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Epigenetic changes induced by Bacteroides fragilis toxin (BFT) [ATAC-seq]

ABSTRACT: Purpose: The goal of this study is to determine how BFT2 alters chromatin accessibility at relatively early time points in colon epithelial cells, and to correlate the changes in chromatin accessibility with changes in gene expression, transcription factor binding sites, and the location of common single nucleotide variants (SNVs) and differentially methylated regions (DMRs) in colorectal cancer. Methods: HT29/C1 cells were plated at low density and allowed to grow for 4 days at 37C and 10% CO2. Afterwards, cells were either left untreated or treated with 100ng/mL BFT2 for 24 or 48 hours. After the specified time point, cells were counted and DNA was collected in order to prepare ATAC-seq libraries. ATAC-seq library was prepared as outlined by Buenrostro et al. (2013). The 4nM pooled ATAC-seq library was sequenced using the Illumina HiSeq. For each sample, three biological replicates were sequenced. After sequencing, the data was analyzed using the pipeline developed by the Kundaje lab, as outlined on ENCODE (Lee et al., 2016). Briefly, reads were trimmed, aligned and filtered using Bowtie2, and peaks were called using MACS2. The three replicate peak files were combined in order to create one consensus file for each treatment condition using an irreproducible discovery rate (IDR) threshold of 0.1. For all analyses, the “optimal set” consensus file was used. Results: Our data show that several genes are differentially expressed after BFT treatment and these changes correlate with changes in chromatin accessibility. This correlation is mediated by an increase in chromatin accessibility at particular transcription factor binding sites. Also, sites of increased chromatin accessibility are associated with a lower frequency of common single nucleotide variants (SNVs) in CRC and with a higher frequency of common differentially methylated regions (DMRs) in CRC. Conclusions: This study adds critical knowledge to our understanding of host-microbe interactions in the gut. While scientists have begun to analyze the effect of specific bacteria on the epigenome of colon epithelial cells, to date, no other studies have surveyed the effect of a specific bacterial toxin on chromatin accessibility in colon epithelial cells. We conclude that BFT2 alters chromatin accessibility in colon epithelial cells, and that these changes correlate with subsequent changes in gene expression. Also, sites of BFT2-induced increased chromatin accessibility are associated with a lower frequency of common single nucleotide variants (SNVs) in CRC and with a higher frequency of common differentially methylated regions (DMRs) in CRC.

ORGANISM(S): Homo sapiens

PROVIDER: GSE113219 | GEO | 2018/04/18

REPOSITORIES: GEO

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Project description:Purpose: The goal of this study is to determine how BFT2 alters chromatin accessibility at relatively early time points in colon epithelial cells, and to correlate the changes in chromatin accessibility with changes in gene expression, transcription factor binding sites, and the location of common single nucleotide variants (SNVs) and differentially methylated regions (DMRs) in colorectal cancer. Methods: HT29/C1 cells were plated at low density and allowed to grow for 4 days at 37C and 10% CO2. Afterwards, cells were either left untreated or treated with 100ng/mL BFT2 for 24 or 48 hours. After the specified time point, cells were counted and RNA was collected in order to prepare RNA-seq libraries. RNA was collected using the Qiagen RNeasy mini kit. mRNA was enriched using the NEBNext Poly(A) mRNA Magnetic Isolation Module. Afterwards, a non-directional RNA-seq library was constructed using the NEBnext Ultra RNA Library Prep kit from Illumina. The 2nM pooled RNA-seq library was sequenced using the Illumina HiSeq. Results: After sequencing, Kallisto was used to perform pseudoalignment of the raw RNA-seq data. Then, Sleuth was used to quantify gene expression and perform differential expression analyses. After treatment with BFT2 for 24 hours, 70 genes were differentially expressed (P-value < 0.01). Of these genes, 41 showed a decrease in gene expression, while 29 showed an increase in gene expression. After BFT2 treatment for 48 hours, we found only 16 differentially expressed genes (P-value < 0.01); 3 showed a decrease in gene expression and 13 showed an increase in gene expression. Conclusions: This study adds critical knowledge to our understanding of host-microbe interactions in the gut. While scientists have begun to analyze the effect of specific bacteria on the epigenome of colon epithelial cells, to date, no other studies have surveyed the effect of a specific bacterial toxin on chromatin accessibility in colon epithelial cells. We conclude that BFT2 alters chromatin accessibility in colon epithelial cells, and that these changes correlate with subsequent changes in gene expression. Also, sites of BFT2-induced increased chromatin accessibility are associated with a lower frequency of common single nucleotide variants (SNVs) in CRC and with a higher frequency of common differentially methylated regions (DMRs) in CRC.

Project description:In colorectal cancer (CRC), chromosomal instability (CIN) is typically studied using comparative-genomic hybridization (CGH) arrays. We studied paired (tumor and surrounding healthy) fresh-frozen tissue from 86 CRC patients using Illumina’s Infinium-based SNP array. This method allowed us to study CIN in CRC, with simultaneous analysis of copy number (CN) and B-allele frequency (BAF), which is a representation of allelic composition. This data helped us to detect mono-allelic and bi-allelic amplifications/deletion, copy neutral loss of heterozygosity, and levels of mosaicism for mixed cell populations, some of which can not be assessed with other methods that do not measure BAF. We identified associations between CN abnormalities and different CRC phenotypes (MSI, histological diagnosis, location, tumor grade, stage, MSI and presence of lymph node metastasis). We showed commonalities between regions of CN change observed in CRC and the regions reported in previous studies of other solid cancers (e.g., amplifications of 20q, 13q, 8q, 5p and deletions of 18q, 17p and 8p). From the Therapeutic Target Database we found relevant drugs, targeted to the genes located in these regions with CN changes, approved or in trials for other cancers and common diseases. These drugs may be considered for future therapeutic trials in CRC, based on personalized cytogenetic diagnosis. We also found many regions harboring genes which are not currently targeted by any relevant drugs that may be considered for future drug discovery studies. Our study shows the application of high-density SNP arrays for cytogenetic study in CRC and its importance for personalized treatment. DNA was extracted from colon tissue samples provided by 86 CRC patients. Each patient provided paired CRC tumor tissue and adjacent normal colon mucosa samples, which were fresh-frozen after resection. Samples were genotyped using Illumina's Infinium-based 610 Quad and CytoSNP 12 microarray chips. The genotype data from paired tumor and normal samples was used to detect tumor-specific chromosomal instabilities in copy number, including copy-neutral LOH, which cannot be assessed by many other cytogenetic methods. Supplementary file "GSE34678_raw_GPL8887.txt" includes the raw data for Samples using GPL8887 (GSM853162-GSM853239); supplementary file "GSE34678_raw_GPL13829.txt" includes the raw data for Samples using GPL13829 (GSM853240-GSM853363).

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Epigenetic changes induced by Bacteroides fragilis toxin (BFT) [ATAC-seq]

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