Name: Methylation profiling by array of human HCT116 colon carcinoma cells to investigate DNA methylationchanges induced by the demethylating drugs 5-azacytidine and CP-4200
Keywords: methylation profiling by array

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Methylation profiling by array of human HCT116 colon carcinoma cells to investigate DNA methylationchanges induced by the demethylating drugs 5-azacytidine and CP-4200

ABSTRACT: array-based analysis of genome-wide DNA methylation changes induced by the demethylating drugs 5-azacytidine and CP-4200 on HCT116 cells for 72 hours

ORGANISM(S): Homo sapiens

DISEASE(S): Colon Carcinoma

SUBMITTER: Maria Rius

PROVIDER: E-MTAB-702 | ArrayExpress | 2013-06-01

REPOSITORIES: ArrayExpress

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Project description:A fundamental challenge in the post-genome era is to understand and annotate the consequences of genetic variation, particularly within the context of human tissues. We describe a set of integrated experiments designed to investigate the effects of common genetic variability on DNA methylation, mRNA expression and microRNA (miRNA) expression in four distinct human brain regions. We show that brain tissues may be readily distinguished based on methylation status or expression profile. We find an abundance of genetic cis regulation mRNA expression and show for the first time abundant quantitative trait loci for DNA CpG methylation. We observe that the largest magnitude effects occur across distinct brain regions. We believe these data, which we have made publicly available, will be useful in understanding the biological effects of genetic variation. Authorized Access data: Mapping of GEO sample accessions to dbGaP subject/sample IDs is available through dbGaP Authorized Access, see http://www.ncbi.nlm.nih.gov/projects/gap/cgi-bin/study.cgi?study_id=phs000249.v1.p1 Because of our interest in genomic regulation of expression and neurological disorders we embarked upon a series of experiments to provide a brain region-specific contextual framework for genetic and epigenetic regulation of gene expression. We obtained frozen brain tissue from the cerebellum, frontal cortex, pons and temporal cortex from 150 subjects (total 600 tissue samples). We undertook four separate assays across this series; first, genome-wide SNP genotyping; second, assay of >27,000 CpG methylation sites in each of the four brain regions; third, mRNA expression profiling of >22,000 transcripts in all four brain regions; and, fourth, miRNA expression profiling of 735 miRNA transcripts. Here we discuss the results of these experiments, particularly in the context of integrated datasets to define expression and CpG methylation quantitative trait loci (eQTL and methQTL) and detailing differences and similarities across brain regions.

Project description:Serrated adenocarcinoma (SAC) is a recently recognized colorectal cancer (CRC) subtype accounting for 7.5 - 8.7% of CRCs. It has been shown that SAC has a worse prognosis and has different molecular and immunohistochemical features compared to conventional carcinoma (CC) but, to date, there is no study analysing its methylome profile. We have investigated the methylation status of 450,000 CpG sites using the Infinium Human Methylation 450 BeadChip array in 103 colorectal specimens from Spanish and Finnish patients. The comparison between the epigenetic signature of 36 SACs and 34 matched CCs was established with the aim of identifying the functions which characterize SAC biology and also the most differentially methylated genes for subsequent validatation by pyrosequencing, methylation-specific PCR, qPCR and immunohistochemistry, including additional cases. Microarray data showed a higher representation of morphogenesis-, neurogenesis-, cytoskeleton- and vesicle-transport-related functions and also significant differential methylation of 15 genes, including the iodothyronine deiodinase DIO3 and the forkhead family transcription factor FOXD2 genes which were validated at the CpG, mRNA and protein level. A quantification study of the methylation status of CpG sequences in FOXD2 demonstrated a novel region controlling gene expression. Moreover, differences in these markers were also evident when comparing SAC with CRC showing molecular and histological features of high level microsatellite instability. This methylome study demonstrates that SAC has a distinct epigenetic regulation pattern resulting in different biological functions and that DIO3 and FOXD2 might be molecular targets for a specific histology-oriented treatment of CRC. HumanMethylation450K BeadChip (Illumina, Inc, San Diego, CA), using Infinium HD Methylation assay for genome-wide DNA methylation screening, was employed. In brief, genomic DNA (1000 ng) from each sample was bisulfite converted with the EZ DNA Methylation Kit (Zymo Research, Orange, CA) according to the manufacturer´s recommendations. Bisulfite-treated DNA was isothermally amplified at 37°C (20-24h) and the DNA product was fragmented by an endpoint enzymatic process, then precipitated, resuspended, applied to an Infinium Human Methylation450K BeadChip (Illumina, San Diego, CA, USA) and hybridized at 48°C (16-24h). The fluorescently-stained chip was imaged by the Illumina i-SCAN and Illumina's Genome Studio program (Methylation Module) was used to analyze BeadArray data to assign site-specific DNA methylation β-values to each CpG site.

			Action	DRS
	E-MTAB-702.README.txt	Txt
	E-MTAB-702.idf.txt	Idf
	E-MTAB-702.raw.1.zip	Raw
	E-MTAB-702.sdrf.txt	Txt

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Methylation profiling by array of human HCT116 colon carcinoma cells to investigate DNA methylationchanges induced by the demethylating drugs 5-azacytidine and CP-4200

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