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Epithelial RNase H2 Maintains Genome Integrity and Prevents Intestinal Tumorigenesis in Mice.

ABSTRACT: BACKGROUND & AIMS:RNase H2 is a holoenzyme, composed of 3 subunits (ribonuclease H2 subunits A, B, and C), that cleaves RNA:DNA hybrids and removes mis-incorporated ribonucleotides from genomic DNA through ribonucleotide excision repair. Ribonucleotide incorporation by eukaryotic DNA polymerases occurs during every round of genome duplication and produces the most frequent type of naturally occurring DNA lesion. We investigated whether intestinal epithelial proliferation requires RNase H2 function and whether RNase H2 activity is disrupted during intestinal carcinogenesis. METHODS:We generated mice with epithelial-specific deletion of ribonuclease H2 subunit B (H2b?IEC) and mice that also had deletion of tumor-suppressor protein p53 (H2b/p53?IEC); we compared phenotypes with those of littermate H2bfl/fl or H2b/p53fl/fl (control) mice at young and old ages. Intestinal tissues were collected and analyzed by histology. We isolated epithelial cells, generated intestinal organoids, and performed RNA sequence analyses. Mutation signatures of spontaneous tumors from H2b/p53?IEC mice were characterized by exome sequencing. We collected colorectal tumor specimens from 467 patients, measured levels of ribonuclease H2 subunit B, and associated these with patient survival times and transcriptome data. RESULTS:The H2b?IEC mice had DNA damage to intestinal epithelial cells and proliferative exhaustion of the intestinal stem cell compartment compared with controls and H2b/p53?IEC mice. However, H2b/p53?IEC mice spontaneously developed small intestine and colon carcinomas. DNA from these tumors contained T>G base substitutions at GTG trinucleotides. Analyses of transcriptomes of human colorectal tumors associated lower levels of RNase H2 with shorter survival times. CONCLUSIONS:In analyses of mice with disruption of the ribonuclease H2 subunit B gene and colorectal tumors from patients, we provide evidence that RNase H2 functions as a colorectal tumor suppressor. H2b/p53?IEC mice can be used to study the roles of RNase H2 in tissue-specific carcinogenesis.

SUBMITTER: Aden K

PROVIDER: S-EPMC6311085 | biostudies-literature | 2019 Jan

REPOSITORIES: biostudies-literature

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Epithelial RNase H2 Maintains Genome Integrity and Prevents Intestinal Tumorigenesis in Mice.

Aden Konrad K Bartsch Kareen K Dahl Joseph J Reijns Martin A M MAM Esser Daniela D Sheibani-Tezerji Raheleh R Sinha Anupam A Wottawa Felix F Ito Go G Mishra Neha N Knittler Katharina K Burkholder Adam A Welz Lina L van Es Johan J Tran Florian F Lipinski Simone S Kakavand Nassim N Boeger Christine C Lucius Ralph R von Schoenfels Witigo W Schafmayer Clemens C Lenk Lennart L Chalaris Athena A Clevers Hans H Röcken Christoph C Kaleta Christoph C Rose-John Stefan S Schreiber Stefan S Kunkel Thomas T Rabe Björn B Rosenstiel Philip P

Gastroenterology 20180928 1

<h4>Background & aims</h4>RNase H2 is a holoenzyme, composed of 3 subunits (ribonuclease H2 subunits A, B, and C), that cleaves RNA:DNA hybrids and removes mis-incorporated ribonucleotides from genomic DNA through ribonucleotide excision repair. Ribonucleotide incorporation by eukaryotic DNA polymerases occurs during every round of genome duplication and produces the most frequent type of naturally occurring DNA lesion. We investigated whether intestinal epithelial proliferation requires RNase H ...[more]

PMID: 30273559

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Project description:Background & aimsIntestinal epithelial cells (IECs) regulate intestinal immune cells, particularly development of T-helper 17 (Th17) cells. Deregulation of this process leads to intestinal inflammation and tumorigenesis, via unknown mechanisms. TANK-binding kinase 1 (TBK1) is expressed by IECs and cells in the innate immune system. We studied the functions of TBK1 in the intestinal immune response and tumorigenesis in mice.MethodsWe performed studies of wild-type mice, mice with conditional disruption of Tbk1 (Tbk1IEC-KO), Tbk1IEC-KO mice crossed with ApcMin/+ mice, and Mt-/- mice crossed with ApcMin/+ mice. Some mice were given intraperitoneal injections of a neutralizing antibody against interleukin 17 (IL17) or IL1β. Intestine tissues were collected from mice and analyzed by histology, for numbers of adenomas and Th17 cells, and expression of inflammatory cytokines by real-time PCR. IECs were isolated from wild-type and Tbk1IEC-KO mice, stimulated with lipopolysaccharide, co-cultured for with bone marrow-derived macrophages, and analyzed by RNA sequencing and biochemical analyses.ResultsCompared to ApcMin/+Tbk1WT mice, ApcMin/+Tbk1IEC-KO mice had significant increases in number and size of intestinal polyps, and significantly more Th17 cells in lamina propria. Administration of an antibody against IL17 reduced the number of intestinal polyps in ApcMin/+Tbk1IEC-KO mice to that observed in ApcMin/+Tbk1WT mice. In culture, TBK1-deficient IECs promoted expression of IL1β by macrophages, which induced differentiation of naïve CD4+ T cells into Th17 cells. RNA sequencing analysis revealed that the TBK1-deficient IECs had increased expression of metallothionein 1 (MT1), an immune regulator that promotes intestinal inflammation. Intestine tissues from ApcMin/+Mt-/- mice had significant fewer Th17 cells than ApcMin/+Mt+/+ mice, and a significantly lower number of polyps. Analyses of colorectal tumors in the Cancer Genome Atlas found colorectal tumors with high levels of MT1 and IL17 mRNAs to be associated with reduced survival times of patients.ConclusionsExpression of TBK1 by IECs suppresses expression of MT1 and prevents expression of IL1β by macrophages and differentiation of Th17 cells, to prevent inflammation and tumorigenesis. Strategies to block this pathway might be developed for colorectal tumorigenesis.

Project description:Two types of RNA:DNA associations can lead to genome instability: the formation of R-loops during transcription and the incorporation of ribonucleotide monophosphates (rNMPs) into DNA during replication. Both ribonuclease (RNase) H1 and RNase H2 degrade the RNA component of R-loops, whereas only RNase H2 can remove one or a few rNMPs from DNA. We performed high-resolution mapping of mitotic recombination events throughout the yeast genome in diploid strains of Saccharomyces cerevisiae lacking RNase H1 (rnh1Δ), RNase H2 (rnh201Δ), or both RNase H1 and RNase H2 (rnh1Δ rnh201Δ). We found little effect on recombination in the rnh1Δ strain, but elevated recombination in both the rnh201Δ and the double-mutant strains; levels of recombination in the double mutant were about 50% higher than in the rnh201 single-mutant strain. An rnh201Δ mutant that additionally contained a mutation that reduces rNMP incorporation by DNA polymerase ε (pol2-M644L) had a level of instability similar to that observed in the presence of wild-type Polε. This result suggests that the elevated recombination observed in the absence of only RNase H2 is primarily a consequence of R loops rather than misincorporated rNMPs. The details of these experiments are in press in Genetics This diploid S.cerevisiae strain used here is a hybrid between W303-1A and YJM789. The backgrounds are diverged by about 50,000 polymorphisms that are surveyed by custom CGH microarrays containing SNP-specific probes (St. Charles et al., 2012; Genetics 190: 1267-1284; PMID 22267500). The whole genome array surveyed loss of heterozygosity (LOH) events throughout the genome of in wild type, pol2-M644L, rnh1Δ, rnh201Δ, rnh201Δ pol2-M644L, and rnh1Δ rnh201Δ mutant backgrounds in 10, 12, 10, 19, 17, and 22 samples, respectively. The names of these strains are KO_198,KO_234, KO_73, KO_75, KO_244, and KO_5, respectively. Another assay was performed mapping reciprocal crossover events on the right arm of chromosome IV in the rnh201Δ (strains KO_135 and KO188) and the rnh1Δrnh201Δ (strain KO_132) mutant backgrounds. A reporter system with a wild type ADE2 on the end of chromosome IV allowed us to detect crossovers by seeing a red-white sectored colony. Each reciprocal event was mapped by doing a microarray specific for the right arm of chromosome IV (St. Charles and Petes, 2013; PLoS Genet. 1267-1284 PMCID PMC3616911) on the red and white sides of sectors. 21 sectors were analyzed for the rnh201Δ mutant while 14 were analyzed for the rnh1Δ rnh201Δ mutant. We mapped crossovers on the right arm of chromosome IV in the rnh201Δ mutant in four different diploid isolates: KO135_5 (1R/1W, 2R/2W, 4R/4W, 5R/5W, 12R/12W, and 13R/13W), KO_135_6 (1R/1W, 4R/4W, 5R/5W, 7R/7W, 9R/9W, 11R/11W, 13R/13W, and 17R/17W), KO 188_1 (1R/1W, and 4R/4W), and KO188_2 (6R/6W, 7R/7W, 11R/11W, and 12R/12W). We mapped crossovers on the right arm of chromosome IV in the rnh1Δ rnh201Δ mutant in two different diploid isolates: KO_132_31 (2R/2W, 6R/6W, 8R/8W, 15R/15W, 17R/17W, 21R/21W, 22R/22W, and 24R/24W) and KO_132_29 (3R/3W, 7R/7W, 18R/18W, 24R/24W, and 34R/34W).

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Epithelial RNase H2 Maintains Genome Integrity and Prevents Intestinal Tumorigenesis in Mice.

Publications

Epithelial RNase H2 Maintains Genome Integrity and Prevents Intestinal Tumorigenesis in Mice.

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