Constitutional Trisomy 8 Mosaicism as a Model for Epigenetic Studies of Aneuploidy (DNA methylation)
ABSTRACT: To increase our understanding of epigenetic patterns associated with aneuploidy we used constitutional trisomy 8 mosaicism as a model, enabling analyses of single cell clones, harboring either trisomy or disomy 8, from the same patient. We profiled gene and miRNA expression as well as genome-wide and promoter specific DNA methylation and hydroxymethylation patterns in trisomic and disomic fibroblasts, using microarrays and methylated DNA immunoprecipitation. comparison of trisomy 8 cells with disomic as well as reference fibroblasts
Project description:We propose that tetraploidy induces epigentic changes including DNA methylation due to the abnormal chromatin in the cells. To test our hypothesis, we employed methylated CpG island recovery assay (MIRA) assisted microarray to determine the DNA methylation profiles in both diploid and tetraploid cells. First, we conducted a methylated-CpG island recovery assay (MIRA). Briefly, we enriched for methylated CpG islands with methylated DNA binding proteins, MBD2/MBD3L1. The pulled down DNA fragments containing CpG islands and input DNA fragments were amplified with real-time PCR. After labeling, they were hybridized to CpG island promoter array. Data were collected and analyzed.
Project description:Astrocytomas are common and lethal human brain tumors. Here, we have analyzed the methylation status of over 28,000 CpG islands and 18,000 promoters in normal human brain and in astrocytomas of various grades using the methylated-CpG island recovery assay (MIRA). We identified six to seven thousand methylated CpG islands in normal human brain. ~5% of the promoter-associated CpG islands in normal brain are methylated. Promoter CpG island methylation is inversely and intragenic methylation is directly correlated with gene expression levels in brain tissue. In astrocytomas, several hundred CpG islands undergo specific hypermethylation relative to normal brain with 428 methylation peaks common to more than 25% of the tumors. Genes involved in brain development and neuronal differentiation, such as POU4F3, GDNF, OTX2, NEFM, CNTN4, OTP, SIM1, FYN, EN1, CHAT, GSX2, NKX6-1, RAX, PAX6, DLX2, were strongly enriched among genes frequently methylated in tumors. There was an overrepresentation of homeobox genes and 31% of the most commonly methylated genes represent targets of the Polycomb complex. We identified several chromosomal loci in which many (sometimes more than 20) consecutive CpG islands were hypermethylated in tumors. Seven of such loci were near homeobox genes, including the HOXC and HOXD clusters, and the BARHL2, DLX1, and PITX2 genes. Two other clusters of hypermethylated islands were at sequences of recent gene duplication events. Our analysis offers mechanistic insights into brain neoplasia suggesting that methylation of genes involved in neuronal differentiation, perhaps in cooperation with other oncogenic events, may shift the balance from regulated differentiation towards gliomagenesis. Comparison of methylation patterns of 30 astrocytomas and 6 controls
Project description:This SuperSeries is composed of the following subset Series: GSE18802: Comparison of gene expression profiles in diploid and transformed tetraploid MEF cells GSE18814: Comparison of DNA methylation profiles in diploid and transformed tetraploid MEF cells Refer to individual Series
Project description:Methylated modifications of genome are common events in carcinogenesis and is involved in the tumorigenesis and progression of various cancers including gastric cancer Methylated DNA immunoprecipitation (MeDIP) combined with a human miRNA tiling microarray analysis demonstrated that there are much methylation differention between gastric cancers and adjacent controls microRNA gene methylation comparison of 3 pairs of gastric cancer and controls
Project description:Emerging evidences indicate that microRNAs (miRNAs) are often deregulated and have fundamental roles in hepatocellular carcinoma (HCC). However, the mechanism underlying miRNA dysregulation in HCC is still elusive. In this report, we used an integrated analysis strategy combining methylated DNA immunoprecipitation chip (MeDIP-chip) and miRNA expression microarray data to study the correlation between aberrant methylation and dysregulation of miRNA in HCC. In all, we showed that global miRNA methylation profiles were significantly different between cancerous and normal hepatocytes, and abnormal methylation was an important mechanism governing miRNA expression in HCC. MeDIP-chip was processed in cancerous hepatocytes SK-HEP-1, HepG2, MHCC97-H and normal hepatocytes PHHC-4-1, PHHC-4-2, PHHC-4-3 (3 technical repeat of PHHC-4). MiRNA microarray were processed for cancerous hepatocytes SK-HEP-1, HepG2, Hep3B, Huh7, MHCC97-H, MHCC97-L, SMMC-7721 and normal hepatocytes PHHC-1, PHHC-2, PHHC-3. Then an integrated analysis strategy combining MeDIP-chip and miRNA expression microarray [GSE20077] were used to study the correlation of aberrant DNA methylation and dysregulation of miRNAs.
Project description:Streptococcus suis is an important zoonotic pathogen that can cause meningitis and sepsis in both pigs and humans. In this study,we evaluated the genetic difference of 40 Streptococcus suis strains belonging to various sequence types by comparative genomic hybridization to identify genes associated with the variation in pathogenicity using NimbleGen’s tilling microarray platform. Application of Comparative Phylogenomics to Identify Genetic Differences Relating to Pathogenicity of Streptococcus suis Comparative genomic analysis on the 40 S.suis strains of different serotypes and ST types through tilling arrays
Project description:E2F2 is essential for the maintenance of T lymphocyte quiescence. To identify the full set of E2F2 target genes, and to gain further understanding of the role of E2F2 in transcriptional regulation, we have performed ChIP-chip analyses across the genome of lymph node-derived T lymphocytes. Here we show that during quiescence, E2F2 binds the promoters of a large number of genes involved in DNA metabolism and cell cycle regulation, concomitant with their transcriptional silencing. We performed 3 ChIP-chip experiments with an antibody against E2F2 and another 3 ChIP-chip experiments with an antibody against SV40TAg (irrelevant antibody).
Project description:Ebf1 is a key determinant of B-lymphocyte specification. In order to identify unknown transcriptional targets, endogenous Ebf1 was isolated by chromatin-IP from primary pro-B cells and the copurified DNA was hybridized to promoter tiling arrays. Ebf1 was ChIPed using a polyclonal antibody directed against an N-terminal peptide from primary pro-B cells grown in the presence of Il7. The resulting DNA was analyzed by tiling array hybridization.
Project description:In diverse eukaryotes, constitutively silent sequences, such as transposons and repeats, are marked by methylation at histone H3 lysine 9 (H3K9me). Despites the conservation and importance in the genome integrity, mechanisms to exclude H3K9m from active genes remained largely unexplored. Here we show in Arabidopsis that the exclusion depends on a histone demethylase gene, IBM1 (increase in BONSAI methylation); loss-of-function ibm1 mutation caused ectopic H3K9me in thousands of genes, which accompanies genic DNA methylation at non-CG sites. The ibm1-induced genic H3K9me depended on both histone methylase KYP/SUVH4 and DNA methylase CMT3, suggesting interdependence of two epigenetic marks – H3K9me and non-CG methylation. Notably, IBM1 enhanced loss of H3K9m in transcriptionally de-repressed sequences. Furthermore, disruption of transcription in genes induced ectopic non-CG methylation, mimicking the loss of IBM1 function. We propose that active chromatin is stabilized by the autocatalytic loop of transcription and H3K9 demethylation. This process counteracts accumulation of silent epigenetic marks, H3K9me and non-CG methylation, which is also autocatalytic. Leaves of 4-week-old plants were fixed as described previously (Saze et al, 2008). Chromatin immunoprecipitation (ChIP) was performed as described previously (Kimura et al, 2008), using antibody against H3K9me2 (CMA307, Kimura et al, 2008, PMID: 18227620). Non-immunoprecipitated DNA (input DNA) and ChIP samples were amplified, labeled, and hybridized to microarray according to the manufacturer’s instruction (Protocols for Chromatin Immunoprecipitation and Amplification, NimbleGen). Input DNA and ChIP DNA were differentially labeled with Cy3 and Cy5, respectively, and competitively hybridized to a microarray chip. We used NimbleGen 2.1M HD2 array covering entire genome of A. thaliana.