Unknown,Transcriptomics,Genomics,Proteomics

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Genome-wide analysis of the rat colon reveals site-specific differences in histone modifications and proto-oncogene expression

ABSTRACT: Goal: Since disease susceptibility of the intestine exhibits an anatomical bias, we propose that the chromatin landscape, especially the site-specific epigenetic differences in histone modification patterns throughout the longitudinal axis, contributes to a differential response to chemoprotective agents. Method: We assessed the chromatin structure associated with gene expression profiles in the rat proximal and distal colon by globally correlating chromatin immunoprecipitation next-generation sequencing analysis (ChIP-Seq) with mRNA transcription (RNA-Seq) data. Crypts were isolated from the proximal and distal colonic regions from rats maintained on a semi-purified diet, and mRNA gene expression profiles were generated using RNA-Seq. The remaining isolated crypts were paraformaldehyde-crosslinked and chromatin immunoprecipitated using antibodies against H3K4me3, H3K9me3, and RNA Polymerase II. Results/Conclusion: Globally, RNA-Seq results indicated that 9,866 genes were actively expressed, of which 540 genes were differentially expressed between the proximal and distal colon. With regard to differentially expressed genes, a high correlation was observed between H3K4me3-Seq and RNA-Seq data, with 96% of the canonical pathways being similarly affected in the H3K4me3-Seq and RNA-Seq data sets. Gene ontology analysis indicated that colonic crypt location significantly impacted both chromatin and transcriptional regulation of genes involved in cell transformation, lipid metabolism, lymphatic development and immune cell trafficking. Gene function analysis indicated that the PI3-Kinase signaling pathway was regulated in a site-specific manner, e.g., pathway proto-oncogenes, c-Jun, c-Fos and ATF, were up-regulated in the distal colon. Middle and long non-coding RNAs (lncRNAs) were also detected in the colon, including select lncRNAs formerly only detected in the rat nervous system. In summary, distinct combinatorial patterns of histone modifications exist in the proximal versus distal colon. These site-specific differences may explain the differential effects of chemoprotective agents on cell transformation in the ascending (proximal) and descending (distal) colon. Examination of mRNA profiles and 2 different histone modifications (H3K4me3 and H3K9me3) in 2 tissue types (proximal and distal colon).

ORGANISM(S): Rattus norvegicus

SUBMITTER: Karen Triff

PROVIDER: E-GEOD-49389 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Goal: Since disease susceptibility of the intestine exhibits an anatomical bias, we propose that the chromatin landscape, especially the site-specific epigenetic differences in histone modification patterns throughout the longitudinal axis, contributes to a differential response to chemoprotective agents. Method: We assessed the chromatin structure associated with gene expression profiles in the rat proximal and distal colon by globally correlating chromatin immunoprecipitation next-generation sequencing analysis (ChIP-Seq) with mRNA transcription (RNA-Seq) data. Crypts were isolated from the proximal and distal colonic regions from rats maintained on a semi-purified diet, and mRNA gene expression profiles were generated using RNA-Seq. The remaining isolated crypts were paraformaldehyde-crosslinked and chromatin immunoprecipitated using antibodies against H3K4me3, H3K9me3, and RNA Polymerase II. Results/Conclusion: Globally, RNA-Seq results indicated that 9,866 genes were actively expressed, of which 540 genes were differentially expressed between the proximal and distal colon. With regard to differentially expressed genes, a high correlation was observed between H3K4me3-Seq and RNA-Seq data, with 96% of the canonical pathways being similarly affected in the H3K4me3-Seq and RNA-Seq data sets. Gene ontology analysis indicated that colonic crypt location significantly impacted both chromatin and transcriptional regulation of genes involved in cell transformation, lipid metabolism, lymphatic development and immune cell trafficking. Gene function analysis indicated that the PI3-Kinase signaling pathway was regulated in a site-specific manner, e.g., pathway proto-oncogenes, c-Jun, c-Fos and ATF, were up-regulated in the distal colon. Middle and long non-coding RNAs (lncRNAs) were also detected in the colon, including select lncRNAs formerly only detected in the rat nervous system. In summary, distinct combinatorial patterns of histone modifications exist in the proximal versus distal colon. These site-specific differences may explain the differential effects of chemoprotective agents on cell transformation in the ascending (proximal) and descending (distal) colon.

Project description:Analysis of non-differentiated Caco-2 intestinal epithelial cell line treated with polydextrose fermentation metabolites fermented for 48 hours in 4-stage in vitro colon simulator, in which the conditions mimic the human proximal, ascending, transverse and distal colon in sequence , as well as with medium, 100 mM NaCl and 5 mM butyrate. Polydextrose, a soluble fiber fermented in colon, was fermented with the in vitro colon simulator in three amounts of 0%, 1% and 2%. Results provide insight into the mechanisms underlying colon cancer cells and a comparison of a complex fiber metabolome to 5 mM butyrate and 100 mM NaCl. Furthermore, the results give insight of dosage effect of increasing the concentration of fiber. High level of dietary fiber has been epidemiologically linked to protection against the risk for developing colon cancer. The mechanisms of this protection are not clear. Fermentation of dietary fiber in the colon results in production of for example butyrate that has drawn attention as a chemopreventive agent. Polydextrose, a soluble fiber that is only partially fermented in colon, was fermented in an in vitro colon simulator, in which the conditions mimic the human proximal, ascending, transverse and distal colon in sequence. The subsequent fermentation metabolome were applied on colon cancer cells, and the gene expression changes studied. Polydextrose fermentation down-regulated classes linked with cell cycle, and affected number of metabolically active cells. Further, up-regulated effects on classes linked with apoptosis implicate that polydextrose fermentation plays a role in induction of apoptosis in colon cancer cells. The up-regulated genes involved also key regulators of lipid metabolism, such as PPARg and PGC-1α. These results offer hypotheses for the mechanisms of two health benefits linked with consumption of dietary fiber, reducing risk of development of colon cancer, and dyslipidemia. Non-differentiated Caco-2 cells were treated with polydextrose fermentation metabolites from the vessels representing different parts of the colon, or with 100 mM NaCl or with 5 mM butyrate for 24 hours. For polydextrose fermentation three concentrations of polydextrose were used: 0%, 1% and 2% for a simulation that lasted for 48 hours. Polydextrose fermentation samples from total of 12 vessels, as well as from medium sample, 5 mM butyrate and 100 mM NaCl were analysed as single replica.

Project description:BACKGROUND: Appendicitis followed by appendectomy (AA) at a young age protects against later inflammatory bowel disease (IBD). Using a novel murine appendicitis model we earlier demonstrated that AA proffered significant protection against subsequent experimental colitis. AIM: To delineate genes and biological pathways involved in the protective effect of AA against subsequent colitis using gene set enrichment analysis (GSEA) of DNA microarray data. METHODS: Appendicitis and appendicectomy was done (5 week old male BALB/c mice) near the most proximal colon (caecal lymphoid follicles) and colonic samples were harvested from the most distal colon. Two consecutive laparotomies were done in control Sham-Sham (SS) mice. RNA was extracted (TRIzol®) from 4 individual colonic samples per group (AA group vs. SS group) with each sample taken independently through Affymetrix® microarray hybridization. For GSEA, data for more than 23,000 genes were exported from Partek and analyzed with GSEA software (with 2852 gene sets encoded) to establish correlates with phenotypes of the gene sets. RESULTS: Distal colonic expression of 636 gene-sets were significantly upregulated in AA group samples (False Discovery Rates (FDR) values < 1 % and p value < 0.001; stringent statistical selection). These were validated by quantitative PCR of 14 selected genes across the immunological spectrum and over time-intervals of 3 days, 14 days and 28 days. CONCLUSIONS: Many key immunological, apoptosis-related and cellular function-associated gene-sets involved in the protective effect of AA in experimental colitis were identified. Further analysis of these profiles and biological pathways will assist utilizing these gene products and manipulating various aspects of these pathways to develop better therapeutic strategies in the management of intractable IBD. Appendicitis and appendicectomy was done (5 week old male BALB/c mice) near the most proximal colon (caecal lymphoid follicles) and colonic samples were harvested from the most distal colon. Two consecutive laparotomies were done in control Sham-Sham (SS) mice. RNA was extracted (TRIzol®) from 4 individual colonic samples per group (AA group vs. SS group) with each sample taken independently through Affymetrix® microarray hybridization. variable_protocol: appendicitis and appendectomy: AA1, AA2, AA3, AA4 variable_protocol: sham/sham surgery: SS1, SS2, SS3, SS4 repeat_biological replicate: AA1, AA2, AA3, AA4 repeat_biological replicate: SS1, SS2, SS3, SS4 Upregulated gene-set linked as supplementary file.

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Genome-wide analysis of the rat colon reveals site-specific differences in histone modifications and proto-oncogene expression

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