Project description:Genome wide DNA methylation profiling of target sites of dnmt3b isoforms in HCT derivative cell line DKO8. The Illumina Infinium 450k Human DNA methylation Beadchip was used to obtain DNA methylation profiles across approximately 450,000 CpGs
Project description:Genome wide DNA methylation profiling of target sites of dnmt3b isoforms in HCT derivative cell line 3BKO followed by 5-Aza-CdR treatment. The Illumina Infinium 450k Human DNA methylation Beadchip was used to obtain DNA methylation profiles across approximately 450,000 CpGs
Project description:Genome wide DNA methylation profiling of target sites of dnmt3b isoforms in HCT derivative cell line 3ABDKO followed by 5-Aza-CdR treatment. The Illumina Infinium 450k Human DNA methylation Beadchip was used to obtain DNA methylation profiles across approximately 450,000 CpGs
Project description:Both DNA methylation and alternative splicing represent mechanisms closely involved in regulation of gene expression and are well known to be involved in cancer susceptibility and development. Human DNA methylation is introduced into DNA by enzymes of the DNA cytosine methyltransferases family, which includes DNMT3B. The DNMT3B protein allows a de novo DNA methylation activity, which is a key mechanism involved in the transformation of normal cell into cancer cell. The DNMT3B3 isoform is overexpressed in cancerous tissues and tumor cell lines, and could act as a dominant negative factor. This isoform was found to be highly expressed in our cohort of non-BRCA1/2 families. Using Infinium Human Methylation 450 BeadChips, we undertook the characterization of the specific methylation profile associated with this DNMT3B3 isoform and its DNMT3B2 wild type counterpart in ER/PR-positive and –negative breast cancer cell lines. A large spectrum of DNMT3B3/DNMT3B2 expression ratio values was observed in cancer and non-cancerous cell lines. Based on their methylation profiles, hierarchical clustering of parental cell lines revealed clustering of cells based on their ER/PR status. Overexpression of DNMT3B3 triggered methylation changes of thousands of CpG sites in MCF7, T-47D, MDA-MB-231 and MCF10A cells. These methylated loci were distributed in similar proportion over the 22 autosomal chromosomes and genomic locations such as promoter, gene body or intergenic regions. Pathways associated with genes containing these differentially methylated CpG sites were also determined regarding their enrichment. Moreover, based on the trend of methylation changes, the results suggest an anti-proliferative effect of the DNMT3B3 isoform through negative effect on its wild-type isoform counterpart DNMT3B2. To our knowledge, this study represents the first exhaustive analysis describing specific methylation profile triggered by modulated expression of DNMT3B isoforms and revealed specific genes and pathways, which could significantly regulate cell growth and proliferation as well as other biological and molecular mechanisms.
Project description:We used microarrays to detail the global programme of gene expression for MCF-7 and MDA-MB-231 and revealed the correlation between the methylation state of various genomic components and gene expression level. The expression analyses of the two breast cancer cell lines are a part of the whole study. The summary of our study is as follows: We establish a technique, called modified methylation-specific digital karyotyping (MMSDK) based on methylation-specific digital karyotyping (MSDK) with a novel sequencing approach. Briefly, after a tandem digestion of genomic DNA with a methylation-sensitive mapping enzyme and a fragmenting enzyme, short sequence tags are obtained. These tags are amplified, followed by direct, massively parallel sequencing (Solexa 1G Genome Analyzer). This method allows high-throughput and low-cost genome-wide DNA methylation mapping. We applied this method to investigate global DNA methylation profiles for widely used breast cancer cell lines, MCF-7 and MDA-MB-231, which are representatives for luminal-like and mesenchymal-like cancer types, respectively. By comparison, a highly similar overall DNA methylation pattern was revealed for the two cell lines. However a cohort of individual genomic loci with significantly different DNA methylation profile between two cell lines was identified. Furthermore, we revealed a genome-wide significant correlation between gene expression and the methylation status of gene promoters with CpG islands (CGIs) in the two cancer cell lines, and a correlation of gene expression and the methylation status of promoters without CGIs in MCF-7 cells. Experiment Overall Design: Breast cancer cell lines, MCF-7 and MDA-MB-231, were selected for the study of the impact of DNA methylation on gene expression regulation. Total RNA extraction was performed for both cell lines and hybridization was carried out using Affymetrix microarrays. We developed a modified methylation-specific digital karyotyping (MSDK) to obtain DNA methylation profiling genome wide. Then, we combined the analysis of DNA methylation data and gene expression data to reveal a correlation between epigenetic and transcriptional features genome wide.
Project description:Genome wide DNA methylation profiling of normal and tumor colorectal cancer samples, HCT116 DKO (DNMT1 (-/-) and DNMT3b (-/-)), M.sssI treated DKO cell lines, and human cervical cancer cell line HeLa. The Illumina Infinium Human DNA methylation EPIC Beadchip was used to obtain DNA methylation profiles across approximately 85,000 CpGs in the above samples.
Project description:DNA methylation has suppressive effects on gene transcription and it is involved in a variety of physiologic processes, including development and cancer. As we and others demonstrated, Dnmt3b is a tumor suppressor in oncogene-driven lymphoid and myeloid malignancies in mice. Due to numerous activities such as methylation-dependent and independent repression and accessory functions, it is difficult to pinpoint physiological processes solely dependent on catalytic activity of Dnmt3b. By utilizing our new mouse model expressing catalytically inactive Dnmt3b at physiological levels we identified genome-wide methylation changes and aberrant gene expression profiles that are specific to catalytic activity of DNMT3b.
Project description:Genome-wide DNA methylation profiling of HCT116 WT, HCT116 DNMT1 and DNMT3B double KO, and breast cancer tumors by next generation Infinium assay
Project description:We performed methylation profiling using RRBS-seq to investigate the role of DNMT3B in MYC-driven tumor maintenance. Comparing tumor cells before and upon DNMT3B knock-down revealed genome-wide changes in the DNA methylation pattern.
Project description:Genome-wide DNA methylation patterns are established and maintained by the coordinated action of three DNA methyltransferases, DNMT1, DNMT3A, and DNMT3B. DNMT3B hypomorphic germline mutations are responsible for two-thirds of Immunodeficiency, Centromere Instability, Facial Anomalies (ICF) syndrome cases. The molecular defects in transcription, DNA methylation, and chromatin structure in ICF cells remain relatively uncharacterized. We used expressing microarrays to define the global program of gene expression to elucidate the role of DNMT3B in these processes using EBV-immortalized lymphoblastoid cell lines (LCLs) derived from ICF syndrome and normal individuals. Keywords: disease-state analysis