Restriction Landmark Genomic Scanning Research Articles

DNA Methylation Profiles of Donor Nuclei Cells and Tissues of Cloned Bovine Fetuses

Methylation of DNA in CpG islands plays an important role during fetal development and differentiation because CpG islands are preferentially located in upstream regions of mammalian genomic DNA, including the transcription start site of housekeeping genes and are also associated with tissue-specific genes. Somatic nuclear transfer (NT) technology has been used to generate live clones in numerous mammalian species, but only a low percentage of nuclear transferred animals develop to term. Abnormal epigenetic changes in the CpG islands of donor nuclei after nuclear transfer could contribute to a high rate of abortion during early gestation and increase perinatal death. These changes have yet to be explored. Thus, we investigated the genome-wide DNA methylation profiles of CpG islands in nuclei donor cells and NT animals. Using Restriction Landmark Genomic Scanning (RLGS), we showed, for the first time, the epigenetic profile formation of tissues from NT bovine fetuses produced from cumulus cells. From approximately 2600 unmethylated NotI sites visualized on the RLGS profile, at least 35 NotI sites showed different methylation statuses. Moreover, we proved that fetal and placental tissues from artificially inseminated and cloned cattle have tissue-specific differences in the genome-wide methylation profiles of the CpG islands. We also found that possible abnormalities occurred in the fetal brain and placental tissues of cloned animals.

Multiple forms of genetic instability within a 2‐Mb chromosomal segment of 3q26.3–q27 are associated with development of esophageal adenocarcinoma

Gene amplification is one of the mechanisms to activate oncogenes in many cancers, including esophageal adenocarcinoma (EA). In the present study, we used two-dimensional restriction landmark genome scanning to clone a NotI/DpnII fragment that showed increased genomic dosage in 1 of 44 EAs analyzed. This fragment maps to 3q26.3-q27, and subsequent experiments identified two intrachromosomal amplicons within a 10-Mb DNA segment in 7 of 75 (9%) EAs. The distal amplified-core region maps centromeric to the PIK3CA locus, and a microsatellite (D3S1754) within this region exhibited significant instability (MSI), in stark contrast to the genomewide microsatellite stability found in EA. D3S1754-MSI arises in premalignant Barrett's dysplastic cells and preceded amplification of the nascent MSI allele in the corresponding EA. Seven ESTs within the amplified-core were overexpressed in amplicon-containing EAs. One of these, EST AW513672, represents a chimeric transcript that initiated from an antisense promoter sequence in the 5'UTR of a full-length LINE-1 element (L1-5'ASP). Similar chimeric transcripts encoding portions of the MET oncogene and the BCAS3 gene also were overexpressed in EAs, suggesting that L1-5'ASP activation may occur at a broad level in primary EAs. Thus, the fine dissection of a 2-Mb amplified DNA segment in 3q26.3-q27 in EA revealed multiple genetic alterations that had occurred sequentially and/or concurrently during EA development.

DNA copy number gains in head and neck squamous cell carcinoma

Gene amplification, a common mechanism for oncogene activation in cancer, has been used as a tag for the identification of novel oncogenes. DNA amplification is frequently observed in head and neck squamous cell carcinoma (HNSCC) and potential oncogenes have already been reported. We applied restriction landmark genome scanning (RLGS) to study gene amplifications and low-level copy number changes in HNSCC in order to locate previously uncharacterized regions with copy number gains in primary tumor samples. A total of 63 enhanced RLGS fragments, indicative of DNA copy number changes, including gains of single alleles, were scored. Enhanced sequences were identified from 33 different chromosomal regions including those previously reported (e.g. 3q26.3 and 11q13.3) as well as novel regions (e.g. 3q29, 8q13.1, 8q22.3, 9q32, 10q24.32, 14q32.32, 17q25.1 and 20q13.33). Furthermore, our data suggest that amplicons 11q13.3 and 3q26.3-q29 may be divided into possibly two and three independent amplicons, respectively, an observation supported by published microarray expression data.

CpG island hypermethylation profiling of lung cancer using restriction landmark genomic scanning (RLGS) analysis

Lung cancer remains the leading cause of cancer related mortality, accounting for almost one-third of cancer deaths in men and one-fourth of cancer deaths in women; 160,440 lung cancer deaths are expected in 2004. Survival from lung cancer depends mainly upon the stage at presentation. As localized tumors generally do not cause symptoms, the disease is usually diagnosed in symptomatic patients at advanced stages when the prognosis is poor. As a result, the overall 5-year lung cancer survival rate is only 15%. It is well known that epigenetic alterations such as DNA methylation of CpG dinucleotides located in CpG islands within the regulatory (promoter) regions of genes are associated with transcriptional silencing in cancer. Promoter hypermethylation of critical pathway genes could identify potential biomarkers for lung cancer risk. Our goal for this study is to identify novel hypermethylated genes in lung cancer. We have investigated the methylation profiles of DNA samples from 14 paired lung tumor and adjacent normal tissues resected from the same individuals using restriction landmark genomic scanning (RLGS). We could assess the DNA methylation status of an average of 2,012 CpG islands for each tumor. We identified 162 differentially methylated loci where CpG islands were hypermethylated in lung tumors but not in adjacent non-cancer tissues. Among 162 sites of differential DNA methylation, detected from at least one tumor/normal pair, 21 hypermethylated genes were identified that were not reported previously as hypermethylated in lung tumor tissue.

TWIST2 Demonstrates Differential Methylation in Immunoglobulin Variable Heavy Chain Mutated and Unmutated Chronic Lymphocytic Leukemia

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. 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. 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. 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.

Association of tissue-specific differentially methylated regions (TDMs) with differential gene expression

Early studies proposed that DNA methylation could have a role in regulating gene expression during development [Riggs, A.D. (1975) Cytogenet. Cell Genet. 14, 9-25]. However, some studies of DNA methylation in known tissue-specific genes during development do not support a major role for DNA methylation. In the results presented here, tissue-specific differentially methylated regions (TDMs) were first identified, and then expression of genes associated with these regions correlated with methylation status. Restriction landmark genomic scanning (RLGS) was used in conjunction with virtual RLGS to identify 150 TDMs [Matsuyama, T., Kimura, M.T., Koike, K., Abe, T., Nakao, T., Asami, T., Ebisuzaki, T., Held, W.A., Yoshida, S. & Nagase, H. (2003) Nucleic Acids Res. 31, 4490-4496]. Analysis of 14 TDMs by methylation-specific PCR and by bisulfite genomic sequencing confirms that the regions identified by RLGS are differentially methylated in a tissue-specific manner. The results indicate that 5% or more of the CpG islands are TDMs, disputing the general notion that all CpG islands are unmethylated. Some of the TDMs are within 5' promoter CpG islands of genes, which exhibit a tissue-specific expression pattern that is consistent with methylation status and a role in tissue differentiation.

Global assessment of promoter methylation in a mouse model of cancer identifies ID4 as a putative tumor-suppressor gene in human leukemia

DNA methylation is associated with malignant transformation, but limitations imposed by genetic variability, tumor heterogeneity, availability of paired normal tissues and methodologies for global assessment of DNA methylation have limited progress in understanding the extent of epigenetic events in the initiation and progression of human cancer and in identifying genes that undergo methylation during cancer. We developed a mouse model of T/natural killer acute lymphoblastic leukemia that is always preceded by polyclonal lymphocyte expansion to determine how aberrant promoter DNA methylation and consequent gene silencing might be contributing to leukemic transformation. We used restriction landmark genomic scanning with this mouse model of preleukemia reproducibly progressing to leukemia to show that specific genomic methylation is associated with only the leukemic phase and is not random. We also identified Idb4 as a putative tumor-suppressor gene that is methylated in most mouse and human leukemias but in only a minority of other human cancers.

306 ANALYSIS OF GLOBAL DNA METHYLATION IN BOVINE SPERMATOZOA

Methylation of cytosine base in the CpG dinucleotide sequence (DNA methylation) is a major epigenetic modification of the genome and plays an important role in gene expression. Recently, global DNA methylation in genome was studied by using a restriction landmark genomic scanning (RLGS) method and/or a representational difference analysis (RDA) method. However, these methods are complicated and need to use restriction enzymes. Therefore, the information derived from those methods is restricted to the region of the DNA sequence which is able to be cleaved by restriction enzymes. In this study, to establish a simple method to estimate global DNA methylation level in bovine spermatozoa, we tried to develop the DNA methylation analyzing method by using immunostaining of 5-methylcytosine. The immunostaining method for 5-methylcytosine in this study was based on the method developed by Benchaib et al. (2003 Fertil. Steril. 80, 947-952) for human spermatozoa. Because of the species difference, we modified some treatments to apply to bovine spermatozoa. Frozen-thawed bovine spermatozoa were washed by using 30 and 45% Percoll gradient solutions. After washing, spermatozoa were treated with 0.25 dithicthreitol M (DTT) and 1% sodium dodecyl sulfate (SDS) at room temperature (RT). Then, treated spermatozoa were spread on a slide glass with Cytospin4 (30g, 5 � 104 cells/mL) and air-dried at RT. Air-dried bovine spermatozoa specimens were fixed in methanol: glacial acetic acid (3:1) solution at RT and treated with 1% Triton X and 1% SDS at RT; DNA was denatured with 6 N HCl at RT. After the denaturation, 5-methylcytosine in sperm DNA was analyzed by immunofluorescence technique with mouse anti 5-methylcytosine antibody and fluorescein isothiocyanate (FITC)-conjugated goat anti mouse IgG antibody. The total sperm DNA was counterstained with propidium iodide (PI). Stained samples were observed with a confocal laser scanning microscope and obtained images were analyzed with fluorescence image analysis software. The area that was clearly stained with PI in each sperm head was designated and measured as the area of total sperm DNA, and the number of the dots that showed FITC fluorescence within the total sperm DNA area was designated and measured as the area of 5-methylcytosine in total sperm DNA. The area measurement was performed with fixed light strength. Three bovine spermatozoa samples derived from different bulls, used daily for calf production by AI, were examined. The ratio of the mean total area of the 5-methylcytosine in sperm DNA to the mean total area of the sperm DNA was 34.1% in bull A (9.13 � 5.66 �m2, 26.75 � 5.29 �m2, n = 57), 45.2% in bull B (16.60 � 3.79 �m2, 36.74 � 5.95 �m2, n = 41) and 43.9% in bull C (14.66 � 4.27 �m2, 33.45 � 7.13 �m2, n = 22). There was significant difference in the ratio between bull A and bulls B and C (P &lt; 0.01). More research is required to evaluate the meaning of this individual difference of DNA methylation between bulls. This work was supported by Wakayama Prefecture Collaboration of Regional Entities for the Advanced of Technological Excellence, Japan, and by a Grant-in-Aid for the 21st Century Center of Excellence Program of the MEXT, Japan.

Global Assessment of Promoter Methylation in a Murine Model of Cancer Identifies ID4 as a Putative Novel Tumor Suppressor Gene in Human Leukemia.

We developed a mouse model of acute T/natural killer (NK) acute lymphoblastic leukemia (ALL) that is always preceded by polyclonal lymphocyte expansion to determine how aberrant promoter DNA methylation and consequent gene silencing might be contributing to leukemic transformation. We demonstrated the non-random nature of methylation in the transformation from benign polyclonal expansion to leukemia, and identified a novel tumor suppressor gene silenced in the majority of human leukemia but not solid tumors. To this, we utilized restriction landmark genomic scanning (RLGS) on eight IL-15 transgenic (tg) mice with T/NK ALL, and performed RLGS on an additional five spleens from IL-15tg mice without ALL, but with polyclonal T/NK expansion, and on four spleens from wild type (wt) mice. A total of 2447 RLGS fragments with good resolution on each RLGS profile were analyzed. Only 1–2 variable changes were detected in either wt or polyclonal T/NK expansion controls. In contrast, the eight spleens with clonal T/NK leukemia had 45 to 209 changes (1.8 to 8.5% of total fragments) consistent with aberrant DNA methylation. The association of RLGS fragment losses or reduced intensity with leukemic transformation versus polyclonal expansion was highly significant (P<0.001). The promoter of Inhibitor of DNA binding 4 (Id4) was found methylated in 87.5% of the mouse T/NK ALLs. ID4 was found methylated in 85.7% (72/84) of primary human acute myeloid leukemias (AML) and in 100% (61/61) of chronic lymphocytic leukemias (CLL). Id4 was silenced secondary to promoter methylation in 100% mouse T/NK ALLs (n=5) and human acute leukemias (n=6). Overexpression of Id4 induced apoptosis in mouse cell line YAC-1 and inhibited tumor growth both in vitro and in vivo. These data, and an analysis of 219 RLGS profiles from different types of human tumors demonstrate that the ID4 promoter is preferentially methylated and functions as a putative novel tumor suppressor gene in mouse and human leukemia. This study highlights the use of a novel animal model, novel mouse NotI-EcoRV arrayed library, and RLGS to examine the role of methylation in leukemic transformation and to identify new targets for prevention and therapeutic strategies.

Methylation of the Tumor Suppressor Gene PTPRO (Receptor-Type Protein Tyrosine Phosphatase) Is Associated with Expression of Important Apoptosis-Related Proteins in Chronic Lymphocytic Leukemia (CLL).

We previously demonstrated that apoptosis-inhibiting proteins (e.g., Bcl-2, Mcl-1, and XIAP) are important in predicting response to chemotherapy and antibody therapy for CLL. Additionally, genetic abnormalities and epigenetic control of gene expression have become increasingly important in understanding the prognosis and potential therapeutic opportunities in this disease. Greater than 80% of patients with CLL have genetic abnormalities detectable by FISH. Important subsets include those with a less favorable prognosis (deletions of 17p, 11q), an intermediate prognosis (trisomy 12), or a favorable outcome (deletion of 13q). PTPRO, encoding a receptor-type tyrosine phosphatase, is a specific tumor suppressor gene located in chromosomal region 12p12.3. PTPRO has been highly characterized as inhibiting anchorage-independent tumor growth in human lung carcinomas. This gene has been found to be silenced by promoter methylation, and can be re-expressed by hypomethylating agents in human lung tumor models. Thus, we examined the methylation profile of the promoter region of PTPRO in primary leukemic cells from patients with CLL, and in B cells obtained from normal volunteers. Analysis of the PTPRO CpG island by methylation-specific PCR and combined bisulfite restriction analysis (COBRA) demonstrated that this region was methylated in 91% of samples from 46 patients, whereas it was unmethylated in control B cells from normal volunteers. Specific quantitative determinations of important proteins related to apoptosis in the leukemic cells from 20 of these patients showed that, in the group with methylation of the PTPRO promoter, there was a significant elevation of Bcl-2 (p=0.079), Mcl-1 (p=0.004), XIAP (p=0.048), Bcl-2/BAX ratio (p=0.0024), and Mcl-1/BAX ratio (p=0.0017). There was no apparent relationship with FISH category or immunoglobulin VH mutational status. Thus, PTPRO promoter methylation analysis may identify a subset of CLL patients less likely to respond to therapy, and provide a potential reversible target for new therapeutic approaches to this disease. Finally, analysis of methylation of CLL samples by restriction landmark genome scanning also revealed methylation of genes for three additional protein tyrosine phosphatases: PTPRN2, PTPRZ2, and PTPN11. Increases in methylation of two of these (PTPRN2 and PTPRZ2) in CLL samples relative to control B cells was confirmed by COBRA. These results provide additional potential molecular targets for CLL therapy.

Identification of an epigenetically silenced gene, RFX1, in human glioma cells using restriction landmark genomic scanning.

To identify the CpG islands differentially methylated in human glioma, we performed restriction landmark genomic scanning with a CpG methylation-sensitive enzyme. We found 12 spots, the intensity of which was entirely lost or decreased in both the human glioma tissues examined as compared with that in matched normal lymphocytes, indicating aberrant methylation of these CpG islands in gliomas. The expression of RFX1, one of the genes associated with the methylated CpG islands, was frequently decreased in human glioma cell lines and tissues. We also demonstrated that the isolated CpG island located in the seventh intron of the RFX1 gene had enhancer activity and was hypermethylated in all of the glioma tissues and cell lines analysed, but not in normal brains or lymphocytes. Treatment of glioma cells with a demethylating agent, 5-azacytidine, resulted in the expression of RFX1, indicating that the silencing of the RFX1 gene may be attributable to its methylation. RFX1 has been implicated in transcriptional downregulation of the proto-oncogene c-myc. By expression of the RFX1 gene, the cellular proliferative activity of glioma cells was suppressed. Taken together, these results suggest that the RFX1 gene may be epigenetically silenced in human gliomas and involved in glioma tumorigenesis.

Preference of DNA methyltransferases for CpG islands in mouse embryonic stem cells.

Many CpG islands have tissue-dependent and differentially methylated regions (T-DMRs) in normal cells and tissues. To elucidate how DNA methyltransferases (Dnmts) participate in methylation of the genomic components, we investigated the genome-wide DNA methylation pattern of the T-DMRs with Dnmt1-, Dnmt3a-, and/or Dnmt3b-deficient ES cells by restriction landmark genomic scanning (RLGS). Approximately 1300 spots were detected in wild-type ES cells. In Dnmt1(-/-) ES cells, additional 236 spots emerged, indicating that the corresponding loci are methylated by Dnmt1 in wild-type ES cells. Intriguingly, in Dnmt3a(-/-)Dnmt3b(-/-) ES cells, the same 236 spots also emerged, and no additional spots appeared differentially. Therefore, Dnmt1 and Dnmt3a/3b share targets in CpG islands. Cloning and virtual image RLGS revealed that 81% of the RLGS spots were associated with genes, and 62% of the loci were in CpG islands. By contrast to the previous reports that demethylation at repeated sequences was severe in Dnmt1(-/-) cells compared with Dnmt3a(-/-)Dnmt3b(-/-) cells, a complete loss of methylation was observed at RLGS loci in Dnmt3a(-/-)Dnmt3b(-/-) cells, whereas methylation levels only decreased to 16% to 48% in the Dnmt1(-/-) cells. We concluded that there are CpG islands with T-DMR as targets shared by Dnmt1 and Dnmt3a/3b and that each Dnmt has target preferences depending on the genomic components.

A NotI– EcoRV promoter library for studies of genetic and epigenetic alterations in mouse models of human malignancies

Aberrant promoter methylation and associated chromatin changes are primarily studied in human malignancies. Thus far, mouse models for human cancer have been rarely utilized to study the role of DNA methylation in tumor onset and progression. It would be advantageous to use mouse tumor models to a greater extent to study the role and mechanism of DNA methylation in cancer because mouse models allow manipulation of the genome, study of samples/populations with a homogeneous genetic background, the possibility of modulating gene expression in vivo, the statistical power of using large numbers of tumor samples, access to various tumor stages, and the possibility of preclinical trials. Therefore, it is likely that the mouse will emerge as an increasingly utilized model to study DNA methylation in cancer. To foster the use of mouse models, we developed an arrayed mouse NotI– EcoRV genomic library, with clones from three commonly used mouse strains (129SvIMJ, FVB/NJ, and C57BL/6J). A total of 23,040 clones representing an estimated three- to fourfold coverage of the mouse genome were arrayed in 60 × 384-well plates. We developed restriction landmark genomic scanning (RLGS) mixing gels with 32 plates to enable the cloning of methylated sequences from RLGS profiles run with NotI– EcoRV– HinfI. RLGS was used to study aberrant methylation in two mouse models that overexpressed IL-15 or c-Myc and developed either T/NK-cell leukemia or T-cell lymphomas, respectively. Careful analysis of 198 sequences showed that 188 (94.9%) identified CpG-island sequences, 132 sequences (66.7%) had homology to the 5′ regions of known genes or mRNAs, and all 132 NotI– EcoRV clones were located at the same CpG islands with the predicted promoter sequences. We have also developed a modified pGL3-based luciferase vector that now contains the NotI, AscI, and EcoRV restriction sites and allows the rapid cloning of NotI– EcoRV library fragments in both orientations. Luciferase assays using NotI– EcoRV clones confirmed that the library is enriched for promoter sequences. Thus, this library will support future genetic and epigenetic studies in mouse models.

Methylation of the mitotic checkpoint genes BUB3 and BUBR1 in lung cancer

9558 Background: The mitotic checkpoint is a failsafe mechanism for the cell to ensure accurate chromosome segregation during mitosis. Mutations in genes encoding essential checkpoint proteins lead to chromosome instability and promote carcinogenesis. The BUB3 and BUBR1 genes are essential components of the mitotic checkpoint pathway. AIM: To determine the role of aberrant promoter methylation of the genes BUB3 and BUBR1 in lung cancer. Materials and Methods: We performed Restriction Landmark Genomic Scanning (RLGS) in 20 Non-Small Cell Lung Cancer (NSCLC) samples and found the mitotic checkpoint gene BUR1 to be methylated. We then decided to look for methylation in other members of the BUB family. DNA was isolated from 53 microdissected freshly frozen NSCLC samples. Bisulphite treated DNA was then subjected to MSP following standard techniques. Results: BUR1 was found to be methylated in 47% (25/53) samples and BUB3 to be methylated in 45% (24/53) samples. It was surprising for us to find that the combination of both genes is methylated in 77% of the lung cancer samples analyzed. Conclusions: methylated of the mitotic checkpoint genes BUB3 and BUBR1 is a common epigenetic event in lung cancer. No significant financial relationships to disclose.

Genetic identity of clones and methods to explore DNA.

Cloning by nuclear transfer has made it possible to produce genetically identical animals in terms of nuclear DNA content. Recent molecular biology tools are offering scientific ways to get an insight into the identity issues, by exploring and comparing genomes of cloned animals in order to test their genetic identity and methylation differences. We have initiated a study to compare genomic DNA of bovine adult clones, of normal phenotype. We have used, in parallel, the AFLP technique (amplification fragment length polymorphism) and one of its variant, MSAP (methylation-sensitive amplification polymorphism). We are also investigating other techniques leading to the detection of sequence polymorphisms between two genomes based on genomes hybridisation. We chose the representational difference analysis (RDA) methods that can be combined with mismatch-specific recognition or mismatch binding property of some proteins (CEL I, MutS). We plan to use these RDA methods for genome-wide detection of subtle mutations, then to focus on changes affecting the methylation status of promoting genomic regions in abnormal clones. This will be achieved using MSAP with NotI and applying, in parallel, the RLGS (restriction landmark genome scanning) technique. This study will hopefully improve the molecular and functional characterizations of these "new animals."

Microarray analysis of epigenetic silencing of gene expression in the KAS-6/1 multiple myeloma cell line.

The epigenetic control of gene transcription in cancer has been the theme of many recent studies and therapeutic approaches. Carcinogenesis is frequently associated with hypermethylation and consequent down-regulation of genes that prevent cancer, e.g., those that control cell proliferation and apoptosis. We used the demethylating drug zebularine to induce changes in DNA methylation, then examined patterns of gene expression using cDNA array analysis and Restriction Landmark Genomic Scanning followed by RNase protection assay and reverse transcription-PCR to confirm the results. Microarray studies revealed that many genes were epigenetically regulated by methylation. We concluded that methylation decreased the expression of, or silenced, several genes, contributing to the growth and survival of multiple myeloma cells. For example, a number of genes (BAD, BAK, BIK, and BAX) involved in apoptosis were found to be suppressed by methylation. Sequenced methylation-regulated DNA fragments identified by Restriction Landmark Genomic Scanning were found to contain CpG islands, and some corresponded to promoters of genes that were regulated by methylation. We also observed that after the removal of the demethylating drug, the addition of interleukin 6 restored CpG methylation and re-established previously silenced gene patterns, thus implicating a novel role of interleukin 6 in processes regulating epigenetic gene repression and carcinogenesis.

Reevaluation of gene amplification in medulloblastomas

Introduction: We have recently described novel gene loci amplified in medulloblastomas detected by Restriction Landmark Genomic Scanning. The relevance of the corresponding genes has thus far not been determined. The rare occurence of amplifications in medulloblastomas makes it necessary to use large „cohorts“ of tumor samples, available only as paraffin sections. To this end we introduced a real-time PCR method examining DNA derived from paraffin sections.

A genome scanning approach to assess the genetic effects of radiation in mice and humans.

We used Restriction Landmark Genome Scanning (RLGS) to assess, on a genome-wide basis, the mutation induction rate in mouse germ cells after radiation exposure. Analyses of 1,115 autosomal NotI DNA fragments per mouse for reduced spot intensity, indicative of loss of one copy, in 506 progeny derived from X-irradiated spermatogonia (190, 237 and 79 mice in 0-, 3-, and 5-Gy groups, respectively), permitted us to identify 16 mutations affecting 23 fragments in 20 mice. The 16 mutations were composed of eight small changes (1-9 bp) at microsatellite sequences, five large deletions (more than 25 kb), and three insertions of SINE B2 or LINE1 transposable elements. The maximum induction rate of deletion mutations was estimated as (0.17 +/- 0.09) x 10(-5)/locus Gy(-1). The estimate is considerably lower than 1 x 10(-5)/locus Gy(-1), the mean induction rate of deletion mutations at Russell's 7 loci, which assumed that deletion mutations comprise 50% of all mutations. We interpret the results as indicating that the mean induction rate of mutations in the whole genome may be substantially lower than that at the 7 loci. We also demonstrate the applicability of RLGS for detection of human mutations, which allows direct comparisons between the two species.

Epigenetic profiling in chronic lymphocytic leukemia reveals novel methylation targets.

CpG island methylation is an epigenetic alteration that contributes to tumorigenesis by transcriptional inactivation of genes. Little is known about the overall levels of CpG island methylation in chronic lymphocytic leukemia (CLL). To provide a baseline estimate of global aberrant methylation and identify target sequences for additional investigation, we performed Restriction Landmark Genomic Scanning on 10 CLL samples. Two methylation-sensitive landmark enzymes were used (NotI and AscI), allowing assessment of over 3000 CpG islands in each sample. Tumor-derived Restriction Landmark Genomic Scanning profiles were compared with profiles from CD19-selected B cells from normal volunteers and matched normal neutrophils from 4 CLL patients. We found 2.5-8.1% (mean 4.8%) of the CpG islands in CLL samples were aberrantly methylated compared with controls, and the methylation events had a nonrandom distribution (P < 0.0001). Furthermore, we identified 193 aberrantly methylated sequences, of which 93% have CpG island characteristics and 90% have homology to genes or expressed sequences. One such gene, the G protein-coupled metabotropic glutamate receptor 7 (GRM7), possibly inhibits cyclic AMP signaling in the induction of apoptosis. Bisulfite sequencing of GRM7 confirmed extensive CpG island methylation, and treatment with 5-aza-2'-deoxycytidine (decitabine) resulted in up-regulated expression of several genes in vitro with concurrent cellular depletion of DNMT1 protein. Our dual-enzyme global methylation study shows that CLL is characterized by widespread nonrandom CpG island methylation similar to other tumors and provides a panel of novel methylation targets that can be used in larger studies designed to assess impact on disease progression and survival.

Distinct sequences on 11q13.5 and 11q23–24 are frequently coamplified with MLL in complexly organized 11q amplicons in AML/MDS patients

Amplification within chromosome arm 11q involving the mixed-lineage leukemia gene (MLL) locus is a rare but recurrent aberration in acute myeloid leukemia and myelodysplastic syndrome (AML/MDS). We and others have observed that 11q amplifications in most AML/MDS cases have not been restricted to the chromosomal region surrounding the MLL gene. Therefore, we implemented a strategy to characterize comprehensively 11q amplicons in a series of 13 AML/MDS patients with MLL amplification. Analysis of 4 of the 13 cases by restriction landmark genomic scanning in combination with virtual genome scan and by matrix-based comparative genomic hybridization demonstrated that the 11q amplicon in these four cases consisted of at least three discontinuous sequences derived from different regions of the long arm of chromosome 11. We defined a maximally 700-kb sequence around the MLL gene that was amplified in all cases. Apart from the core MLL amplicon, we detected two additional 11q regions that were coamplified. Using fluorescence in situ hybridization (FISH) analysis, we demonstrated that sequences in 11q13.5 and 11q23-24 were amplified in 8 of 13 and 10 of 12 AML/MDS cases, respectively. Both regions harbor a number of potentially oncogenic genes. In all 13 cases, either one or both of these regions were coamplified with the MLL amplicon. Thus, we demonstrated that 11q amplicons in AML/MDS patients display a complex organization and have provided evidence for coamplification of two additional regions on the long arm of chromosome 11 that may harbor candidate target genes.