Decoding of translation-regulating entities reveals heterogeneous translation deficiency patterns in cellular senescence.

Cellular senescence constitutes a generally irreversible proliferation barrier, accompanied by macromolecular damage and metabolic rewiring. Several senescence types have been identified based on the initiating stimulus, such as replicative (RS), stress-induced (SIS) and oncogene-induced senescence (OIS). These senescence subtypes are heterogeneous and often develop subset-specific phenotypes. Reduced protein synthesis is considered a senescence hallmark, but whether this trait pertains to various senescence subtypes and if distinct molecular mechanisms are involved remain largely unknown. Here, we analyze large published or experimentally produced RNA-seq and Ribo-seq datasets to determine whether major translation-regulating entities such as ribosome stalling, the presence of uORFs/dORFs and IRES elements may differentially contribute to translation deficiency in senescence subsets. We show that translation-regulating mechanisms may not be directly relevant to RS, however uORFs are significantly enriched in SIS. Interestingly, ribosome stalling, uORF/dORF patterns and IRES elements comprise predominant mechanisms upon OIS, strongly correlating with Notch pathway activation. Our study provides for the first time evidence that major translation dysregulation mechanisms/patterns occur during cellular senescence, but at different rates depending on the stimulus type. The degree at which those mechanisms accumulate directly correlates with translation deficiency levels. Our thorough analysis contributes to elucidating crucial and so far unknown differences in the translation machinery between senescence subsets.

Enhanced frequency of transcription pre-initiation complexes assembly after exposure to UV irradiation results in increased repair activity and reduced probabilities for mutagenesis.

In addition to being essential for gene expression, transcription is crucial for the maintenance of genome integrity. Here, we undertook a systematic approach, to monitor the assembly kinetics of the pre-initiating RNA Polymerase (Pol) II at promoters at steady state and different stages during recovery from UV irradiation-stress, when pre-initiation and initiation steps have been suggested to be transiently shut down. Taking advantage of the reversible dissociation of pre-initiating Pol II after high salt treatment, we found that de novo recruitment of the available Pol II molecules at active promoters not only persists upon UV at all times tested but occurs significantly faster in the early phase of recovery (2 h) than in unexposed human fibroblasts at the majority of active genes. Our method unveiled groups of genes with significantly different pre-initiation complex (PIC) assembly dynamics after UV that present distinct rates of UV-related mutational signatures in melanoma tumours, providing functional relevance to the importance of keeping transcription initiation active during UV recovery. Our findings uncover novel mechanistic insights further detailing the multilayered transcriptional response to genotoxic stress and link PIC assembly dynamics after exposure to genotoxins with cancer mutational landscapes.

Cockayne Syndrome Group B (CSB): The Regulatory Framework Governing the Multifunctional Protein and Its Plausible Role in Cancer.

Cockayne syndrome (CS) is a DNA repair syndrome characterized by a broad spectrum of clinical manifestations such as neurodegeneration, premature aging, developmental impairment, photosensitivity and other symptoms. Mutations in Cockayne syndrome protein B (CSB) are present in the vast majority of CS patients and in other DNA repair-related pathologies. In the literature, the role of CSB in different DNA repair pathways has been highlighted, however, new CSB functions have been identified in DNA transcription, mitochondrial biology, telomere maintenance and p53 regulation. Herein, we present an overview of identified structural elements and processes that impact on CSB activity and its post-translational modifications, known to balance the different roles of the protein not only during normal conditions but most importantly in stress situations. Moreover, since CSB has been found to be overexpressed in a number of different tumors, its role in cancer is presented and possible therapeutic targeting is discussed.

MAP3K8 Regulates Cox-2-Mediated Prostaglandin E2 Production in the Lung and Suppresses Pulmonary Inflammation and Fibrosis.

Idiopathic pulmonary fibrosis (IPF) is characterized by exuberant deposition of extracellular matrix components, leading to the deterioration of lung architecture and respiratory functions. Profibrotic mechanisms are controlled by multiple regulatory molecules, including MAPKs, in turn regulated by multiple phosphorylation cascades. MAP3K8 is an MAPK kinase kinase suggested to pleiotropically regulate multiple pathogenic pathways in the context of inflammation and cancer; however, a possible role in the pathogenesis of IPF has not been investigated. In this report, MAP3K8 mRNA levels were found decreased in the lungs of IPF patients and of mice upon bleomycin-induced pulmonary fibrosis. Ubiquitous genetic deletion of Map3k8 in mice exacerbated the modeled disease, whereas bone marrow transfer experiments indicated that although MAP3K8 regulatory functions are active in both hematopoietic and nonhematopoietic cells, Map3k8 in hematopoietic cells has a more dominant role. Macrophage-specific deletion of Map3k8 was further found to be sufficient for disease exacerbation thus confirming a major role for macrophages in pulmonary fibrotic responses and suggesting a main role for Map3k8 in the homeostasis of their effector functions in the lung. Map3k8 deficiency was further shown to be associated with decreased Cox-2 expression, followed by a decrease in PGE2 production in the lung; accordingly, exogenous administration of PGE2 reduced inflammation and reversed the exacerbated fibrotic profile of Map3k8 -/- mice. Therefore, MAP3K8 has a central role in the regulation of inflammatory responses and Cox-2-mediated PGE2 production in the lung, and the attenuation of its expression is integral to pulmonary fibrosis development.

Prox1 inhibits neurite outgrowth during central nervous system development.

During central nervous system (CNS) development, proper and timely induction of neurite elongation is critical for generating functional, mature neurons, and neuronal networks. Despite the wealth of information on the action of extracellular cues, little is known about the intrinsic gene regulatory factors that control this developmental decision. Here, we report the identification of Prox1, a homeobox transcription factor, as a key player in inhibiting neurite elongation. Although Prox1 promotes acquisition of early neuronal identity and is expressed in nascent post-mitotic neurons, it is heavily down-regulated in the majority of terminally differentiated neurons, indicating a regulatory role in delaying neurite outgrowth in newly formed neurons. Consistently, we show that Prox1 is sufficient to inhibit neurite extension in mouse and human neuroblastoma cell lines. More importantly, Prox1 overexpression suppresses neurite elongation in primary neuronal cultures as well as in the developing mouse brain, while Prox1 knock-down promotes neurite outgrowth. Mechanistically, RNA-Seq analysis reveals that Prox1 affects critical pathways for neuronal maturation and neurite extension. Interestingly, Prox1 strongly inhibits many components of Ca2+ signaling pathway, an important mediator of neurite extension and neuronal maturation. In accordance, Prox1 represses Ca2+ entry upon KCl-mediated depolarization and reduces CREB phosphorylation. These observations suggest that Prox1 acts as a potent suppressor of neurite outgrowth by inhibiting Ca2+ signaling pathway. This action may provide the appropriate time window for nascent neurons to find the correct position in the CNS prior to initiation of neurites and axon elongation.

Global unleashing of transcription elongation waves in response to genotoxic stress restricts somatic mutation rate.

Complex molecular responses preserve gene expression accuracy and genome integrity in the face of environmental perturbations. Here we report that, in response to UV irradiation, RNA polymerase II (RNAPII) molecules are dynamically and synchronously released from promoter-proximal regions into elongation to promote uniform and accelerated surveillance of the whole transcribed genome. The maximised influx of de novo released RNAPII correlates with increased damage-sensing, as confirmed by RNAPII progressive accumulation at dipyrimidine sites and by the average slow-down of elongation rates in gene bodies. In turn, this transcription elongation 'safe' mode guarantees efficient DNA repair regardless of damage location, gene size and transcription level. Accordingly, we detect low and homogenous rates of mutational signatures associated with UV exposure or cigarette smoke across all active genes. Our study reveals a novel advantage for transcription regulation at the promoter-proximal level and provides unanticipated insights into how active transcription shapes the mutagenic landscape of cancer genomes.

Nucleotide Excision Repair: From Neurodegeneration to Cancer.

DNA damage poses a constant threat to genome integrity taking a variety of shapes and arising by normal cellular metabolism or environmental insults. Human syndromes, characterized by increased cancer pre-disposition or early onset of age-related pathology and developmental abnormalities, often result from defective DNA damage responses and compromised genome integrity. Over the last decades intensive research worldwide has made important contributions to our understanding of the molecular mechanisms underlying genomic instability and has substantiated the importance of DNA repair in cancer prevention in the general population. In this chapter, we discuss Nucleotide Excision Repair pathway, the causative role of its components in disease-related pathology and recent technological achievements that decipher mutational landscapes and may facilitate pathological classification and personalized therapy.

Proteins of nucleotide and base excision repair pathways interact in mitochondria to protect from loss of subcutaneous fat, a hallmark of aging.

Defects in the DNA repair mechanism nucleotide excision repair (NER) may lead to tumors in xeroderma pigmentosum (XP) or to premature aging with loss of subcutaneous fat in Cockayne syndrome (CS). Mutations of mitochondrial (mt)DNA play a role in aging, but a link between the NER-associated CS proteins and base excision repair (BER)-associated proteins in mitochondrial aging remains enigmatic. We show functional increase of CSA and CSB inside mt and complex formation with mtDNA, mt human 8-oxoguanine glycosylase (mtOGG)-1, and mt single-stranded DNA binding protein (mtSSBP)-1 upon oxidative stress. MtDNA mutations are highly increased in cells from CS patients and in subcutaneous fat of aged Csb(m/m) and Csa(-/-) mice. Thus, the NER-proteins CSA and CSB localize to mt and directly interact with BER-associated human mitochondrial 8-oxoguanine glycosylase-1 to protect from aging- and stress-induced mtDNA mutations and apoptosis-mediated loss of subcutaneous fat, a hallmark of aging found in animal models, human progeroid syndromes like CS and in normal human aging.

Association between transcriptional activity, local chromatin structure, and the efficiencies of both subpathways of nucleotide excision repair of melphalan adducts.

The repair of melphalan-induced N-alkylpurine monoadducts and interstrand cross-links was examined in different repair backgrounds, focusing on four genes (beta-actin, p53, N-ras, and delta-globin) with dissimilar transcription activities. Adducts were found to be substrates for both global genome repair (GGR) and transcription-coupled repair (TCR), with TCR being less efficient than GGR. In nucleotide excision repair-deficient cells, adducts accumulated to similar levels in all four genes. The repair efficiency in different gene loci varied in a qualitatively and quantitatively similar way in both GGR-deficient and TCR-deficient backgrounds and correlated with transcriptional activity and local chromatin condensation. No strand-specific repair was found in GGR(+)/TCR(+) cells, implying that GGR dominated. Adducts were lost over two sharply demarcated phases: a rapid phase resulting in the removal within 1 hour of up to approximately 80% of the adducts, and a subsequent phase with t(1/2) approximately 36 to 48 hours. Following pretreatment of cells with alpha-amanitin, the rate of transcription, the state of chromatin condensation, and the repair efficiencies (both TCR and GGR) of the transcribed beta-actin, p53, and N-ras genes became similar to those of the nontranscribed delta-globin gene. In conclusion, a continuous, parallel variation of the state of transcription and local chromatin condensation, on one hand, and the rates of both GGR and TCR, on the other hand, have been shown.

Persistent transcription-blocking DNA lesions trigger somatic growth attenuation associated with longevity.

The accumulation of stochastic DNA damage throughout an organism's lifespan is thought to contribute to ageing. Conversely, ageing seems to be phenotypically reproducible and regulated through genetic pathways such as the insulin-like growth factor-1 (IGF-1) and growth hormone (GH) receptors, which are central mediators of the somatic growth axis. Here we report that persistent DNA damage in primary cells from mice elicits changes in global gene expression similar to those occurring in various organs of naturally aged animals. We show that, as in ageing animals, the expression of IGF-1 receptor and GH receptor is attenuated, resulting in cellular resistance to IGF-1. This cell-autonomous attenuation is specifically induced by persistent lesions leading to stalling of RNA polymerase II in proliferating, quiescent and terminally differentiated cells; it is exacerbated and prolonged in cells from progeroid mice and confers resistance to oxidative stress. Our findings suggest that the accumulation of DNA damage in transcribed genes in most if not all tissues contributes to the ageing-associated shift from growth to somatic maintenance that triggers stress resistance and is thought to promote longevity.

Transcription-coupled nucleotide excision repair in mammalian cells: molecular mechanisms and biological effects.

The encounter of elongating RNA polymerase II (RNAPIIo) with DNA lesions has severe consequences for the cell as this event provides a strong signal for P53-dependent apoptosis and cell cycle arrest. To counteract prolonged blockage of transcription, the cell removes the RNAPIIo-blocking DNA lesions by transcription-coupled repair (TC-NER), a specialized subpathway of nucleotide excision repair (NER). Exposure of mice to UVB light or chemicals has elucidated that TC-NER is a critical survival pathway protecting against acute toxic and long-term effects (cancer) of genotoxic exposure. Deficiency in TC-NER is associated with mutations in the CSA and CSB genes giving rise to the rare human disorder Cockayne syndrome (CS). Recent data suggest that CSA and CSB play differential roles in mammalian TC-NER: CSB as a repair coupling factor to attract NER proteins, chromatin remodellers and the CSA- E3-ubiquitin ligase complex to the stalled RNAPIIo. CSA is dispensable for attraction of NER proteins, yet in cooperation with CSB is required to recruit XAB2, the nucleosomal binding protein HMGN1 and TFIIS. The emerging picture of TC-NER is complex: repair of transcription-blocking lesions occurs without displacement of the DNA damage-stalled RNAPIIo, and requires at least two essential assembly factors (CSA and CSB), the core NER factors (except for XPC-RAD23B), and TC-NER specific factors. These and yet unidentified proteins will accomplish not only efficient repair of transcription-blocking lesions, but are also likely to contribute to DNA damage signalling events.

Send in the clamps: control of DNA translesion synthesis in eukaryotes.

The replication of damaged DNA templates by translesion synthesis (TLS) is associated with mutagenesis and carcinogenesis. This perspective discusses the different levels at which TLS may be controlled and proposes a model for TLS of severely helix-distorting DNA lesions that includes a decisive role for the Rad9-Hus1-Rad1 DNA-damage-signaling clamp. The dual involvement of this clamp in both DNA-damage signaling and TLS may have profound implications in determining cellular responses to DNA damage.

Enhanced DDB2 expression protects mice from carcinogenic effects of chronic UV-B irradiation.

UV-damaged DNA-binding protein (UV-DDB) is essential for global genome repair (GGR) of UV-induced cyclobutane pyrimidine dimers (CPD). Unlike human cells, rodent epidermal cells are deficient in GGR of CPDs and express a subunit of UV-DDB, DDB2, at a low level. In this study, we generated mice (K14-DDB2) ectopically expressing mouse DDB2 at elevated levels. Enhanced expression of DDB2 both delayed the onset of squamous cell carcinoma and decreased the number of tumors per mouse in chronically UV-B light-exposed hairless mice. Enhanced expression of DDB2 improved repair of both CPDs and pyrimidine(6-4)pyrimidone photoproducts (6-4PP) in dermal fibroblasts. However, GGR of CPDs in K14-DDB2 mice did not reach the level of efficiency of human cells, suggesting that another repair protein may become rate limiting when DDB2 is abundantly present. To complement these studies, we generated mice in which the DDB2 gene was disrupted. DDB2-/- and DDB2+/- mice were found to be hypersensitive to UV-induced skin carcinogenesis. On the cellular level, we detected a delay in the repair of 6-4PPs in DDB2-/- dermal fibroblasts. Neither the absence nor the enhanced expression of DDB2 affected the levels of UV-induced apoptosis in epidermal keratinocytes or cultured dermal fibroblasts. Our results show an important role for DDB2 in the protection against UV-induced cancer and indicate that this protection is most likely mediated by accelerating the repair of photolesions.

Transcription-associated breaks in xeroderma pigmentosum group D cells from patients with combined features of xeroderma pigmentosum and Cockayne syndrome.

Defects in the XPD gene can result in several clinical phenotypes, including xeroderma pigmentosum (XP), trichothiodystrophy, and, less frequently, the combined phenotype of XP and Cockayne syndrome (XP-D/CS). We previously showed that in cells from two XP-D/CS patients, breaks were introduced into cellular DNA on exposure to UV damage, but these breaks were not at the sites of the damage. In the present work, we show that three further XP-D/CS patients show the same peculiar breakage phenomenon. We show that these breaks can be visualized inside the cells by immunofluorescence using antibodies to either gamma-H2AX or poly-ADP-ribose and that they can be generated by the introduction of plasmids harboring methylation or oxidative damage as well as by UV photoproducts. Inhibition of RNA polymerase II transcription by four different inhibitors dramatically reduced the number of UV-induced breaks. Furthermore, the breaks were dependent on the nucleotide excision repair (NER) machinery. These data are consistent with our hypothesis that the NER machinery introduces the breaks at sites of transcription initiation. During transcription in UV-irradiated XP-D/CS cells, phosphorylation of the carboxy-terminal domain of RNA polymerase II occurred normally, but the elongating form of the polymerase remained blocked at lesions and was eventually degraded.

Repair of DNA lesions in chromosomal DNA impact of chromatin structure and Cockayne syndrome proteins.

Decondensation of chromatin is essential to facilitate access to DNA metabolizing processes such as transcription and DNA repair. Disruption of histone-DNA contacts by histone modification or by ATP dependent chromatin remodelling allows DNA-binding proteins to compete with histones for DNA. The efficiency of global genome nucleotide excision repair (GGR) that removes a variety of helix distorting DNA lesions is known to be affected by chromatin structure most notably demonstrated by the slow repair of heterochromatin. In addition, the efficiency of GGR to repair lesions in transcriptionally active genes requires functional CSA and B proteins. We found that repair of UV-photolesions in both strands of the active adenosine deaminase gene was delayed in CS cells when compared to normal human fibroblasts. We suggest that the lack of transcription recovery characteristic for CS cells exposed to DNA damaging agents, might lead to changes in the chromatin structure of active genes, causing less efficient repair of lesions in these genes when compared to normal cells.

Composition and architecture of the Schizosaccharomyces pombe Rad18 (Smc5-6) complex.

The rad18 gene of Schizosaccharomyces pombe is an essential gene that is involved in several different DNA repair processes. Rad18 (Smc6) is a member of the structural maintenance of chromosomes (SMC) family and, together with its SMC partner Spr18 (Smc5), forms the core of a high-molecular-weight complex. We show here that both S. pombe and human Smc5 and -6 interact through their hinge domains and that four independent temperature-sensitive mutants of Rad18 (Smc6) are all mutated at the same glycine residue in the hinge region. This mutation abolishes the interactions between the hinge regions of Rad18 (Smc6) and Spr18 (Smc5), as does mutation of a conserved glycine in the hinge region of Spr18 (Smc5). We purified the Smc5-6 complex from S. pombe and identified four non-SMC components, Nse1, Nse2, Nse3, and Rad62. Nse3 is a novel protein which is related to the mammalian MAGE protein family, many members of which are specifically expressed in cancer tissue. In initial steps to understand the architecture of the complex, we identified two subcomplexes containing Rad18-Spr18-Nse2 and Nse1-Nse3-Rad62. The subcomplexes are probably bridged by a weaker interaction between Nse2 and Nse3.