Ataxia Telangiectasia Mutated Signaling Delays Skin Pigmentation upon UV Exposure by Mediating MITF Function toward DNA Repair Mode.

Skin pigmentation is paused after sun exposure; however, the mechanism behind this pausing is unknown. In this study, we found that the UVB-induced DNA repair system, led by the ataxia telangiectasia mutated (ATM) protein kinase, represses MITF transcriptional activity of pigmentation genes while placing MITF in DNA repair mode, thus directly inhibiting pigment production. Phosphoproteomics analysis revealed ATM to be the most significantly enriched pathway among all UVB-induced DNA repair systems. ATM inhibition in mouse or human skin, either genetically or chemically, induces pigmentation. Upon UVB exposure, MITF transcriptional activation is blocked owing to ATM-dependent phosphorylation of MITF on S414, which modifies MITF activity and interactome toward DNA repair, including binding to TRIM28 and RBBP4. Accordingly, MITF genome occupancy is enriched in sites of high DNA damage that are likely repaired. This suggests that ATM harnesses the pigmentation key activator for the necessary rapid, efficient DNA repair, thus optimizing the chances of the cell surviving. Data are available from ProteomeXchange with the identifier PXD041121.

Accelerated replicative senescence of ataxia-telangiectasia skin fibroblasts is retained at physiologic oxygen levels, with unique and common transcriptional patterns.

The genetic disorder, ataxia-telangiectasia (A-T), is caused by loss of the homeostatic protein kinase, ATM, and combines genome instability, tissue degeneration, cancer predisposition, and premature aging. Primary fibroblasts from A-T patients exhibit premature senescence when grown at ambient oxygen concentration (21%). Here, we show that reducing oxygen concentration to a physiological level range (3%) dramatically extends the proliferative lifespan of human A-T skin fibroblasts. However, they still undergo senescence earlier than control cells grown under the same conditions and exhibit high genome instability. Comparative RNA-seq analysis of A-T and control fibroblasts cultured at 3% oxygen followed by cluster analysis of differentially expressed genes and functional enrichment analysis, revealed distinct transcriptional dynamics in A-T fibroblasts senescing in physiological oxygen concentration. While some transcriptional patterns were similar to those observed during replicative senescence of control cells, others were unique to the senescing A-T cells. We observed in them a robust activation of interferon-stimulated genes, with undetected expression the interferon genes themselves. This finding suggests an activation of a non-canonical cGAS-STING-mediated pathway, which presumably responds to cytosolic DNA emanating from extranuclear micronuclei detected in these cells. Senescing A-T fibroblasts also exhibited a marked, intriguely complex alteration in the expression of genes associated with extracellular matrix (ECM) remodeling. Notably, many of the induced ECM genes encode senescence-associated secretory phenotype (SASP) factors known for their paracrine pro-fibrotic effects. Our data provide a molecular dimension to the segmental premature aging observed in A-T patients and its associated symptoms, which develop as the patients advance in age.

Genomic Amplification of UBQLN4 Is a Prognostic and Treatment Resistance Factor.

Ubiquilin-4 (UBQLN4) is a proteasomal shuttle factor that directly binds to ubiquitylated proteins and delivers its cargo to the 26S proteasome for degradation. We previously showed that upregulated UBQLN4 determines the DNA damage response (DDR) through the degradation of MRE11A. However, the regulatory mechanism at DNA level, transcriptionally and post-transcriptional levels that control UBQLN4 mRNA levels remains unknown. In this study, we screened 32 solid tumor types and validated our findings by immunohistochemistry analysis. UBQLN4 is upregulated at both mRNA and protein levels and the most significant values were observed in liver, breast, ovarian, lung, and esophageal cancers. Patients with high UBQLN4 mRNA levels had significantly poor prognoses in 20 of 32 cancer types. DNA amplification was identified as the main mechanism promoting UBQLN4 upregulation in multiple cancers, even in the early phases of tumor development. Using CRISPR screen datasets, UBQLN4 was identified as a common essential gene for tumor cell viability in 81.1% (860/1,060) of the solid tumor derived cell lines. Ovarian cancer cell lines with high UBQLN4 mRNA levels were platinum-based chemotherapy resistant, while they were more sensitive to poly (adenosine diphosphate-ribose) polymerase inhibitors (PARPi). Our findings highlight the utilities of UBQLN4 as a significant pan-cancer theranostic factor and a precision oncology biomarker for DDR-related drug resistance.

New hallmarks of ageing: a 2022 Copenhagen ageing meeting summary.

Genomic instability, telomere attrition, epigenetic alterations, mitochondrial dysfunction, loss of proteostasis, deregulated nutrient-sensing, cellular senescence, stem cell exhaustion, and altered intercellular communication were the original nine hallmarks of ageing proposed by López-Otín and colleagues in 2013. The proposal of these hallmarks of ageing has been instrumental in guiding and pushing forward research on the biology of ageing. In the nearly past 10 years, our in-depth exploration on ageing research has enabled us to formulate new hallmarks of ageing which are compromised autophagy, microbiome disturbance, altered mechanical properties, splicing dysregulation, and inflammation, among other emerging ones. Amalgamation of the 'old' and 'new' hallmarks of ageing may provide a more comprehensive explanation of ageing and age-related diseases, shedding light on interventional and therapeutic studies to achieve healthy, happy, and productive lives in the elderly.

The hallmarks of aging in Ataxia-Telangiectasia.

Ataxia-telangiectasia (A-T) is caused by absence of the catalytic activity of ATM, a protein kinase that plays a central role in the DNA damage response, many branches of cellular metabolism, redox and mitochondrial homeostasis, and cell cycle regulation. A-T is a complex disorder characterized mainly by progressive cerebellar degeneration, immunodeficiency, radiation sensitivity, genome instability, and predisposition to cancer. It is increasingly recognized that the premature aging component of A-T is an important driver of this disease, and A-T is therefore an attractive model to study the aging process. This review outlines the current state of knowledge pertaining to the molecular and cellular signatures of aging in A-T and proposes how these new insights can guide novel therapeutic approaches for A-T.

It takes three to the DNA damage response tango.

The DNA damage response is robustly activated by DNA double-strand breaks and controlled by three apical protein kinases of the PI3-kinase-related protein kinase (PIKK) family: ataxia-telangiectasia, mutated (ATM), ataxia-telangiectasia and Rad3-related (ATR) and DNA-dependent protein kinase (DNA-PK). Phosphoproteomic analysis reveals the relative share of these PIKKs in coordinating this network, and compensation by ATR and DNA-PK for ATM absence in the genetic disorder, ataxia-telangiectasia (A-T).

Regulation of MRE11A by UBQLN4 leads to cisplatin resistance in patients with esophageal squamous cell carcinoma.

Resistance to standard cisplatin-based chemotherapies leads to worse survival outcomes for patients with esophageal squamous cell carcinoma (ESCC). Therefore, there is an urgent need to understand the aberrant mechanisms driving resistance in ESCC tumors. We hypothesized that ubiquilin-4 (UBQLN4), a protein that targets ubiquitinated proteins to the proteasome, regulates the expression of Meiotic Recombination 11 Homolog A (MRE11A), a critical component of the MRN complex and DNA damage repair pathways. Initially, immunohistochemistry analysis was conducted in specimens from patients with ESCC (n = 120). In endoscopic core ESCC biopsies taken from 61 patients who underwent neoadjuvant chemotherapy (NAC) (5-fluorouracil and cisplatin), low MRE11A and high UBQLN4 protein levels were associated with reduced pathological response to NAC (P < 0.001 and P < 0.001, respectively). Multivariable analysis of surgically resected ESCC tissues from 59 patients revealed low MRE11A and high UBLQN4 expression as independent factors that can predict shorter overall survival [P = 0.01, hazard ratio (HR) = 5.11, 95% confidence interval (CI), 1.45-18.03; P = 0.02, HR = 3.74, 95% CI, 1.19-11.76, respectively]. Suppression of MRE11A expression was associated with cisplatin resistance in ESCC cell lines. Additionally, MRE11A was found to be ubiquitinated after cisplatin treatment. We observed an amplification of UBQLN4 gene copy numbers and an increase in UBQLN4 protein levels in ESCC tissues. Binding of UBQLN4 to ubiquitinated-MRE11A increased MRE11A degradation, thereby regulating MRE11A protein levels following DNA damage and promoting cisplatin resistance. In summary, MRE11A and UBQLN4 protein levels can serve as predictors for NAC response and as prognostic markers in ESCC patients.

Phosphoproteomics reveals novel modes of function and inter-relationships among PIKKs in response to genotoxic stress.

The DNA damage response (DDR) is a complex signaling network that relies on cascades of protein phosphorylation, which are initiated by three protein kinases of the family of PI3-kinase-related protein kinases (PIKKs): ATM, ATR, and DNA-PK. ATM is missing or inactivated in the genome instability syndrome, ataxia-telangiectasia (A-T). The relative shares of these PIKKs in the response to genotoxic stress and the functional relationships among them are central questions in the genome stability field. We conducted a comprehensive phosphoproteomic analysis in human wild-type and A-T cells treated with the double-strand break-inducing chemical, neocarzinostatin, and validated the results with the targeted proteomic technique, selected reaction monitoring. We also matched our results with 34 published screens for DDR factors, creating a valuable resource for identifying strong candidates for novel DDR players. We uncovered fine-tuned dynamics between the PIKKs following genotoxic stress, such as DNA-PK-dependent attenuation of ATM. In A-T cells, partial compensation for ATM absence was provided by ATR and DNA-PK, with distinct roles and kinetics. The results highlight intricate relationships between these PIKKs in the DDR.

The cerebellar degeneration in ataxia-telangiectasia: A case for genome instability.

Research on the molecular pathology of genome instability disorders has advanced our understanding of the complex mechanisms that safeguard genome stability and cellular homeostasis at large. Once the culprit genes and their protein products are identified, an ongoing dialogue develops between the research lab and the clinic in an effort to link specific disease symptoms to the functions of the proteins that are missing in the patients. Ataxi A-T elangiectasia (A-T) is a prominent example of this process. A-T's hallmarks are progressive cerebellar degeneration, immunodeficiency, chronic lung disease, cancer predisposition, endocrine abnormalities, segmental premature aging, chromosomal instability and radiation sensitivity. The disease is caused by absence of the powerful protein kinase, ATM, best known as the mobilizer of the broad signaling network induced by double-strand breaks (DSBs) in the DNA. In parallel, ATM also functions in the maintenance of the cellular redox balance, mitochondrial function and turnover and many other metabolic circuits. An ongoing discussion in the A-T field revolves around the question of which ATM function is the one whose absence is responsible for the most debilitating aspect of A-T - the cerebellar degeneration. This review suggests that it is the absence of a comprehensive role of ATM in responding to ongoing DNA damage induced mainly by endogenous agents. It is the ensuing deterioration and eventual loss of cerebellar Purkinje cells, which are very vulnerable to ATM absence due to a unique combination of physiological features, which kindles the cerebellar decay in A-T.

The Role of E3, E4 Ubiquitin Ligase (UBE4B) in Human Pathologies.

The genome is exposed daily to many deleterious factors. Ubiquitination is a mechanism that regulates several crucial cellular functions, allowing cells to react upon various stimuli in order to preserve their homeostasis. Ubiquitin ligases act as specific regulators and actively participate among others in the DNA damage response (DDR) network. UBE4B is a newly identified member of E3 ubiquitin ligases that appears to be overexpressed in several human neoplasms. The aim of this review is to provide insights into the role of UBE4B ubiquitin ligase in DDR and its association with p53 expression, shedding light particularly on the molecular mechanisms of carcinogenesis.

The EXPANDER Integrated Platform for Transcriptome Analysis.

Genome-wide analysis of cellular transcriptomes using RNA-seq or expression arrays is a major mainstay of current biological and biomedical research. EXPANDER (EXPression ANalyzer and DisplayER) is a comprehensive software package for analysis of expression data, with built-in support for 18 different organisms. It is designed as a "one-stop shop" platform for transcriptomic analysis, allowing for execution of all analysis steps starting with gene expression data matrix. Analyses offered include low-level preprocessing and normalization, differential expression analysis, clustering, bi-clustering, supervised grouping, high-level functional and pathway enrichment tests, and networks and motif analyses. A variety of options is offered for each step, using established algorithms, including many developed and published by our laboratory. EXPANDER has been continuously developed since 2003, having to date over 18,000 downloads and 540 citations. One of the innovations in the recent version is support for combined analysis of gene expression and ChIP-seq data to enhance the inference of transcriptional networks and their functional interpretation. EXPANDER implements cutting-edge algorithms and makes them accessible to users through user-friendly interface and intuitive visualizations. It is freely available to users at http://acgt.cs.tau.ac.il/expander/.

UBQLN4 Represses Homologous Recombination and Is Overexpressed in Aggressive Tumors.

Genomic instability can be a hallmark of both human genetic disease and cancer. We identify a deleterious UBQLN4 mutation in families with an autosomal recessive syndrome reminiscent of genome instability disorders. UBQLN4 deficiency leads to increased sensitivity to genotoxic stress and delayed DNA double-strand break (DSB) repair. The proteasomal shuttle factor UBQLN4 is phosphorylated by ATM and interacts with ubiquitylated MRE11 to mediate early steps of homologous recombination-mediated DSB repair (HRR). Loss of UBQLN4 leads to chromatin retention of MRE11, promoting non-physiological HRR activity in vitro and in vivo. Conversely, UBQLN4 overexpression represses HRR and favors non-homologous end joining. Moreover, we find UBQLN4 overexpressed in aggressive tumors. In line with an HRR defect in these tumors, UBQLN4 overexpression is associated with PARP1 inhibitor sensitivity. UBQLN4 therefore curtails HRR activity through removal of MRE11 from damaged chromatin and thus offers a therapeutic window for PARP1 inhibitor treatment in UBQLN4-overexpressing tumors.

Inactive Atm abrogates DSB repair in mouse cerebellum more than does Atm loss, without causing a neurological phenotype.

The genome instability syndrome, ataxia-telangiectasia (A-T) is caused by null mutations in the ATM gene, that lead to complete loss or inactivation of the gene's product, the ATM protein kinase. ATM is the primary mobilizer of the cellular response to DNA double-strand breaks (DSBs) - a broad signaling network in which many components are ATM targets. The major clinical feature of A-T is cerebellar atrophy, characterized by relentless loss of Purkinje and granule cells. In Atm-knockout (Atm-KO) mice, complete loss of Atm leads to a very mild neurological phenotype, suggesting that Atm loss is not sufficient to markedly abrogate cerebellar structure and function in this organism. Expression of inactive ("kinase-dead") Atm (AtmKD) in mice leads to embryonic lethality, raising the question of whether conditional expression of AtmKD in the murine nervous system would lead to a more pronounced neurological phenotype than Atm loss. We generated two mouse strains in which AtmKD was conditionally expressed as the sole Atm species: one in the CNS and one specifically in Purkinje cells. Focusing our analysis on Purkinje cells, the dynamics of DSB readouts indicated that DSB repair was delayed longer in the presence of AtmKD compared to Atm loss. However, both strains exhibited normal life span and displayed no gross cerebellar histological abnormalities or significant neurological phenotype. We conclude that the presence of AtmKD is indeed more harmful to DSB repair than Atm loss, but the murine central nervous system can reasonably tolerate the extent of this DSB repair impairment. Greater pressure needs to be exerted on genome stability to obtain a mouse model that recapitulates the severe A-T neurological phenotype.

The Ubiquitin E3/E4 Ligase UBE4A Adjusts Protein Ubiquitylation and Accumulation at Sites of DNA Damage, Facilitating Double-Strand Break Repair.

Double-strand breaks (DSBs) are critical DNA lesions that robustly activate the elaborate DNA damage response (DDR) network. We identified a critical player in DDR fine-tuning: the E3/E4 ubiquitin ligase UBE4A. UBE4A's recruitment to sites of DNA damage is dependent on primary E3 ligases in the DDR and promotes enhancement and sustainment of K48- and K63-linked ubiquitin chains at these sites. This step is required for timely recruitment of the RAP80 and BRCA1 proteins and proper organization of RAP80- and BRCA1-associated protein complexes at DSB sites. This pathway is essential for optimal end resection at DSBs, and its abrogation leads to upregulation of the highly mutagenic alternative end-joining repair at the expense of error-free homologous recombination repair. Our data uncover a critical regulatory level in the DSB response and underscore the importance of fine-tuning the complex DDR network for accurate and balanced execution of DSB repair.

Nuclear poly(A)-binding protein 1 is an ATM target and essential for DNA double-strand break repair.

The DNA damage response (DDR) is an extensive signaling network that is robustly mobilized by DNA double-strand breaks (DSBs). The primary transducer of the DSB response is the protein kinase, ataxia-telangiectasia, mutated (ATM). Here, we establish nuclear poly(A)-binding protein 1 (PABPN1) as a novel target of ATM and a crucial player in the DSB response. PABPN1 usually functions in regulation of RNA processing and stability. We establish that PABPN1 is recruited to the DDR as a critical regulator of DSB repair. A portion of PABPN1 relocalizes to DSB sites and is phosphorylated on Ser95 in an ATM-dependent manner. PABPN1 depletion sensitizes cells to DSB-inducing agents and prolongs the DSB-induced G2/M cell-cycle arrest, and DSB repair is hampered by PABPN1 depletion or elimination of its phosphorylation site. PABPN1 is required for optimal DSB repair via both nonhomologous end-joining (NHEJ) and homologous recombination repair (HRR), and specifically is essential for efficient DNA-end resection, an initial, key step in HRR. Using mass spectrometry analysis, we capture DNA damage-induced interactions of phospho-PABPN1, including well-established DDR players as well as other RNA metabolizing proteins. Our results uncover a novel ATM-dependent axis in the rapidly growing interface between RNA metabolism and the DDR.

Monitoring the ATM-Mediated DNA Damage Response in the Cerebellum Using Organotypic Cultures.

The ATM gene and its protein product, the ATM protein kinase, were identified as a result of attempts to understand the molecular basis of the genetic disorder, ataxia-telangiectasia (A-T). The cardinal symptom of A-T is neurodegeneration expressed primarily as progressive cerebellar atrophy. A major tool in the investigation of ATM functions in the cerebellum is cerebellar organotypic cultures, which allow cerebellar slices to live in culture for several weeks without losing their viability and organization. These cultures are amenable to various treatments and manipulations and provide a close look at Purkinje cells in their almost natural environment. We optimized the protocol for establishing and maintaining these cultures and provide here examples of readouts of the DNA damage response in cerebellar organotypic cultures treated with a DNA-damaging agent.

Genome instability: Linking ageing and brain degeneration.

Ageing is a multifactorial process affected by cumulative physiological changes resulting from stochastic processes combined with genetic factors, which together alter metabolic homeostasis. Genetic variation in maintenance of genome stability is emerging as an important determinant of ageing pace. Genome instability is also closely associated with a broad spectrum of conditions involving brain degeneration. Similarities and differences can be found between ageing-associated decline of brain functionality and the detrimental effect of genome instability on brain functionality and development. This review discusses these similarities and differences and highlights cell classes whose role in these processes might have been underestimated-glia and microglia.