Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

RNA-sequencing of coding RNA from human purified intestinal epithelial samples and organoids derived from foetal and paediatric small and large intestine

ABSTRACT: Human intestinal epithelial organoids (IEO) culture models are rapidly emerging as novel experimental tools to investigate fundamental aspects of intestinal epithelial (patho)physiology. Cellular source and culture protocols vary between different IEO models and reliable markers for their characterization/validation are currently limited. Here, we provide the following reference datasets of transcriptomic profiling by RNA-sequencing: Purified intestinal epithelial cells (EpCAM+) from paediatric ileum and colon, Intestinal organoid cultures from paediatric ileum and colon, Purified intestinal epithelial cells (EpCAM+) from foetal small intestine and foetal large intestine, Intestinal organoid cultures from foetal small intestine and foetal large intestine, Intestinal organoid cultures derived from induced pluripotent stem cells.
Complementary data from methylation profiling on the same samples have been deposited at ArrayExpress under accession number E-MTAB-4957 ( https://www.ebi.ac.uk/arrayexpress/experiments/E-MTAB-4957 ).

INSTRUMENT(S): Illumina HiSeq 2500

ORGANISM(S): Homo sapiens

SUBMITTER: Judith Kraiczy

PROVIDER: E-MTAB-5015 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

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

Kraiczy Judith J Nayak Komal M KM Howell Kate J KJ Ross Alexander A Forbester Jessica J Salvestrini Camilla C Mustata Roxana R Perkins Sally S Andersson-Rolf Amanda A Leenen Esther E Liebert Anke A Vallier Ludovic L Rosenstiel Philip C PC Stegle Oliver O Dougan Gordon G Heuschkel Robert R Koo Bon-Kyoung BK Zilbauer Matthias M

Gut 20171115 1

<h4>Objective</h4>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.<h4>Design</h4>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 wer ...[more]

PMID: 29141958

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Project description:Human intestinal epithelial organoids (IEO) culture models are rapidly emerging as novel experimental tools to investigate fundamental aspects of intestinal epithelial (patho)physiology. Cellular source and culture protocols vary between different IEO models and reliable markers for their characterization/validation are currently limited. DNA methylation has been demonstrated to play a key role in regulating gene expression and cellular function. This epigenetic mark is comparably stable and has been shown to reflect cellular identity such as tissue origin, developmental stage and age. Our genome wide DNA methylation datasets of purified human epithelium represent a unique resource, which can be used by other researchers to validate their model systems We provide the following datasets of genome-wide DNA methylation profiling by Infinium HumanMethylation450 BeadChip: Purified intestinal epithelial cells (EpCAM+) from paediatric ileum and colon, Non-epithelial mucosal cells (EpCAM-), Whole colonic mucosal biopsies, Intestinal organoid cultures from paediatric ileum and colon, Purified intestinal epithelial cells (EpCAM+) from foetal small intestine and foetal large intestine, Intestinal organoid cultures from foetal small intestine and foetal large intestine, Intestinal organoid cultures derived from induced pluripotent stem cells.<br>Note:</br><br>Some samples in this data set were previously submitted to ArrayExpress under accession number E-MTAB-3709. They're clearly marked in the âDescriptionâ field in sample annotations. </br><br>Information about batch effect during sample handling is included in the experimental variable âblockâ. Batch effect is not massive but present, so it is recommended that batch correction is carried out in data analysis.</br><br>Complementary RNA-seq data on the purified cells and organoids can be found in ArrayExpress at E-MTAB-5015 ( https://www.ebi.ac.uk/arrayexpress/experiments/E-MTAB-5015/ ). </br>

Project description:Background & Aims: Hierarchical organization of intestine relies on their stem cells by self-renew and producing committed progenitors. Although signals like Wnt are known to animate the continued renewal by maintaining intestinal stem cells (ISCs) activity, molecular mechanisms especially E3 ubiquitin ligases that modulate ISCs ‘stemness’ and supportive niche have not been well understood. Here, we investigated the role of Cullin 4B (Cul4b) in regulating ISC functions. Methods: We generated mice with intestinal epithelial-specific disruption of Cul4b (pVillin-cre; Cul4bfn/Y), inducible disruption of Cul4b (Lgr5-creERT2; Cul4bfn/Y, CAG-creERT2; Cul4bfn/Y) and their control (Cul4bfn/Y). Intestinal tissues were analyzed by histology, immunofluorescence, RNA sequencing and mass spectrum. Intestinal organoids deprived from mice with pVillin-Cre; Cul4bfn/Y, Lgr5-Cre; Cul4bfn/Y, Tg-Cul4b and their controls were used in assays to measure intestinal self-renewal, proliferation and differentiation. Wnt signaling and intestinal markers were analyzed by immunofluorescence and immunoblot assays. Differential proteins upon Cul4b ablation or Cul4b-interacting proteins were identified by mass spectrometry. Results: Cul4b specifically located at ISCs zone. Block of Cul4b impaired intestinal homeostasis maintenance by reduced self-renewal and proliferation. Transcriptome analysis revealed that Cul4b-null intestine lose ISC characterization and showed disturbed ISC niche. Mechanistically, reactivated Wnt pathway could recover intestinal dysfunction of Cul4b knockout mice. Analysis of differential total and ubiquitylated proteins uncovered the novel targeting substrate of Cullin-Ring ubiquitin ligase 4b (CRL4b), immunity-related GTPase family M member 1 (Irgm1) in intestine. Decreased Irgm1 rescued abnormally interferon signaling, overemphasized autophagy and downstream phosphate proteins in Cul4b knockout mice. Conclusion: We conclude that Cul4b is essential for ISC self-renewal and Paneth cell function by targeting Irgm1 and modulating Wnt signaling. Our results demonstrate that Cul4b is a novel ISC stemness and niche regulator.

Dataset Information

RNA-sequencing of coding RNA from human purified intestinal epithelial samples and organoids derived from foetal and paediatric small and large intestine

Publications

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

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