewed pattern in their normal and lesional tissues. The authors also found that digestion with HpaII, but not HhaI, failed to generate a nonrandom X inactivation pattern in some of the cases, suggesting that the methylation status at the HpaII sites was altered in some lesions, and that HhaI should be used to verify results and to avoid incorrect conclusions.

X chromosome inactivation patterns in obligate carriers of X-linked lymphoproliferative syndrome

To determine whether the gene defect that causes X-linked lymphoproliferative syndrome (XLP) results in a selective disadvantage in proliferation or survival of leukocytes, we analyzed X chromosome inactivation patterns in neutrophils, T cells, and B cells from two unrelated obligate carriers of XLP. Analysis of DNA methylation patterns and production of somatic cell hybrids demonstrated that all three cell lines from both women exhibited normal, random X chromosome inactivation. These findings indicate that the XLP gene defect does not result in a global defect in proliferation or survival of T cells or B cells. It remains possible that a subset of T or B cells or natural killer cells may be selectively affected. It is also possible that the gene defect alters function but not proliferation or survival of T or B cells.

Intracisternal A particles: RNA expression and DNA methylation in murine teratocarcinoma cell lines.

Two main intracisternal-A-particle-specific RNA species (29 to 30S and 35 to 36S) are variably expressed in particle-producing and particle-nonproducing murine teratocarcinoma cell lines. An analysis of DNA methylation patterns after hybridization with an intracisternal-A-particle-specific probe showed an apparent direct correlation between DNA methylation and RNA expression. However, when the methylation assay was performed on the excised intracisternal A particle genes in one of the particle-rich cell lines (PCC6), some undermethylated sequences were detectable. These results are consistent with the concept that only few of the genes are transcriptionally active.