Methylation Of Repetitive Sequences Research Articles

MicroRNA-29b/142-5p contribute to the pathogenesis of biliary atresia by regulating the IFN-\u03b3 gene

Biliary atresia is one of the most common liver disease in infancy. The cause and pathogenesis remain largely unknown. This study aimed to investigate the potential regulatory effect of miR-29b/142-5p on IFN-γ gene methylation. miRNAs microarray was performed on four pairs of liver and blood specimens from biliary atresia and choledochal cysts. We found the overexpression of miR-142-5p and mRNA level of DNA methyltransferase (DNMT) 1, and miR-29b and DNMT3a/DNMT3b were significantly negatively correlated in biliary atresia livers. Meanwhile, the methylation of the LINE-1, ALU and SAT2 repetitive sequences and the IFN-γ promoter was lower, but the expression of IFN-γ was upregulated. After transfected with DNMTs siRNAs, downregulation of DNMTs exerted a significant hypomethylating effect on the repetitive sequences, which led to upregulation of IFN-γ in Jurkat cells. The direct interactions between miR-29b and DNMT3a/3b, and miR-142-5p and DNMT1 were identified using luciferase reporter assays. By transfecting mimics of miR-29b/142-5p into Jurkat cells, we found overexpression of miR-29b/142-5p markedly suppressed expression of DNMTs. Furthermore, the methylation of repetitive sequences and the IFN-γ promoter region were remarkably downregulated, and with elevated IFN-γ expression. After transfecting the miRNA inhibitors, the levels of DNMTs and the methylation of the IFN-γ gene promoter region was upregulated, while levels of IFN-γ were markedly suppressed. Our study suggested that miRNA-29b/142-5p overexpression and targeted inhibition of DNMTs expression resulted in decreased overall gene methylation and overexpression of the methylation-sensitive IFN-γ gene.

Birthweight, Maternal Weight Trajectories and Global DNA Methylation of LINE-1 Repetitive Elements

Low birthweight, premature birth, intrauterine growth retardation, and maternal malnutrition have been related to an increased risk of cardiovascular disease, type 2 diabetes mellitus, obesity, and neuropsychiatric disorders later in life. Conversely, high birthweight has been linked to future risk of cancer. Global DNA methylation estimated by the methylation of repetitive sequences in the genome is an indicator of susceptibility to chronic diseases. We used data and biospecimens from an epigenetic birth cohort to explore the association between trajectories of fetal and maternal weight and LINE-1 methylation in 319 mother-child dyads. Newborns with low or high birthweight had significantly lower LINE-1 methylation levels in their cord blood compared to normal weight infants after adjusting for gestational age, sex of the child, maternal age at delivery, and maternal smoking during pregnancy (p = 0.007 and p = 0.036, respectively), but the magnitude of the difference was small. Infants born prematurely also had lower LINE-1 methylation levels in cord blood compared to term infants, and this difference, though small, was statistically significant (p = 0.004). We did not find important associations between maternal prepregnancy BMI or gestational weight gain and global methylation of the cord blood or fetal placental tissue. In conclusion, we found significant differences in cord blood LINE-1 methylation among newborns with low and high birthweight as well as among prematurely born infants. Future studies may elucidate whether chromosomal instabilities or other functional consequences of these changes contribute to the increased risk of chronic diseases among individuals with these characteristics.

Biological and clinical relevance of quantitative global methylation of repetitive DNA sequences in chronic lymphocytic leukemia

Global DNA hypomethylation affecting repeat sequences has been reported in different cancer types. Herein, we investigated the methylation levels of repetitive DNA elements in chronic lymphocytic leukemia (CLL), their correlation with the major cytogenetic and molecular features, and clinical relevance in predicting therapy-free survival (TFS). A quantitative bisulfite-PCR Pyrosequencing method was used to evaluate methylation of Alu, long interspersed nuclear elements-1 (LINE-1) and satellite-α (SAT-α) sequences in 77 untreated early-stage (Binet A) CLL patients. Peripheral B-cells from 7 healthy donors were used as controls. Methylation levels (median %5mC) were lower in B-CLLs compared with controls (21.4 vs. 25.9; 66.8 vs. 85.7; 84.0, vs. 88.2 for Alu, LINE-1 and SAT-α, respectively) (p < 0.001). Among CLL patients, a significant association was observed with 17p13.1 deletion (16.8 vs. 22.4; 51.2 vs. 68.5; 52.6 vs. 85.0, for Alu, LINE-1 and SAT-α) but not with other major genetic lesions, IgVH mutation status, CD38 or ZAP-70 expression. Follow-up analyses showed that lower SAT-α methylation levels appeared to be an independent prognostic marker significantly associated with shorter TFS. Our study extended previous limited evidences in methylation of repetitive sequences in CLL suggesting an important biological and clinical relevance in the disease.

Abstract 181: Biological and clinical relevance of quantitative global methylation in repetitive DNA sequences in B-cell chronic lymphocytic leukemia

Abstract Background: B-cell chronic lymphocytic leukemia (B-CLL) is characterized by highly clinical heterogeneity; the identification of reliable prognostic factors useful in predicting patient outcome and planning therapeutic strategies represent a crucial task. Global DNA hypomethylation affecting repeat sequences, such as long interspersed nuclear elements-1 (LINE-1), Alu and satellite α DNA (SAT-α DNA), has been reported in different cancer types and associated with chromosomal instability. Aim: In order to investigate the role of global DNA methylation in B-CLL we quantified the methylation levels of Alu, LINE-1 and SAT-α repetitive in B-CLL patients and correlated them with the major cytogenetic and molecular features and clinical relevance in predicting therapy-free survival (TFS). Patients and methods: A quantitative bisulfite-PCR Pyrosequencing method was used to evaluate methylation of Alu, LINE-1, and SAT-α in a panel of highly purified (&amp;gt;90%) peripheral mononuclear CD19+ cells from 7 healthy donors and 77 B-CLL untreated well-characterized patients in early stage disease (Binet stage A). Results: Forty-eight patients had unmutated IgVH genes; ZAP-70 and CD38 were positive in 28 and 35 cases, respectively; 13q14.3 deletion was present as a sole abnormality in 21/33 patients while in the remaining cases it was combined with 17p13.1 (4 pts) or 11q22.3 deletions (7 pts) or both (1 pt). The 11q22.3 and 17p13.1 deletions were detected as sole abnormality in 6 and 7 patients, respectively. Trisomy 12 occurred in 17 patients, as a sole abnormality in all cases. Methylation levels (median %5mC) were lower in B-CLLs compared with controls (21.4 vs. 25.9; 66.8 vs. 85.7; and 84.0, vs. 88.2; for Alu, LINE-1 and SAT-α, respectively). A significant association was observed with 17p13.1 deletion (15.4 vs. 22.4; 42.9 vs. 68.5; 32.7 vs. 85.0, for Alu, LINE-1 and SAT-α) but not with other major genetic lesions, IgVH mutation status, CD38 or ZAP-70 expression. Notably, lower SAT-α methylation levels appeared to be an independent prognostic marker significantly associated with a shorter TFS. Conclusion: Our study extended limited evidence in methylation of repetitive sequences in B-CLL suggesting an important biological and clinical relevance in the disease. 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 181.

A Study of Aberrant DNA Methylation in Refractory and Relapsed Childhood Acute Lymphoblastic Leukaemia.

Abstract 3456Poster Board III-344Refractory and relapsed Acute Lymphoblastic Leukaemia (ALL) raise difficult clinical challenges and represent a significant cause of death in childhood and adolescence. This study examines epigenetic changes in relapsed paediatric ALL to increase our understanding of the mechanisms of treatment failure in these patients. Aberrant DNA methylation, a frequent phenomenon in paediatric ALL, has been identified as an independent factor of poor prognosis. In this study, using a CGI microarray consisting of 12K clones representing 5411 unique chromosomal sequences spread throughout the genome, we analysed a cohort of 22 childhood B-precursor ALL patient samples, 11 diagnosis samples and 11 relapse samples (unmatched) from patients who experienced a hematologic relapse, and developed genome-wide methylation profiles of leukaemic cells, at diagnosis and relapse. Comparison of these profiles identified 227 genes with statistically significant methylation changes – increase and decrease of methylation levels - between the two patient groups. The methylation status and the methylation change of 5 genes (ECRG4, CDH5, IRX3, FOXD3 and ERBB4) were independently verified, using pyrosequencing, in 6 ALL patients, 3 diagnosis and 3 relapse samples. The relationship between methylation and expression of one of these genes (ECRG4) was examined in ALL cell lines (CEMC1 and Jurkat) before and after 5-Aza-2'-deoxycytidine treatment. More genes are currently under investigation. Methylation studies on the repetitive element LINE-1 showed a significant increase in methylation of repetitive sequences in relapse samples (mean 79% vs. 89%, respectively; p=0.003, Mann Whitney test). Chromosomal mapping of the 227 genes revealed significant clustering of differentially methylated targets in chromosomes 11, 13 and 15, chromosomal locations that were previously found to be associated with ALL prognosis. Functional gene relation analysis revealed a connection between 11 genes (FOXD3, HPGD, NR4A2, CAST, TBRG4, XRCC6, NAB2, SEZL6, CDH13, NR2F1, SP1) that show increased methylation in relapse ALL and NR3C1, the gene coding for glucocorticoid receptor. Currently we are investigating the role of methylation in glucocorticoid resistance in ALL cell lines. Our data indicate an underlying epigenetic mechanism responsible for the chemoresistance observed in relapsed paediatric ALL patients. DisclosuresNo relevant conflicts of interest to declare.

Sequence organisation in nuclear DNA from Physarum polycephalum: methylation of repetitive sequences

Nuclear DNA from the slime mould Physarum polycephalum is digested by the restriction endonuclease HpaII to generate a high molecular weight and a low molecular weight component. These are referred to as the M+ and the M- compartment, respectively. Sequences that are present in the M+ compartment are cleaved by MspI, the restriction enzyme isoschizomer of HpaII, thus showing that the recognition sequences for these enzymes in M+ DNA contain methylated CpG doublets. The distribution of repetitive sequences in the M+ and M- DNA compartments was investigated by comparison of the 'fingerprint' patterns of total Physarum DNA and isolated M+ DNA after digestion using different restriction endonucleases, and by probing for the presence of specific repetitive sequences in Southern blots of M+ and M- DNA by the use of cloned DNA segments. Both types of experiment indicate that many repetitive sequences are shared by both compartments, though some repetitive sequences appear to be considerably enriched, or are present exclusively, either in M+ DNA or in M- DNA.