Mouse Intestinal HMGCR complex
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Orbitrap Fusion
ORGANISM(S): Mus Musculus (ncbitaxon:10090)
SUBMITTER: 
William R Lagor
PROVIDER: MSV000087589 | MassIVE | Tue Jun 08 14:58:00 BST 2021
SECONDARY ACCESSION(S): PXD026572
REPOSITORIES: MassIVE
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Project description:Objective: The intestine occupies the critical interface between cholesterol absorption and excretion. Despite this, surprisingly little is known about the role of de novo cholesterol synthesis in this organ, and its relationship to whole body cholesterol homeostasis. In addition to cholesterol, the mevalonate pathway is responsible for the synthesis of numerous non-sterol isoprenoids. Here we investigate the physiological importance of the mevalonate pathway within the intestine, through genetic deletion of the rate-limiting enzyme. Approach and Results: Mice lacking 3-hydroxy-3-methylglutaryl-coenzyme A reductase (Hmgcr) in intestinal villus and crypt epithelial cells were generated using a Villin-Cre transgene. In contrast to intestine-specific Srebp-2 and Scap knockouts, mice with intestinal-specific loss of Hmgcr are viable through adulthood and fertile. Hmgcr was efficiently deleted based on mRNA levels, as well as quantitative analysis of Hmgcr alleles by droplet digital PCR. Lipidomics revealed substantial reductions in the abundance of numerous non-sterol isoprenoids and sterol intermediates within the epithelial layer, while cholesterol levels were preserved. Although the intestinal knockout mice are born smaller, there is no net defect in feed efficiency or triglyceride absorption due to compensatory changes in bile acid composition and intestinal growth. At the cellular level, loss of Hmgcr is compensated for quickly after weaning through a dramatic expansion of the stem cell compartment within the crypts. Conclusions: Genetic loss of Hmgcr in the intestine is compatible with life, through mechanisms involving compensatory changes in bile acid composition, increased absorptive surface area, and expansion of the resident stem cell compartment.
2021-01-12 | GSE164589 | GEO
Project description:To assess the role of LSD1 in mouse small intestinal epithelium, we grew small intestinal organoids in vitro from mice with an epithelial specific deletion of LSD1 (Villin-Cre+; Lsd1f/f) and from wild type (Villin-Cre-; Lsd1f/f) mice. This experiment uses a new Cre strain with 100% recombination efficiency. Similar to intestinal epithelium from mice with an intestinal epithelium specific LSD1-KO, Paneth cells are not present in LSD1-KO small intestinal organoids. We used these sequencing data to show intrinsic epithelial changes in the intestinal epithelium caused by LSD1 deletion in the absence of microbiota and surrounding in vivo cell types.
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Project description:To study the epigenetic regulation of intestinal epithelium we focus on the role of chromatin modulators. Lysine-specific histone demethylase 1a (KDM1A, LSD1) is one of the enzymes that can erase the H3K4me1/2 mark. To assess the role of LSD1 in intestinal epithelium we studied wild type (WT) (Villin-Cre -; Lsd1f/f) and intestinal-epithelial-specific knock-out (KO) (Villin-Cre+; Lsd1f/f) mice. We found that KO mice completely lack Paneth cells, and have altered stem cell characteristics compared to WT littermates. To assess genome-wide ATAC levels in WT and KO small intestines, we isolated intestinal epithelium tissue from wild type mice and LSD1 KO mice. This tissue was digested to single cells and performed ATAC seq as described in the protocols.
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Project description:To assess the role of LSD1 in mouse small intestinal epithelium, we isolated small intestinal crypts from wild type (WT) (Villin-Cre -; Lsd1f/f) and intestinal-epithelial-specific knock-out (KO) (Villin-Cre+; Lsd1f/f) mice. We dissociated crypts into single cells, and FACS sorted Epcam+ cells, to avoid immune-cell contamination. RNA was directly isolated from these sorted cells, and this was used for RNA seq. As KO crypts are different from WT crypts (KO crypts lack Paneth cells), identifying genes specifically regulated by LSD1 helps us to identify how LSD1 regulates intestinal crypt biology. Specifically, because we were able to combine this with ChIP-seq of the same cells, to identify where H3K4me1 levels (target of the histone demethylase LSD1) were different in the genome.
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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.
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Project description:We generated RNAseq expression analyses of PCGF6 WT and KO intestinal crypts in two independent replicates using two distinct transgenic models (AhCre and Villin Cre-ERT2) We then performed differential expression analyses of PCGF6 KO vs. WT independently in the AhCre and Villin transgenic models
2023-12-18 | GSE228478 | GEO
Project description:This study aims to measure the transcriptome profile of ilium tissues from intestinal epithelial conditional Ifnar1 KO and control littermates following cryptosporidial infection. Neonates of the conditional intestinal epithelial Ifnar1 knockout mice and control littlermates were exposed to C. parvum infection for 48 and 72h. Ilium tissues were collected and total RNA was isolated for the genome-wide analysis.
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Project description:The goal of the present study was to determine whether loss of the insulin receptor alters the molecular landscape of the intestinal mucosa, using intestinal-epithelial insulin receptor knockout (IE-irKO) mice and both genetic (IRfl/fl and Villin-cre) controls. Quantitative proteomic analysis by Liquid Chromatography Mass Spectrometry (LC-MS) was deployed on jejunal and colonic mucosa from mice fed a chow- or Western diet (WD). Jejunal mucosa from IE-irKO mice demonstrated alterations in all intestinal cell linages, Paneth, goblet, absorptive and enteroendocrine cells, whereas only goblet and absorptive cells were affected in the colon. There was also a significant effect of the WD on the gut proteome. A significant reduction was detected in Paneth cell proteins with anti-microbial activity, including lysozyme C-1, angiogenin-4, cryptdin-related sequence1C-3 and -2, a-defensin 17 and intelectin-1a. The key protein expressed by goblet cells, mucin-2, was also reduced in the IE-irKO mice. Proteins involved in lipid metabolism, including aldose reductase-related protein 1, 15-hydroxyprostaglandin dehydrogenase [NAD(+)], apolipoprotein A-II and pyruvate dehydrogenase kinase isozyme 4, were increased in the mucosa of WD-fed IE-irKO mice as compared to controls. In contrast, expression of the nutrient-responsive gut hormones, glucose-dependent insulinotropic polypeptide and neurotensin, was reduced in the jejunal mucosa of IE-irKO mice, and there was a reduction in proteins of the P-type ATPases and the solute carrier-transporter family in the colon of WD-fed IE-irKO mice. In conclusion, IE-irKO mice display a distinct molecular phenotype, suggesting a biological role of insulin and its receptor in determining differentiated cell-specificity in the intestinal epithelium.
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