ABSTRACT
RNA splicing, transcription and the DNA damage response are intriguingly linked in mammals but the underlying mechanisms remain poorly understood. Using an in vivo biotinylation tagging approach in mice, we show that the splicing factor XAB2 interacts with the core spliceosome and that it binds to spliceosomal U4 and U6 snRNAs and pre-mRNAs in developing livers. XAB2 depletion leads to aberrant intron retention, R-loop formation and DNA damage in cells. Studies in illudin S-treated cells and Csbm/m developing livers reveal that transcription-blocking DNA lesions trigger the release of XAB2 from all RNA targets tested. Immunoprecipitation studies reveal that XAB2 interacts with ERCC1-XPF and XPG endonucleases outside nucleotide excision repair and that the trimeric protein complex binds RNA:DNA hybrids under conditions that favor the formation of R-loops. Thus, XAB2 functionally links the spliceosomal response to DNA damage with R-loop processing with important ramifications for transcription-coupled DNA repair disorders.
Subject(s)
DNA Repair , DNA-Binding Proteins/metabolism , Endonucleases/metabolism , Nuclear Proteins/metabolism , RNA Splicing Factors/metabolism , Transcription Factors/metabolism , Animals , Cell Line , DNA Damage/drug effects , Female , Gene Expression Regulation, Developmental , Gene Knock-In Techniques , Gene Knockdown Techniques , Liver/growth & development , Liver/metabolism , Male , Mice , Mice, Transgenic , Mouse Embryonic Stem Cells , Polycyclic Sesquiterpenes/pharmacology , R-Loop Structures/genetics , RNA Precursors/genetics , RNA Precursors/metabolism , RNA Splicing Factors/genetics , RNA, Small Nuclear , RNA-Seq , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Spliceosomes/metabolism , Transcription, GeneticABSTRACT
Inborn defects in DNA repair are associated with complex developmental disorders whose causal mechanisms are poorly understood. Using an in vivo biotinylation tagging approach in mice, we show that the nucleotide excision repair (NER) structure-specific endonuclease ERCC1-XPF complex interacts with the insulator binding protein CTCF, the cohesin subunits SMC1A and SMC3 and with MBD2; the factors co-localize with ATRX at the promoters and control regions (ICRs) of imprinted genes during postnatal hepatic development. Loss of Ercc1 or exposure to MMC triggers the localization of CTCF to heterochromatin, the dissociation of the CTCF-cohesin complex and ATRX from promoters and ICRs, altered histone marks and the aberrant developmental expression of imprinted genes without altering DNA methylation. We propose that ERCC1-XPF cooperates with CTCF and cohesin to facilitate the developmental silencing of imprinted genes and that persistent DNA damage triggers chromatin changes that affect gene expression programs associated with NER disorders.
Subject(s)
Cell Cycle Proteins/metabolism , Chromosomal Proteins, Non-Histone/metabolism , DNA Repair , DNA-Binding Proteins/metabolism , Endonucleases/metabolism , Gene Silencing , Genomic Imprinting , Repressor Proteins/metabolism , Age Factors , Animals , Animals, Newborn , CCCTC-Binding Factor , Cell Cycle Proteins/genetics , Cells, Cultured , Chondroitin Sulfate Proteoglycans/genetics , Chondroitin Sulfate Proteoglycans/metabolism , Chromosomal Proteins, Non-Histone/genetics , Coculture Techniques , DNA Damage , DNA Helicases/genetics , DNA Helicases/metabolism , DNA-Binding Proteins/genetics , Endonucleases/genetics , Fibroblasts/enzymology , Gene Expression Regulation, Developmental , Genotype , Histones/metabolism , Liver/enzymology , Mice, 129 Strain , Mice, Inbred C57BL , Mice, Transgenic , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Phenotype , Promoter Regions, Genetic , Repressor Proteins/genetics , X-linked Nuclear Protein , CohesinsABSTRACT
Mutant dwarf and calorie-restricted mice benefit from healthy aging and unusually long lifespan. In contrast, mouse models for DNA repair-deficient progeroid syndromes age and die prematurely. To identify mechanisms that regulate mammalian longevity, we quantified the parallels between the genome-wide liver expression profiles of mice with those two extremes of lifespan. Contrary to expectation, we find significant, genome-wide expression associations between the progeroid and long-lived mice. Subsequent analysis of significantly over-represented biological processes revealed suppression of the endocrine and energy pathways with increased stress responses in both delayed and premature aging. To test the relevance of these processes in natural aging, we compared the transcriptomes of liver, lung, kidney, and spleen over the entire murine adult lifespan and subsequently confirmed these findings on an independent aging cohort. The majority of genes showed similar expression changes in all four organs, indicating a systemic transcriptional response with aging. This systemic response included the same biological processes that are triggered in progeroid and long-lived mice. However, on a genome-wide scale, transcriptomes of naturally aged mice showed a strong association to progeroid but not to long-lived mice. Thus, endocrine and metabolic changes are indicative of "survival" responses to genotoxic stress or starvation, whereas genome-wide associations in gene expression with natural aging are indicative of biological age, which may thus delineate pro- and anti-aging effects of treatments aimed at health-span extension.
