Heterozygous RPA2 variant as a novel genetic cause of telomere biology disorders.

Premature telomere shortening or telomere instability is associated with a group of rare and heterogeneous diseases collectively known as telomere biology disorders (TBDs). Here we identified two unrelated individuals with clinical manifestations of TBDs and short telomeres associated with the identical monoallelic variant c.767A>G; Y256C in RPA2 Although the replication protein A2 (RPA2) mutant did not affect ssDNA binding and G-quadruplex-unfolding properties of RPA, the mutation reduced the affinity of RPA2 with the ubiquitin ligase RFWD3 and reduced RPA ubiquitination. Using engineered knock-in cell lines, we found an accumulation of RPA at telomeres that did not trigger ATR activation but caused short and dysfunctional telomeres. Finally, both patients acquired, in a subset of blood cells, somatic genetic rescue events in either POT1 genes or TERT promoters known to counteract the accelerated telomere shortening. Collectively, our study indicates that variants in RPA2 represent a novel genetic cause of TBDs. Our results further support the fundamental role of the RPA complex in regulating telomere length and stability in humans.

Inflammasome Regulates Hematopoiesis through Cleavage of the Master Erythroid Transcription Factor GATA1.

Chronic inflammatory diseases are associated with altered hematopoiesis that could result in neutrophilia and anemia. Here we report that genetic or chemical manipulation of different inflammasome components altered the differentiation of hematopoietic stem and progenitor cells (HSPC) in zebrafish. Although the inflammasome was dispensable for the emergence of HSPC, it was intrinsically required for their myeloid differentiation. In addition, Gata1 transcript and protein amounts increased in inflammasome-deficient larvae, enforcing erythropoiesis and inhibiting myelopoiesis. This mechanism is evolutionarily conserved, since pharmacological inhibition of the inflammasome altered erythroid differentiation of human erythroleukemic K562 cells. In addition, caspase-1 inhibition rapidly upregulated GATA1 protein in mouse HSPC promoting their erythroid differentiation. Importantly, pharmacological inhibition of the inflammasome rescued zebrafish disease models of neutrophilic inflammation and anemia. These results indicate that the inflammasome plays a major role in the pathogenesis of neutrophilia and anemia of chronic diseases and reveal druggable targets for therapeutic interventions.

mTert induction in p21-positive cells counteracts capillary rarefaction and pulmonary emphysema.

Lung diseases develop when telomeres shorten beyond a critical point. We constructed a mouse model in which the catalytic subunit of telomerase (mTert), or its catalytically inactive form (mTertCI), is expressed from the p21Cdkn1a locus. Expression of either TERT or TERTCI reduces global p21 levels in the lungs of aged mice, highlighting TERT non-canonical function. However, only TERT reduces accumulation of very short telomeres, oxidative damage, endothelial cell (ECs) senescence and senile emphysema in aged mice. Single-cell analysis of the lung reveals that p21 (and hence TERT) is expressed mainly in the capillary ECs. We report that a fraction of capillary ECs marked by CD34 and endowed with proliferative capacity declines drastically with age, and this is counteracted by TERT but not TERTCI. Consistently, only TERT counteracts decline of capillary density. Natural aging effects are confirmed using the experimental model of emphysema induced by VEGFR2 inhibition and chronic hypoxia. We conclude that catalytically active TERT prevents exhaustion of the putative CD34 + EC progenitors with age, thus protecting against capillary vessel loss and pulmonary emphysema.

STIM1 translocation to the nucleus protects cells from DNA damage.

DNA damage represents a challenge for cells, as this damage must be eliminated to preserve cell viability and the transmission of genetic information. To reduce or eliminate unscheduled chemical modifications in genomic DNA, an extensive signaling network, known as the DNA damage response (DDR) pathway, ensures this repair. In this work, and by means of a proteomic analysis aimed at studying the STIM1 protein interactome, we have found that STIM1 is closely related to the protection from endogenous DNA damage, replicative stress, as well as to the response to interstrand crosslinks (ICLs). Here we show that STIM1 has a nuclear localization signal that mediates its translocation to the nucleus, and that this translocation and the association of STIM1 to chromatin increases in response to mitomycin-C (MMC), an ICL-inducing agent. Consequently, STIM1-deficient cell lines show higher levels of basal DNA damage, replicative stress, and increased sensitivity to MMC. We show that STIM1 normalizes FANCD2 protein levels in the nucleus, which explains the increased sensitivity of STIM1-KO cells to MMC. This study not only unveils a previously unknown nuclear function for the endoplasmic reticulum protein STIM1 but also expands our understanding of the genes involved in DNA repair.

RPA-Mediated Recruitment of the E3 Ligase RFWD3 Is Vital for Interstrand Crosslink Repair and Human Health.

Defects in the repair of DNA interstrand crosslinks (ICLs) are associated with the genome instability syndrome Fanconi anemia (FA). Here we report that cells with mutations in RFWD3, an E3 ubiquitin ligase that interacts with and ubiquitylates replication protein A (RPA), show profound defects in ICL repair. An amino acid substitution in the WD40 repeats of RFWD3 (I639K) found in a new FA subtype abolishes interaction of RFWD3 with RPA, thereby preventing RFWD3 recruitment to sites of ICL-induced replication fork stalling. Moreover, single point mutations in the RPA32 subunit of RPA that abolish interaction with RFWD3 also inhibit ICL repair, demonstrating that RPA-mediated RFWD3 recruitment to stalled replication forks is important for ICL repair. We also report that unloading of RPA from sites of ICL induction is perturbed in RFWD3-deficient cells. These data reveal important roles for RFWD3 localization in protecting genome stability and preserving human health.

A clickable melphalan for monitoring DNA interstrand crosslink accumulation and detecting ICL repair defects in Fanconi anemia patient cells.

Fanconi anemia (FA) is a genetic disorder associated with developmental defects, bone marrow failure and cancer. The FA pathway is crucial for the repair of DNA interstrand crosslinks (ICLs). In this study, we have developed and characterized a new tool to investigate ICL repair: a clickable version of the crosslinking agent melphalan which we name click-melphalan. Our results demonstrate that click-melphalan is as effective as its unmodified counterpart in generating ICLs and associated toxicity. The lesions induced by click-melphalan can be detected in cells by post-labelling with a fluorescent reporter and quantified using flow cytometry. Since click-melphalan induces both ICLs and monoadducts, we generated click-mono-melphalan, which only induces monoadducts, in order to distinguish between the two types of DNA repair. By using both molecules, we show that FANCD2 knock-out cells are deficient in removing click-melphalan-induced lesions. We also found that these cells display a delay in repairing click-mono-melphalan-induced monoadducts. Our data further revealed that the presence of unrepaired ICLs inhibits monoadduct repair. Finally, our study demonstrates that these clickable molecules can differentiate intrinsic DNA repair deficiencies in primary FA patient cells from those in primary xeroderma pigmentosum patient cells. As such, these molecules may have potential for developing diagnostic tests.

Karyomegalic interstitial nephritis and DNA damage-induced polyploidy in Fan1 nuclease-defective knock-in mice.

The Fan1 endonuclease is required for repair of DNA interstrand cross-links (ICLs). Mutations in human Fan1 cause karyomegalic interstitial nephritis (KIN), but it is unclear whether defective ICL repair is responsible or whether Fan1 nuclease activity is relevant. We show that Fan1 nuclease-defective (Fan1(nd/nd)) mice develop a mild form of KIN. The karyomegalic nuclei from Fan1(nd/nd) kidneys are polyploid, and fibroblasts from Fan1(nd/nd) mice become polyploid upon ICL induction, suggesting that defective ICL repair causes karyomegaly. Thus, Fan1 nuclease activity promotes ICL repair in a manner that controls ploidy, a role that we show is not shared by the Fanconi anemia pathway or the Slx4-Slx1 nuclease also involved in ICL repair.

Zebrafish Models to Study Inflammasome-Mediated Regulation of Hematopoiesis.

Hematopoiesis is a complex process through which immature bone marrow precursor cells mature into all types of blood cells. Although the association of hematopoietic lineage bias (including anemia and neutrophilia) with chronic inflammatory diseases has long been appreciated, the causes involved are obscure. Recently, cytosolic multiprotein inflammasome complexes were shown to activate inflammatory and immune responses, and directly regulate hematopoiesis in zebrafish models; this was deemed to occur via cleavage and inactivation of the master erythroid transcription factor GATA1. Herein summarized are the zebrafish models that are currently available to study this unappreciated role of inflammasome-mediated regulation of hematopoiesis. Novel putative therapeutic strategies, for the treatment of hematopoietic alterations associated with chronic inflammatory diseases in humans, are also proposed.

Cooperative control of holliday junction resolution and DNA repair by the SLX1 and MUS81-EME1 nucleases.

Holliday junctions (HJs) are X-shaped DNA structures that arise during homologous recombination, which must be removed to enable chromosome segregation. The SLX1 and MUS81-EME1 nucleases can both process HJs in vitro, and they bind in close proximity on the SLX4 scaffold, hinting at possible cooperation. However, the cellular roles of mammalian SLX1 are not yet known. Here, we use mouse genetics and structure function analysis to investigate SLX1 function. Disrupting the murine Slx1 and Slx4 genes revealed that they are essential for HJ resolution in mitotic cells. Moreover, SLX1 and MUS81-EME1 act together to resolve HJs in a manner that requires tethering to SLX4. We also show that SLX1, like MUS81-EME1, is required for repair of DNA interstrand crosslinks, but this role appears to be independent of HJ cleavage, at least in mouse cells. These findings shed light on HJ resolution in mammals and on maintenance of genome stability.

USP45 deubiquitylase controls ERCC1-XPF endonuclease-mediated DNA damage responses.

Reversible protein ubiquitylation plays important roles in various processes including DNA repair. Here, we identify the deubiquitylase USP45 as a critical DNA repair regulator. USP45 associates with ERCC1, a subunit of the DNA repair endonuclease XPF-ERCC1, via a short acidic motif outside of the USP45 catalytic domain. Wild-type USP45, but not a USP45 mutant defective in ERCC1 binding, efficiently deubiquitylates ERCC1 in vitro, and the levels of ubiquitylated ERCC1 are markedly enhanced in USP45 knockout cells. Cells lacking USP45 are hypersensitive specifically to UV irradiation and DNA interstrand cross-links, similar to cells lacking ERCC1. Furthermore, the repair of UV-induced DNA damage is markedly reduced in USP45-deficient cells. ERCC1 translocation to DNA damage-induced subnuclear foci is markedly impaired in USP45 knockout cells, possibly accounting for defective DNA repair. Finally, USP45 localises to sites of DNA damage in a manner dependent on its deubiquitylase activity, but independent of its ability to bind ERCC1-XPF. Together, these results establish USP45 as a new regulator of XPF-ERCC1 crucial for efficient DNA repair.

Distinct functional roles for the two SLX4 ubiquitin-binding UBZ domains mutated in Fanconi anemia.

Defects in SLX4, a scaffold for DNA repair nucleases, result in Fanconi anemia (FA), due to the defective repair of inter-strand DNA crosslinks (ICLs). Some FA patients have an SLX4 deletion removing two tandem UBZ4-type ubiquitin-binding domains that are implicated in protein recruitment to sites of DNA damage. Here, we show that human SLX4 is recruited to sites of ICL induction but that the UBZ-deleted form of SLX4 in cells from FA patients is not. SLX4 recruitment does not require either the ubiquitylation of FANCD2 or the E3 ligases RNF8, RAD18 and BRCA1. We show that the first (UBZ-1) but not the second UBZ domain of SLX4 binds to ubiquitin polymers, with a preference for K63-linked chains. Furthermore, UBZ-1 is required for SLX4 recruitment to ICL sites and for efficient ICL repair in murine fibroblasts. The SLX4 UBZ-2 domain does not bind to ubiquitin in vitro or contribute to ICL repair, but it is required for the resolution of Holliday junctions in vivo. These data shed light on SLX4 recruitment, and they point to the existence of currently unidentified ubiquitylated ligands and E3 ligases that are crucial for ICL repair.

Beyond current treatment of Fanconi Anemia: What do advances in cell and gene-based approaches offer?

Fanconi anemia (FA) is a rare inherited disorder that mainly affects the bone marrow. This condition causes decreased production of all types of blood cells. FA is caused by a defective repair of DNA interstrand crosslinks and to date, mutations in over 20 genes have been linked to the disease. Advances in science and molecular biology have provided new insight between FA gene mutations and the severity of clinical manifestations. Here, we will highlight the current and promising therapeutic options for this rare disease. The current standard treatment for FA patients is hematopoietic stem cell transplantation, a treatment associated to exposure to radiation or chemotherapy, immunological complications, plus opportunistic infections from prolonged immune incompetence or increased risk of morbidity. New arising treatments include gene addition therapy, genome editing using CRISPR-Cas9 nuclease, and hematopoietic stem cell generation from induced pluripotent stem cells. Finally, we will also discuss the revolutionary developments in mRNA therapeutics as an opportunity for this disease.

ZAKα/P38 kinase signaling pathway regulates hematopoiesis by activating the NLRP1 inflammasome.

Chronic inflammatory diseases are associated with hematopoietic lineage bias, including neutrophilia and anemia. We have recently identified that the canonical inflammasome mediates the cleavage of the master erythroid transcription factor GATA1 in hematopoietic stem and progenitor cells (HSPCs). We report here that genetic inhibition of Nlrp1 resulted in reduced number of neutrophils and increased erythrocyte counts in zebrafish larvae. We also found that the NLRP1 inflammasome in human cells was inhibited by LRRFIP1 and FLII, independently of DPP9, and both inhibitors regulated hematopoiesis. Mechanistically, erythroid differentiation resulted in ribosomal stress-induced activation of the ZAKα/P38 kinase axis which, in turn, phosphorylated and promoted the assembly of NLRP1 in both zebrafish and human. Finally, inhibition of Zaka with the FDA/EMA-approved drug Nilotinib alleviated neutrophilia in a zebrafish model of neutrophilic inflammation and promoted erythroid differentiation and GATA1 accumulation in K562 cells. In conclusion, our results reveal that the NLRP1 inflammasome regulates hematopoiesis and pave the way to develop novel therapeutic strategies for the treatment of hematopoietic alterations associated with chronic inflammatory and rare diseases.

UFMylation of MRE11 is essential for telomere length maintenance and hematopoietic stem cell survival.

Ubiquitin-fold modifier 1 (UFM1) is involved in neural and erythroid development, yet its biological roles in these processes are unknown. Here, we generated zebrafish models deficient in Ufm1 and Ufl1 that exhibited telomere shortening associated with developmental delay, impaired hematopoiesis and premature aging. We further report that HeLa cells lacking UFL1 have instability of telomeres replicated by leading-strand synthesis. We uncover that MRE11 UFMylation is necessary for the recruitment of the phosphatase PP1-α leading to dephosphorylation of NBS1. In the absence of UFMylation, NBS1 remains phosphorylated, thereby reducing MRN recruitment to telomeres. The absence of MRN at telomeres favors the formation of the TRF2-Apollo/SNM1 complex consistent with the loss of leading telomeres. These results suggest that MRE11-UFMylation may serve as module to recruit PP1-α. Last, zebrafish expressing Mre11 that cannot be UFMylated phenocopy Ufm1-deficient zebrafish, demonstrating that UFMylation of MRE11 is a previously undescribed evolutionarily conserved mechanisms regulating telomere length.

Ubiquitinated Fancd2 recruits Fan1 to stalled replication forks to prevent genome instability.

Mono-ubiquitination of Fancd2 is essential for repairing DNA interstrand cross-links (ICLs), but the underlying mechanisms are unclear. The Fan1 nuclease, also required for ICL repair, is recruited to ICLs by ubiquitinated (Ub) Fancd2. This could in principle explain how Ub-Fancd2 promotes ICL repair, but we show that recruitment of Fan1 by Ub-Fancd2 is dispensable for ICL repair. Instead, Fan1 recruitment--and activity--restrains DNA replication fork progression and prevents chromosome abnormalities from occurring when DNA replication forks stall, even in the absence of ICLs. Accordingly, Fan1 nuclease-defective knockin mice are cancer-prone. Moreover, we show that a Fan1 variant in high-risk pancreatic cancers abolishes recruitment by Ub-Fancd2 and causes genetic instability without affecting ICL repair. Therefore, Fan1 recruitment enables processing of stalled forks that is essential for genome stability and health.

Rapid generation of endogenously driven transcriptional reporters in cells through CRISPR/Cas9.

CRISPR/Cas9 technologies have been employed for genome editing to achieve gene knockouts and knock-ins in somatic cells. Similarly, certain endogenous genes have been tagged with fluorescent proteins. Often, the detection of tagged proteins requires high expression and sophisticated tools such as confocal microscopy and flow cytometry. Therefore, a simple, sensitive and robust transcriptional reporter system driven by endogenous promoter for studies into transcriptional regulation is desirable. We report a CRISPR/Cas9-based methodology for rapidly integrating a firefly luciferase gene in somatic cells under the control of endogenous promoter, using the TGFß-responsive gene PAI-1. Our strategy employed a polycistronic cassette containing a non-fused GFP protein to ensure the detection of transgene delivery and rapid isolation of positive clones. We demonstrate that firefly luciferase cDNA can be efficiently delivered downstream of the promoter of the TGFß-responsive gene PAI-1. Using chemical and genetic regulators of TGFß signalling, we show that it mimics the transcriptional regulation of endogenous PAI-1 expression. Our unique approach has the potential to expedite studies on transcription of any gene in the context of its native chromatin landscape in somatic cells, allowing for robust high-throughput chemical and genetic screens.

Anterior cruciate ligament reconstruction using four-strand semitendinosus and gracilis tendon grafts and metal interference screw fixation.

PURPOSE: The purpose of this study was to determine the outcome of 200 anterior cruciate ligament (ACL) reconstructions using hamstring tendons. TYPE OF STUDY: This is a case series reporting on 200 endoscopic procedures for reconstruction of the ACL. METHODS: This study included patients over 18 years old with a healthy controlateral knee, intact posterior cruciate ligament, and without any peripheral surgical procedure or cartilage injury. Patients having undergone prior ligament reconstruction were excluded from the study. The minimum follow-up was 1 year. Clinical review allowed for documentation of International Knee Documentation Committee (IKDC), KT-1000 arthrometer laxity measurement, and isokinetic dynamometric analysis. RESULTS: Quadriceps and hamstring muscle strength loss was less than 17%. At review, overall IKDC evaluation found that 50% of patients graded A, 44% graded B, and 6% were C or D. The differential anterior laxity was graded A for 157 patients with a median of 1 mm. Of the 113 high-performance athletes, 98 (86%) had resumed a preinjury level of sporting activity. CONCLUSIONS: Endoscopic reconstruction using 4-strand hamstring autograft may be considered safe, reliable, and reproducible. Preliminary outcome is fulfilling and this technique corresponds completely to therapeutic fields regarding ACL reconstructions.

Improved genome editing in human cell lines using the CRISPR method.

The Cas9/CRISPR system has become a popular choice for genome editing. In this system, binding of a single guide (sg) RNA to a cognate genomic sequence enables the Cas9 nuclease to induce a double-strand break at that locus. This break is next repaired by an error-prone mechanism, leading to mutation and gene disruption. In this study we describe a range of refinements of the method, including stable cell lines expressing Cas9, and a PCR based protocol for the generation of the sgRNA. We also describe a simple methodology that allows both elimination of Cas9 from cells after gene disruption and re-introduction of the disrupted gene. This advance enables easy assessment of the off target effects associated with gene disruption, as well as phenotype-based structure-function analysis. In our study, we used the Fan1 DNA repair gene as control in these experiments. Cas9/CRISPR-mediated Fan1 disruption occurred at frequencies of around 29%, and resulted in the anticipated spectrum of genotoxin hypersensitivity, which was rescued by re-introduction of Fan1.

Lipo-chitooligosaccharidic symbiotic signals are recognized by LysM receptor-like kinase LYR3 in the legume Medicago truncatula.

While chitooligosaccharides (COs) derived from fungal chitin are potent elicitors of defense reactions, structurally related signals produced by certain bacteria and fungi, called lipo-chitooligosaccharides (LCOs), play important roles in the establishment of symbioses with plants. Understanding how plants distinguish between friend and foe through the perception of these signals is a major challenge. We report the synthesis of a range of COs and LCOs, including photoactivatable probes, to characterize a membrane protein from the legume Medicago truncatula. By coupling photoaffinity labeling experiments with proteomics and transcriptomics, we identified the likely LCO-binding protein as LYR3, a lysin motif receptor-like kinase (LysM-RLK). LYR3, expressed heterologously, exhibits high-affinity binding to LCOs but not COs. Homology modeling, based on the Arabidopsis CO-binding LysM-RLK AtCERK1, suggests that LYR3 could accommodate the LCO in a conserved binding site. The identification of LYR3 opens up ways for the molecular characterization of LCO/CO discrimination.

DVC1 (C1orf124) recruits the p97 protein segregase to sites of DNA damage.

Ubiquitin-binding domains (UBDs) are crucial for recruiting many proteins to sites of DNA damage. Here we characterize C1orf124 (Spartan; referred to as DVC1), which has an UBZ4-type UBD found predominantly in DNA repair proteins. DVC1 associates with DNA replication factories and localizes to sites of DNA damage in human cells, in a manner that requires the ability of the DVC1 UBZ domain to bind to ubiquitin polymers in vitro and a conserved PCNA-interacting motif. DVC1 interacts with the p97 protein 'segregase'. We show that DVC1 recruits p97 to sites of DNA damage, where we propose that p97 facilitates the extraction of the translesion synthesis (TLS) polymerase (Pol) η during DNA repair to prevent excessive TLS and limit the incidence of mutations induced by DNA damage. We introduce DVC1 as a regulator of cellular responses to DNA damage that prevents mutations when DNA damage occurs.