Global methylation analysis of development and progression of colorectal carcinoma
Ontology highlight
ABSTRACT: Colorectal tumorigenesis proceedes through well defined clinical stages assoicated with charateristic mutations. Besides genetic alterations, epi-driver genes that are aberrantly expressed in cancers in a fashion that confers a seletive growth advantage can also contribute to tumor evolution. To gain a global view of methylation patterns in normal and maliganant colorectal epithelia, we performed genome-wide DNA methylation analysis on DNAs from 48 fresh frozen CRC samples at different stages of CRC progression. We used IlluminaHumanMethylation450 Beadchip to get a broad view of genome-wide DNA methylationdata during CRC progression and identified significantly differentially methylated genes during CRC progression A total of 48 macro-dissected tissues including normal colon tissue, adenomas, carcinomas and metastases were collected by the Department of Pathology at the University of Virginia under the supervision of an experienced pathologist. DNA was extracted and run on IlluminaHumanMethylation450 Beadchip by Expression Analyisis.
Project description:An integrative functional genomics of multiple forms of data is vital for discovering molecular drivers of cancer development and progression. Here, we present an integrated genomic strategy utilizing DNA methylation and transcriptome profile data to discover epigenetically regulated genes implicated in cancer development and invasive progression. More specifically, this analysis identified Fibromodulin (FMOD) as a glioblastoma (GBM) upregulated gene due to the loss of promoter methylation. Secreted FMOD promotes glioma cell migration through its ability to induce filamentous actin stress fiber formation. Treatment with Cytochalasin D, an actin polymerization inhibitor, significantly reduced the FMOD induced glioma cell migration. siRNA and small molecule inhibitor-based studies identified that FMOD-induced glioma cell migration is dependent on Integrin-FAK-Src-Rho-ROCK signaling pathway. FMOD lacking C terminus LRR11 domain (FMOD), which does not bind collagen type I, failed to induce integrin and promote glioma cell migration. Further, FMOD-induced integrin activation and migration was abrogated by a 9-mer wild type peptide from the FMOD C-terminus. However, the same peptide with mutation in two residues essential for FMOD interaction with collagen type I failed to compete with FMOD, thus signifying the importance of collagen type I-FMOD interaction in integrin activation. ChIP-PCR experiments revealed that TGF-Ã1 regulates FMOD expression through epigenetic remodeling of FMOD promoter that involved demethylation and gain of active histone marks with a simultaneous loss of DNMT3A and EZH2 occupancy, but enrichment of SMAD2 and CBP. FMOD silencing inhibited the TGF-Ã1 mediated glioma cell migration significantly. In univariate and multivariate cox regression analysis, both FMOD promoter methylation and transcript levels predicted prognosis in GBM. Thus, the present study identified several epigenetically regulated alterations responsible for cancer development and progression. Specifically, we found that secreted FMOD as an important regulator of glioma cell migration downstream of TGF-Ã1 pathway and forms a potential basis for therapeutic intervention in GBM. We used DNA isolated from 17 diffuse astrocytoma, 16 anaplastic astrocytoma, 36 glioblastoma and 9 control brain tissue for the array. P53 status: All cases were subjected to histopathology and the diagnosis of GBM was confirmed. Paraffin sections (4 μm) from the tissues were collected on silane-coated slides, and the protein expression of p53 was assessed by immunohistochemistry (IHC). Antigen retrieval was done by heat treatment at 850W in citrate buffer or at 700W. After the initial processing steps, sections were incubated overnight with primary antibody for p53 (Mouse Monoclonal DO-7; Biogenex, USA)at 4°C with 1: 200 dilution. MIB: Paraffin blocks of all the cases were retrieved, and histological features were reviewed on freshly cut and stained (hematoxylin and eosin) sections. After confirming the diagnosis of glioblastoma, 4 µm thick sections were collected on silane-coated slides, and IHC was performed using MIB-1 (anti Ki-67 monoclonal BGX 297, diluted to 1:30) EGFR status: Paraffin blocks of all the cases were retrieved, and histological features were reviewed on freshly cut and stained (hematoxylin and eosin) sections. After confirming the diagnosis of glioblastoma, 4 µm thick sections were collected on silane-coated slides, and IHC was performed using EGFR (monoclonal E-30, diluted to 1:50) GFAP status: Paraffin blocks of all the cases were retrieved, and histological features were reviewed on freshly cut and stained (hematoxylin and eosin) sections. After confirming the diagnosis of glioblastoma, 4 µm thick sections were collected on silane-coated slides, and IHC was performed using glial fibrillary acidic protein (GFAP, monoclonal GA-5, diluted to 1:100).
Project description:Prenatal exposure to inorganic arsenic (iAs) is detrimental to the health of newborns and increases the risk of disease development later in life. Here we examined a subset of newborn cord blood leukocyte samples collected from mothers enrolled in the Biomarkers of Exposure to ARsenic (BEAR) pregnancy cohort in Gomez Palacio, Mexico who were exposed to a range of drinking water arsenic concentrations (0.456-236 µg/L). Changes in iAs-associated DNA 5-methyl cytosine methylation were assessed across 424,935 CpG sites representing 18,761 genes and were compared to corresponding expression levels and birth outcomes. In the context of arsenic exposure, a total of 2,705 genes were identified with iAs-associated differences in DNA methylation. Site-specific analyzes identified DNA methylation changes that were most predictive of gene expression levels. Specifically, CpG methylation within CpG islands positioned within the first exon and 200bp upstream of the transcription start site yielded the most significant association with gene expression levels. A set of 16 genes was identified with correlated iAs-associated changes in DNA methylation and mRNA expression and all were highly enriched for binding sites of the early growth response (EGR) and CCCTC-binding factor (CTCF) transcription factors. Furthermore, DNA methylation levels of seven of these genes were associated with differences in birth outcomes including gestational age, placental weight and head circumference. These data highlight the complex interplay between DNA methylation and functional changes in gene expression and health outcomes and underscore the need for functional analyzes coupled to epigenetic assessments. 48 DNA methylation profiles were obtained but only 38 samples corresponding to samples also analyzed for gene expression (see Rager 2014) were further normalized and used in the publication from the Biomarkers of Arsenic (BEAR) cohort were analyzed for DNA methylation profiles.
Project description:Miscarriage occurs in 15-20% of clinical pregnancies. While chromosomal errors are observed in over 50%, causes of karyotypically normal losses are poorly understood. DNA methylation undergoes reprogramming during development and must be appropriately set to maintain a healthy pregnancy. We hypothesize that aberrant DNA methylation may cause karyotypically normal miscarriage, particularly among women experiencing recurrent miscarriage (RM). DNA methylation in first trimester chorionic villi was assessed in chromosomally normal miscarriages from women with RM (N=33) or isolated miscarriage (M, N=21), and elective terminations (TA, N=16). Differentially methylated candidate loci were identified using the Illumina Infinium HumanMethylation27 BeadChip array by comparing 10 RM to 10 TA samples. Follow up showed a significant increase in methylation in RM and M compared to TA placentae at the CYP1A2 (p=0.002) and AXL (p=0.02) promoters and decrease at the DEFB1 (p=0.008) promoter. Gene function analysis showed an enrichment of imprinted genes (p=9.53E-10) and genes previously associated with RM (p=9.51E-06). DNA methylation was evaluated at 7 imprinted loci using bisulfite pyrosequencing. An increase of outliers at these loci was observed in RM (3.9%) compared to M (0%) and TA (0.9%) (p=0.02), with increased average methylation at the H19/IGF2 ICR1 in M samples (p<0.0001). Altered DNA methylation in the placenta at specific loci, as well as global dysregulation in specific cases, may contribute to or be a consequence of placental insufficiency in karyotypically normal miscarriage. First-trimester placental villi samples from karyotypically normal miscarriages from recurrent miscarriage patients (N, N=10) and chromosomally normal elective terminations (PZET, N=10).
Project description:Genome wide DNA methylation profiling of somatic and pluripotent cells from different lineages (mesoderm, endoderm and parthenogenetic germ cells) The Illumina Infinium 27k Human DNA methylation Beadchip v1.2 was used to obtain DNA methylation profiles across approximately 27,000 CpGs. Samples included 1 Human ES cell line, 2 beta cells, 2 beta-iPS cells, 1 fibroblast, 2 fibroblast-iPS cells, 2 parthenogenetic cells and 3 parthenogenetic-iPS cells. Molecular reprogramming of somatic cells into human induced pluripotent stem cells (iPSCs) is accompanied by extensive changes in gene expression patterns and epigenetic marks. To better understand the link between gene expression and DNA methylation, we have profiled human somatic cells from different embryonic cell types (endoderm, mesoderm and parthenogenetic germ cells) and the iPSCs generated from them. We show that reprogramming is accompanied by extensive DNA methylation in CpG-poor promoters, sparing CpG-rich promoters. Intriguingly, methylation in CpG-poor promoters occurred not only in downregulated genes, but also in genes that are not expressed in the parental somatic cells or their respective iPSCs. These genes are predominantly tissue-specific genes of other cell types from different lineages. Our results suggest a role of DNA methylation in the silencing of the somatic cell identity by global non-specific methylation of tissue-specific genes from all lineages, regardless of their expression in the parental somatic cells. Genomic DNA from each sample was bisulfite converted, DNA was applied to BeadChips (Illumina). 13 samples included, Human ES cell as control.
Project description:To evaluate the methylation profiles of breast cell lines, we performed methylation profiling of 55 well-characterized breast cancer cell lines on the Illumina HumanMethylation27 (HM27) platform and made use of publicly available methylation profiles of primary breast tumors for comparison. The available annotation for each cell line includes estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) status, as well as the tumor type, and the age of each patient. Additionally, recent publications have described genome-wide mRNA expression profiles for most of these lines, and samples were classified on the basis of the expression profile into Basal A (BaA), Basal B/Claudin Low (BaB/CLDNlow) and Luminal (Lu) subtypes. Finally, GI50 has been calculated for these cell lines for 77 approved therapeutic agents. We find that the DNA methylation profiles of breast cancer cell lines largely retain the features that characterize primary tumors, although there are crucial differences as well. We assayed DNA methylation in 55 breast cancer cell lines. DNA extracted from breast cell lines was bisulfite treated and hybridized to Illumina HM27 arrays.
Project description:Elderly AA volunteers confirmed MCI assigned into a six-month program of aerobic exercise (eleven participants) underwent a 40-minute supervised-training 3-times/week and controls (eight participants) performed stretch training. Participants had maximal oxygen consumption (VO2max) test and Genome-wide methylation levels at CpG sites using the Infinium HumanMethylation450 BeadChip assay at baseline and after a six-month exercise program.
Project description:Since we have found that the RhoC gene causes extensive changes to gene expression, a methylation array was carried out to determine if the observed transcriptional changes are due to methylation changes in the genome.
Project description:Identification of promoter methylation profile associated with cervical cancer progression. The Ilumina Infinium 27k Human DNA methylation Beadchip v1.2 was used to obtain DNA methylation profiles across approximately 27,000 CpGs from 19 cervical samples (including normal, CIN I-II, in situ and invasive cervical cancer tissues). Bisulphite converted DNA from the 19 samples were hybridised to the Illumina Infinium 27k Human Methylation Beadchip v1.2