Nucleotide Excision Repair and Transcription-Associated Genome Instability.

Transcription is a potential threat to genome integrity, and transcription-associated DNA damage must be repaired for proper messenger RNA (mRNA) synthesis and for cells to transmit their genome intact into progeny. For a wide range of structurally diverse DNA lesions, cells employ the highly conserved nucleotide excision repair (NER) pathway to restore their genome back to its native form. Recent evidence suggests that NER factors function, in addition to the canonical DNA repair mechanism, in processes that facilitate mRNA synthesis or shape the 3D chromatin architecture. Here, these findings are critically discussed and a working model that explains the puzzling clinical heterogeneity of NER syndromes highlighting the relevance of physiological, transcription-associated DNA damage to mammalian development and disease is proposed.

DNA damage and innate immunity: links and trade-offs.

To counteract DNA damage, cells employ genome maintenance pathways that are directed inward, relentlessly to scan and repair the genome. Adaptive and innate immune mechanisms are often directed outward, protecting self against pathogens. Recent work has revealed direct links between innate immune signaling and the DNA damage response (DDR). Here we review current understanding of the mechanism by which cells sense damaged and foreign DNA. We examine the functional role of DNA damage signaling in immune activation and discuss the relevance of these processes to DNA damage-driven chronic inflammation in disease and in aging.

The Nucleolus: In Genome Maintenance and Repair.

The nucleolus is the subnuclear membrane-less organelle where rRNA is transcribed and processed and ribosomal assembly occurs. During the last 20 years, however, the nucleolus has emerged as a multifunctional organelle, regulating processes that go well beyond its traditional role. Moreover, the unique organization of rDNA in tandem arrays and its unusually high transcription rates make it prone to unscheduled DNA recombination events and frequent RNA:DNA hybrids leading to DNA double strand breaks (DSBs). If not properly repaired, rDNA damage may contribute to premature disease onset and aging. Deregulation of ribosomal synthesis at any level from transcription and processing to ribosomal subunit assembly elicits a stress response and is also associated with disease onset. Here, we discuss how genome integrity is maintained within nucleoli and how such structures are functionally linked to nuclear DNA damage response and repair giving an emphasis on the newly emerging roles of the nucleolus in mammalian physiology and disease.

Transcription stress at telomeres leads to cytosolic DNA release and paracrine senescence.

Transcription stress has been linked to DNA damage -driven aging, yet the underlying mechanism remains unclear. Here, we demonstrate that Tcea1-/- cells, which harbor a TFIIS defect in transcription elongation, exhibit RNAPII stalling at oxidative DNA damage sites, impaired transcription, accumulation of R-loops, telomere uncapping, chromatin bridges, and genome instability, ultimately resulting in cellular senescence. We found that R-loops at telomeres causally contribute to the release of telomeric DNA fragments in the cytoplasm of Tcea1-/- cells and primary cells derived from naturally aged animals triggering a viral-like immune response. TFIIS-defective cells release extracellular vesicles laden with telomeric DNA fragments that target neighboring cells, which consequently undergo cellular senescence. Thus, transcription stress elicits paracrine signals leading to cellular senescence, promoting aging.

The DREAM complex functions as conserved master regulator of somatic DNA-repair capacities.

The DNA-repair capacity in somatic cells is limited compared with that in germ cells. It has remained unknown whether not only lesion-type-specific, but overall repair capacities could be improved. Here we show that the DREAM repressor complex curbs the DNA-repair capacities in somatic tissues of Caenorhabditis elegans. Mutations in the DREAM complex induce germline-like expression patterns of multiple mechanisms of DNA repair in the soma. Consequently, DREAM mutants confer resistance to a wide range of DNA-damage types during development and aging. Similarly, inhibition of the DREAM complex in human cells boosts DNA-repair gene expression and resistance to distinct DNA-damage types. DREAM inhibition leads to decreased DNA damage and prevents photoreceptor loss in progeroid Ercc1-/- mice. We show that the DREAM complex transcriptionally represses essentially all DNA-repair systems and thus operates as a highly conserved master regulator of the somatic limitation of DNA-repair capacities.

XPF interacts with TOP2B for R-loop processing and DNA looping on actively transcribed genes.

Co-transcriptional RNA-DNA hybrids can not only cause DNA damage threatening genome integrity but also regulate gene activity in a mechanism that remains unclear. Here, we show that the nucleotide excision repair factor XPF interacts with the insulator binding protein CTCF and the cohesin subunits SMC1A and SMC3, leading to R-loop-dependent DNA looping upon transcription activation. To facilitate R-loop processing, XPF interacts and recruits with TOP2B on active gene promoters, leading to double-strand break accumulation and the activation of a DNA damage response. Abrogation of TOP2B leads to the diminished recruitment of XPF, CTCF, and the cohesin subunits to promoters of actively transcribed genes and R-loops and the concurrent impairment of CTCF-mediated DNA looping. Together, our findings disclose an essential role for XPF with TOP2B and the CTCF/cohesin complex in R-loop processing for transcription activation with important ramifications for DNA repair-deficient syndromes associated with transcription-associated DNA damage.

The splicing factor XAB2 interacts with ERCC1-XPF and XPG for R-loop processing.

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.

Tissue-infiltrating macrophages mediate an exosome-based metabolic reprogramming upon DNA damage.

DNA damage and metabolic disorders are intimately linked with premature disease onset but the underlying mechanisms remain poorly understood. Here, we show that persistent DNA damage accumulation in tissue-infiltrating macrophages carrying an ERCC1-XPF DNA repair defect (Er1F/-) triggers Golgi dispersal, dilation of endoplasmic reticulum, autophagy and exosome biogenesis leading to the secretion of extracellular vesicles (EVs) in vivo and ex vivo. Macrophage-derived EVs accumulate in Er1F/- animal sera and are secreted in macrophage media after DNA damage. The Er1F/- EV cargo is taken up by recipient cells leading to an increase in insulin-independent glucose transporter levels, enhanced cellular glucose uptake, higher cellular oxygen consumption rate and greater tolerance to glucose challenge in mice. We find that high glucose in EV-targeted cells triggers pro-inflammatory stimuli via mTOR activation. This, in turn, establishes chronic inflammation and tissue pathology in mice with important ramifications for DNA repair-deficient, progeroid syndromes and aging.

Glycosaminoglycans and PDGF signaling in mesenchymal cells.

Platelet derived growth factor (PDGF) is involved in the autocrine growth stimulation of normal and malignant cells, the stimulation of angiogenesis, and the recruitment and regulation of tumor fibroblasts. PDGF has been shown to physically interact with glycosaminoglycans which are abundant in the extracellular microenvironment. The present review discusses the effects of glycosaminoglycans on the functions mediated by the PDGF on cells of mesenchymal origin. Recent studies have demonstrated that both soluble and surface bound glycosaminoglycan chains can modulate PDGF-BB isoform signaling depending on the cell type. These data demonstrated that the microenvironment rich in GAGs/PGs is able to significantly modify the cellular response to PDGF-BB signaling in a critical way for cell growth and differentiation.

ERCC1-XPF cooperates with CTCF and cohesin to facilitate the developmental silencing of imprinted genes.

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.

DNA damage triggers a chronic autoinflammatory response, leading to fat depletion in NER progeria.

Lipodystrophies represent a group of heterogeneous disorders characterized by loss of fat tissue. However, the underlying mechanisms remain poorly understood. Using mice carrying an ERCC1-XPF DNA repair defect systematically or in adipocytes, we show that DNA damage signaling triggers a chronic autoinflammatory response leading to fat depletion. Ercc1-/- and aP2-Ercc1F/- fat depots show extensive gene expression similarities to lipodystrophic Pparγ(ldi/+) animals, focal areas of ruptured basement membrane, the reappearance of primary cilia, necrosis, fibrosis, and a marked decrease in adiposity. We find that persistent DNA damage in aP2-Ercc1F/- fat depots and in adipocytes ex vivo triggers the induction of proinflammatory factors by promoting transcriptionally active histone marks and the dissociation of nuclear receptor corepressor complexes from promoters; the response is cell autonomous and requires ataxia telangiectasia mutated (ATM). Thus, persistent DNA damage-driven autoinflammation plays a causative role in adipose tissue degeneration, with important ramifications for progressive lipodystrophies and natural aging.

Heparin--a unique stimulator of human colon cancer cells' growth.

The cancer microenvironment and the interactions between cancer and surrounding tissue cells are thought to play a pivotal role in tumor development and progression. Glycosaminoglycans (GAGs)/proteoglycans (PGs) are major constituents of the extracellular matrix, the composition of which may affect various cellular functions. In the present study, the effects of GAGs on the proliferation of HT29, SW1116, and HCT116 human colon cancer cell lines were examined using exogenously added GAGs, an inhibitor of endogenous GAG sulfation and specific glycosidase digestions. Our results demonstrate that colon cancer cell growth was exclusively stimulated by exogenously added heparin and insensitive to endogenous GAGs/PGs production, in a sulfation pattern-related manner. Treatment of the tested cell lines with the FGF-2 neutralizing antibody showed that the stimulatory effect of heparin on the cells' growth was not FGF-2-dependent. Responsiveness of colon cancer cell lines to exogenous heparin/heparan sulfate may play a role in their growth and metastasis.