DNA methylation landscape and signature of CD4+ lymphocytes of lamina propria in Crohn's patients by reduced representation and bisulfite sequencing [BiSulfite-seq]
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE184312 | GEO | 2023/09/01
REPOSITORIES: GEO
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Project description:A genome-wide DNA methylation study was conducted using base resolution bisulfite sequencing for purified intestinal CD4+ cells (resected or biopsied mucosa of a terminal ileum) from 21 patients with Crohn’s disease and 12 age/sex matched controls. The sequencing data was analyzed for differentially methylated CpGs (DMCs) and differentially methylated regions (DMRs). Integration was performed with RNA-sequencing data for the same set of samples to verify the functional impact of DNA methylation changes.
2023-09-01 | GSE184307 | GEO
Project description:This SuperSeries is composed of the SubSeries listed below.
2023-09-01 | GSE184314 | GEO
Project description:BackgroundAbnormal handling of E. coli by lamina propria (LP) macrophages may contribute to Crohn's disease (CD) pathogenesis. We aimed to determine LP macrophage phenotypes in CD, ulcerative colitis (UC) and healthy controls (HC), and in CD, to compare macrophage phenotypes according to E. coli carriage.MethodsMucosal biopsies were taken from 35 patients with CD, 9 with UC and 18 HCs. Laser capture microdissection was used to isolate E. coli-laden and unladen LP macrophages from ileal or colonic biopsies. From these macrophages, mRNA was extracted and cytokine and activation marker expression measured using RT-qPCR.ResultsE. coli-laden LP macrophages were identified commonly in mucosal biopsies from CD patients (25/35, 71 %), rarely in UC (1/9, 11 %) and not at all in healthy controls (0/18). LP macrophage cytokine mRNA expression was greater in CD and UC than healthy controls. In CD, E. coli-laden macrophages expressed high IL-10 & CD163 and lower TNFα, IL-23 & iNOS irrespective of macroscopic inflammation. In inflamed tissue, E. coli-unladen macrophages expressed high TNFα, IL-23 & iNOS and lower IL-10 & CD163. In uninflamed tissue, unladen macrophages had low cytokine mRNA expression, closer to that of healthy controls.ConclusionIn CD, intra-macrophage E. coli are commonly found and LP macrophages express characteristic cytokine mRNA profiles according to E. coli carriage. Persistence of E. coli within LP macrophages may provide a stimulus for chronic inflammation.
| S-EPMC4490755 | biostudies-literature
Project description:To investigate the global DNA methylation changes in mouse hematopoietic stem cell aging, we performed whole-genome bisulfite sequencing (WGBS). We generated 1,494 million (4mo HSCs), and 1,493 million (24mo HSCs) raw reads; about 82.6% and 84.3%, respectively, were successfully aligned to either strand of the reference genome (mm9). Of all the cytosines present in the reference genome sequence, about 93% of Cs and 99% of CGs were covered in both datasets, with an average coverage of 46-fold (4mo) and 50-fold (24mo). In contrast to the age-associated hypomethylation observed in studies of somatic cells, mentioned above, HSCs showed an increase of methylation with age. The average methylation level over all 16 million covered CpGs increased from 83.5% in young (4mo) HSC to 84.6% in old (24mo) HSC. We observed a total of 448,166 differentially methylated CpGs (DMCs), defined as those having a 20% or more difference in methylation ratio, of which 38.5% (172,609) were hypomethylated (hypo-DMCs) and 61.5% (275,557) were hypermethylated (hyper-DMCs) with aging. For different genomic features, a slightly greater DNA methylation ratio increase was observed for the gene body, LINEs and SINEs, while CGIs and promoters showed balanced increases and decreases. Localization analysis of DMCs indicates that DNA encoding for ribosome RNA (rDNA) is primarily a hotspot for hypo-DMCs, while promoters without CpG islands, CpG island shores and LINE repetitive elements exhibit both hypo- and hyper-DMCs. Mouse hematopoietic stem cell DNA methylation profiles of 4 month and 24 month old WT mice were generated generated by deep sequencing, in duplicate, using Illumina Hiseq 2000.
2014-04-30 | E-GEOD-47815 | biostudies-arrayexpress
Project description:To investigate the global DNA methylation changes in mouse hematopoietic stem cell aging, we performed whole-genome bisulfite sequencing (WGBS). We generated 1,494 million (4mo HSCs), and 1,493 million (24mo HSCs) raw reads; about 82.6% and 84.3%, respectively, were successfully aligned to either strand of the reference genome (mm9). Of all the cytosines present in the reference genome sequence, about 93% of Cs and 99% of CGs were covered in both datasets, with an average coverage of 46-fold (4mo) and 50-fold (24mo). In contrast to the age-associated hypomethylation observed in studies of somatic cells, mentioned above, HSCs showed an increase of methylation with age. The average methylation level over all 16 million covered CpGs increased from 83.5% in young (4mo) HSC to 84.6% in old (24mo) HSC. We observed a total of 448,166 differentially methylated CpGs (DMCs), defined as those having a 20% or more difference in methylation ratio, of which 38.5% (172,609) were hypomethylated (hypo-DMCs) and 61.5% (275,557) were hypermethylated (hyper-DMCs) with aging. For different genomic features, a slightly greater DNA methylation ratio increase was observed for the gene body, LINEs and SINEs, while CGIs and promoters showed balanced increases and decreases. Localization analysis of DMCs indicates that DNA encoding for ribosome RNA (rDNA) is primarily a hotspot for hypo-DMCs, while promoters without CpG islands, CpG island shores and LINE repetitive elements exhibit both hypo- and hyper-DMCs.
2014-04-30 | GSE47815 | GEO
Project description:CX(3)CR1(+) and CD103(+) dendritic cells (DCs) in intestinal lamina propria play a key role in mucosal immunity. However, the origin and the developmental pathways that regulate their differentiation in the lamina propria remain unclear. We showed that monocytes gave rise exclusively to CD103(-)CX(3)CR1(+) lamina propria DCs under the control of macrophage-colony-stimulating factor receptor (M-CSFR) and Fms-like thyrosine kinase 3 (Flt3) ligands. In contrast, common DC progenitors (CDP) and pre-DCs, which give rise to lymphoid organ DCs but not to monocytes, differentiated exclusively into CD103(+)CX(3)CR1(-) lamina propria DCs under the control of Flt3 and granulocyte-macrophage-colony-stimulating factor receptor (GM-CSFR) ligands. CD103(+)CX(3)CR1(-) DCs but not CD103(-)CX(3)CR1(+) DCs in the lamina propria constitutively expressed CCR7 and were the first DCs to transport pathogenic Salmonella from the intestinal tract to the mesenteric lymph nodes. Altogether, these results underline the diverse origin of the lamina propria DC network and identify mucosal DCs that arise from pre-DCs as key sentinels of the gut immune system.
| S-EPMC2778256 | biostudies-literature
Project description:BackgroundThe aetiology of Crohn's disease [CD] involves immune dysregulation in a genetically susceptible individual. Genome-wide association studies [GWAS] have identified 200 loci associated with CD, ulcerative colitis, or both, most of which fall within non-coding DNA regions. Long non-coding RNAs [lncRNAs] regulate gene expression by diverse mechanisms and have been associated with disease activity in inflammatory bowel disease. However, disease-associated lncRNAs have not been characterised in pathogenic immune cell populations.MethodsTerminal ileal samples were obtained from 22 CD patients and 13 controls. RNA from lamina propria CD4+ T cells was sequenced and long intergenic non-coding RNAs [lincRNAs] were detected. Overall expression patterns, differential expression [DE], and pathway and gene enrichment analyses were performed. Knockdown of novel lincRNAs XLOC_000261 and XLOC_000014 was performed. Expression of Th1 or Th17-associated transcription factors, T-bet and RORγt, respectively, was assessed by flow cytometry.ResultsA total of 6402 lincRNAs were expressed, 960 of which were novel. Unsupervised clustering and principal component analysis showed that the lincRNA expression discriminated patients from controls. A total of 1792 lincRNAs were DE, and 295 [79 novel; 216 known] mapped to 267 of 5727 DE protein-coding genes. The novel lincRNAs were enriched in inflammatory and Notch signalling pathways [p <0.05]. Furthermore, DE lincRNAs in CD patients were more frequently found in DNA regions with known inflammatory bowel disease [IBD]-associated loci. The novel lincRNA XLOC_000261 negatively regulated RORγt expression in Th17 cells.ConclusionsWe describe a novel set of DE lincRNAs in CD-associated CD4+ cells and demonstrate that novel lincRNA XLOC_000261 appears to negatively regulate RORγt protein expression in Th17 cells.
| S-EPMC6930003 | biostudies-literature
Project description:DNA methylation landscape and signature of CD4+ lymphocytes of lamina propria in Crohn's patients by reduced representation and bisulfite sequencing [BiSulfite-seq]
| PRJNA763988 | ENA
Project description:This data was generated by ENCODE. If you have questions about the data, contact the submitting laboratory directly (Florencia Pauli mailto:fpauli@hudsonalpha.org). If you have questions about the Genome Browser track associated with this data, contact ENCODE (mailto:genome@soe.ucsc.edu). This track is produced as part of the ENCODE project. The track reports the percentage of DNA molecules that exhibit cytosine methylation at specific CpG dinucleotides. In general, DNA methylation within a gene's promoter is associated with gene silencing, and DNA methylation within the exons and introns of a gene is associated with gene expression. Proper regulation of DNA methylation is essential during development and aberrant DNA methylation is a hallmark of cancer. DNA methylation status is assayed at more than 500,000 CpG dinucleotides in the genome using Reduced Representation Bisulfite Sequencing (RRBS). Genomic DNA is digested with the methyl-insensitive restriction enzyme MspI, small genomic DNA fragments are purified by gel electrophoresis, and then used to construct an Illumina sequencing library. The library fragments are treated with sodium bisulfite and amplified by PCR to convert every unmethylated cytosine to a thymidine while leaving methylated cytosines intact. The sequenced fragments are aligned to a customized reference genome sequence and for each assayed CpG we report the number of sequencing reads covering that CpG and the percentage of those reads that are methylated. For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf DNA methylation at CpG sites was assayed with a modified version of Reduced Representation Bisulfite Sequencing (RRBS; Meissner et al., 2008). RRBS was performed on cell lines grown by many ENCODE production groups. The production group that grew the cells and isolated genomic DNA is indicated in the "obtainedBy" field of the metadata. When a cell type was provided by more than one lab, the data for the cells from only one lab are displayed in the table above. However, the data for every cell type from every lab is available from the Downloads page. RRBS was carried out by the Myers production group at the HudsonAlpha Institute for Biotechnology. Isolation of genomic DNA Genomic DNA is isolated from biological replicates of each cell line using the QIAGEN DNeasy Blood & Tissue Kit according to the instructions provided by the manufacturer. DNA concentrations for each genomic DNA preparation are determined using fluorescent DNA binding dye and a fluorometer (Invitrogen Quant-iT dsDNA High Sensitivity Kit and Qubit Fluorometer). Typically, 1 µg of DNA is used to make an RRBS library; however, we have also had success in making libraries with 200 ng genomic DNA from rare or precious samples. RRBS library construction and sequencing RRBS library construction starts with MspI digestion of genomic DNA , which cuts at every CCGG regardless of methylation status. Klenow exo- DNA Polymerase is then used to fill in the recessed end of the genomic DNA and add an adenosine as a 3prime overhang. Next, a methylated version of the Illumina paired-end adapters is ligated onto the DNA. Adapter ligated genomic DNA fragments between 105 and 185 basepairs are selected using agarose gel electrophoresis and Qiagen Qiaquick Gel Extraction Kit. The selected adapter-ligated fragments are treated with sodium bisulfite using the Zymo Research EZ DNA Methylation Gold Kit, which converts unmethylated cytosines to uracils and leaves methylated cytosines unchanged. Bisulfite treated DNA is amplified in a final PCR reaction which has been optimized to uniformly amplify diverse fragment sizes and sequence contexts in the same reaction. During this final PCR reaction uracils are copied as thymines resulting in a thymine in the PCR products wherever an unmethylated cytosine existed in the genomic DNA. The sample is now ready for sequencing on the Illumina sequencing platform. These libraries were sequenced with an Illumina Genome Analyzer IIx according to the manufacturer's recommendations. Data analysis To analyze the sequence data, a reference genome is created that contains only the 36 base pairs adjacent to every MspI site and every C in those sequences is changed to T. A converted sequence read file is then created by changing each C in the original sequence reads to a T. The converted sequence reads are aligned to the converted reference genome, and only reads that map uniquely to the reference genome are kept. Once reads are aligned the percent methylation is calculated for each CpG using the original sequence reads. The percent methylation and number of reads is reported for each CpG.
2011-03-01 | E-GEOD-27584 | biostudies-arrayexpress
Project description:Intestinal lymphoepithelial interactions occur in the epithelium and the subepithelial space. We asked whether normal, Crohn's disease (CD), or ulcerative colitis (UC) lamina propria lymphocytes (LPL) could promote intestinal epithelial cell (IEC) growth and differentiation.T84 cells were cocultured with isolated LPL. IECs were then lysed and subjected to measurement of intestinal alkaline phosphatase (IAP) activity; Western blot analysis for MAPK and Akt activation; and real-time polymerase chain reaction to assess caudal-related homeoprotein 2 (CDX2) messenger RNA (mRNA) levels. Tissue sections were immunostained for evidence of mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K) activation, CDX2, and IAP; and CDX2 mRNA expression was assessed in human colonic biopsy specimens.IAP activity was increased in T84 cells cocultured for 8 days with normal LPL (P < .05) and even greater with CD LPL (P < .001). Crypt IECs in active CD mucosa expressed IAP ex vivo. Phospho-MAPK (extracellular signal-regulated kinase 1/2, p38, and c-Jun-N-terminal kinase) and phospho-Akt were seen as early as 30 minutes after coculture. MAPK activation was greatest in T84 cells cocultured with CD LPL. There was a specific increase in Phospho-p38 MAPK and Phospho-Akt staining in the nuclei of crypt IECs in active vs inactive CD, normal mucosa, and UC mucosa. CDX2 mRNA expression was increased in CD LPL cocultured T84 cells, which did not correlate with CDX2 protein localization ex vivo.There is cross talk between LPL and IECs, which leads to IEC differentiation. The differentiation is accelerated in CD mucosa.
| S-EPMC2975902 | biostudies-literature