Transcriptome analysis and Genome-wide DNA methylation maps in chronic lymphocytic leukemia cells determined by next-generation sequencing
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ABSTRACT:
ORGANISM(S): Homo sapiens
SUBMITTER: Huidong Shi
PROVIDER: E-GEOD-66167 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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Project description:We conducted a genome-wide DNA methylation analysis in CD19+ B-cells from CLL patient and normal control samples using reduced representation bisulfite sequencing (RRBS). The methylation status of 1.8-2.3 million CpGs in the CLL genome was determined; about 45% of these CpGs were located in more than 23,000 CpG islands (CGIs). While global CpG methylation was similar between CLL and normal B-cells, 1764 gene promoters were identified as being differentially methylated between the two groups. Aberrant hypermethylation was found in all HOX gene clusters and a significant number of WNT signaling pathway genes. The genes that were frequently hypermethylated were typically associated with histone H3 lysine 27 tri-methylation or bivalent domains in normal B-cells. An additional 152 genes were found to be differentially methylated between normal naïve and memory B-cells. Of these 152 genes, 123 were hypomethylated in memory B-cells when compared to naïve B-cells. Overall, CLL B-cells had methylation patterns more similar to memory B-cells than naïve B-cells. Cluster analysis showed that the tissue-specific methylated genes separated CLL samples into two groups with differential ZAP70 methylation status. Hypomethylation occurred more frequently in the gene body including introns, exons, and 3'-UTRs in CLL. The hypomethylation in the NFATc1 P2 promoter and first intron correlated with up-regulation of both NFATc1 RNA and protein expression levels in CLL suggesting that an epigenetic mechanism is involved in the constitutive activation of NFAT activity in CLL cells. This comprehensive DNA methylation map will further our understanding of the epigenetic contribution to cellular dysfunction in CLL. To perform a genome-wide analysis of DNA methylation in CLL, we applied the Reduced Representation Bisulfite Sequencing (RRBS) to CD19+ B-cells isolated from normal control and CLL peripheral blood samples. The genomic DNA from each sample was digested with the methylation-insensitive restriction enzyme MspI (restriction site, CCGG) and ligated to Illumina sequencing adaptors containing methylated cytosine residues. The ligated MspI fragments were size-selected, treated with sodium bisulfite, and amplified by PCR. The PCR products were purified and sequenced using Illumina GAIIx sequencer with a read length of 52 or 76bp. 11 CLL B-cell samples, 3 normal control samples including one each of normal CD19+, CD19+/ IgD+ naïve, and CD19+/CD27+ memory B-cell sample and three CLL cell lines (Mec-1, Mec-2, and Wac-3) were used. We generated 20-30 million Illumina sequencing reads for each sample.
2012-05-10 | E-GEOD-32698 | biostudies-arrayexpress
Project description:At sites of inflammation, certain Foxp3+ Tregs have the ability to alter their phenotype and become pro-inflammatory helper/effector cells, without losing Foxp3 expression. We show that this functional reprogramming is controlled by the transcription factor Eos (Ikzf4), an obligate co-repressor for Foxp3. The ability to reprogram was restricted to a specific subset of Foxp3+ Tregs, arising as early as the thymus and identifiable by short half-life of Eos at rest, characteristic cell-surface markers (CD38+CD69+CD103NEG) and a distinct pattern of DNA methylation. Mice made selectively deficient in this subset of Eos-labile Tregs became markedly impaired in their ability to cross-present new antigens and prime CD8+ T cells. Downregulation of Eos and consequent Treg reprogramming was prevented by the immunoregulatory enzyme IDO, via activation of the aryl hydrocarbon receptor (AhR). Thus, the Foxp3+ lineage contains a committed subset of Tregs that are constitutively primed for conversion into biologically important helper cells. Cells from thymus or spleen were incubated for 1 hr with cycloheximide (CHX), then CD4+GFP+ Tregs were FACS-sorted into Eos-labile (CD38+CD103NEG) and Eos-stable (CD103+CD38NEG) subsets. Control CD4+GFPNEG (non Treg) cells were sorted from spleen. Genome-wide differential methylation analysis was performed using Reduced Representation Bisulfite Sequencing (RRBS). The genomic DNA from each sample was digested with the methylation-insensitive restriction enzyme MspI (restriction site, CCGG) and ligated to Illumina sequencing adaptors containing methylated cytosine residues. The ligated MspI fragments were size-selected, treated with sodium bisulfite, and amplified by PCR. The PCR products were purified and sequenced using Illumina HiSeq 2000 sequencer with a read length of 100bp.
2013-02-20 | E-GEOD-44380 | biostudies-arrayexpress
Project description:Glioblastoma (GBM) is the most common and most aggressive primary brain tumor in adults. The existence of a small population of stem-like tumor cells that efficiently propagate tumors and resist cytotoxic therapy is one proposed mechanism leading to the resilient behavior of tumor cells and poor prognosis. In this study, we performed an in-depth analysis of the DNA methylation landscape in GBM-derived cancer stem cells (GSCs). Parallel comparisons of primary tumors and GSC lines derived from these tumors with normal controls (a neural stem cell (NSC) line and normal brain tissue) identified groups of hyper- and hypomethylated genes that display a trend of either increasing or decreasing methylation levels in the order of controls, primary GBMs, and their counterpart GSC lines, respectively. Interestingly, concurrent promoter hypermethylation and gene body hypomethylation were observed in a subset of genes including MGMT, AJAP1 and PTPRN2. These unique DNA methylation signatures were also found in primary GBM-derived xenograft tumors indicating that they are not tissue culture-related epigenetic changes. Integration of GSC-specific epigenetic signatures with gene expression analysis further identified candidate tumor suppressor genes that are frequently down regulated in GBMs such as SPINT2, NEFM and PENK. Forced re-expression of SPINT2 reduced glioma cell proliferative capacity, anchorage independent growth, cell motility, and tumor sphere formation in vitro. The results from this study demonstrate that GSCs possess unique epigenetic signatures that may play important roles in the pathogenesis of GBM. The reduced representation bisulfite sequencing (RRBS) approach (Meissner et al., 2008) was used to generate genome-wide single-base resolution CpG methylation profiles of three primary GBMs (1063T, 1133T, and 1142T) and three GSC lines (1063S, 1133S, 1142S) derived from these primary GBM tumors. The NSC line and the normal brain (NB) tissue sample were used as controls for comparison purposes. In addition, we analyzed three GBM xenograft tumor tissue samples (Mayo22, Mayo39, Mayo59) developed by Dr. Jann N. Sarkaria of Mayo Clinic.
2015-06-24 | E-GEOD-70175 | biostudies-arrayexpress
Project description:DCIS is a non-invasive precursor lesion to invasive breast carcinoma. We still have no understanding on why only some DCIS lesions evolve to invasive cancer while others appear not to do so during the life span of the patient. Here, we performed full exome (tumor vs. matching normal), transcriptome and methylome analysis of 30 pure high-grade DCIS (HG-DCIS) and 10 normal breast epithelial samples. Sixty two percent of HG-DCIS cases displayed mutations affecting cancer driver genes or potential drivers. Mutations were observed affecting PIK3CA (21% of cases), TP53 (17%), GATA3 (7%), MLL3 (7%) and single cases of mutations affecting CDH1, MAP2K4, TBX3, NF1, ATM and ARID1A. Significantly, 83% of lesions displayed numerous large chromosomal copy number alterations, suggesting they might precede selection of cancer driver mutations. Integrated pathway-based modeling analysis of RNA-seq data allowed us to identify two DCIS subgroups (DCIS-C1 and DCIS-C2) based on their tumor intrinsic subtypes, proliferative, immune scores and in the activity of specific signaling pathways. The more aggressive DCIS-C1 (highly proliferative, basal-like or ERBB2+) displayed signatures characteristic of activated Treg cells (CD4+/CD25+/FOXP3+) and CTLA4+/CD86+ complexes indicative of a tumor-associated immune suppressive phenotype. Strikingly, all lesions showed evidence of TP53 pathway inactivation. Similarly ncRNA and methylation profiles reproduce changes observed post-invasion. Among the most significant findings we observed upregulation of lncRNA HOTAIR in DCIS-C1 lesions and hypermethylation of HOXA5 and specific SOX genes. We conclude that most HG-DCIS lesions, in spite of representing a pre-invasive stage of tumor progression, displayed molecular profiles indistinguishable from invasive breast cancer. DNA from 24 out of 30 (80%) HG-DCIS samples and 5 normal breast organoids (total 29 samples) were subjected to reduced representation bisulfite sequencing analysis (RRBS) by using Illumina HiSeq2000 platform. Please note that description of samples employed for the NGS analyses including age, race, ER/PR immunohistochemistry results, ITIL/STIL scores and PAM50 classification is provided the 'Supplementary Data1_Samples data.xlsx' (available on Superseries record)
2015-07-25 | E-GEOD-69993 | biostudies-arrayexpress
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Project description:Nearly all CpG-dense promoters are occupied by the multi-domain chromosomal protein FBXL10. We show here that complete inactivation of the Fbxl10 gene leads to dense de novo methylation only of the promoters that are co-occupied by both FBXL10 and by Polycomb Repressive Complexes; this results in pervasive defects in embryonic development and death of homozygous Fbxl10 mutant embryos at midgestation. Deletion of key components of Polycomb Repressive Complexes 1 and 2 did not lead to ectopic de novo methylation. These results indicate that FBXL10 defends Polycomb-occupied promoters against ectopic de novo methylation. FBXL10 is the first reported factor whose loss leads to a gain in genomic DNA methylation. DNA methylation analysis using RRBS and expression analysis using RNA-seq was performed on WT and Fbxl10T/T ES cells.
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