An upstream protein-coding region in enteroviruses modulates virus infection in gut epithelial cells.

Enteroviruses comprise a large group of mammalian pathogens that includes poliovirus. Pathology in humans ranges from sub-clinical to acute flaccid paralysis, myocarditis and meningitis. Until now, all of the enteroviral proteins were thought to derive from the proteolytic processing of a polyprotein encoded in a single open reading frame. Here we report that many enterovirus genomes also harbour an upstream open reading frame (uORF) that is subject to strong purifying selection. Using echovirus 7 and poliovirus 1, we confirmed the expression of uORF protein in infected cells. Through ribosome profiling (a technique for the global footprinting of translating ribosomes), we also demonstrated translation of the uORF in representative members of the predominant human enterovirus species, namely Enterovirus A, B and C. In differentiated human intestinal organoids, uORF protein-knockout echoviruses are attenuated compared to the wild-type at late stages of infection where membrane-associated uORF protein facilitates virus release. Thus, we have identified a previously unknown enterovirus protein that facilitates virus growth in gut epithelial cells-the site of initial viral invasion into susceptible hosts. These findings overturn the 50-year-old dogma that enteroviruses use a single-polyprotein gene expression strategy and have important implications for the understanding of enterovirus pathogenesis.This work was supported by Wellcome Trust grant [106207] and European Research Council grant [646891] to A.E.F; and Wellcome Trust grants 097997/Z/11/Z and 207498/Z/17/Z to I.G

DNA Methylation and Transcription Patterns in Intestinal Epithelial Cells From Pediatric Patients With Inflammatory Bowel Diseases Differentiate Disease Subtypes and Associate With Outcome.

BACKGROUND & AIMS: We analyzed DNA methylation patterns and transcriptomes of primary intestinal epithelial cells (IEC) of children newly diagnosed with inflammatory bowel diseases (IBD) to learn more about pathogenesis. METHODS: We obtained mucosal biopsies (N = 236) collected from terminal ileum and ascending and sigmoid colons of children (median age 13 years) newly diagnosed with IBD (43 with Crohn's disease [CD], 23 with ulcerative colitis [UC]), and 30 children without IBD (controls). Patients were recruited and managed at a hospital in the United Kingdom from 2013 through 2016. We also obtained biopsies collected at later stages from a subset of patients. IECs were purified and analyzed for genome-wide DNA methylation patterns and gene expression profiles. Adjacent microbiota were isolated from biopsies and analyzed by 16S gene sequencing. We generated intestinal organoid cultures from a subset of samples and genome-wide DNA methylation analysis was performed. RESULTS: We found gut segment-specific differences in DNA methylation and transcription profiles of IECs from children with IBD vs controls; some were independent of mucosal inflammation. Changes in gut microbiota between IBD and control groups were not as large and were difficult to assess because of large amounts of intra-individual variation. Only IECs from patients with CD had changes in DNA methylation and transcription patterns in terminal ileum epithelium, compared with controls. Colon epithelium from patients with CD and from patients with ulcerative colitis had distinct changes in DNA methylation and transcription patterns, compared with controls. In IECs from patients with IBD, changes in DNA methylation, compared with controls, were stable over time and were partially retained in ex-vivo organoid cultures. Statistical analyses of epithelial cell profiles allowed us to distinguish children with CD or UC from controls; profiles correlated with disease outcome parameters, such as the requirement for treatment with biologic agents. CONCLUSIONS: We identified specific changes in DNA methylation and transcriptome patterns in IECs from pediatric patients with IBD compared with controls. These data indicate that IECs undergo changes during IBD development and could be involved in pathogenesis. Further analyses of primary IECs from patients with IBD could improve our understanding of the large variations in disease progression and outcomes

DNA methylation defines regional identity of human intestinal epithelial organoids and undergoes dynamic changes during development.

OBJECTIVE: Human intestinal epithelial organoids (IEOs) are increasingly being recognised as a highly promising translational research tool. However, our understanding of their epigenetic molecular characteristics and behaviour in culture remains limited. DESIGN: We performed genome-wide DNA methylation and transcriptomic profiling of human IEOs derived from paediatric/adult and fetal small and large bowel as well as matching purified human gut epithelium. Furthermore, organoids were subjected to in vitro differentiation and genome editing using CRISPR/Cas9 technology. RESULTS: We discovered stable epigenetic signatures which define regional differences in gut epithelial function, including induction of segment-specific genes during cellular differentiation. Established DNA methylation profiles were independent of cellular environment since organoids retained their regional DNA methylation over prolonged culture periods. In contrast to paediatric and adult organoids, fetal gut-derived organoids showed distinct dynamic changes of DNA methylation and gene expression in culture, indicative of an in vitro maturation. By applying CRISPR/Cas9 genome editing to fetal organoids, we demonstrate that this process is partly regulated by TET1, an enzyme involved in the DNA demethylation process. Lastly, generating IEOs from a child diagnosed with gastric heterotopia revealed persistent and distinct disease-associated DNA methylation differences, highlighting the use of organoids as disease-specific research models. CONCLUSIONS: Our study demonstrates striking similarities of epigenetic signatures in mucosa-derived IEOs with matching primary epithelium. Moreover, these results suggest that intestinal stem cell-intrinsic DNA methylation patterns establish and maintain regional gut specification and are involved in early epithelial development and disease.This work was supported by funding from the following charitable organizations: Crohn’s in Childhood Research Association (CICRA), the Evelyn Trust, Crohn’s and Colitis in Childhood (“3Cs”), Addenbrooke’s Charitable Trust (ACT), and the Newlife Foundation for Disabled Children. J.K. was funded by a CICRA PhD studentship, K.H. was funded by an EBPOD EMBL-EBI/Cambridge Computational Biomedical Postdoctoral Fellowship. B-K.K. was supported by a Sir Henry Dale Fellowship from the Wellcome Trust and the Royal Society [101241/Z/13/Z] and received core support from the Wellcome Trust and MRC to the WT - MRC Cambridge Stem Cell Institute. GD and JF received support from the Wellcome Trust. J.F. was supported by a studentship from the MRC. P.R was supported by the Deutsche Forschungsgemeinschaft EXC306 Cluster “Inflammation at Interfaces” and BMBF IHEC DEEP TP5.2

DNA methylation analysis in the intestinal epithelium-effect of cell separation on gene expression and methylation profile.

Epigenetic signatures are highly cell type specific. Separation of distinct cell populations is therefore desirable for all epigenetic studies. However, to date little information is available on whether separation protocols might influence epigenetic and/or gene expression signatures and hence might be less beneficial. We investigated the influence of two frequently used protocols to isolate intestinal epithelium cells (IECs) from 6 healthy individuals.Epithelial cells were isolated from small bowel (i.e. terminal ileum) biopsies using EDTA/DTT and enzymatic release followed by magnetic bead sorting via EPCAM labeled microbeads. Effects on gene/mRNA expression were analyzed using a real time PCR based expression array. DNA methylation was assessed by pyrosequencing of bisulfite converted DNA and methylated DNA immunoprecipitation (MeDIP).While cell purity was >95% using both cell separation approaches, gene expression analysis revealed significantly higher mRNA levels of several inflammatory genes in EDTA/DTT when compared to enzymatically released cells. In contrast, DNA methylation of selected genes was less variable and only revealed subtle differences. Comparison of DNA methylation of the epithelial cell marker EPCAM in unseparated whole biopsy samples with separated epithelium (i.e. EPCAM positive and negative fraction) demonstrated significant differences in DNA methylation between all three tissue fractions indicating cell type specific methylation patterns can be masked in unseparated tissue samples.Taken together, our data highlight the importance of considering the potential effect of cell separation on gene expression as well as DNA methylation signatures. The decision to separate tissue samples will therefore depend on study design and specific separation protocols

Culture-Associated DNA Methylation Changes Impact on Cellular Function of Human Intestinal Organoids.

BACKGROUND & AIMS: Human intestinal epithelial organoids (IEOs) are a powerful tool to model major aspects of intestinal development, health, and diseases because patient-derived cultures retain many features found in vivo. A necessary aspect of the organoid model is the requirement to expand cultures in vitro through several rounds of passaging. This is of concern because the passaging of cells has been shown to affect cell morphology, ploidy, and function. METHODS: Here, we analyzed 173 human IEO lines derived from the small and large bowel and examined the effect of culture duration on DNA methylation (DNAm). Furthermore, we tested the potential impact of DNAm changes on gene expression and cellular function. RESULTS: Our analyses show a reproducible effect of culture duration on DNAm in a large discovery cohort as well as 2 publicly available validation cohorts generated in different laboratories. Although methylation changes were seen in only approximately 8% of tested cytosine-phosphate-guanine dinucleotides (CpGs) and global cellular function remained stable, a subset of methylation changes correlated with altered gene expression at baseline as well as in response to inflammatory cytokine exposure and withdrawal of Wnt agonists. Importantly, epigenetic changes were found to be enriched in genomic regions associated with colonic cancer and distant to the site of replication, indicating similarities to malignant transformation. CONCLUSIONS: Our study shows distinct culture-associated epigenetic changes in mucosa-derived human IEOs, some of which appear to impact gene transcriptomic and cellular function. These findings highlight the need for future studies in this area and the importance of considering passage number as a potentially confounding factor

Influence of isolation methods on gene expression profiles.

<p>A) Differentially regulated genes in IEC isolated with EDTA/DTT compared to enzymatic release. Data is expressed as box and whisker plots. The length of the boxes represents the interquartile range and the whiskers the 3^rd and 97^th percentile of the data. Significance testing was performed using paired Students T-test and values for <i>p</i><0.05 were considered to be statistically significant. B) Cluster analysis of all analyzed patients. Individuals are numbered. An L signifies IEC isolated using enzymatic release. An E signifies IEC isolated using the EDTA/DTT-based approach. All six patients were included in this analysis. Tissue samples for EDTA/DTT and enzymatic release were obtained from the same side in each patient and all analyses were performed in triplicate.</p

Cytokeratin 8 and CD3γ copy numbers in intestinal epithelial cells.

<p>A) FACS analysis of T cell contamination. Positive selection of IEC was performed using CD326 Microbeads followed by staining using T-cell receptor antibodies and Pacific Blue Annexin V to detect apoptotic cells. Panel A1 shows an example of IEC isolated using the enzyme based protocol, panel A2 using the EDTA/DTT based protocol. T-cell receptor positive cells are located in quadrant Q1 and Q2. Analysis of absolute mRNA copy numbers of cytokeratin 8 and CD3γ in intestinal epithelial cells separated by positive selection using CD326 Microbeads cells. B) Expression of mRNA copy numbers of Cytokeratin 8 and CD3γ. Data is expressed as box and whisker plots. The length of the boxes represents the interquartile range and the whiskers the 3^rd and 97^th percentile of the data. Significance testing was performed using paired Students T-test and values for <i>p</i><0.05 were considered to be statistically significant.</p

Analysis of cytosine methylation of promoter regions of selected genes.

<p>A) CpG methylation of promoter regions of selected genes analysed by MeDIP. B) CpG methylation of promoter regions of selected genes analysed by pyrosequencing. Data is expressed as box and whisker plots. The length of the boxes represents the interquartile range and the whiskers the 3^rd and 97^th percentile of the data. Significance testing was performed using paired Students T-test and values for <i>p</i><0.05 were considered to be statistically significant.</p

DNA methylation of EPCAM in whole biopsy samples compared to separated epithelium and negative cell fraction.

<p>A) DNA methylation of 5 CpGs within the EPCAM gene using a pyrosequencing assay. B) Total CpG methylation comparing EPCAM versus NOD1. Assays included 5 CpG sites for EPCAM and 2 for NOD1. C) EPCAM mRNA expression in whole biopsies compared to epithelial positive and negative separated cell fraction. Data is expressed as mean +/− SEM of 5 tissue samples. Differences were considered as statistically significant if p<0.05 (** p<0.001, ***p<0.0001).</p