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PubMed2006

Promoter hypermethylation of the potential tumor suppressor DAL-1/4.1B gene in renal clear cell carcinoma.

Renal clear cell carcinoma (RCCC) is a malignant tumor with poor prognosis caused by the high incidence of metastasis to distal organs. Although metastatic RCCC cells frequently show aberrant cytoskeletal organization, the underlying mechanism has not been elucidated. DAL-1/4.1B is an actin-binding protein implicated in the cytoskeleton-associated processes, while its inactivation is frequently observed in lung and breast cancers and meningiomas, suggesting that 4.1B is a potential tumor suppressor. We studied a possible involvement of 4.1B in RCCCs and evaluated it as a clinical indicator. 4.1B protein was detected in the proximal convoluted tubules of human kidney, the presumed cell of origin of RCCC. On the other hand, loss or marked reduction of its expression was observed in 10 of 19 (53%) renal cell carcinoma (RCC) cells and 12 of 19 (63%) surgically resected RCCC by reverse transcription-PCR. Bisulfite sequencing or bisulfite SSCP analyses revealed that the 4.1B promoter was methylated in 9 of 19 (47%) RCC cells and 25 of 55 (45%) surgically resected RCCC, and inversely correlated with 4.1B expression (p < 0.0001). Aberrant methylation appeared to be a relatively early event because more than 40% of the tumors with pT1a showed hypermethylation. Furthermore, 4.1B methylation correlated with a nuclear grade (p = 0.017) and a recurrence-free survival (p = 0.0036) and provided an independent prognostic factor (p = 0.038, relative risk 10.5). These results indicate that the promoter methylation of the 4.1B is one of the most frequent epigenetic alterations in RCCC and could predict the metastatic recurrence of the surgically resected RCCC.

Aged↗

PubMed2006

Qualitative and quantitative polymerase chain reaction-based methods for DNA methylation analyses.

DNA methylation can be analyzed easily by qualitative or quantitative polymerase chain reaction (PCR)-based methods, including methylation-specific PCR (MSP), bisulfite sequencing, methylation-sensitive restriction enzyme PCR, combined bisulfite restriction analysis (COBRA), methylation-sensitive single nucleotide primer extension (Ms-SNuPE), and quantitative real-time MSP. MSP, which couples the bisulfite modification of DNA and PCR, is fast, highly sensitive, specific, and widely applied for DNA methylation analyses. Bisulfite modification converts unmethylated cytosine to uracil, whereas methylcytosine remains unmodified. Most of these methods require specific PCR primers that are designed to distinguish between methylated and unmethylated DNA sequences. Bisulfite sequencing is comparatively time-consuming. Methylation-sensitive restriction enzyme PCR combines methylation-sensitive restriction enzyme digestion and PCR. After enzyme digestion, PCR products are obtained if the enzyme does not digest the methylated CpG sites within the specified DNA region. COBRA, Ms-SNuPE, and quantitative real-time MSP allow the quantitative analyses of DNA methylation.

DNA↗

PubMed2026

DNA methylation profiles of quail blood cells by whole-genome bisulfite and Oxford Nanopore sequencing.

Whole Genome Bisulfite Sequencing (WGBS) has been the gold standard DNA methylation mapping and quantification for over a decade. Oxford Nanopore Technologies (ONT) sequencing directly measures nucleotide modifications. In this study, we have compared DNA methylation levels (5-methylcytosine) at CpG sites in the quail genome using WGBS and ONT. Samples were collected to investigate transgenerational DNA methylation changes in Japanese quail following ancestral exposure to a phytoestrogen. Blood samples from 24 third-generation (G3) individuals-descendants of either treated or untreated ancestors-were sequenced after bisulfite conversion. Both methods revealed broadly consistent methylation patterns. ONT reads covered more CpG sites and detected a higher number of differentially methylated cytosines (DMCs). Principal component analyses showed that both sex and ancestral treatment groups accounted for a portion of the observed epigenetic variation, for both technologies. Strong concordance between WGBS and ONT results supports the reliability of ONT sequencing for epigenomic research, including in quails. These data pave the way for further investigation into whether genistein induces epigenetic changes for several generations.

Animals↗

PubMed2001

Bisulfite genomic sequencing of microdissected cells.

Mapping of methylation patterns in CpG islands has become an important tool for understanding tissue-specific gene expression in both normal and pathological situations. However, the inherent cellular heterogeneity of any given tissues can affect the outcome and interpretation of molecular studies. In order to analyse genomic DNA methylation on a pure cell population from tissue sample, we have developed a simple technique of single-cell microdissection from cryostat sections which can be combined with bisulfite-mediated sequencing of 5-methylcytosine. We report here our results on the methylation status of the androgen receptor gene studied by bisulfite genomic sequencing on purified cells isolated from human testis.

Cells, Cultured↗

PubMed2005

Mapping of the methylation pattern of the MUC2 promoter in pancreatic cancer cell lines, using bisulfite genomic sequencing.

Expression of the MUC2 gene is controlled by the methylation of CpG sites in the promoter region, but the detailed methylation status of this region has yet to be reported. We have mapped the complete methylation status of the MUC2 promoter from position -1989 to position +288 upstream, a region that contains 59 CpG sites, using bisulfite genomic sequencing in two pancreatic cancer cell lines (PANC1, BxPC3) and in isolated normal colon crypts as a control. The MUC2 promoter in PANC1, a cell line that does not express MUC2, was highly methylated (average 87%, complete methylation at 28 of the 59 CpG sites), while the promoter region in the MUC2-expressing BxPC3 cell line (average 43%, complete methylation at 2 of 59 CpG sites) and in MUC2-expressing normal colon crypts (average 33%, no CpG site was completely methylated) were only partially methylated (P<0.0001). 5-Aza-2'-deoxycytidine treatment of PANC1 cells reduced the methylation level (average 36%) and induced MUC2 mRNA expression. However, mRNA expression of AP2, SP1 and CDX2 was not affected by this treatment. Our data provide the first detailed methylation map of the MUC2 promoter region for the first time, using the conversion-specific bisulfite genomic sequencing. Previously unproven methylation sites were detected, and some AP2 and SP1 binding sites showed different methylation levels among PANC1, BxPC3 and colonic crypt cells. Our mapping data provide an essential basis for further studies of methylation-regulated MUC2 inactivation.

Azacitidine↗

PubMed2004

Profiling DNA methylation by bisulfite genomic sequencing: problems and solutions.

The surge of interest in DNA methylation during the last two decades has triggered an urgent need for an effective method to detect the methylation status of the cytosines in the genome. Bisulfite genomic sequencing is the most attractive choice so far for many laboratories. Various protocols have been established, but difficulties are often encountered, particularly by individuals who have limited experience in this field. This analysis presents a simple protocol that has consistently worked well in our laboratory. Discussions of potential technical problems and corresponding solutions are also included to facilitate the reproducibility of this protocol.

Chemical Precipitation↗

PubMed1996

Methylation analysis of a marsupial X-linked CpG island by bisulfite genomic sequencing.

Paternal X chromosome inactivation occurs in rodent extraembryonic membranes and in all tissues of marsupials. Methylation of CpG islands occurs on the inactive X in eutherians and is considered to be a stabilizing mechanism. The only previous study of a marsupial X-linked CpG island was of the G6PD gene of the Virginia opossum, in which the paternally derived allele is not completely repressed. We have cloned the 5' end of the G6PD gene from an Australian marsupial, the common wallaroo, and sequenced the associated CpG island. The paternally derived G6PD allele is completely repressed in tissues of this species. Methylation analysis using HpaII and Cfol restriction enzymes and bisulfite genomic sequencing of 47 CpG dinucleotides in a 613-bp region reveals hypomethylation of male and female DNA from tissues, cultured fibroblasts (in which the paternal allele is partially expressed) and sperm. This suggests that methylation of CpG islands is not required for maintenance of X inactivation in marsupials even where repression of the paternal allele is complete.

Animals↗

PubMed2006

Mapping of the methylation pattern of the hMSH2 promoter in colon cancer, using bisulfite genomic sequencing.

The detailed methylation status of CpG sites in the promoter region of hMSH2 gene has yet not to be reported. We have mapped the complete methylation status of the hMSH2 promoter, a region that contains 75 CpG sites, using bisulfite genomic sequencing in 60 primary colorectal cancers. And the expression of hMSH2 was detected by immunohistochemistry. The hypermethylation of hMSH2 was detected in 18.33% (11/60) of tumor tissues. The protein of hMSH2 was detected in 41.67% (25/60) of tumor tissues. No hypermethylation of hMSH2 was detected in normal tissues. The protein of hMSH2 was detected in all normal tissues. Our study demonstrated that hMSH2 hypermethylation and protein expression were associated with the development of colorectal cancer.

Journal Article↗

PubMedDate not supplied

Mapping the methylation pattern by bisulfite genomic sequencing of the E-cadherin promoter CpG island in nasopharyngeal carcinoma.

BACKGROUND: It is hypothesised that hypermethylation of the CpG island in the promoter region in several tumour suppressor genes contributes one of the mechanisms for silencing or down-regulating the expression of the tumour suppressor genes. One of the putative tumour suppressor genes, the E-cadherin gene, has shown down-regulation in nasopharyngeal carcinoma (NPC). The aim of this study was to explore the methylation profiles of the E-cadherin promoter region in NPC patients. MATERIALS AND METHODS: Fifteen NPC paraffin-embedded tissue sections were included in the study accompanied by two cell lines, MCF7 and MDA435, as controls. The technique of bisulfite genomic sequencing was used to determine the methylation patterns of the 29 CpG dinucleotides along the E-cadherin promoter region. DNAs were extracted from the samples, and then were subject to bisulfite conversion. Nested polymerase chain reactions were performed on the modified DNAs, the sequences of which were determined by direct sequencing. RESULTS: A pilot study ascertained the correlation between reduced E-cadherin expression and hypermethylation of the E-cadherin promoter region from the E-cadherin (+) MCF7 and the E-cadherin (-) MDA435 cell lines. The experimental study revealed that, among 10 NPC cases in which DNA could be successfully extracted, 6 (60%) displayed methylation patterns at two or more CpG sites (range: 2-11, 6.8-37.9%). Five of them (83.3%) were comprised of at least 2 transcription enhancing factor sites. Two of the 4 cases (50%) with methylated 4th CpG site (HpaII site) and 3 of the 4 cases (75%) with methylated 11th CpG site (GC region) had grades 3 or 4 level of methylation. CONCLUSION: The data indicated that hypermethylation of the E-cadherin promoter region occurred in a large proportion of NPC patients, and frequent methylation of specific CpG sites associated with transcription enhancing elements suggested that this phenomenon may contribute part of the mechanisms for silencing or down-regulating E-cadherin gene expression in NPC.

Base Sequence↗

PubMed2026

ADCY2 promoter hypomethylation in adolescents with borderline personality disorder: An RRBS study with targeted BSP replication.

BACKGROUND: Borderline Personality Disorder (BPD) is characterized by emotional dysregulation, impulsivity, and interpersonal instability. Although genetic and environmental contributions to BPD have been investigated, epigenetic correlates in adolescents remain poorly understood. The cyclic adenosine monophosphate (cAMP) signaling pathway is implicated in stress responsivity and emotion regulation, but its epigenetic variation in adolescent BPD remains understudied. METHODS: DNA methylation profiling was performed using Reduced Representation Bisulfite Sequencing (RRBS) in buccal epithelial DNA from adolescents with BPD (n = 15) and healthy controls (HC; n = 15). Differentially methylated regions (DMRs) were identified using metilene, a computational tool for detecting DMRs from bisulfite sequencing data, and subjected to Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses. Associations between methylation signals and borderline personality features were examined. Adenylate cyclase 2 (ADCY2) promoter methylation was examined using targeted bisulfite sequencing PCR (BSP) in an independent cohort (BPD n = 5; HC n = 5). RESULTS: RRBS identified 7641 DMRs between BPD and HC, with enrichment in pathways related to neuronal signaling and synaptic organization. A hypomethylated DMR was detected in the ADCY2 promoter, a gene implicated in cAMP signaling. Lower ADCY2 promoter methylation was associated with greater clinical severity, including emotional dysregulation, borderline traits, anxiety symptoms, and self-injury behaviors (r=-0.71 to -0.83; all p < 0.0001). Hypomethylation of the ADCY2 promoter was replicated in the independent cohort. CONCLUSIONS: This exploratory two-stage epigenetic study suggests that ADCY2 promoter hypomethylation in peripheral buccal epithelial DNA is associated with core symptom dimensions of adolescent BPD. These findings support a role for cAMP-related epigenetic variation in BPD and warrant replication in longitudinal studies.

ADCY2↗

PubMed2002

Hypermethylation of the p14(ARF) gene in ulcerative colitis-associated colorectal carcinogenesis.

The p14(ARF) protein directly inhibits the MDM-2 oncoprotein, which mediates degradation of the p53 protein. It has been shown that p14(ARF) expression is frequently down-regulated by p14(ARF) gene hypermethylation in colorectal cancer. To determine whether p14(ARF) inactivation was involved in ulcerative colitis (UC)-associated carcinogenesis, the frequency and timing of p14(ARF) methylation was investigated in four different histological stages of UC-associated carcinogenesis. Methylation-specific PCR and bisulfite sequencing were used to determine the prevalence of p14(ARF) gene methylation. p14(ARF) methylation was observed in 19 of 38 (50%) adenocarcinomas, 4 of 12 (33%) dysplasias, and 3 of the 5 (60%) nonneoplastic UC mucosae. In contrast, 3 of 40 (3.7%) normal tissues showed p14(ARF) methylation (chi(2) test: P = 0.0003). Bisulfite sequencing was used to analyze 28 CpGs of p14(ARF) gene in 20 samples. The number of methylated CpGs ranged from 0 to 4, 0 to 20, and 0 to 28 in the normal, dysplastic, and carcinomatous samples, respectively (Kruskall-Wallis test: P = 0.0005). Densely methylated alleles were detected only in carcinomas by bisulfite sequencing. In conclusion, our data suggest that methylation of p14(ARF) is a relatively common early event in UC-associated carcinogenesis. p14(ARF) offers potential as a biomarker for the early detection of cancer or dysplasia in UC. Finally, analyses of p14(ARF) methylation in other organs should explore not only frank cancers but other premalignant lesions.

Base Sequence↗

PubMed2005

Methylation status analysis of cell cycle regulatory genes (p16INK4A, p15INK4B, p21Waf1/Cip1, p27Kip1 and p73) in natural killer cell disorders.

Natural killer (NK) cell disorders are rare diseases. Genetic abnormalities of the several tumor suppressor genes, including p15INK4B, p16INK4A/p14ARF, p53, p73, and Rb genes have been reported. Deletions and point mutations of these genes are frequently detected in these diseases. It has been reported that tumor suppressor genes are inactivated by DNA methylation of the promoter region and/or first exon of the genes in a variety of human cancers. In this study we analyze the methylation status of the genes associated with cell cycle regulation, including p16INK4A, p15INK4B, p21/Waf1/Cip1, p27/Kip1, p73, and p14ARF, by methylation specific (MS) PCR and/or bisulfite sequencing. We examined 29 cases of NK cell disorders (five aggressive NK cell leukemia/lymphoma, three blastic NK cell lymphoma/leukemia, five nasal NK cell lymphoma, three myeloid/NK cell precursor acute leukemia, 13 chronic NK lymphocytosis). We found methylation of the first exon of the p16INK4A gene in two cases (one aggressive, one blastic), and methylation of the p14ARF gene in one aggressive NK cell leukemia. Bisulfite sequencing revealed that methylation of the p15 and p27 genes was rare in these disorders. MS-PCR suggested that the p73 and p21 genes were methylated in seven cases, respectively (p73: one blastic, one nasal, five chronic; p21: one myeloid/NK, one aggressive, one nasal, and four chronic); bisulfite sequencing confirmed that methylated alleles of these genes were dominant in the samples except three cases (one myeloid/NK, one aggressive, and one chronic) in which methylated alleles of the p21 genes were less than 34% of all alleles. These results suggested that inactivation of the cell cycle regulatory genes by DNA methylation could be associated with tumorigenesis in NK cell disorders, not only aggressive subtypes but also chronic subtype.

Base Sequence↗

PubMed2016

A probabilistic generative model for quantification of DNA modifications enables analysis of demethylation pathways.

We present a generative model, Lux, to quantify DNA methylation modifications from any combination of bisulfite sequencing approaches, including reduced, oxidative, TET-assisted, chemical-modification assisted, and methylase-assisted bisulfite sequencing data. Lux models all cytosine modifications (C, 5mC, 5hmC, 5fC, and 5caC) simultaneously together with experimental parameters, including bisulfite conversion and oxidation efficiencies, as well as various chemical labeling and protection steps. We show that Lux improves the quantification and comparison of cytosine modification levels and that Lux can process any oxidized methylcytosine sequencing data sets to quantify all cytosine modifications. Analysis of targeted data from Tet2-knockdown embryonic stem cells and T cells during development demonstrates DNA modification quantification at unprecedented detail, quantifies active demethylation pathways and reveals 5hmC localization in putative regulatory regions.

5-Methylcytosine↗

PubMed2005

Epigenetic silencing of the protocadherin family member PCDH-gamma-A11 in astrocytomas.

In a microarray-based methylation analysis of astrocytomas [World Health Organization (WHO) grade II], we identified a CpG island within the first exon of the protocadherin-gamma subfamily A11 (PCDH-gamma-A11) gene that showed hypermethylation compared to normal brain tissue. Bisulfite sequencing and combined bisulfite restriction analysis (COBRA) was performed to screen low- and high-grade astrocytomas for the methylation status of this CpG island. Hypermethylation was detected in 30 of 34 (88%) astrocytomas (WHO grades II and III), 20 of 23 (87%) glioblastomas (WHO grade IV), and 8 of 8 (100%) glioma cell lines. There was a highly significant correlation (P = .00028) between PCDH-gamma-A11 hypermethylation and decreased transcription as determined by competitive reverse transcription polymerase chain reaction in WHO grades II and III astrocytomas. After treatment of glioma cell lines with a demethylating agent, transcription of PCDH-gamma-A11 was restored. In summary, we have identified PCDH-gamma-A11 as a new target silenced epigenetically in astrocytic gliomas. The inactivation of this cell-cell contact molecule might be involved in the invasive growth of astrocytoma cells into normal brain parenchyma.

Astrocytoma↗

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↗

PubMed2025

Immune Cell Type-Specific DNA Methylation Regions Associate With 24-Hour Blood Pressure Regulation in Black People.

BACKGROUND: DNA methylation and immune cells have been linked to blood pressure (BP) regulation and the development of hypertension. However, the immune cell profiles and the cell type-specific DNA methylation associated with BPs remain unclear. METHODS: This study evaluates the 19 cell type deconvolution algorithms using reduced representation bisulfite sequencing data, comparing them to in silico mixtures derived from whole-genome bisulfite sequencing. The top-performing algorithm, Epigenetic Dissection of Intra-Sample Heterogeneity (EpiDISH)-Robust Partial Correlations, was applied to 281 Black inpatients with 24-hour BP monitoring. The immune cell profiles and cell type-specific DNA methylation regions associated with these BP phenotypes were further investigated using regression analysis. RESULTS: In patients with hypertension, B-cell and CD4 effector memory T-cell abundances were significantly elevated. Monocyte and CD8 effector memory T-cell fractions positively correlated with nighttime BP, and CD3 T cells were inversely associated with office BP. These associations remained robust after covariate adjustments and were partially validated in the Medical Information Mart for Intensive Care-IV cohort. For the first time, we identified several cell type-specific DNA methylation regions as being associated with BP phenotypes and patterns across 13 immune cells, with approximately one third predominantly found in effector CD8 T cells. CONCLUSIONS: These findings provide novel insights into the epigenetically regulated immune mechanisms underlying BP regulation and identify potential targets for hypertension management.

Humans↗

PubMed2026

Multi-omics integrative analysis provides insight into potential molecular responses to sustained high water flow in common carp (Cyprinus carpio) cultured in recirculating aquaculture.

To investigate the potential molecular responses by which water flow intensity affects the growth of common carp (Cyprinus carpio) in a recirculating aquaculture system (RAS), a control group (CG, actual water velocity 0.3 cm/s) and three sustained flow treatment groups were established, including a low-flow group (LF, 1 body length per second, bl/s), a medium-flow group (MF, 2 bl/s), and a high-flow group (HF, 3 bl/s). After 12 weeks of culture in the RAS, growth performance was compared among groups under different flow intensities. The best-performing group and the control group were then selected for the determination of intestinal digestive enzyme activities, as well as transcriptomic and whole-genome bisulfite sequencing analyses of muscle tissue. The results showed that the specific growth rate and feed intake of the HF group were significantly higher than those of the other groups (P < 0.05), whereas no significant difference in feed conversion ratio was observed among groups. Compared with the CG group, lipase activity was significantly higher in the HF group (P < 0.05), while α-amylase and trypsin activities showed increasing trends without significant differences. RNA-seq identified a total of 273 differentially expressed genes, including 72 upregulated genes and 201 downregulated genes in the HF group relative to the CG group. These genes were mainly enriched in glycolysis, pyruvate metabolism, ATP metabolism, the pentose phosphate pathway, the insulin signaling pathway, the PPAR signaling pathway, and the adipocytokine signaling pathway, indicating that sustained high water flow induced a muscle transcriptional response characterized by remodeling of energy metabolism and substrate utilization. Whole-genome bisulfite sequencing analysis showed that DNA methylation in common carp muscle occurred predominantly in the CpG context. Differentially methylated regions between the HF and CG groups were mainly distributed in transcription-related regulatory regions, including promoters, CpG islands, and CpG island shores. In promoter regions, the number of hypermethylated regions in the HF group relative to the CG group was markedly higher than that of hypomethylated regions. Integrated analysis further identified two candidate genes showing both promoter differential methylation and differential expression, namely LOC109094644 and bcorl1, suggesting that adaptation to high water flow may involve IGF-related growth regulation and remodeling of upstream transcriptional programs. The qPCR results were consistent with the transcriptomic data. Taken together, within the tested range, a sustained water flow of 3 bl/s was more conducive to the growth of common carp in the RAS, which may be associated with enhanced lipid digestion and utilization, remodeling of the muscle energy metabolic network, changes in promoter methylation, and the coordinated regulation of key candidate genes. This study provides a theoretical basis for clarifying the exercise adaptation mechanism of common carp in recirculating aquaculture and for optimizing flow velocity parameters.

Animals↗

PubMed2006

Methylation status of the Epstein-Barr virus (EBV) BamHI W latent cycle promoter and promoter activity: analysis with novel EBV-positive Burkitt and lymphoblastoid cell lines.

The Epstein-Barr virus (EBV) latent cycle promoter Wp, present in each tandemly arrayed copy of the BamHI W region in the EBV genome, drives expression of the EB viral nuclear antigens (EBNAs) at the initiation of virus-induced B-cell transformation. Thereafter, an alternative EBNA promoter, Cp, becomes dominant, Wp activity declines dramatically, and bisulfite sequencing of EBV-transformed lymphoblastoid cell lines (LCLs) shows extensive Wp methylation. Despite this, Wp is never completely silenced in LCLs. Here, using a combination of bisulfite sequencing and methylation-specific PCR, we show that in standard LCLs transformed with wild-type EBV isolates, some Wp copies always remain unmethylated, and in LCLs transformed with a recombinant EBV carrying just two BamHI W copies, Wp is completely unmethylated. Furthermore, we have analyzed rare LCLs, recently established using wild-type EBV isolates, and rare Burkitt lymphoma (BL) cell clones, recently established from tumors carrying EBNA2-deleted EBV genomes, which express EBNAs exclusively from Wp-initiated transcripts. Here, in sharp contrast to standard LCL and BL lines, all resident copies of Wp appear to be predominantly hypomethylated. Thus, studies of B cells with atypical patterns of Wp usage emphasize the strong correlation between the presence of unmethylated Wp sequences and promoter activity.

B-Lymphocytes↗