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PubMed1998

The Epstein-Barr virus major latent promoter Qp is constitutively active, hypomethylated, and methylation sensitive.

Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA1) is indispensable for viral DNA replication and episome maintenance in latency. Four promoters, Cp, Wp, Qp, and Fp, are known to drive EBNA1 expression. Here we show that the TATA-less Qp is constitutively active in a variety of EBV-positive [EBV(+)] tumors and cell lines, irrespective of the activities of other EBNA1 promoters, the type of viral latency, and the cell type. The transcription of highly regulated promoters such as the EBV Cp is known to be directly regulated by CpG methylation. To characterize the role of CpG methylation in the regulation of the constitutively active Qp, we performed bisulfite genomic sequencing and functional analyses using a methylation cassette transcriptional reporter assay. Twenty consecutive CpG sites (16 proximal to the Qp initiation site and 4 upstream of the adjacent Fp initiation site) were studied by bisulfite sequencing of DNA extracted from EBV(+) tumors and cell lines. Eighteen EBV(+) tumors of lymphoid (B, T, and NK cell) or epithelial origin and five Burkitt's lymphoma cell lines were studied. The 16 CpG sites proximal to Qp were virtually all unmethylated, but the 4 CpG sites upstream of the Fp initiation site were variably methylated. The methylation cassette assay showed that in vitro methylation of the Qp cassette (-172 to +32) resulted in strong repression of Qp activity in transient transfection. Thus, Qp is susceptible to repression by methylation but was found to be consistently hypomethylated and expressed in all tumors and tumor-derived cell lines studied.

Base Sequence↗
PubMed2005

Epigenetic silencing of DSC3 is a common event in human breast cancer.

INTRODUCTION: Desmocollin 3 (DSC3) is a member of the cadherin superfamily of calcium-dependent cell adhesion molecules and a principle component of desmosomes. Desmosomal proteins such as DSC3 are integral to the maintenance of tissue architecture and the loss of these components leads to a lack of adhesion and a gain of cellular mobility. DSC3 expression is down-regulated in breast cancer cell lines and primary breast tumors; however, the loss of DSC3 is not due to gene deletion or gross rearrangement of the gene. In this study, we examined the prevalence of epigenetic silencing of DSC3 gene expression in primary breast tumor specimens. METHODS: We used bisulfite genomic sequencing to analyze the methylation state of the DSC3 promoter region from 32 primary breast tumor specimens. We also used a quantitative real-time RT-PCR approach, and analyzed all breast tumor specimens for DSC3 expression. Finally, in addition to bisulfite sequencing and RT-PCR, we used an in vivo nuclease accessibility assay to determine the chromatin architecture of the CpG island region from DSC3-negative breast cancer cells lines. RESULTS: DSC3 expression was downregulated in 23 of 32 (72%) breast cancer specimens comprising: 22 invasive ductal carcinomas, 7 invasive lobular breast carcinomas, 2 invasive ductal carcinomas that metastasized to the lymph node, and a mucoid ductal carcinoma. Of the 23 specimens showing a loss of DSC3 expression, 13 (56%) were associated with cytosine hypermethylation of the promoter region. Furthermore, DSC3 expression is limited to cells of epithelial origin and its expression of mRNA and protein is lost in a high proportion of breast tumor cell lines (79%). Lastly, DNA hypermethylation of the DSC3 promoter is highly correlated with a closed chromatin structure. CONCLUSION: These results indicate that the loss of DSC3 expression is a common event in primary breast tumor specimens, and that DSC3 gene silencing in breast tumors is frequently linked to aberrant cytosine methylation and concomitant changes in chromatin structure.

Breast Neoplasms↗
PubMed2003

Hepatocyte growth factor activator inhibitor 2/placental bikunin (HAI-2/PB) gene is frequently hypermethylated in human hepatocellular carcinoma.

To identify methylation-mediated silencing of genes in hepatocellular carcinoma (HCC), we surveyed genes induced by treatment with 5-aza-2'-deoxycytidine (5-Aza-CdR) in six human hepatoma cell lines using cDNA microarray analysis and determined the methylation status of 5' CpG islands by bisulfite DNA sequencing or methylation-specific PCR. Fifty genes exhibited a >5-fold induction in response to treatment with 5-Aza-CdR in at least one of the hepatoma cell lines examined. Among these genes, the hepatocyte growth factor activator inhibitor 2/placental bikunin (HAI-2/PB) gene was maximally induced by 5-Aza-CdR in three of six cell lines studied (HLE, HuH7, and Hep3B). Bisulfite sequencing revealed that the 5' CpG island of this gene was densely methylated in HLE, HuH7, and Hep3B cells. After treatment with 5-Aza-CdR, re-expression and demethylation of HAI-2/PB gene were detected in these cells. These findings suggest that HAI-2/PB expression may be inappropriately repressed by promoter hypermethylation in HCC. Methylation-specific PCR analysis demonstrated that HAI-2/PB hypermethylation occurred in 21 of 26 HCC tumors (80.8%), whereas in the corresponding nontumorous liver tissues, it was found in 7 of 26 samples (26.9%). In addition, HAI-2/PB hypermethylation was not detected in any of the seven normal liver samples from individuals without HCC. Reverse transcription-PCR analysis demonstrated that promoter hypermethylation was associated with the reduced expression of the HAI-2/PB gene in HCC tumors. In conclusion, we have found that the HAI-2/PB gene is silenced by promoter hypermethylation in human hepatoma cells by means of cDNA microarray analysis after 5-Aza-CdR treatment, and that HAI-2/PB hypermethylation occurs frequently in primary HCC tumors.

Antimetabolites, Antineoplastic↗
PubMed2005

Consistent transcriptional silencing of 35S-driven transgenes in gentian.

In this study, no transgenic gentian (Gentiana triflora x Gentiana scabra) plants produced via Agrobacterium-mediated transformation exhibited transgene (GtMADS, gentian-derived MADS-box genes or sGFP, green fluorescent protein) expression in their leaf tissues, despite the use of constitutive Cauliflower mosaic virus (CaMV) 35S promoter. Strikingly, no expression of the selectable marker gene (bar) used for bialaphos selection was observed. To investigate the possible cause of this drastic transgene silencing, methylation-specific sequences were analysed by bisulfite genomic sequencing using tobacco transformants as a control. Highly methylated cytosine residues of CpG and CpWpG (W contains A or T) sites were distinctively detected in the promoter and 5' coding regions of the transgenes 35S-bar and 35S-GtMADS in all gentian lines analysed. These lines also exhibited various degrees of cytosine methylation in asymmetrical sequences. The methylation frequencies in the other transgene, nopaline synthase (NOS) promoter-driven nptII, and the endogenous GtMADS gene coding region, were much lower and were variable compared with those in the 35S promoter regions. Transgene methylation was observed in the bialaphos-selected transgenic calluses expressing the transgenes, and methylation sequences were distributed preferentially around the as-1 element in the 35S promoter. Calluses derived from leaf tissues of silenced transgenic gentian also exhibited transgene suppression, but expression was recovered by treatment with the methylation inhibitor 5-aza-2'-deoxycytidine (aza-dC). These results indicated that cytosine methylation occurs exclusively in the 35S promoter regions of the expressed transgenes during selection of gentian transformants, causing transcriptional gene silencing.

Base Sequence↗
PubMed1999

Altered expression of retinoblastoma (RB) protein in acute myelogenous leukemia does not result from methylation of the Rb promotor.

Prior studies demonstrated that expression of the retinoblastoma (RB) protein in acute myelogenous leukemia (AML) is heterogeneous with low expression conferring a poor prognosis. The molecular change(s) responsible for low RB expression in AML are unknown. Since methylation of the RB promoter has been shown to result in decreased expression we hypothesized that this might explain some cases of low RB expression in AML. To investigate this hypothesis Southern blotting and PCR sequencing after bisulfite conversion were used to study the methylation status of the RB gene promoter. DNA and protein lysates were prepared from the mononuclear cell fraction from peripheral blood or bone marrow samples from 46 patients with newly diagnosed AML. By Western blot 16, 22 and 8 patients had low, elevated and hyperphosphorylated patterns of RB expression respectively using previously defined criteria. The SacI endonuclease cuts a 5.7-kb or 6.8 -kb fragment, depending on polymorphism, containing the RB promoter, detected by the probe p123M1.8 that covers the RB promoter region and exon 1. The methylation sensitive endonuclease SacII cuts twice within a key hairpin loop structure in the RB promoter that contains binding sites for AP1, Sp1 and RBF1. Others have demonstrated that methylation within this hairpin loop can decrease RB mRNA transcription by up to 92%. Comparison of the SacI and SacI + SacII digestion fragments showed no evidence of methylation in the promoter region of RB in any of the patients studied. DNA from the promoter region of 11 patients with no/low RB expression was subjected to bisulfite conversion and PCR sequencing. No evidence of methylation was seen by this method either. These results suggests that hypermethylation of the RB promoter region is at best an infrequent event in AML and that RB promoter hypermethylation is not the predominant cause of the low levels of RB expression observed in 20% of AML patients.

Base Sequence↗
PubMed2003

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.

Animals↗
PubMed2006

Effects of Helicobacter pylori eradication on methylation status of E-cadherin gene in noncancerous stomach.

PURPOSE: Promoter hypermethylation of E-cadherin plays an important role on gastric cancer development. Whereas E-cadherin methylation was frequently detected in the stomach of Helicobacter pylori-infected individuals, we tested whether eradication of H. pylori alters the methylation status of the noncancerous gastric epithelium. EXPERIMENTAL DESIGN: Endoscopic biopsies were taken from the antrum and corpus of H. pylori-infected subjects without gastric cancer. Presence of methylated E-cadherin sequences in the gastric specimens was detected by methylation-specific PCR. Bisulfite DNA sequencing was done to determine the topographical distribution and changes in methylation profiles with H. pylori eradication. RESULTS: Among the 28 H. pylori-infected subjects (median age, 44.5 years), 15 (53.6%) had E-cadherin methylation detected in stomach at baseline. Discordant methylation patterns between the antrum and corpus were noted in six patients. One year after successful H. pylori eradication, there was a significant reduction in the methylation density of the promoter region and exon 1 of the E-cadherin gene as detected by bisulfite DNA sequencing (P < 0.001). CONCLUSION: Promoter methylation in E-cadherin was frequently detected in the stomach of H. pylori-infected individuals. Eradication of H. pylori might possibly reduce the methylation density in E-cadherin gene and the chance of subsequent neoplastic transformation.

Adult↗
PubMed2003

Methylation of the thyroid-stimulating hormone receptor gene in epithelial thyroid tumors: a marker of malignancy and a cause of gene silencing.

Thyroid-stimulating hormone receptor (TSHR) expression is frequently silenced in epithelial thyroid cancers associated with decreased or absent TSH-promoted iodine uptake. To study the underlying molecular mechanism of decreased TSHR expression, we examined the methylation status of the TSHR gene promoter by sequencing bisulfite-treated DNA from thyroid tumors. After identification of methylated sites by sequencing bisulfite-treated DNA, we used methylation-specific polymerase chain reaction and found frequent CpG methylation in papillary thyroid cancer (23 of 39 patients; 59%) and follicular thyroid cancers (7 of 15 patients; 47%). In contrast, we saw no methylation in normal thyroid tissues and benign adenomas (0 of 8 patients; 0%). In human thyroid tumor cell lines, we observed that TSHR was normally expressed at the protein and mRNA level in cells where the TSHR gene was unmethylated, whereas it was silenced in cell lines where the TSHR promoter was hypermethylated. Treatment of the latter cells with a demethylating agent partially restored TSHR expression. We thus demonstrate aberrant methylation of human TSHR as a likely molecular pathway responsible for the silencing of this gene in thyroid cancers. We propose that methylation of TSHR may provide a novel diagnostic marker of malignancy and a basis for potential use of demethylating agents in conjunction with TSH-promoted radioiodine therapy for epithelial thyroid cancers.

Adenoma↗
PubMed2006

Does urea promote the bisulfite-mediated deamination of cytosine in DNA? Investigation aiming at speeding-up the procedure for DNA methylation analysis.

Methylation of cytosine in DNA at position 5 plays important roles in gene functions. Changes in the methylation status are linked to cancer. These studies have been developed on the basis of determining 5-methylcytosine residues [mC] in DNA. This analytical procedure uses the principle that bisulfite deaminates cytosine [C] but it deaminates mC only very slowly. Thus, 'bisulfite genomic sequencing' involves treatment of a given DNA sample with bisulfite followed by PCR amplification and sequencing, through which C residues in the original DNA are found as T and mC as C. In this procedure, a treatment with 3-5 M sodium bisulfite for 12-16 hr at 55 degrees C has been conventionally used. Recently, we were able to improve the efficiency of this procedure by introducing a highly concentrated (10 M) bisulfite solution. Aiming at further improvement of the procedure, we have now explored the effect of adding urea in this bisulfite treatment, as urea was reported to improve the deamination efficiency. Using 7.5 M ammonium bisulfite (pH 5.4) at 70 degrees C with or without the presence of 6 M urea, we performed deamination and sequencing of a DNA sample having known multiple CpG sites with mC. The deaminated DNAs were then subjected to PCR amplification followed by sequencing. In the 15 min-treated sample, the deamination extents were; C 96.5%, mC 1.1% for "bisulfite-only"; and C 90.3%, mC 1.4% for "bisulfite + urea". In the 30 min-treated sample, these values were; C 99.7%, mC 3.6% for "bisulfite only"; and C 99.7%, mC 2.1% for "bisulfite + urea". These results indicate that urea did not enhance the deamination efficiency. In the PCR, we did not observe significant improvements regarding the amounts of DNA necessary to obtain adequate amplification. Urea at 2 M, 4 M, and 8 M, showed no improvements. We conclude that urea gave no significant effect in the bisulfite genomic sequencing of the DNA used.

5-Methylcytosine↗
PubMed1998

DNA methylation regulates p27kip1 expression in rodent pituitary cell lines.

We previously reported loss of expression of p27Kip1 (p27) protein in rat GH3 and mouse GHRH-CL1 pituitary tumor cells compared with normal pituitary (NP). The molecular basis for the loss of expression of p27 protein in GH3 and GHRH-CL1 cells is unknown. To determine the role of p27 gene methylation in the regulation of the expression of this cell cycle protein, the methylation patterns of p27 in normal and neoplastic pituitary cells was analyzed. Inhibition of DNA methyltransferase (DNA-MTase) with 5-aza-2'-deoxycytidine (AZAdC) induced expression of both p27 protein and mRNA when GH3 and GHRH-CL1 cells were treated for 7 days in vitro. DNA methylation correlated inversely with the expression of p27 gene products in NP and pituitary tumor cell lines. Bisulfite genomic sequencing analysis showed that the normally unmethylated cytosines in exon 1 in NP and AtT20 cells were extensively methylated in GH3 and GHRH-CL1 cells. After treatment of GH3 and GHRH-CL1 cells with 10 micromol/L AZAdC, there were decreased numbers of methylated cytosines (by 60% to 90%/o) with variable methylation patterns observed by bisulfite genomic sequencing. Analysis of genomic DNA with methylation-sensitive enzymes showed that all SmaI, HhaI, and AvaI enzyme sites of the p27 gene in exon 1 were methylated in GH3 cells but not in NP, confirming the bisulfite genomic sequencing results. AtT20 cells and a human pituitary null cell adenoma cell line (HP75), which expressed abundant p27, had a methylation pattern similar to the NP. DNA-MTase activity was elevated fourfold in GH3 cells and twofold in GHRH-CL1 cells compared with DNA-MTase activity in NP and AtT20 cells. These results suggest that increased DNA methylation is another mechanism of silencing of the p27 gene in some pituitary tumors and possibly in other types of neoplasms.

Animals↗
PubMed2004

Accelerated bisulfite-deamination of cytosine in the genomic sequencing procedure for DNA methylation analysis.

Understanding the biological consequences of DNA methylation is a current focus of intensive studies. A standard method for analyzing the methylation at position 5 of cytosines in genomic DNA involves chemical modification of the DNA with bisulfite, followed by PCR amplification and sequencing. Bisulfite deaminates cytosine, but it deaminates 5-methylcytosine only very slowly, thereby allowing determination of the methylated sites. The determination is usually performed using sodium bisulfite solutions of 3-5 M concentration with an incubation period of 12-16 hr at 50 degrees C. We demonstrate here that this deamination can be speeded up significantly by increasing the bisulfite concentration and the temperature with which the reaction is performed. In an experiment, in which denatured DNA was treated with 9 M bisulfite for 10 min at pH 5.4 and 90 degrees C, deamination of cytosines occurred to an extent of 99.6%, while 5-methylcytosine residues in the DNA were deaminated at less than 10%. Using a plasmid DNA fragment, we observed that the DNA can serve as a template for PCR amplification after the bisulfite treatment. This new procedure is expected to offer an improved genomic sequencing method, leading to the promotion of research on understanding the biological and medical significance of DNA methylation.

Animals↗
PubMed2002

DNA methylation and epigenetic inheritance.

Mammalian cell lines silence genes at low frequency by the methylation of promoter sequences. These silent genes can be reactivated at high frequency by the demethylating agent 5-azacytidine (5-aza-CR). The inactive and active epigenetic states of such genes are stably inherited. A method for silencing genes is now available. It involves treatment of permeabilized cells with 5-methyl deoxycytidine triphosphate (5-methyl dCTP) which is incorporated into DNA. The methylation of promoter sequences has been confirmed using the bisulfite genomic sequencing procedure. Methylated oligonucleotides homologous to promoter sequences might be used to specifically target and silence given genes, but results so far have not been conclusive. Treatments that silence or reactivate genes by changing DNA methylation can be referred to as epimutagens, as distinct from mutagens that act by changing DNA sequences. The epimutagen 5-aza-CR reactivates genes but has little mutagenic activity, whereas standard mutagens (such as ethyl methane sulfonate and ultraviolet light) have little reactivation activity. Nevertheless, much more information is required about the effects of DNA-damaging agents in changing DNA methylation and gene activity and also about the role of epimutations in tumor progression.

Animals↗
PubMed2003

CpG dinucleotide methylation patterns in the human androgen receptor gene and X-chromosome inactivation in peripheral blood leukocytes of phenotypically normal women.

To evaluate methylation patterns in CpG dinucleotides (CpGs) of the human androgen receptor gene ( HUMARA) and X-chromosome inactivation (XCI) status in phenotypically normal women in a general population, bisulfite genomic sequencing and methylation-specific PCR of genomic DNA extracted from peripheral blood samples of 124 phenotypically normal women were examined. CpGs methylation patterns were based on bisulfite genomic sequencing of the region containing nine CpGs in the HUMARA exon 1. The results of methylation status in CpGs from 43 independent colonies of 14 women revealed that not all CpGs were methylated even in highly methylated HUMARA alleles, and that the methylation status in CpGs varied between clones, by the position of CpGs methylation and in each subject. Evaluation of XCI was based on the method of an HUMARA (CAG)n polymorphism assay after bisulfite modification of DNA samples. The HUMARA allele size ratios of the women (82 heterozygotes) varied over a wide range and the distribution patterns of the ratios approached a 'normal distribution'. Since excessive skewing of XCI was observed in 11-12% of women, female carriers of an X-linked hereditary disease manifest its clinical symptoms or signs possibly in maximum 5-6%.

Adult↗
PubMed2004

Mutation analysis of the KIF21A gene in an Indian family with CFEOM1: implication of CpG methylation for most frequent mutations.

PURPOSE: To carry out the mutation analysis of the KIF21A gene in a four-generation Indian family affected with CFEOM1 and to find out the molecular basis of the most frequent mutations c.2860C>T and c.2861G>A in exon 21 of the KIF21A gene. METHODS: Mutational analysis was carried out by direct automated sequencing of the PCR products from exons 8, 20, and 21 of the KIF21A gene. Allele specific oligo hybridization analysis was carried out to study the segregation of the mutation within the family. Methylation status of the mutated CpG dinucleotide in exon 21 was detected using bisulfite genomic sequencing technique on genomic DNA isolated from blood and sperms. RESULTS: We found a previously reported missense mutation c.2860C>T (p.954R>W) in exon 21 of the KIF21A gene in our family. This mutation was found in a CpG dinucleotide. Bisulfite genomic sequencing revealed that all the CpG dinucleotides in exon 21 including the one which harbored the two most frequent mutations were methylated both in the genomic DNA from blood and sperms. CONCLUSIONS: CFEOM1 phenotype in our family was caused by a previously reported most frequent missense mutation, c.2860C>T. This mutation occurred at the C residue in a CpG dinucleotide, which was found to be methylated. Previous work has demonstrated that this CpG dinucleotide is a mutational hotspot in the KIF21A gene, and our finding suggests that its high mutability may result, in part, from its methylated state.

CpG Islands↗
PubMed2004

Tamoxifen-resistant breast cancers show less frequent methylation of the estrogen receptor beta but not the estrogen receptor alpha gene.

No reports have examined the association between tamoxifen resistance and the methylation of the estrogen receptor (ER) alpha and beta genes. Therefore we investigated the methylation patterns of the ER genes in the tamoxifen-resistant tumors. We used bisulfite genomic sequencing and reverse transcriptase PCR to determine the methylation patterns and mRNA expression of the two ER genes from control (n = 68) and tamoxifen-resistant tissues (n = 34) chosen by an age-matched sampling method. Bisulfite genomic sequencing allowed us to reveal the methylation of the ER alpha gene in 15 of the control tumors (22.1%) and in 11 tumors of the resistant group (32.4%). The methylation of ER beta was observed in 40 control tumors (58.8%) and in 8 recurrent tumors (23.5%). The methylation rate of the ER beta but not the ER alpha in the control group was significantly higher than in its counterpart (ER alpha, P = 0.261; ER beta, P = 0.001). Among the methylated tumors mean methylation density of ER alpha and ER beta in the resistant cases was significantly elevated (ER alpha, P = 0.014; ER beta, P < 0.001). Furthermore, the expression rate of ER beta mRNA was higher among the tumor in the resistant group than in the control with marginal significance (77.8% vs. 38.1%, P = 0.109). Additionally, in the cancers from the resistant cases, the cells showed a higher percentage of positive staining for Ki67 than those from the control group (P = 0.001). Our study indicates that there is an inverse relationship between the methylation rate of the ER beta gene and tamoxifen resistance. The tamoxifen-resistant tumors showed more dense methylation of the ER beta gene than control tumors. Although the number of case samples was limited, our results support the hypothesis that hypermethylation of the ER beta gene negatively affects the development of tamoxifen resistance.

Antineoplastic Agents, Hormonal↗
PubMed2005

Epigenetic processes play a major role in B-cell-specific gene silencing in classical Hodgkin lymphoma.

Many B-lineage-specific genes are down-regulated in Hodgkin and Reed-Sternberg (HRS) cells of classical Hodgkin lymphoma (cHL). We investigated the involvement of epigenetic modifications in gene silencing in cHL cell lines and in microdissected primary HRS cells. We assessed the expression and methylation status of CD19, CD20, CD79B, SYK, PU.1, BOB.1/OBF.1, BCMA, and LCK, all of which are typically down-regulated in cHL. We could reactivate gene expression in cHL cell lines with the DNA demethylating agent 5-aza-deoxycytidine (5-aza-dC). Using methylation-specific polymerase chain reaction (MSP), bisulfite genomic sequencing, and digestion with methylation-sensitive endonuclease followed by polymerase chain reaction (PCR), we determined the methylation status of promoter regions of PU.1, BOB.1/OBF.1, CD19, SYK, and CD79B. Down-regulation of transcription typically correlated with hypermethylation. Using bisulfite genomic sequencing we found that in microdissected HRS cells of primary cHL SYK, BOB.1/OBF.1, and CD79B promoters were also hypermethylated. Ectopic expression of both Oct2 and PU.1 in a cHL cell line potentiated endogenous PU.1 and SYK expression after 5-aza-dC treatment. These observations indicate that silencing of the B-cell-specific genes in cHL may be the consequence of a compromised regulatory network where down-regulation of a few master transcription factors results in silencing of numerous genes.

Azacitidine↗
PubMed2000

MethTools--a toolbox to visualize and analyze DNA methylation data.

The Bisulfite Genomic Sequencing technique has found wide acceptance for the generation of DNA-methylation maps with single-base resolution. The method is based on the selective deamination of cytosine to uracil (and subsequent conversion to thymine via PCR), whereas 5-methylcytosine residues remain unchanged. Methylation maps are created by the comparison of bisulfite converted sequences with the untreated genomic sequence. 'MethTools' is a collection of software tools that replaces the time-consuming manual comparison process, generates graphical outputs of methylation patterns and methyl-ation density, estimates the systematic error of the experiment and searches for conserved methylated nucleotide patterns. The programs are written in Perl 5 and C, and the source code can be downloaded. All tools run independently but the programs are interfaced. Thus, a script can perform the entire analysis procedure automatically. In addition, a web-based remote analysis service is offered. Both the source code and the remote analysis are available at http://genome.imb-jena.de/methtools/

Animals↗
PubMed2002

Association of H19 promoter methylation with the expression of H19 and IGF-II genes in adrenocortical tumors.

Low H19 and abundant IGF-II expression may have a role in the development of adrenocortical carcinomas. In the mouse, the H19 promoter area has been found to be methylated when transcription of the H19 gene is silent and unmethylated when it is active. We used PCR-based methylation analysis and bisulfite genomic sequencing to study the cytosine methylation status of the H19 promoter region in 16 normal adrenals and 30 pathological adrenocortical samples. PCR-based analysis showed higher methylation status at three HpaII-cutting CpG sites of the H19 promoter in adrenocortical carcinomas and in a virilizing adenoma than in their adjacent normal adrenal tissues. Bisulfite genomic sequencing revealed a significantly higher mean degree of methylation at each of 12 CpG sites of the H19 promoter in adrenocortical carcinomas than in normal adrenals (P < 0.01 for all sites) or adrenocortical adenomas (P < 0.01, except P < 0.05 for site 12 and P > 0.05 for site 11). The mean methylation degree of the 12 CpG sites was significantly higher in the adrenocortical carcinomas (mean +/- SE, 76 +/- 7%) than in normal adrenals (41 +/- 2%) or adrenocortical adenomas (45 +/- 3%; both P < 0.005). RNA analysis indicated that the adrenocortical carcinomas expressed less H19 but more IGF-II RNAs than normal adrenal tissues did. The mean methylation degree of the 12 H19 promoter CpG sites correlated negatively with H19 RNA levels (r = -0.550; P < 0.01), but positively with IGF-II mRNA levels (r = 0.805; P < 0.001). In the adrenocortical carcinoma cell line NCI-H295R, abundant IGF-II, but minimal H19, RNA expression was detected by Northern blotting. Treatment with a cytosine methylation inhibitor, 5-aza-2'-deoxycytidine, increased H19 RNA expression, whereas it decreased IGF-II mRNA accumulation dose- and time-dependently (both P < 0.005) and reduced cell proliferation to 10% in 7 d. Our results suggest that altered DNA methylation of the H19 promoter is involved in the abnormal expression of both H19 and IGF-II genes in human adrenocortical carcinomas.

Adenoma↗