Unknown,Transcriptomics,Genomics,Proteomics

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RNA-sequencing of mRNAs from control and CAP-D3 deficient Salmonella infected HT-29 cells

ABSTRACT: BACKGROUND & AIMS- More frequent interaction of bacteria with the colonic epithelium is associated with ulcerative colitis (UC). The identities of all proteins which promote bacterial clearance in colonic epithelial cells are unknown. Previously, we discovered that dCAP-D3 (Chromosome Associated Protein-D3), regulates responses to bacterial infection. We examined whether CAP-D3 promotes bacterial clearance in human colonic epithelium. METHODS- Clearance of Salmonella or adherent-invasive Escherichia coli LF82 was assessed by gentamycin protection assays in HT-29 and Caco-2 cells expressing CAP-D3 shRNA. CAP-D3 levels in colonic epithelial cells from healthy and UC patient tissues were analyzed by immunoblot. RNA-sequencing identified bacterially-induced CAP-D3 target genes. The role of CAP-D3 target genes in bacterial clearance was analyzed by gentamycin protection assays, immunofluorescent staining, and by using pharmacologic inhibitors. RESULTS- CAP-D3 expression was reduced in colonic epithelial cells from UC patients with active disease. Reduction of CAP-D3 expression inhibited autophagy and decreased intracellular bacterial clearance. The components of the heterodimeric SLC7A5/SLC3A2 amino acid transporter were identified as CAP-D3 target genes; their levels increased in infected, CAP-D3 deficient cell lines and in cells from UC patients. In HT-29 cells, this resulted in earlier SLC7A5 recruitment to Salmonella-containing vacuoles, increased mTOR activity, and enhanced bacterial survival. Inhibition of SLC7A5/SLC3A2 or mTOR activity rescued the bacterial clearance defect in CAP-D3 deficient cells. CONCLUSIONS- CAP-D3 attenuates amino acid transporter transcription to promote bacterial autophagy in colon epithelial cells. CAP-D3 protein levels are decreased in patients with active UC, suggesting that CAP-D3 is a potential therapeutic target to restore mucosal homeostasis in UC patients. Three RNA samples from 3 independent experiments including timepoints taken at 0, 0.5 and 7 hours post-infection were analyzed on a bioanalyzer for quality; one of the 0.5 hour post-infection samples was excluded at this time due to poor RNA purity. Directional, cDNA libraries made from cellular mRNAs were generated from the other 8 samples and sequenced (paired-end sequencing of 100 bp reads) in the Genomics Core at the University of Chicago on an Illumina HiSeq2000.

ORGANISM(S): Homo sapiens

SUBMITTER: Michelle Longworth

PROVIDER: E-GEOD-62520 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Chromosome-associated protein D3 promotes bacterial clearance in human intestinal epithelial cells by repressing expression of amino acid transporters.

Schuster Andrew T AT Homer Craig R CR Kemp Jacqueline R JR Nickerson Kourtney P KP Deutschman Emily E Kim Yeojung Y West Gail G Sadler Tammy T Stylianou Eleni E Krokowski Dawid D Hatzoglou Maria M de la Motte Carol C Rubin Brian P BP Fiocchi Claudio C McDonald Christine C Longworth Michelle S MS

Gastroenterology 20150218 7

<h4>Background & aims</h4>Defects in colonic epithelial barrier defenses are associated with ulcerative colitis (UC). The proteins that regulate bacterial clearance in the colonic epithelium have not been completely identified. The Drosophila chromosome-associated protein D3 (dCAP-D3) regulates responses to bacterial infection. We examined whether CAP-D3 promotes bacterial clearance in human colonic epithelium.<h4>Methods</h4>Clearance of Salmonella or adherent-invasive Escherichia coli LF82 was ...[more]

PMID: 25701737

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Project description:Mouse models have dramatically improved our understanding of cancer development and tumor biology. However, these models have shown limited efficacy as tractable systems for unbiased genetic experimentation. Here, we report the adaptation of loss of function screening to mouse models of cancer. Using this approach, we have been able to screen nearly 1000 genetic alterations in the context of an individual tumor-bearing mouse. Results from these experiments have identified a central role for regulators of actin dynamics and cell motility in lymphoma cell homeostasis in vivo, and validation experiments confirmed that these proteins represent bona fide lymphoma drug targets. Additionally, suppression of one of these targets, Rac2, potentiated the action of the front-line chemotherapeutic vincristine in vivo, suggesting a critical relationship between tumor cell motility and tumor relapse in hematopoietic malignancies. Eu-myc Arf-/- lymphoma cells were transduced with a GFP-tagged, pooled library of 2250 shRNAs targeting 1000 genes with known or putative roles in cancer. Infected cells were GFP sorted. Acutely after sorting, lymphoma cells were tail-vein injected into three syngeneic recipient mice, maintained in three separate culture dishes for two weeks, or collected immediately to serve as a reference sample. At the time of lymphoma presentation, approximately two weeks after injection, tumors were harvested and genomic DNA was extracted from primary tumors and cultured cells. shRNAs were PCR amplified from genomic DNA using common primers, and hairpin representation was analyzed by high throughput sequencing (454 sequencing was used to determine the relative abundance of shRNAs before and after a period of cancer cell growth.). 8 samples were examined - the retroviral plasmid library, cells that were collected immediately after retroviral infection and subsequent GFP-sorting, the three samples that were maintained in vitro for two weeks, and tumors from three individual mice.

Dataset Information

RNA-sequencing of mRNAs from control and CAP-D3 deficient Salmonella infected HT-29 cells

Publications

Chromosome-associated protein D3 promotes bacterial clearance in human intestinal epithelial cells by repressing expression of amino acid transporters.

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