Restriction Landmark Genomic Scanning Research Articles

Sperm DNA methylome abnormalities occur both pre- and post-treatment in men with Hodgkin disease and testicular cancer

BackgroundCombination chemotherapy has contributed to increased survival from Hodgkin disease (HD) and testicular cancer (TC). However, questions concerning the quality of spermatozoa after treatment have arisen. While studies have shown evidence of DNA damage and aneuploidy in spermatozoa years following anticancer treatment, the sperm epigenome has received little attention. Our objectives here were to determine the impact of HD and TC, as well as their treatments, on sperm DNA methylation. Semen samples were collected from community controls (CC) and from men undergoing treatment for HD or TC, both before initiation of chemotherapy and at multiple times post-treatment. Sperm DNA methylation was assessed using genome-wide and locus-specific approaches.ResultsImprinted gene methylation was not affected in the sperm of HD or TC men, before or after treatment. Prior to treatment, using Illumina HumanMethylation450 BeadChip (450 K) arrays, a subset of 500 probes was able to distinguish sperm samples from TC, HD and CC subjects; differences between groups persisted post-treatment. Comparing altered sperm methylation between HD or TC patients versus CC men, twice as many sites were affected in TC versus HD men; for both groups, the most affected CpGs were hypomethylated. For TC patients, the promoter region of GDF2 contained the largest region of differential methylation. To assess alterations in DNA methylation over time/post-chemotherapy, serial samples from individual patients were compared. With restriction landmark genome scanning and 450 K array analyses, some patients who underwent chemotherapy showed increased alterations in DNA methylation, up to 2 to 3 years post-treatment, when compared to the CC cohort. Similarly, a higher-resolution human sperm-specific assay that includes assessment of environmentally sensitive regions, or “dynamic sites,” also demonstrated persistently altered sperm DNA methylation in cancer patients post-treatment and suggested preferential susceptibility of “dynamic” CpG sites.ConclusionsDistinct sperm DNA methylation signatures were present pre-treatment in men with HD and TC and may help explain increases in birth defects reported in recent clinical studies. Epigenetic defects in spermatozoa of some cancer survivors were evident even up to 2 years post-treatment. Abnormalities in the sperm epigenome both pre- and post-chemotherapy may contribute to detrimental effects on future reproductive health.

Stability of the human sperm DNA methylome to folic acid fortification and short-term supplementation.

Do short-term and long-term exposures to low-dose folic acid supplementation alter DNA methylation in sperm? No alterations in sperm DNA methylation patterns were found following the administration of low-dose folic acid supplements of 400 μg/day for 90 days (short-term exposure) or when pre-fortification of food with folic acid and post-fortification sperm samples (long-term exposure) were compared. Excess dietary folate may be detrimental to health and DNA methylation profiles due to folate's role in one-carbon metabolism and the formation of S-adenosyl methionine, the universal methyl donor. DNA methylation patterns are established in developing male germ cells and have been suggested to be affected by high-dose (5 mg/day) folic acid supplementation. This is a control versus treatment study where genome-wide sperm DNA methylation patterns were examined prior to fortification of food (1996-1997) in men with no history of infertility at baseline and following 90-day exposure to placebo (n = 9) or supplement containing 400 μg folic acid/day (n = 10). Additionally, pre-fortification sperm DNA methylation profiles (n = 19) were compared with those of a group of post-fortification (post-2004) men (n = 8) who had been exposed for several years to dietary folic acid fortification. Blood and seminal plasma folate levels were measured in participants before and following the 90-day treatment with placebo or supplement. Sperm DNA methylation was assessed using the whole-genome and genome-wide techniques, MassArray epityper, restriction landmark genomic scanning, methyl-CpG immunoprecipitation and Illumina HumanMethylation450 Bead Array. Following treatment, supplemented individuals had significantly higher levels of blood and seminal plasma folates compared to placebo. Initial first-generation genome-wide analyses of sperm DNA methylation showed little evidence of changes when comparing pre- and post-treatment samples. With Illumina HumanMethylation450 BeadChip arrays, no significant changes were observed in individual probes following low-level supplementation; when compared with those of the post-fortification cohort, there were also few differences in methylation despite exposure to years of fortified foods. Illumina HumanMethylation450 BeadChip data from this study have been submitted to the NCBI Gene Expression Omnibus under the accession number GSE89781. This study was limited to the number of participants available in each cohort, in particular those who were not exposed to early (pre-1998) fortification of food with folic acid. While genome-wide DNA methylation was assessed with several techniques that targeted genic and CpG-rich regions, intergenic regions were less well interrogated. Overall, our findings provide evidence that short-term exposure to low-dose folic acid supplements of 400 μg/day, over a period of 3 months, a duration of time that might occur during infertility treatments, has no major impact on the sperm DNA methylome. This work was supported by a grant to J.M.T. from the Canadian Institutes of Health Research (CIHR: MOP-89944). The authors have no conflicts of interest to declare.

Left-sided early-onset vs late-onset colorectal carcinoma: histologic, clinical, and molecular differences.

Carcinomas of the left colon represent a neoplasm of older patients (late onset), but epidemiologic evidence has been showing an increasing incidence in patients 50 years or younger (early onset). In this study, we investigate pathologic and molecular features of early- and late-onset carcinoma of the left colon. We selected 22 patients 50 years or younger and 21 patients 70 years or older with left-sided colorectal carcinoma (CRC). All samples were evaluated for pathologic features, microsatellite instability, and KRAS and BRAF mutations. Moreover, both groups were analyzed to identify CpG island methylator phenotype features and assessed with restriction landmark genome scanning (RLGS) to unveil differential DNA methylation patterns. Early-onset patients had advanced pathologic stages compared with late-onset patients (P = .0482). All cases showed a microsatellite stable profile and BRAF wild-type sequence. Early-onset patients (43%) more frequently had mutations at KRAS codon 12 compared with late-onset patients (14%) (P =.0413). RLGS showed that patients younger than 50 years who had CRC had a significantly lower percentage of methylated loci than did patients 70 years or older (P = .04124), and differential methylation of several genomic loci was observed in the two groups. Our results suggest that left-sided CRCs may present differential patterns of aberrant DNA methylation when they are separated by age.

Epigenetic silencing of BTB and CNC homology 2 and concerted promoter CpG methylation in gastric cancer

BTB and CNC homology 2 (BACH2) is a lymphoid-specific transcription factor with a prominent role in B-cell development. Genetic polymorphisms within a single locus encoding BACH2 are associated with various autoimmune diseases and allergies. In this study, restriction landmark genomic scanning revealed methylation at a NotI site in a CpG island covering the BACH2 promoter in gastric cancer cell lines and primary gastric tumors. Increased methylation of the BACH2 promoter was observed in 52% (43/83) of primary gastric tumors, and BACH2 hypermethylation was significantly associated with decreased gene expression. Treatment with 5-aza-2′-deoxycytidine and/or trichostatin. A restored BACH2 expression in BACH2-silenced gastric cancer cell lines, and knockdown of BACH2 using short hairpin RNA (i.e. RNA interference) increased cell proliferation in gastric cancer cells. Clinicopathologic data showed that decreased BACH2 expression occurred significantly more frequently in intestinal-type (27/44, 61%) compared with diffuse-type (13/50, 26%) gastric cancers (P<0.001). Furthermore, BACH2 promoter methylation paralleled that of previously identified targets, such as LRRC3B, LIMS2, PRKD1 and POPDC3, in a given set of gastric tumors. We propose that concerted methylation in many promoters plays a role in accelerating gastric tumor formation and that methylated promoter loci may be targets for therapeutic treatment, such as the recently introduced technique of epigenetic editing.

More of a good thing or less of a bad thing: gene copy number variation in polyploid cells of the placenta.

More of a good thing or less of a bad thing: gene copy number variation in polyploid cells of the placenta.

Genome‐wide screening of aberrant DNA methylation which associated with gene expression in mouse skin cancers

Epigenetic alteration of genomic DNA is a common and key process in carcinogenesis. There is considerable evidence indicating that some of the somatic alterations occurring during carcinogenesis in humans also involve the same processes as those observed in mice. Therefore, we analyzed mouse skin cancer tissues induced by the 2-stage carcinogenesis model to identify skin tumor-specific differentially methylated regions (ST-DMRs) during the multistep carcinogenesis process. We have previously identified ST-DMRs using the restriction landmark genomic scanning (RLGS) technique and reported that some of the mouse ST-DMRs were also epigenetically modified in human cancers, such as melanoma, neuroblastoma, and brain tumor. These results encouraged us to pursue global methylation screening in mouse skin carcinogenesis. Using the methylated DNA immunoprecipitation (MeDIP) method combined with the NimbleGen promoter plus CpG island (CpGi) array, we identified 615 ST-DMRs. In combination with global gene expression analysis, 91 of these ST-DMRs were shown to be located on or around the genes differentially expressed between normal skin and tumor tissues, including a candidate human tumor suppressor gene Tfap2e. As observed in human colorectal cancers, Tfap2e was methylated at a CpGi located in intron 3 and downregulated in skin tumors. Our results identified aberrant methylated regions that were associated with gene expression regulation during carcinogenesis, which may indicate critical genetic regions also involved in human carcinogenesis. © 2013 Wiley Periodicals, Inc.

The Genetic Risk in Mice from Radiation: An Estimate of the Mutation Induction Rate per Genome

Restriction Landmark Genome Scanning (RLGS) is a method that uses end-labeled (32)P NotI sites that are mostly associated with coding genes to visualizes thousands of DNA fragments as spots in two-dimensional autoradiograms. This approach allows direct detection of autosomal deletions as spots with half normal intensity. The method was applied to mouse offspring derived from spermatogonia exposed to 4 Gy of X rays. A genome-wide assessment of the mutation induction rate was estimated from the detected deletions. Examinations were made of 1,007 progeny (502 derived from control males and 505 from irradiated males) and 1,190 paternal and 1,240 maternal spots for each mouse. The results showed one deletion mutation in the unirradiated paternal genomes of 502 offspring (0.2%) and 5 deletions in the irradiated paternal genomes of 505 offspring (1%). The difference was marginally significant, with the deletion sizes ranged from 2-13 Mb. If the frequencies are taken at face value, the net increase was 0.8% after an exposure of 4 Gy, or 0.2% per Gy per individual if a linear dose response is assumed. Since the present RLGS analysis examined 1,190 NotI sites, while the mouse genome contains ∼25,000 genes, the genomic probability of any gene undergoing a deletion mutation would be 25× 0.2%, or 5% per Gy. Furthermore, since the present RLGS screened about 0.2% of the total genome, the probability of detecting a deletion anywhere in the total genome would be estimated to be 500 times 0.2% or 100% (i.e., 1 deletion per Gy). These results are discussed with reference to copy number variation in the human genome.

Delta-like 3 is silenced by methylation and induces apoptosis in human hepatocellular carcinoma

The genetic and epigenetic events of hepatocarcinogenesis are relatively poorly understood. By analyzing genes from human hepatocellular carcinoma (HCC) with restriction landmark genomic scanning, several aberrantly methylated genes, including Delta-like 3 (DLL3), have been isolated. In this study, we investigated the function of DLL3 in hepatocarcinogenesis. Methylation of the DLL3 gene in HCC cell lines was investigated with methylation-specific PCR and expression of DLL3 mRNA in HCC cells was examined by RT-PCR. Reactivation of DLL3 expression by treatment with a demethylating agent was examined in methylation-silenced HuH2 cells. Human DLL3 cDNA was cloned and DLL3 function was examined by restoring DLL3 expression in HuH2 cells. The effects of DLL3 on cell growth were evaluated by colony formation assay. Induction of cell death by overexpression of DLL3 was examined by flow cytometric assay using Annexin V and PI. Apoptotic cells were detected by TUNEL staining and the amount of single-stranded DNA was measured by ELISA. As a result, the promoter region of the DLL3 gene was methylated in four of ten HCC cell lines. This aberrant methylation correlated well with the suppression of RNA expression and a demethylating agent reactivated DLL3 expression in methylation-silenced HCC cells. Interestingly, the restoration of DLL3 in the methylation-silenced HuH2 cells led to growth suppression on colony formation assay. Flow cytometric assay with Annexin V and PI showed that this growth suppression by DLL3 expression is associated with the induction of apoptosis. Furthermore, these apoptotic effects were confirmed by TUNEL staining and measurement of single-stranded DNA. These results suggest that DLL3 was silenced by methylation in human HCC and that it negatively regulates the growth of HCC cells.

Identification of differentially methylated regions during vernalization revealed a role for RNA methyltransferases in bolting

Sugar beet (Beta vulgaris altissima) is a biennial root crop with an absolute requirement for cold exposure to bolt and flower, a process called vernalization. Global DNA methylation variations have been reported during vernalization in several plants. However, few genes targeted by DNA methylation during vernalization have been described. The objectives of this study were to identify differentially methylated regions and to study their involvement in bolting induction and tolerance. Restriction landmark genome scanning was applied to DNA from shoot apical meristems of sugar beet genotypes, providing a direct quantitative epigenetic assessment of several CG methylated genes without prior knowledge of gene sequence. Several differentially methylated regions exhibiting variations of gene-body DNA methylation and expression during cold exposure and/or between genotypes were identified, including an AROGENATE DEHYDRATASE and two RNA METHYLCYTOSINE TRANSFERASE sequences. One RNA METHYLCYTOSINE TRANSFERASE sequence displayed gene-body hypermethylation and activation of expression, while the other was hypomethylated and inhibited by cold exposure. Global RNA methylation and phenolic compound levels changed during cold exposure in a genotype-dependent way. The use of methyl RNA immunoprecipitation of total RNA and reverse transcription-PCR analysis revealed mRNA methylation in a vernalized bolting-resistant genotype for the FLOWERING LOCUS 1 gene, a repressor of flowering. Finally, Arabidopsis mutants for RNA METHYLCYTOSINE TRANSFERASE and AROGENATE DEHYDRATASE were shown to exhibit, under different environmental conditions, early or late bolting phenotypes, respectively. Overall, the data identified functional targets of DNA methylation during vernalization in sugar beet, and it is proposed that RNA methylation and phenolic compounds play a role in the floral transition.

Gene silencing of SLC5A8 identified by genome-wide methylation profiling in lung cancer

BackgroundAberrant DNA hypermethylation has been implicated as a component of an epigenetic mechanism that silences genes in cancers. MethodsWe performed a genome-wide search to identify differentially methylated loci between 26 tumor and adjacent non-tumor paired tissues from same lung cancer patients using restriction landmark genomic scanning (RLGS) analysis. Among 229 loci which were hypermethylated in lung tumors as compared to adjacent non-tumor tissues, solute carrier family 5, member 8 (SLC5A8) was one of the hypermethylated genes, and known as a tumor suppressor gene which is silenced by epigenetic changes in various tumors. We investigated the significance of DNA methylation in SLC5A8 expression in lung cancer cell lines, and 23 paired tumor and adjacent non-tumor lung tissues by reverse transcription-PCR (RT-PCR), quantitative methylation specific PCR (QMSP) and bisulfite modified DNA sequencing analyses. ResultsReduced or lost expression of SLC5A8 was observed in 39.1% (9/23) of the tumor tissues as compared with paired adjacent non-tumor tissues. Bisulfite sequencing results of lung cancer cell lines and tissues which did not express SLC5A8 showed a densely methylated promoter region of SLC5A8. SLC5A8 was reactivated by treatment with DNA methyltransferase inhibitor, 5-Aza and/or HDAC inhibitor, trichostatin A (TSA) in lung cancer cell lines, which did not express SLC5A8. Hypermethylation was detected at the promoter region of SLC5A8 in primary lung tumor tissues as compared with adjacent non-tumor tissues (14/23, 60.9%). ConclusionThese results suggest that DNA methylation in the SLC5A8 promoter region may suppress the expression of SLC5A8 in lung tumor.

Epigenetic alteration of CCDC67 and its tumor suppressor function in gastric cancer

In this study, the promoter of the gene coiled-coil domain-containing 67 (CCDC67) was found to be frequently methylated in gastric cancer cell lines and in primary gastric tumors, as examined by restriction landmark genomic scanning. In addition, CCDC67 expression was down-regulated in 72.7% of gastric cancer cell lines tested. In most cases, gene down-regulation was associated with CpG hypermethylation in the CCDC67 promoter. Treatment with 5-aza-2'-deoxycytidine and/or trichostatin A restored CCDC67 expression in down-regulated cell lines. Pyrosequencing analysis of 150 paired primary gastric cancer samples revealed that promoter CpG methylation was increased in 74% of tested tumors compared with paired adjacent normal tissues, and this hypermethylation correlated significantly with down-regulation of CCDC67. CCDC67 protein was localized to the cell membrane by immunocytochemistry. Stable transfection of a CCDC67 gene in one gastric cancer cell line inhibited adhesion-dependent and -independent colony formation, and CCDC67 expression suppressed tumorigenesis in nude mice. We suggest that CCDC67 is a putative tumor suppressor gene that is silenced in gastric cancers by promoter CpG methylation and that it may play an important role in cell signaling and migration related to tumorigenesis.

Genic DNA methylation changes during in vitro organogenesis: organ specificity and conservation between parental lines of epialleles

During differentiation, in vitro organogenesis calls for the adjustment of the gene expression program toward a new fate. The role of epigenetic mechanisms including DNA methylation is suggested but little is known about the loci affected by DNA methylation changes, particularly in agronomic plants for witch in vitro technologies are useful such as sugar beet. Here, three pairs of organogenic and non-organogenic in vitro cell lines originating from different sugar beet (Beta vulgaris altissima) cultivars were used to assess the dynamics of DNA methylation at the global or genic levels during shoot or root regeneration. The restriction landmark genome scanning for methylation approach was applied to provide a direct quantitative epigenetic assessment of several CG methylated genes without prior knowledge of gene sequence that is particularly adapted for studies on crop plants without a fully sequenced genome. The cloned sequences had putative roles in cell proliferation, differentiation or unknown functions and displayed organ-specific DNA polymorphism for methylation and changes in expression during in vitro organogenesis. Among them, a potential ubiquitin extension protein 6 (UBI6) was shown, in different cultivars, to exhibit repeatable variations of DNA methylation and gene expression during shoot regeneration. In addition, abnormal development and callogenesis were observed in a T-DNA insertion mutant (ubi6) for a homologous sequence in Arabidopsis. Our data showed that DNA methylation is changed in an organ-specific way for genes exhibiting variations of expression and playing potential role during organogenesis. These epialleles could be conserved between parental lines opening perspectives for molecular markers.

Abstract 4814: Detection of DNAH1 fragment closely related to human colorectal cancer by RLGS and functional analysis of DNAH1 in cancer cells

Abstract [Introduction] Many cancer related genes in human cancer have been isolated by recent development of techniques in molecular biology, however, the existence of unknown genes still have been expected. Restriction landmark genomic scanning (RLGS) is one of the most powerful methods for the identification of a DNA methylation which correlate with protein expression level. This DNA analysis method is 2-dimensional gel electrophoresis of genomic DNA fragment digested with specific restriction enzyme. We have evaluated clinical samples such as breast cancer, hepatocellular carcinoma (HCC), glioma and colorectal cancer for more than 10 years. In this study, we evaluated RLGS to find genetic alterations relative to human colorectal cancer and cloned some of altered spots. We have found that one of these clone codes a part of dynein axonemal heavy chain 1 (DNAH1). Dyneins are large motor protein complexes that generate the force for the movement of eukaryotic cilia and flagella. [Materials and Methods] High molecular weight genomic DNAs were extracted from 25 colorectal cancerous tissues and corresponding normal mucosae. To analysis alteration spot, each DNA was cleaved with the restriction enzyme Not I, size fractionated by 1st-dimensional electrophoresis using Pvu II, and the DNA fragments were then cleaved by Pst I as the third enzyme in gel and separated by 2-dimensional electrophoresis. Various cell lines, such as A431, AsPC-1, TE10, MCF7, and WiDr were used for in vitro analysis. To examine gene expression of DNAH1, quantitative RT-PCR was performed using TaqMan® Gene Expression Assays. Cell migration was measured using Boyden chamber migration assay for vertical migration and EZ-TAXIScan (GE Healthcare Japan) for horizontal migration. AsPC-1 cells and AsPC-1 shRNA DNAH1 stable cells were used for xenograft model to evaluate function of DNAH1 in vivo. [Results] 19 of 25 (76.0%) colorectal cancer cases showed a spot with the same alteration, which never appeared in normal mucosae. A 410-bp DNA fragment corresponding to this spot was cloned. This sequence was found to be 98% homologous to part of dynein axonemal heavy chain 1 (DNAH1). Gene expression level of DNAH1 was correlated to cell motility in vitro migration assays. In addition, DNAH1 expression level and cell motility were altered by treatment of 5-azacytidin (a protein inhibitor of DNA methyltransferase) or siRNA of DNAH1. Histological and histopathological evaluation of xenogragt model showed that AsPC-1 is INF beta and shRNA DNAH1 stable AsPC-1 is INF alpha. [Conclusion] We have identified the genomic alteration specific in human colorectal cancer by RLGS. The expression of homologpus gene to DNAH1 is closely related to colorectal cancer. Moreover, DNAH1 might not be express only in colorectal cancer but also other cancers. Expression level of DNAH1 related to cancer cell motility and metastasis in vivo and in vitro. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4814. doi:10.1158/1538-7445.AM2011-4814

Abstract 4989: The role of Runx2 in epigenetic silencing of a tumor suppressor BMP-3B/GDF10 in lung cancer cells

Abstract Lung cancer is one of the major causes of cancer-related deaths. Several genetic and epigenetic alterations are now established for lung cancer progression. However, the regulatory mechanisms of these alterations are still unclear. Here, we show that the transcription factor Runx2 is highly expressed in lung cancer cells and downregulates a tumor suppressor bone morphogenetic protein-3B (BMP-3B/GDF10). The Runx2 transcription factor, an essential bone cell differentiation factor, is previously implicated in breast and prostate cancer progression by activating cancer related genes and promoting invasive properties. Our studies in Runx2 deficient animals demonstrated a significant increase in BMP-3B expression levels compared to wild type littermates as examined by cDNA arrays and real time PCR analysis. BMP-3B was previously identified as a silenced gene by DNA methylation in restriction landmark genomic scanning in lung cancer cells, thus suggesting an important function linked to maintaining the normal cell phenotype. Characterization of two normal lung fibroblasts (WI-38, IMR-90) and three lung cancer cells lines (A549, H720, H1299) showed an inverse relationship between Runx2 and BMP-3B expression as examined by real time PCR and western blot analysis. The ectopic expression of Runx2 in normal lung fibroblasts showed a significant downregulation of BMP-3B levels. Expression studies with point and deletion mutants of Runx2 in normal lung fibroblasts and lung cancer cells showed that the C-terminal domain of Runx2 is essential for BMP-3B downregulation. Chromatin immunoprecipitation assays in H1299 cells confirmed the mechanism of Runx2 mediated suppression of BMP-3B. Our results demonstrated Runx2 and H3-K9 specific histone methyltransferase SUV39H1 recruitment on the BMP-3B proximal promoter and concomitant increase in histone methylation (H3-K9) status by Runx2 to mediate repression of the BMP-3B gene locus. Furthermore, co-immunoprecipitation studies showed a direct interaction of Runx2 and SUV39H1 in lung cancer cells. Depletion of Runx2 in H1299 cells by stable expression of shRunx2 resulted in reduced H3-K9 methylation of the BMP-3B promoter and increased expression levels. Modulation of Runx2 levels in H1299 cells modestly altered cell growth properties as examined by MTT cell proliferation assay. Taken together, our studies identified BMP-3B as a novel Runx2 target gene and showed that Runx2 interaction with SUV39H1 down-regulates BMP-3B by affecting local chromatin histone methylation at BMP-3B regulatory regions. Our studies suggest that Runx2 not only activates cancer related genes but also plays a critical role in epigenetic silencing of tumor suppressors during cancer progression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4989. doi:10.1158/1538-7445.AM2011-4989

Folate Deficiency and Supplementation Result in DNA Methylation Defects in Sperm.

Folate is a nutrient that is essential for cell proliferation and involved in the biosynthesis of nucleotides and S-adenosylmethionine (SAM). SAM is the methyl donor for numerous cellular biochemical reactions including DNA methylation, an important epigenetic modification implicated in the control of gene expression, genomic imprinting and normal fertility. Although folate supplementation is used as a treatment for human male infertility, little is known about the effects of low or high dietary folate concentrations on sperm DNA methylation patterns. Our goal was to use a mouse model to examine the effects of folic acid deficiency and supplementation on epigenetic patterns in male germ cells. Male Balb/c strain mice were fed, from weaning age (day 21) to one year of age, either a control diet containing an adequate amount of folic acid (2 mg/kg diet), a 7X low folic acid diet (0.3 mg/kg diet) or a 20X high folic acid diet (40 mg/kg diet). The mice were sacrificed at 12 months and reproductive organs (testes, epididymides and seminal vesicles) collected and weighed. Sperm were collected from the cauda epididymides and genomic DNA extracted. Restriction landmark genomic scanning (RLGS) was used to assess genome-wide levels and patterns of DNA methylation (n=4 samples/group). Although weight gain and reproductive organ weights were not affected by the folate deficiency or supplementation, for both treatment groups, RLGS demonstrated evidence of epigenetic abnormalities in sperm. Mice in the folic acid supplemented group exhibited six consistent changes in their methylation profiles, all of which were hypermethylation. Interestingly, among the 14 loci showing methylation alterations in all of the folic acid deficient mice, 10 were hypermethylated and four were hypomethylated. The loci with altered methylation are in the process of being identified using a virtual RLGS profile. Together, the results indicate that DNA methylation patterns in mouse sperm can be altered by long term exposure to both low and high dietary folate. (Supported by CIHR) (poster)

Tissue specific DNA methylation of CpG islands in normal human adult somatic tissues distinguishes neural from non-neural tissues

Although most CpG islands are generally thought to remain unmethylated in all adult somatic tissues, recent genome-wide approaches have found that some CpG islands have distinct methylation patterns in various tissues, with most differences being seen between germ cells and somatic tissues. Few studies have addressed this among human somatic tissues and fewer still have studied the same sets of tissues from multiple individuals. In the current study, we used Restriction Landmark Genomic Scanning to study tissue specific methylation patterns in a set of twelve human tissues collected from multiple individuals. We identified 34 differentially methylated CpG islands among these tissues, many of which showed consistent patterns in multiple individuals. Of particular interest were striking differences in CpG island methylation, not only among brain regions, but also between white and grey matter of the same region. These findings were confirmed for selected loci by quantitative bisulfite sequencing. Cluster analysis of the RLGS data indicated that several tissues clustered together, but the strongest clustering was in brain. Tissues from different brain regions clustered together, and, as a group, brain tissues were distinct from either mesoderm or endoderm derived tissues which demonstrated limited clustering. These data demonstrate consistent tissue specific methylation for certain CpG islands, with clear differences between white and grey matter of the brain. Furthermore, there was an overall pattern of tissue specifically methylated CpG islands that distinguished neural tissues from non-neural.

Strain-Specific Defects in Testicular Development and Sperm Epigenetic Patterns in 5,10-Methylenetetrahydrofolate Reductase-Deficient Mice

Methylenetetrahydrofolate reductase (MTHFR) is a crucial folate pathway enzyme that contributes to the maintenance of cellular pools of S-adenosylmethionine, the universal methyl donor for several reactions including DNA methylation. Whereas Mthfr(-/-) BALB/c mice show growth retardation, developmental delay, and spermatogenic defects and infertility, C57BL/6 mice appear to have a less severe phenotype. In the present study, we investigated the effects of MTHFR deficiency on early germ cell development in both strains and assessed whether MTHFR deficiency results in DNA methylation abnormalities in sperm. The reproductive phenotype associated with MTHFR deficiency differed strikingly between the two strains, with BALB/c mice showing an early postnatal loss of germ cell number and proliferation that was not evident in the C57BL/6 mice. As a result, the BALB/c MTHFR-deficient mice were infertile, whereas the C57BL/6 mice had decreased sperm numbers and altered testicular histology but showed normal fertility. Imprinted genes and sequences that normally become methylated during spermatogenesis were unaffected by MTHFR deficiency in C57BL/6 mice. In contrast, a genome-wide restriction landmark genomic scanning approach revealed a number of sites of hypo- and hypermethylation in the sperm of this mouse strain. These results showing strain-specific defects in MTHFR-deficient mice may help to explain population differences in infertility among men with common MTHFR polymorphisms.

Abstract 4911: HPV-independent methylation of specific TGF-B pathway-relevant genes in head and neck squamous cell carcinomas associated with prior radiation therapy and exposure to alcohol and tobacco

Abstract Head and neck squamous cell carcinoma (HNSCC) is an aggressive malignancy with more than half a million people being diagnosed with the disease annually. Therefore, it is critical to identify early detection markers, such as DNA methylation, for more timely diagnosis. In a recent study, we identified five tumor suppressive genes (IRX1, EBF3, SLC5A8, SEPT9, and FUSSEL18) as frequently methylated in HNSCC biopsies using a global methylation analysis via restriction landmark genomic scanning (RLGS). Interestingly, we found these candidates are all potentially involved in the TGF-B signaling pathway, which is often disrupted in HNSCC. Therefore, we sought to determine coordinated epigenetic silencing of these candidate genes in HNSCC as potential key disruptors of TGF-B signaling, which could ultimately result in HNSCC progression. Through immunoprecipitation studies, all five of the investigated candidate genes were found to interact with components of the TGF-B pathway. Also, overexpression of SLC5A8, EBF3, and IRX1 resulted in decreased mitotic activity and increased apoptosis. These findings are significant because they reveal a set of genes that interact with components of the TGF-B pathway, and their silencing via methylation in HNSCC results in coordinated decrease in apoptosis, increased proliferation, and decreased differentiation. We went on to define the methylation of these genes in relationship to HPV16-positivity, radiation therapy, and alcohol and tobacco exposure. By utilizing the available clinical information linked to the patient specimens, we found a strong association between promoter methylation of FUSSEL18, IRX1, and EBF3 and prior radiation therapy (P &amp;lt; 0.0001) irrespective of HPV status. Also, promoter methylation of FUSSEL18 and SEPTIN9 were found to correlate significantly with exposure to alcohol and tobacco (P = 0.021). Importantly, in this study we preliminarily show a trend between HPV16 positivity and specific target gene hypermethylation of IRX1, EBF3, SLC5A8, and SEPT9. Equally notable and independent of HPV status, hypermethylation of the promoters of a subset of these genes in recurrences especially in the setting of prior radiation or in the setting of alcohol and tobacco use might help guide adjunctive inclusion or exclusion or epigenetic therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4911.

DNA methylation changes in a human cell model of breast cancer progression

Epigenetic inactivation of genes by DNA hypermethylation plays an important role in carcinogenesis. An in vitro model of human breast epithelial cell transformation was used to study epigenetic changes induced by estradiol during the neoplastic process. Different stages of tumor initiation and progression are represented in this model being MCF-10F the normal stage; trMCF cells, the transformed stage; bsMCF cells, the invasive stage and, caMCF cells, the tumor stage. Global methylation studies by restriction landmark genomic scanning (RLGS) showed an increased DNA methylation during the in the invasive and tumor stages. Expression studies showed that NRG1 (neuregulin 1), CSS3 (chondroitin sulfate synthase 3) and SNIP (SNAP-25-interacting protein) were downregulated in the invasive and tumor cells. The transformed cells showed low expression of STXBP6 (amysin) compared to the parental cells MCF-10F. The treatment of these cells with the demethylating agent 5-aza-dC alone or in combination with the histone deacetylase inhibitor trichostatin increased the expression of NRG1, STXBP6, CSS3 and SNIP confirming that DNA methylation plays an important role in the regulation of the expression of these genes. The NRG1 exon 1 has a region located between −136 and +79 (considering +1, the translational initiation site) rich in CpG sites that was analyzed by methylation specific PCR (MSP). NRG1 exon 1 showed progressive changes in the methylation pattern associated with the progression of the neoplastic process in this model; NRG1 exon 1 was unmethylated in MCF-10F and trMCF cells, becoming hypermethylated in the invasive (bsMCF) and tumor (caMCF) stages. Studies of human breast tissue samples showed that NRG1 exon 1 was partially methylated in 14 out of 17 (82.4%) invasive carcinomas although it was unmethylated in normal tissues (8 out of 10 normal breast tissue samples). Furthermore, NRG1 exon 1 was partially methylated in 9 out of 14 (64.3%) morphologically normal tissue samples adjacent to invasive carcinomas.

183 A DNA signature to identify high-risk small node-negative breast cancer patients

The human p73 gene is a homolog of p53, which has been localized to chromosome 1p36 in a region that is frequently deleted in neuroblastoma. Transfection of the p73 gene into neuroblastoma cells that lack detectable p73 protein has been shown to result in growth suppression and to induce neuronal differentiation. In this study, we have identified by means of restriction landmark genome scanning. (RLGS) a genomic fragment that was frequently reduced in intensity in neuroblastomas. The cloned fragment contained exon 1 of p73 as well as intronic and promoter sequences. We investigated the genomic and expression status of p73 and N-myc in 34 neuroblastoma tumors and 12 neuroblastoma cell lines. Approximately a third of neuroblastomas in our series exhibited deletion of p73. Most tumors analyzed exhibited reduced expression of p73, as determined by quantitative RT-PCR, in the absence of detectable p73 gene deletion. The reduced expression of p73 correlated with overexpression of N-myc in a statistically significant manner. The N-myc gene was transfected into two neuroblastoma cell lines that lacked N-myc amplification to determine its effect on p73 RNA levels. p73 was detectable at low level by RTPCR in untransfected SK-N-AS cells and became undetectable following N-myc transfection, whereas in SH-EP1 cells, p73 levels were substantially reduced following transfection but remained detectable. Our data suggest that the N-myc gene modulates expression of p73, allowing neuroblastoma cells to escape the growth suppressing properties of p73.