Global Methylation Changes Research Articles

Synergistic Combinations of Histone Deacetylase Inhibitors and Decitabine Induce a Unique Gene Expression and Epigenetic Profile In Models of Diffuse Large B-Cell Lymphoma

AbstractAbstract 435Diffuse large B-cell lymphoma (DLBCL) is the most common type of lymphoid malignancy, representing approximately 30–40% of all lymphomas. While significant progress has been made in treating this disease over the past decade, it is still regarded as a heterogeneous disease which, after being classified as relapsed or refractory, is fatal in about one-third of patients. Histone deacetylase inhibitors (HDACI) are presently approved for the treatment of relapsed or refractory cutaneous T- cell lymphomas (CTCL), and have marked activity in peripheral T-cell lymphomas (PTCL), though their effectiveness in DLBCL is less established. DNA methyltransferases (DNMTs) are known to recruit and cooperate with histone deacetylases to induce gene silencing. Combinations of drugs affecting these pathways have emerged as active and important, mostly in myeloid leukemias. We hypothesized that the combination of HDACI and DNMT inhibitors (DNMTI) in DLBCL may be active only in combination and not as single agents. We examined the interaction between a broad range of HDACI including vorinostat, depsipeptide, panobinostat and DNMTI using in vivo and in vitro models of DLBCLs, clearly confirming that these agents are in fact synergistic with decitabine. Synergy was measured by relative risk ratio (RRR) and the values obtained were as low as 0.01, representing very strong synergy. This combination of drugs, specifically panobinostat and decitabine, was also shown to be strongly synergistic in a murine xenograft model of DLBCL. In addition, we analyzed the molecular basis for this synergistic effect by evaluating the global gene expression and methylation using microarrays on the cells treated with the single agents and combination in DLBCL. Three DLBCL lines (OCI-Ly1, OCI-Ly10 and Su-DHL6) were treated with decitabine alone (2.5 μ M), panobinostat alone (2.5 nM) or their combination for 48h hours. DNA and RNA from untreated and treated cells were used for genome wide methylation analysis through Illumina Humanmethyation27 platform and gene expression profiling analysis with Illumina HumanHT-12 v3 Expression arrays. 3D principal component analysis clearly clustered the samples treated with panobinostat and combination therapy together and at greater distances from untreated samples and samples treated by decitabine alone. Therefore, the contribution to the gene expression phenotype of the combination was greater from the HDACI than with DNMTI. Consistent with this observation, the top network of genes differentially expressed (p<0.05) by panobinostat involved critical transcription factors like GATA1, GATA4, SMAD and DNMT3A. Additionally, network-functional analysis of genes perturbed by the combination treatments enriched for critical pathways involved in cell death, cell development and cellular proliferation. Surprisingly, differentially expressed genes and networks identified by each of the treatment conditions and by combination therapy were unique with few overlapping genes as shown in Venn diagram in Figure 1a. Genome wide methylation analysis produced similar results with greater contribution to global methylation changes in cells treated by the combination therapy and decitabine as compared to HDACI. Again, methylation status of a distinct set of genes was altered by combination therapy as compared to the individual drugs (Figure 1b). Correlation between genome wide methylation analysis and gene expression profiling identified 16 overlapping genes in the samples treated by the combination of panobinostat and decitabine including known tumor suppressor genes like VHL, DIRAS3 and WT1. Taken together, integrative genomic analysis has provided insights into the relative contribution of independent epigenetic therapies to the combination phenotype. These findings may provide important leads in identifying unique biomarkers of response specific to the combination of panobinostat and decitabine in DLBCL. [Display omitted] Disclosure:O'Connor:Millennium Pharmaceuticals, Inc.: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Phase I trial of low dose decitabine targeting DNA hypermethylation in patients with chronic lymphocytic leukaemia and non‐Hodgkin lymphoma: dose‐limiting myelosuppression without evidence of DNA hypomethylation

Targeting aberrant DNA hypermethylation in chronic lymphocytic leukaemia (CLL) and non-Hodgkin lymphoma (NHL) with decitabine may reverse epigenetic silencing in B-cell malignancies. Twenty patients were enrolled in two phase I trials to determine the minimum effective pharmacological dose of decitabine in patients with relapsed/refractory CLL (n = 16) and NHL (n = 4). Patients received 1-3 cycles of decitabine. Dose-limiting toxicity (DLT) was observed in 2 of 4 CLL and 2 of 2 NHL patients receiving decitabine at 15 mg/m(2) per d days 1-10, consisting of grade 3-4 thrombocytopenia and hyperbilirubinaemia. Six patients with CLL received decitabine at 10 mg/m(2) per d days 1-10 without DLT; however, re-expression of methylated genes or changes in global DNA methylation were not observed. Therefore, a 5-day decitabine schedule was examined. With 15 mg/m(2) per d decitabine days 1-5, DLT occurred in 2 of 6 CLL and 2 of 2 NHL patients, consisting of grade 3-4 neutropenia, thrombocytopenia, and febrile neutropenia. Eight patients had stable disease. In 17 patients, there were no significant changes in genome-wide methylation or in target gene re-expression. In conclusion, dose-limiting myelosuppression and infectious complications prevented dose escalation of decitabine to levels associated with changes in global methylation or gene re-expression in CLL and NHL.

Folate supplementation in pregnancy and gene specific DNA methylation

ObjectivesPericonceptual folate supplementation is recommended to reduce the risk of fetal neural tube defect and other congenital defects. Folate is a source of methyl donors, and experimental data on rodents...

Abstract 4924: Gene-specific hyper and hypomethylation in follicular lymphoma progression

Abstract Epigenetic mechanisms involved in the origin and transformation of indolent lymphomas to aggressive forms are poorly understood. Based on the comparison of methylation states of follicular lymphoma (FL) pre and post- transformation, it has been suggested that DNA methylation changes occur during the early stages of lymphomagenesis. To obtain a more complete understanding of methylation aberrations responsible for progression and transformation of FL, we analyzed 20 untreated FL biopsies (10 low grade, 10 high grade), 5 diffuse large B cell lymphoma (DLBCL) biopsies, and 5 benign reactive lymph node (RLN) biopsies by using methyl-DNA immunoprecipitation coupled with analysis with Agilent 244K human promoter ChIP-on-chip microarrays. Our analysis revealed statistically significant differences in methylation status for 120 genes between FL samples and RLN samples and for 23 genes between low and high grade FL samples. To investigate the biological significance of these findings, we used DAVID functional annotation clustering algorithms and Gominer software. We found that the most methylation-enriched functional groups were comprised of genes with roles in cell cycle control, adhesion and motility, transcription regulation, homeobox, and developmental genes. In order to validate the DNA methylation differences of the significant gene regions, we utilized the MassARRAY EpiTYPER assay. Our data identified 13 genes (CYP26B1, GRM7, HS3ST2, DOCK5, GRB10, SST, TAC1, PALM2-AKAP, FLJ40125, ST6GAL2, WDR35, NCAM1, SATB2) with increased levels of DNA methylation in FL samples compared to controls; 3 of these genes (ST6GAL2, WDR35, NCAM1) exhibited increased methylation in high grades FLs and 1 gene (SATB2) in low grades FLs. We are currently investigating gene specific DNA hypomethylation in low versus high grade FL and FL versus DLBCL. Our data are the first to characterize the global changes in methylation that occur in FL progression. Our findings suggest that high grade FL are associated with hypermethylation of a specific subset of genes compared to low grade subtypes. Ongoing efforts include investigating the functional relevance of these methylation changes by treating, in vitro, FL cell lines with demethylating agents to evaluate changes in gene expression levels and correlating with phenotype alterations. 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 4924.

Abstract 3220: Doxorubicin-induced senescence in hepatocellular carcinoma cells

Abstract Introduction: Doxorubicin (DOX, adriamycin) is an anti-cancer drug commonly used in treatment of several human solid cancers including unresectable hepatocellular carcinoma (HCC). Therapeutic effects of DOX have been linked to DNA intercalation and topoisomerase inhibition as well as oxidative stress induction. As DNA damage and oxidative stress have been identified as potential mediators of cellular senescence, we tested whether DOX is able to trigger senescence arrest in HCC cells. Methods: HepG2 (wt p53) and Huh7 (p53-Y220C) cell lines were exposed to different doses of DOX and their responses were studied over a period of 6 days. Senescence was tested by morphological changes and senescence-associated beta-galactosidase (SABG) activity. DNA damage response was studied at the levels phospho-H2AX, phospho-ATM, phospho-ATR, phospho-CHK1, phospho-CHK2 and 53BP1. Cell cycle analysis was performed by flow cytometry and cell cycle protein expression analysis. We also analyzed histone methylation changes in response to DOX. Results: High dose DOX (500ng/ml) treatment caused p53 accumulation, induced p21 levels and triggered apoptosis, as shown by flow cytometry. Low dose (LD) DOX treatment (25ng/ml and 50ng/ml) did not induce apoptosis, but triggered a senescence response at day 3 in both HepG2 and Huh-7 HCC cell lines. Senescence phenotype was characterised with flattened and enlarged shape, and strong positivity of SABG activity (&amp;gt; 80% in three days, &amp;gt;90% in six days). LD DOX treatment caused a slight increase in p53 and p21 protein levels in correlation with SABG positivity in HepG2 cells. After treating cells 3 days with LD 50ng/ml of DOX, we found that 53BP1 and p-H2AX localized to DNA damage foci. ATM-Chk2-Cdc25-A signal transduction pathway but not the ATR-Chk1-Cdc25 pathway was activated to arrest the cell cycle following DNA damage in G2 phase both in HepG2 and Huh-7 cells. We obtained G2/M arrest reaching as high as 90% for both cell lines. In correlation with this observation, we also showed downregulation of cdc2, and cyclin B1 proteins, key modulators of G2/M cell cycle transition, using immunoblotting while cyclin A1 protein levels remained unchanged. Using this model, we also explored the global epigenetic histone methylation marks in LD DOX treated cells using both immunofluorescence and immunoblotting technique. Upon treatment with LD DOX, we observed increase in H3K27Me3, H3K36Me3 and H4K20Me3 levels and a corresponding decrease in monomethyl forms of these histones, H3K27Me1, H3K36Me1 and H4K20Me1. We did not detect any change on H3K4Me3, H4K36Me2 and H4K20Me2 methylation levels. Taken together, our observations indicate that LD DOX treatment of HCC cells induces p53-independent senescence arrest at phase G2/M. This was associated with a DNA damage response associated with global changes in histone methylation. 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 3220.

A Nucleosome Surface Formed by Histone H4, H2A, and H3 Residues Is Needed for Proper Histone H3 Lys36 Methylation, Histone Acetylation, and Repression of Cryptic Transcription

Set2-mediated H3 Lys(36) methylation is a histone modification that has been demonstrated to function in transcriptional elongation by recruiting the Rpd3S histone deacetylase complex to repress intragenic cryptic transcription. Recently, we identified a trans-histone pathway in which the interaction between the N terminus of Set2 and histone H4 Lys(44) is needed to mediate trans-histone H3 Lys(36) di- and trimethylation. In the current study, we demonstrate that mutation of the lysine 44 residue in histone H4 or the Set2 mutant lacking the histone H4 interaction motif leads to intragenic cryptic transcripts, indicating that the Set2 and histone H4 interaction is important to repress intragenic cryptic transcription. We also determine that histone H2A residues (Leu(116) and Leu(117)), which are in close proximity to histone H4 Lys(44), are needed for proper trans-histone H3 Lys(36) methylation. Similar to H4 Lys(44) mutants, histone H2A Leu(116) and Leu(117) mutations exhibited decreased H3 Lys(36) di- and trimethylation, increased histone H4 acetylation, increased resistance to 6-azauracil, and cryptic transcription. Interestingly, the combined histone H4 Lys(44) and H2A mutations have more severe methylation defects and increased H4 acetylation levels. Furthermore, we identify that additional histone H2A and H3 core residues are also needed for H3 Lys(36) di- and trimethylation. Overall, our results show and suggest that multiple H4, H2A, and H3 residues contribute to and form a Set2 docking/recognition site on the nucleosomal surface so that proper Set2-mediated H3 Lys(36) di- and trimethylation, histone acetylation, and transcriptional elongation can occur.

Methylation profiling in individuals with Russell–Silver syndrome

Russell-Silver syndrome (RSS) is a heterogeneous disorder associated with pre- and post-natal growth restriction and relative macrocephaly. Involvement of imprinted genes on both chromosome 7 and 11p15.5 has been reported. To further characterize the role of epimutations in RSS we evaluated the methylation status at both 11p15.5 imprinting control regions (ICRs): ICR1 associated with H19/IGF2 expression and ICR2 (KvDMR1) associated with CDKN1C expression in a series of 35 patients with RSS. We also evaluated methylation at the promoter regions of other imprinted genes involved in growth such as PLAGL1 (6q24), GCE (7q21), and PEG10 (7q21) in this series of 35 patients with RSS. Thirteen of the 35 patient samples, but none of 22 controls, showed methylation levels at ICR1 that were more than 2 SD below the mean for controls. Three RSS patients were highly methylated at the SCGE promoter, all of which were diagnosed with upd(7)mat. To identify further potential global methylation changes in RSS patients, a subset of 22 patients were evaluated at 1505 CpG sites by the Illumina GoldenGate methylation array. Among the few CpG sites displaying a significant difference between RSS patients and controls, was a CpG associated with the H19 promoter. No other sites associated with known imprinted genes were identified as abnormally methylated in RSS patients by this approach. While the association of hypomethylation of the H19/IGF2 ICR1 is clear, the continuous distribution of methylation values among the patients and controls complicates the establishment of clear cut-offs for clinical diagnosis.

KMT1E Mediated H3K9 Methylation Is Required for the Maintenance of Embryonic Stem Cells by Repressing Trophectoderm Differentiation

Dynamic regulation of histone methylation by methyltransferases and demethylases plays a central role in regulating the fate of embryonic stem (ES) cells. The histone H3K9 methyltransferase KMT1E, formerly known as ESET or Setdb1, is essential to embryonic development as the ablation of the Setdb1 gene results in peri-implantation lethality and prevents the propagation of ES cells. However, Setdb1-null blastocysts do not display global changes in H3K9 methylation or DNA methylation, arguing against a genome-wide defect. Here we show that conditional deletion of the Setdb1 gene in ES cells results in the upregulation of lineage differentiation markers, especially trophectoderm-specific factors, similar to effects observed upon loss of Oct3/4 expression in ES cells. We demonstrate that KMT1E deficiency in ES cells leads to a decrease in histone H3K9 methylation at and derepression of trophoblast-associated genes such as Cdx2. Furthermore, we find genes that are derepressed upon Setdb1 deletion to overlap with known targets of polycomb mediated repression, suggesting that KMT1E mediated H3K9 methylation acts in concert with polycomb controlled H3K27 methylation. Our studies thus demonstrate an essential role for KMT1E in the control of developmentally regulated gene expression programs in ES cells.

MLL5, a trithorax homolog, indirectly regulates H3K4 methylation, represses cyclin A2 expression, and promotes myogenic differentiation

Most cells in adult tissues are nondividing. In skeletal muscle, differentiated myofibers have exited the cell cycle permanently, whereas satellite stem cells withdraw transiently, returning to active proliferation to repair damaged myofibers. We have examined the epigenetic mechanisms operating in conditional quiescence by analyzing the function of a predicted chromatin regulator mixed lineage leukemia 5 (MLL5) in a culture model of reversible arrest. MLL5 is induced in quiescent myoblasts and regulates both the cell cycle and differentiation via a hierarchy of chromatin and transcriptional regulators. Knocking down MLL5 delays entry of quiescent myoblasts into S phase, but hastens S-phase completion. Cyclin A2 (CycA) mRNA is no longer restricted to S phase, but is induced throughout G(0)/G(1), with activation of the cell cycle regulated element (CCRE) in the CycA promoter. Overexpressed MLL5 physically associates with the CCRE and impairs its activity. MLL5 also regulates CycA indirectly: Cux, an activator of CycA promoter and S phase is induced in RNAi cells, and Brm/Brg1, CCRE-binding repressors that promote differentiation are repressed. In knockdown cells, H3K4 methylation at the CCRE is reduced, reflecting quantitative global changes in methylation. MLL5 appears to lack intrinsic histone methyl transferase activity, but regulates expression of histone-modifying enzymes LSD1 and SET7/9, suggesting an indirect mechanism. Finally, expression of muscle regulators Pax7, Myf5, and myogenin is impaired in MLL5 knockdown cells, which are profoundly differentiation defective. Collectively, our results suggest that MLL5 plays an integral role in novel chromatin regulatory mechanisms that suppress inappropriate expression of S-phase-promoting genes and maintain expression of determination genes in quiescent cells.

Azacitidine-Induced Changes in the MDS Methylome Are Associated with Clinical Responses

Azacitidine (AZA) has been shown to improve overall survival in patients with myelodysplastic syndrome (Silverman LR et al. J Clin Oncol 2002; 20: 2429–40). AZA is postulated to work, in part, by inducing hypomethylation and thereby altering gene expression. As a correlative analysis to our phase II study of IV AZA we assessed the baseline methylome of MDS and the changes in global methylation on cycle 2 day 25 (c2d25) of treatment with AZA (75mg/m2 d1–5 of a 28 d cycle). DNA from bone marrow (BM) and peripheral blood (PB) underwent bisulfite conversion and 1500ng of DNA was hybridized to Infinium Human Methylation27 BeadArrays. Arrays were processed by the Genome Sequencing Center at Washington University. The Infinium human Methylation27 Bead Array interrogates >27,000 well annotated CpG sites analyzing 14,500 promoters with single nucleotide resolution. Data was analyzed using BeadStudio version 3 and GraphPad Prism version 5. To compare the average percentages of methylated loci, we used a mixed model for repeated measurement data which allowed us to account for potential correlations among measurements taken from the same subjects. Loci were considered methylated if their average beta value was ≥0.80, hemi-methylated if their average beta value was <0.80 but >0.20, and unmethylated if it was ≤0.20. Loci on the X and Y chromosomes were excluded from analyses due to gender imbalances between responders and non-responders. Fifteen patients had BM and/or PB samples at baseline and/or on c2d25 and a response assessment. Median age was 69 years (range 53–82). IPSS categories were low (7%), Int-1 (27%), Int-2 (40%) and High (7%); FAB classifications were RA (26%), CMML (7%), RAEB (47%) and RAEB-t (20%). Best responses were: 2 patients with CR, 2 with a marrow CR, 1 with a PR, 8 with SD and 2 suffered PD. Patients with CR, mCR and PR were classified as responders whereas all others were classified as non-responders. For all patients the percentage of methylated loci decreased from baseline (13.4%) to c2d25 (9.9%) (p=0.0043). This effect was driven by a decrease in methylated loci in responders (15.1% to 8.9%) (p=0.0109). The decrease in methylated loci in non-responders was not statistically significant (12.4% to 10.5%) (p=0.1083). The difference in baseline methylation between responders and non-responders was not significant (p=0.1719). Baseline methylation was not associated with age (p=0.1475), IPSS (p=0.0934) or High- vs. Low-grade MDS (p=0.3575). Interestingly, the c2d25 assessment anteceded best clinical response in 4 out of 5 responders (all that achieved a mCR or CR) (81 days v. a mean of 202 days, SD 129 days to best response). Demethylation was likewise seen in the PB (18.6% to 11.7% of loci in responders and 18.8% to 13.6% of loci in non-responders) and overall there was good correlation between the mean beta values across the loci for BM and PB (r2=0.9937). But this correlation deteriorated when only methylated loci were included (r2=0.7936), demonstrating that the correlation is driven by hemi- or unmethylated loci, which compose ≥80% of loci, and suggesting that methylation in the PB is unlikely to be a good surrogate for BM methylation. Loci specific changes were considered potentially significant if average beta scores changed by ≥0.20 from baseline to c2d25. 11 loci met this criteria in responders and the changes in several of these were confirmed by t-tests (DNTT p=0.08; APOBEC2 p=0.0015, RGPD5 p=0.0138, TMSF4 p=0.0040). These differences and others between the baseline methylation status of loci in responders and non-responders will be correlated with mRNA expression levels (such as BCL2 p=0.08, HP p=0.0057, SHANK2 p=0.003). This study represents the first methylome-wide description of MDS and the changes induced by azacitidine. If validated in larger cohorts, BM methylation changes prior to cycle 3 may be able to help discriminate responders from non-responders earlier. Further studies are needed that include a larger sample size and that correlate changes in methylation status with changes in transcription and ultimately translation.

Effects of Particulate Matter on Genomic DNA Methylation Content and iNOS Promoter Methylation

BackgroundAltered patterns of gene expression mediate the effects of particulate matter (PM) on human health, but mechanisms through which PM modifies gene expression are largely undetermined.ObjectivesWe aimed at identifying short- and long-term effects of PM exposure on DNA methylation, a major genomic mechanism of gene expression control, in workers in an electric furnace steel plant with well-characterized exposure to PM with aerodynamic diameters < 10 μm (PM10).MethodsWe measured global genomic DNA methylation content estimated in Alu and long interspersed nuclear element-1 (LINE-1) repeated elements, and promoter DNA methylation of iNOS (inducible nitric oxide synthase), a gene suppressed by DNA methylation and induced by PM exposure in blood leukocytes. Quantitative DNA methylation analysis was performed through bisulfite PCR pyrosequencing on blood DNA obtained from 63 workers on the first day of a work week (baseline, after 2 days off work) and after 3 days of work (postexposure). Individual PM10 exposure was between 73.4 and 1,220 μg/m3.ResultsGlobal methylation content estimated in Alu and LINE-1 repeated elements did not show changes in postexposure measures compared with baseline. PM10 exposure levels were negatively associated with methylation in both Alu [β = −0.19 %5-methylcytosine (%5mC); p = 0.04] and LINE-1 [β = −0.34 %5mC; p = 0.04], likely reflecting long-term PM10 effects. iNOS promoter DNA methylation was significantly lower in postexposure blood samples compared with baseline (difference = −0.61 %5mC; p = 0.02).ConclusionsWe observed changes in global and gene specific methylation that should be further characterized in future investigations on the effects of PM.

Intra-individual Change Over Time in DNA Methylation With Familial Clustering

Changes over time in epigenetic marks, which are modifications of DNA such as by DNA methylation, may help explain the late onset of common human diseases. However, changes in methylation or other epigenetic marks over time in a given individual have not yet been investigated. To determine whether there are longitudinal changes in global DNA methylation in individuals and to evaluate whether methylation maintenance demonstrates familial clustering. We measured global DNA methylation by luminometric methylation assay, a quantitative measurement of genome-wide DNA methylation, on DNA sampled at 2 visits on average 11 years apart in 111 individuals from an Icelandic cohort (1991 and 2002-2005) and on average 16 years apart in 126 individuals from a Utah sample (1982-1985 and 1997-2005). Global methylation changes over time. Twenty-nine percent of Icelandic individuals showed greater than 10% methylation change over time (P < .001). The family-based Utah sample also showed intra-individual changes over time, and further demonstrated familial clustering of methylation change (P = .003). The family showing the greatest global methylation loss also demonstrated the greatest loss of gene-specific methylation by a separate methylation assay. These data indicate that methylation changes over time and suggest that methylation maintenance may be under genetic control.

Global and Regional CpG Methylation in Pheochromocytomas and Abdominal Paragangliomas: Association to Malignant Behavior

This study aims to quantitatively assess promoter and global methylation changes in pheochromocytomas and abdominal paragangliomas and its relation to tumor phenotypes. A panel of 53 primary tumors (42 benign, 11 malignant) was analyzed by quantitative bisulfite pyrosequencing. Based on methylation levels in the tumor suppressor genes, p16(INK4A), CDH1, DCR2, RARB, RASSF1A, NORE1A, TP73, APC, DAPK1, p14(ARF), and PTEN, a CpG island methylator phenotype (CIMP) was defined as concerted hypermethylation in three or more genes. Mean Z scores for the hypermethylated promoters were calculated to characterize overall promoter methylation. Global DNA methylation was quantified for LINE-1 promoter sequences and by using luminescent methylation analysis. Five primary tumors (9.4%) exhibited a CIMP phenotype, four of which were malignant paragangliomas. CIMP was significantly associated with malignant behavior (P = 0.005) and younger age at presentation (P < 0.007) but did not result from BRAF V600E mutation. Global hypomethylation of LINE-1 elements was observed in tumors compared with normal adrenal samples (P < 0.02). We here describe the identification of CIMP in abdominal paragangliomas and a strong association of this phenotype with malignant behavior, as well as young age at presentation. The findings raise a prospective for potential benefits of epigenetically acting drugs for a subgroup of young abdominal paraganglioma patients with adverse prognosis.

Tandem repeat mutation, global DNA methylation, and regulation of DNA methyltransferases in cultured mouse embryonic fibroblast cells chronically exposed to chemicals with different modes of action

Mutations at expanded simple tandem repeat (ESTR) DNA sequences provide a useful tool for screening germline mutation. However, the mechanisms resulting in induced mutations are unknown and provide an impediment to the utility of the method. Induced ESTR mutations arise through a nontargeted mechanism resulting in destabilization of the repeat locus. We hypothesized that alterations in DNA methylation, or in DNA methyltransferase expression, may be associated with this indirect mechanism of mutation. DNA mutation frequency was measured in C3H/10T1/2 mouse embryonic fibroblast cells following chronic exposure to six chemicals exhibiting different modes of genotoxic action: N-nitroso-N-ethylurea (ENU); benzo(a)pyrene (BaP); etoposide (ETOP); okadaic acid (OA); cisplatin (CisPt); and 5-azacytidine (5azadC). Induced mutation ranged from 2-fold (ENU, BaP, ETOP), to 1.3-1.4 fold (OA, 5azadC), to nonresponsive (CisPt). Global DNA methylation, measured using the cytosine extension assay, revealed hypomethylation following exposure to ENU and 5azadC, hypermethylation following BaP and OA exposure, and no change following treatment with ETOP or CisPt. DNA methyltransferase transcription (Dnmt1, Dnmt3a, Dnmt3b) was significantly affected by all treatments except ETOP, with the vast majority of changes being downregulation. There was no direct correlation between ESTR mutation, global methylation, or DNA methyltransferase transcription. However, 4/5 ESTR mutagens caused changes in global methylation, while the noninducer (CisPt) did not cause changes in methylation. We hypothesize that chemicals that modify chromatin conformation through changes in methylation may compromise the ability of mismatch repair enzymes (or other enzymes) to access and repair secondary structures that may form across ESTR loci resulting in mutation.

Aberrant DNA methylation in porcine in vitro-, parthenogenetic-, and somatic cell nuclear transfer-produced blastocysts.

Early embryonic development in the pig requires DNA methylation remodeling of the maternal and paternal genomes. Aberrant remodeling, which can be exasperated by in vitro technologies, is detrimental to development and can result in physiological and anatomic abnormalities in the developing fetus and offspring. Here, we developed and validated a microarray based approach to characterize on a global scale the CpG methylation profiles of porcine gametes and blastocyst stage embryos. The relative methylation in the gamete and blastocyst samples showed that 18.5% (921/4,992) of the DNA clones were found to be significantly different (P < 0.01) in at least one of the samples. Furthermore, for the different blastocyst groups, the methylation profile of the in vitro-produced blastocysts was less similar to the in vivo-produced blastocysts as compared to the parthenogenetic- and somatic cell nuclear transfer (SCNT)-produced blastocysts. The microarray results were validated by using bisulfite sequencing for 12 of the genomic regions in liver, sperm, and in vivo-produced blastocysts. These results suggest that a generalized change in global methylation is not responsible for the low developmental potential of blastocysts produced by using in vitro techniques. Instead, the appropriate methylation of a relatively small number of genomic regions in the early embryo may enable early development to occur.

Serial cloning of pigs by somatic cell nuclear transfer: Restoration of phenotypic normality during serial cloning

Somatic cell nuclear transfer (scNT) is a useful way to create cloned animals. However, scNT clones exhibit high levels of phenotypic instability. This instability may be due to epigenetic reprogramming and/or genomic damage in the donor cells. To test this, we produced transgenic pig fibroblasts harboring the truncated human thrombopoietin (hTPO) gene and used them as donor cells in scNT to produce first-generation (G1) cloned piglets. In this study, 2,818 scNT embryos were transferred to 11 recipients and five G1 piglets were obtained. Among them, a clone had a dimorphic facial appearance with severe hypertelorism and a broad prominent nasal bridge. The other clones looked normal. Second-generation (G2) scNT piglets were then produced using ear cells from a G1 piglet that had an abnormal nose phenotype. We reasoned that, if the phenotypic abnormality of the G1 clone was not present in the G2 and third-generation (G3) clones, or was absent in the G2 clones but reappeared in the G3 clones, the phenotypic instability of the G1 clone could be attributed to faulty epigenetic reprogramming rather than to inherent/accidental genomic damage to the donor cells. Blastocyst rates, cell numbers in blastocyst, pregnancy rates, term placenta weight and ponderal index, and birth weight between G1 and G2 clones did not differ, but were significantly (P < 0.05) lower than control age- and sex-matched piglets. Next, we analyzed global methylation changes during development of the preimplantation embryos reconstructed by donor cells used for the production of G1 and G2 clones and could not find any significant differences in the methylation patterns between G1 and G2 clones. Indeed, we failed to detect the phenotypic abnormality in the G2 and G3 clones. Thus, the phenotypic abnormality of the G1 clone is likely to be due to epigenetic dysregulation. Additional observations then suggested that expression of the hTPO gene in the transgenic clones did not appear to be the cause of the phenotypic abnormality in the G1 clones and that the abnormality was acquired by only a few of the G1 clone's cells during its gestational development.

Moderate folate depletion modulates the expression of selected genes involved in cell cycle, intracellular signaling and folate uptake in human colonic epithelial cell lines

Folate deficiency may affect gene expression by disrupting DNA methylation patterns or by inducing base substitution, DNA breaks, gene deletions and gene amplification. Changes in expression may explain the inverse relationship observed between folate status and risk of colorectal cancer. Three cell lines derived from the normal human colon, HCEC, NCM356 and NCM460, were grown for 32–34 days in media containing 25, 50, 75 or 150 nM folic acid, and the expression of genes involved in cell-cycle checkpoints, intracellular signaling, folate uptake and cell adhesion and migration was determined. Expression of Folate Receptor 1 was increased with decreasing media folate in all cell lines, as was p53, p21, p16 and β-catenin. With decreasing folate, the expression of both E-cadherin and SMAD-4 was decreased in NCM356. APC was elevated in NCM356 but unchanged in the other lines. No changes in global methylation were detected. A significant increase in p53 exon 7–8 strand breaks was observed with decreasing folate in NCM460 cells. The changes observed are consistent with DNA damage-induced activation of cell-cycle checkpoints and cellular adaptation to folate depletion. Folate-depletion-induced changes in the Wnt/APC pathway as well as in genes involved in cell adhesion, migration and invasion may underlie observed relationships between folate status and cancer risk.

Lack of Increased Gene Promoter Methylation at Relapse in Acute Promyelocytic Leukemia (APL).

Increasing methylation of gene-regulatory DNA sequences has been associated with the progression of malignancies, and, in APL, the pathogenetic PML-RARα fusion gene has been associated with DNA methyltransferase activity, which might contribute to this process. We, therefore, hypothesized that promoter methylation levels would be increased in APL samples at disease relapse. Preliminary studies of a few CpG-enriched gene promoters, using highly quantitative pyrosequencing technology, demonstrated considerable individual-specific variation in DNA methylation. Hence, we used matched pretreatment and relapse bone marrow and/or peripheral blood samples with >85% promyeloblasts after low-density gradient enrichment from 4 APL patients. High quality DNA was prepared from fresh samples by a guanidinium-cesium chloride gradient procedure. Global analysis of CpG-enriched DNA was performed by the HpaII tiny fragment Enrichment by Ligation-mediated PCR (HELP) assay (Khulan, et al, Genome Res. 16, 1046Khulan, et al, Genome Res. 16, 2006), using a microarray representing the promoters of 24,000 human genes. The HELP assay relies upon a comparative genomic hybridization of HpaII (methylation-sensitive) versus MspI (methylation-insensitive) digested genomic DNA and is able to distinguish populations of methylated loci from hypomethylated (HO) loci. Based on the mean centered log ratio (MCLR) of HpaII/MpaI (Khulan, et al), there were negligible global changes in methylation between the 4 matched pretreatment and relapse samples. Using cut-off values to select for hypermethylated (HR) DNA (MCLR ≤0.0) and more completely HO DNA (MCLR ≥1.0) revealed some decrease in the ratio of HR:HO DNA at relapse in 3 of the 4 patients, primarily due to an increased fraction of HO DNA (see table). [Display omitted] We then assessed relapse-associated changes in methylation in 3,908 promoters in which the change from pretreatment to relapse was associated with p <0.05 (paired t-tests) across all 4 data sets. A change from HO to HR was defined as a decrease in the MCLR by ≥2-fold in the relapse group; a change from HR to HO was the opposite, i.e., an increase in MCLR by ≥2-fold. There was a change from pretreatment HO to relapse HR for 563 loci (7.5% of effective HO promoters) and from pretreatment HR to relapse HO for 949 promoters (8.6% of effective promoters). More stringently, when promoters were selected for a change in MCLR from pretreatment HO ≥1.0 to relapse HR ≤0.0 or, conversely from pretreatment HR ≤0.0 to relapse HO ≥1.0, 40 and 82 promoters, respectively, were identified with p ≤0.05. These results indicate that changes in gene promoter methylation from pretreatment HR to relapse HO are more common than the reverse, and they are contrary to our pre-study hypothesis that increased global DNA methylation might be a major contributor to molecular progression processes in APL cells leading to clinical relapse.

DNA methylation levels in porcine fetal fibroblasts induced by an inhibitor of methylation, 5-azacytidine

Removal of the somatic DNA methylation pattern from donor cells and remodeling of embryonic status have been suggested as integral processes for successful nuclear transfer (NT) reprogramming. This study has investigated the effects of 5-azacytidine (5-azaC), a DNA methylation inhibitor, on global methylation changes in porcine fetal fibroblasts (PFF); this may improve NT attributable to the potential reprogramming of the methyl groups. PFF in 5th passage cultures were treated with 0, 0.5, 1.0, 2.0, and 3.0 microM 5-azaC for 96 h; 5-azaC inhibited the growth at all tested concentrations. At the higher concentrations of 5-azaC used, cells appeared to exhibit morphological changes and to become apoptotic as observed by TUNEL assay. Thus, cells were negatively affected by 5-azaC. Differences in cellular ploidy were also observed at higher concentrations. Analysis showed no considerable changes in the proportion of cells at the G1-phase of the cell cycle with 5-azaC concentrations. The fractional part of the methylated DNA of these cells was significantly reduced by 5-azaC treatment. Confocal microscopy confirmed the inhibition of methylation levels in PFF with increased concentrations of 5-azaC. Exposure to 5-azaC altered the expression of genes involved in imprinting (IGF2) or pro-apoptosis (BAX), whereas there was a reduction in the expression of the main enzyme responsible for replicating the DNA methylation pattern (DNMT1) and anti-apoptosis (BCL2L1). Therefore, 5-azaC induces a relative reduction in methylation in PFF, and cells treated with 0.5 microM 5-azaC may have enhanced potential for porcine NT.

Progressive alterations in global and GC-rich DNA methylation during tumorigenesis.

DNA methylation plays a key role in the regulation of gene expression, and failure to maintain normal patterns of methylation often contributes to carcinogenesis. We have characterized progressive methylation changes during the promotion stage of carcinogenesis using a SENCAR mouse skin initiation/promotion tumorigenesis model. Mice were initiated with a dermal application of 75 microg dimethylbenz[a]anthracene (DMBA) and promoted with 9, 18, 27, and 36 mg cigarette smoke condensate (CSC) thrice weekly for time periods up to 29 weeks, when a large increase in tumor number was produced by the highest three doses. Global and GC-specific methylation were assessed using SssI methylase and arbitrarily primed PCR, respectively. Changes in GC-specific methylation were dose- and time-dependent. CSC doses required to detect these changes were 27 mg at 6 weeks and 18 mg at 9 weeks. This effect appears to be reversible; changes in GC-specific methylation were less marked after 9 weeks promotion with 27 mg CSC followed by 6 weeks of recovery in comparison to 9 and 15 weeks promotion with 27 mg CSC and no recovery period. Both tumor and non-tumor tissue promoted with 27 mg CSC for 29 weeks exhibited changes in GC-specific methylation that were more pronounced in tumors. Tumor tissue was globally hypomethylated, whereas non-tumor tissue did not exhibit changes in global methylation. In conclusion, as expected for a mechanism underlying tumor promotion, CSC alters methylation in a threshold-exhibiting, reversible, progressive fashion during promotion. Progressive alterations in global and GC-rich methylation appear to be mechanistically important during tumor promotion.