Characterization of AKTIP, a new protein involved in human DNA replication and telomere metabolism

Telomeres are nucleoprotein structures that protect the ends of linear chromosomes. They are composed of long tracts of TTAGGG repeats, telomere specific proteins that form the shelterin complex and several telomere accessory proteins that co-operate to telomere metabolism. Proper telomere maintenance is a crucial process to protect the genome against instability and telomere dysfunction has been linked to tumorigenesis and premature aging. AKTIP gene is the human homologue of Drosophila peo, a gene that was recently linked to telomere metabolism. The aim of this study was to understand if AKTIP could have a role in human telomere metabolism, in analogy with the telomeric function of its homologous in fly. For this purpose we have analyzed the phenotype of human cells in which AKTIP expression was downregulated by RNA interference. In human primary cells AKTIP downregulation triggered the reduction of the mitotic index, proliferation impairment and premature senescence. AKTIP reduction induced a strong DNA damage response proved by the accumulation of the phosphorylated form of proteins involved in DNA damage sensing and signaling such as ATM, p53 and Chk1, by the accumulation of p21 mRNA and by the formation of foci containing DNA damage response proteins. About half of these foci were located at telomeres (TIFs) indicating the presence of dysfunctional telomeres in AKTIP knocked down cells. These data were consistent with the accumulation of aberrant telomeres in MEFs p53-/- observed following the downregulation of murine homologue of AKTIP (named Ft1). AKTIP involvement in telomere metabolism was further suggested by its interaction with telomeric repeats observed by ChIP analysis. Altogether, these findings indicate that AKTIP takes part in telomere maintenance. Interestingly, immunostaining assays showed that AKTIP is not a stable component of telomeres but was found located in the nucleus, mainly at nuclear rim. This particular localization, in addition with the telomeric role outlined for AKTIP, suggest that AKTIP is a telomeric nonshelterin protein. Consistent with this hypothesis, we observed that Ft1 downregulation caused the formation of chromosomal aberrations in addition to telomeric abnormalities, indicating that AKTIP/Ft1 plays a role not only in telomere maintenance but also in the overall genomic stability, possibly contributing to DNA replication. Indeed, the most prominent telomeric aberration observed in Ft1 downregulated MEFs was the formation of multiple telomeric signals at the ends of chromosomes, also known as fragile telomeres, indicative of replication impairment. In addition, AKTIP downregulation was found to induce an S-phase block of cell cycle progression and a strong reduction of PCNA positive cells in primary fibroblasts, along with an increased sensitivity to drugs that impair DNA replication, as aphidicolin. Collectively, these data demonstrate that AKTIP is a protein needed for proper DNA maintenance in mammalian cells. In the telomeric context AKTIP likely is a telomeric accessory protein, rather than a shelterin-like protein, because it’s conserved in fly, differently from shelterin proteins, has a role in telomere maintenance but is not stably located at telomeres. AKTIP, in addition to its telomeric function, seems to have a more general role in cellular metabolism, as all the other telomeric nonshelterin proteins. In particular our data indicate that AKTIP could be involved in DNA replication. Considering all the collected data together, our current hypothesis is that AKTIP plays a role in replication of complex DNA structures, including telomeric repeats. Its downregulation could impair the replication fork progression through these DNA regions leading to chromosomal aberrations, DNA damage response and cell cycle alterations, the most prominent phenotypic traits of AKTIP knocked down cells

Mammalian telomeres and their partnership with lamins

Chromosome ends are complex structures, which require a panel of factors for their elongation, replication, and protection. We describe here the mechanics of mammalian telomeres, dynamics and maintainance in relation to lamins. Multiple biochemical connections, including association of telomeres to the nuclear envelope and matrix, of telomeric proteins to lamins, and of lamin-associated proteins to chromosome ends, underline the interplay between lamins and telomeres. Paths toward senescence, such as defective telomere replication, altered heterochromatin organization, and impaired DNA repair, are common to lamins' and telomeres' dysfunction. The convergence of phenotypes can be interpreted through a model of dynamic, lamin-controlled functional platforms dedicated to the function of telomeres as fragile sites. The features of telomeropathies and laminopathies, and of animal models underline further overlapping aspects, including the alteration of stem cell compartments. We expect that future studies of basic biology and on aging will benefit from the analysis of this telomere-lamina interplay

Genomic instability and DNA replication defects in progeroid syndromes

Progeroid syndromes induced by mutations in lamin A or in its interactors – named progeroid laminopathies – are model systems for the dissection of the molecular pathways causing physio- logical and premature aging. A large amount of data, based mainly on the Hutchinson Gilford Progeria syndrome (HGPS), one of the best characterized progeroid laminopathy, has highlighted the role of lamins in multiple DNA activities, including replication, repair, chromatin organization and telomere function. On the other hand, the phenotypes generated by mutations affecting genes directly acting on DNA function, as mutations in the helicases WRN and BLM or in the polymerase polδ, share many of the traits of progeroid laminopathies. These evidences support the hypothesis of a concerted implication of DNA function and lamins in aging. We focus here on these aspects to contribute to the comprehension of the driving forces acting in progeroid syndromes and premature aging

The analysis of pendolino (peo) mutants reveals differences in the fusigenic potential among Drosophila telomeres

Drosophila telomeres are sequence-independent structures that are maintained by transposition to chromosome ends of three specialized retroelements (HeT-A, TART and TAHRE; collectively designated as HTT) rather than telomerase activity. Fly telomeres are protected by the terminin complex (HOAP-HipHop-Moi-Ver) that localizes and functions exclusively at telomeres and by non-terminin proteins that do not serve telomere-specific functions. Although all Drosophila telomeres terminate with HTT arrays and are capped by terminin, they differ in the type of subtelomeric chromatin; the Y, XR, and 4L HTT are juxtaposed to constitutive heterochromatin, while the XL, 2L, 2R, 3L and 3R HTT are linked to the TAS repetitive sequences; the 4R HTT is associated with a chromatin that has features common to both euchromatin and heterochromatin. Here we show that mutations in pendolino (peo) cause telomeric fusions (TFs). The analysis of several peo mutant combinations showed that these TFs preferentially involve the Y, XR and 4th chromosome telomeres, a TF pattern never observed in the other 10 telomere-capping mutants so far characterized. peo encodes a non-terminin protein homologous to the E2 variant ubiquitin-conjugating enzymes. The Peo protein directly interacts with the terminin components, but peo mutations do not affect telomeric localization of HOAP, Moi, Ver and HP1a, suggesting that the peodependent telomere fusion phenotype is not due to loss of terminin from chromosome ends. peo mutants are also defective in DNA replication and PCNA recruitment. However, our results suggest that general defects in DNA replication are unable to induce TFs in Drosophila cells. We thus hypothesize that DNA replication in Peodepleted cells results in specific fusigenic lesions concentrated in heterochromatinassociated telomeres. Alternatively it is possible that Peo plays a dual function being independently required for DNA replication and telomere capping

The Relationship Between Vitamin B6, Diabetes and Cancer

Pyridoxal 5\u2032-phosphate (PLP), the active form of vitamin B6, works as cofactor in numerous enzymatic reactions and it behaves as antioxidant molecule. PLP deficiency has been associated to many human pathologies including cancer and diabetes and the mechanism behind this connection is now becoming clearer. Inadequate intake of this vitamin increases the risk of many cancers; furthermore, PLP deprivation impairs insulin secretion in rats, whereas PLP supplementation prevents diabetic complications and improves gestational diabetes. Growing evidence shows that diabetes and cancer are correlated not only because they share same risk factors but also because diabetic patients have a higher risk of developing tumors, although the underlying mechanisms remain elusive. In this review, we will explore data obtained in Drosophila revealing the existence of a connection between vitamin B6, DNA damage and diabetes, as flies in the past decade turned out to be a promising model also for metabolic diseases including diabetes. We will focus on recent studies that revealed a specific role for PLP in maintaining chromosome integrity and glucose homeostasis, and we will show that these aspects are correlated. In addition, we will discuss recent data identifying PLP as a putative linking factor between diabetes and cancer

p53-sensitive epileptic behavior and inflammation in Ft1 hypomorphic mice

Epilepsy is a complex clinical condition characterized by repeated spontaneous seizures. Seizures have been linked to multiple drivers including DNA damage accumulation. Investigation of epilepsy physiopathology in humans imposes ethical and practical limitations, for this reason model systems are mostly preferred. Among animal models, mouse mutants are particularly valuable since they allow conjoint behavioral, organismal, and genetic analyses. Along with this, since aging has been associated with higher frequency of seizures, prematurely aging mice, simulating human progeroid diseases, offer a further useful modeling element as they recapitulate aging over a short time-window. Here we report on a mouse mutant with progeroid traits that displays repeated spontaneous seizures. Mutant mice were produced by reducing the expression of the gene Ft1 (AKTIP in humans). In vitro, AKTIP/Ft1 depletion causes telomere aberrations, DNA damage, and cell senescence. AKTIP/Ft1 interacts with lamins, which control nuclear architecture and DNA function. Premature aging defects of Ft1 mutant mice include skeletal alterations and lipodystrophy. The epileptic behavior of Ft1 mutant animals was age and sex linked. Seizures were observed in 18 mutant mice (23.6% of aged ≥ 21 weeks), at an average frequency of 2.33 events/mouse. Time distribution of seizures indicated non-random enrichment of seizures over the follow-up period, with 75% of seizures happening in consecutive weeks. The analysis of epileptic brains did not reveal overt brain morphological alterations or severe neurodegeneration, however, Ft1 reduction induced expression of the inflammatory markers IL-6 and TGF-β. Importantly, Ft1 mutant mice with concomitant genetic reduction of the guardian of the genome, p53, showed no seizures or inflammatory marker activation, implicating the DNA damage response into these phenotypes. This work adds insights into the connection among DNA damage, brain function, and aging. In addition, it further underscores the importance of model organisms for studying specific phenotypes, along with permitting the analysis of genetic interactions at the organismal level

The telomeric protein AKTIP interacts with A- and B-type lamins and is involved in regulation of cellular senescence

AKTIP is a shelterin-interacting protein required for replication of telomeric DNA. Here, we show that AKTIP biochemically interacts with A- and B-type lamins and affects lamin A, but not lamin C or B, expression. In interphase cells, AKTIP localizes at the nuclear rim and in discrete regions of the nucleoplasm just like lamins. Double immunostaining revealed that AKTIP partially co-localizes with lamin B1 and lamin A/C in interphase cells, and that proper AKTIP localization requires functional lamin A. In mitotic cells, AKTIP is enriched at the spindle poles and at the midbody of late telophase cells similar to lamin B1. AKTIP-depleted cells show senescence-associated markers and recapitulate several aspects of the progeroid phenotype. Collectively, our results indicate that AKTIP is a new player in lamin-related processes, including those that govern nuclear architecture, telomere homeostasis and cellular senescence

AKTIP/Ft1, a new shelterin-interacting factor required for telomere maintenance

Telomeres are nucleoprotein complexes that protect the ends of linear chromosomes from incomplete replication, degradation and detection as DNA breaks. Mammalian telomeres are protected by shelterin, a multiprotein complex that binds the TTAGGG telomeric repeats and recruits a series of additional factors that are essential for telomere function. Although many shelterin-associated proteins have been so far identified, the inventory of shelterin-interacting factors required for telomere maintenance is still largely incomplete. Here, we characterize AKTIP/Ft1 (humanAKTIP and mouse Ft1 are orthologous), a novel mammalian shelterin-bound factoridentified on the basis of its homology with the Drosophila telomere protein Pendolino. AKTIP/Ft1 shares homology with the E2 variant ubiquitin-conjugating (UEV) enzymes and has been previously implicated in the control of apoptosis and in vesicle trafficking. RNAi-mediated depletion of AKTIP results in formation of telomere disfunction foci (TIFs). Consistent with these results, AKTIP interacts with telomeric DNA and binds the shelterin components TRF1 and TRF2 both in vivo and in vitro. Analysis of AKTIP- depleted human primary fibroblasts showed that they are defective in PCNA recruiting and arrest in the S phase due to the activation of the intra S checkpoint. Accordingly, AKTIP physically interacts with PCNA and the RPA70 DNA replication factor. Ft1-depleted p53-/- MEFs did not arrest in the S phase but displayed significant increases in multiple telomeric signals (MTS) and sister telomere associations (STAs), two hallmarks of defective telomere replication. In addition, we found an epistatic relation for MST formation between Ft1 and TRF1, which has been previously shown to be required for replication fork progression through telomeric DNA. Ch-IP experiments further suggested that in AKTIP-depleted cells undergoing the S phase, TRF1 is less tightly bound to telomeric DNA than in controls. Thus, our results collectively suggest that AKTIP/Ft1 works in concert with TRF1 to facilitate telomeric DNA replication

Mice with reduced expression of the telomere-associated protein Ft1 develop p53-sensitive progeroid traits

Human AKTIP and mouse Ft1 are orthologous ubiquitin E2 variant proteins involved in telomere maintenance and DNA replication. AKTIP also interacts with A- and B-type lamins. These features suggest that Ft1 may be implicated in aging regulatory pathways. Here, we show that cells derived from hypomorph Ft1 mutant (Ft1kof/kof ) mice exhibit telomeric defects and that Ft1kof/kof animals develop progeroid traits, including impaired growth, skeletal and skin defects, abnormal heart tissue, and sterility. We also demonstrate a genetic interaction between Ft1 and p53. The analysis of mice carrying mutations in both Ft1 and p53 (Ft1kof/kof ; p53ko/ko and Ft1kof/kof ; p53+/ko ) showed that reduction in p53 rescues the progeroid traits of Ft1 mutants, suggesting that they are at least in part caused by a p53-dependent DNA damage response. Conversely, Ft1 reduction alters lymphomagenesis in p53 mutant mice. These results identify Ft1 as a new player in the aging process and open the way to the analysis of its interactions with other progeria genes using the mouse model

Transcriptional Response of Human Neurospheres to helper-dependent CAV-2 vectors involves the modulation of DNA damage response, microtubule and centromere gene groups

Brain gene transfer using viral vectors will likely become a therapeutic option for several disorders. Helper-dependent (HD) canine adenovirus type 2 vectors (CAV-2) are well suited for this goal. These vectors are poorly immunogenic, efficiently transduce neurons, are retrogradely transported to afferent structures in the brain and lead to long-term transgene expression. CAV-2 vectors are being exploited to unravel behavior, cognition, neural networks, axonal transport and therapy for orphan diseases. With the goal of better understanding and characterizing HD-CAV-2 for brain therapy, we analyzed the transcriptomic modulation induced by HD-CAV-2 in human differentiated neurospheres derived from midbrain progenitors. This 3D model system mimics several aspects of the dynamic nature of human brain. We found that differentiated neurospheres are readily transduced by HDCAV- 2 and that transduction generates two main transcriptional responses: a DNA damage response and alteration of centromeric and microtubule probes. Future investigations on the biochemistry of processes highlighted by probe modulations will help defining the implication of HD-CAV-2 and CAR receptor binding in enchaining these functional pathways. We suggest here that the modulation of DNA damage genes is related to viral DNA, while the alteration of centromeric and microtubule probes is possibly enchained by the interaction of the HD-CAV-2 fibre with CAR