Abf1 negatively regulates the expression of EPA1 and affects adhesion in Candida glabrata.

Introduction. Adherence is a major virulence trait in Candida glabrata that, in many strains, depends on the EPA (epithelial adhesin) genes, which confer the ability to adhere to epithelial and endothelial cells of the host. The EPA genes are generally found at subtelomeric regions, which makes them subject to subtelomeric silencing. In C. glabrata, subtelomeric silencing depends on different protein complexes, such as silent information regulator and yKu complexes, and other proteins, such as Repressor/activator protein 1 (Rap1) and Abf1. At the EPA1 locus, which encodes the main adhesin Epa1, we previously found at least two cis-acting elements, the protosilencer Sil2126 and the negative element, that contribute to the propagation of silencing from the telomere to the subtelomeric region.Hypothesis. Abf1 binds to the regulatory regions of EPA1 and other regions at the telomere E-R, thereby negatively regulating EPA1 transcription.Aim. To determine whether Abf1 and Rap1 silencing proteins bind to previously identified cis-acting elements on the right telomere of chromosome E (E-R subtelomeric region), resulting in negative regulation of EPA1 transcription and infer Abf1 and Rap1 recognition sites in C. glabrata.Methodology. We used chromatin immunoprecipitation (ChIP) followed by quantitative PCR to determine the binding sites for Abf1 and Rap1 in the intergenic regions between EPA1 and EPA2 and HYR1 and EPA1, and mutants were used to determine the silencing level of the EPA1 promoter region.Results. We found that Abf1 predominantly binds to the EPA1 promoter region, leading to negative regulation of EPA1 expression. Furthermore, the mutant abf1-43, which lacks the last 43 amino acids at its C-terminal end and is defective for subtelomeric silencing, exhibits hyperadherence to epithelial cells in vitro compared to the parental strain, suggesting that EPA1 is derepressed. We also determined the motif-binding sequences for Abf1 and Rap1 in C. glabrata using data from the ChIP assays.Conclusion. Together these data indicate that Abf1 negatively regulates EPA1 expression, leading to decreased adhesion of C. glabrata to epithelial cells.

Dysfunction of Telomeric Cdc13-Stn1-Ten1 Simultaneously Activates DNA Damage and Spindle Checkpoints.

Telomeres, the ends of eukaryotic linear chromosomes, are composed of repeated DNA sequences and specialized proteins, with the conserved telomeric Cdc13/CTC1-Stn1-Ten1 (CST) complex providing chromosome stability via telomere end protection and the regulation of telomerase accessibility. In this study, SIZ1, coding for a SUMO E3 ligase, and TOP2 (a SUMO target for Siz1 and Siz2) were isolated as extragenic suppressors of Saccharomyces cerevisiae CST temperature-sensitive mutants. ten1-sz, stn1-sz and cdc13-sz mutants were isolated next due to being sensitive to intracellular Siz1 dosage. In parallel, strong negative genetic interactions between mutants of CST and septins were identified, with septins being noticeably sumoylated through the action of Siz1. The temperature-sensitive arrest in these new mutants of CST was dependent on the G2/M Mad2-mediated and Bub2-mediated spindle checkpoints as well as on the G2/M Mec1-mediated DNA damage checkpoint. Our data suggest the existence of yet unknown functions of the telomeric Cdc13-Stn1-Ten1 complex associated with mitotic spindle positioning and/or assembly that could be further elucidated by studying these new ten1-sz, stn1-sz and cdc13-sz mutants.

Low-power red laser and blue LED modulate telomere maintenance and length in human breast cancer cells.

Cancer cells have the ability to undergo an unlimited number of cell divisions, which gives them immortality. Thus, the cancer cell can extend the length of its telomeres, allowing these cells to divide unlimitedly and avoid entering the state of senescence or cellular apoptosis. One of the main effects of photobiomodulation (PBM) is the increase in the production of adenosine triphosphate (ATP) and free radicals, mainly reactive oxygen species (ROS). Existent data indicates that high levels of ROS can cause shortening and dysfunctional telomeres. Therefore, a better understanding of the effects induced by PBM on cancer cell telomere maintenance is needed. This work aimed to evaluate the effects of low-power red laser (658 nm) and blue LED (470 nm) on the TRF1 and TRF2 mRNA levels and telomere length in human breast cancer cells. MCF-7 and MDA-MB-231 cells were irradiated with a low-power red laser (69 J cm-2, 0.77 W/cm-2) and blue LED (482 J cm-2, 5.35 W/cm-2), alone or in combination, and the relative mRNA levels of the genes and telomere length were assessed by quantitative reverse transcription polymerase chain reaction. The results suggested that exposure to certain red laser and blue LED fluences decreased the TRF1 and TRF2 mRNA levels in both human breast cancer cells. Telomere length was increased in MCF-7 cells after exposure to red laser and blue LED. However, telomere length in MDA-MB-231 was shortened after exposure to red laser and blue LED at fluences evaluated. Our research suggests that photobiomodulation induced by red laser and low-power blue LED could alter telomere maintenance and length.

GbHSP90 act as a dual functional role regulated in telomere stability in Ginkgo biloba.

The heat shock protein 90 (HSP90) family members are not only widely involved in animal cellular immune response and signal transduction pathway regulation, but also play an important role in plant development and environmental stress response. Here,we identified a HSP90 family member in Ginkgo biloba, designated as GbHSP90, which performs a dual functional role to regulate telomere stability. GbHSP90 was screened by a yeast one-hybrid library using the Ginkgo biloba telomeric DNA (TTTAGGG)5. Fluorescence polarization, surface plasmon resonance(SPR) and EMSA technologyies revealed a specific interaction between GbHSP90 and the double-stranded telomeric DNA via its N-CR region, with no affinity for the single-stranded telomeric DNA or human double-stranded telomeric DNA. Furthermore, yeast two-hybrid system and Split-LUC assay demonstrated that GbHSP90 can interacts with two telomere end-binding proteins:the ginkgo telomerase reverse transcriptase (GbTERT) and the ginkgo Structural Maintenance of Chromosomes protein 1 (GbSMC1). Overexpression of GbHSP90 in human 293 T and HeLa cells increased cell growth rate, the content of telomerase reverse transcriptase (TERT), and promote cell division and inhibit cell apoptosis. Our results indicated GbHSP90 have dually functions: as a telomere-binding protein that binds specifically to double-stranded telomeric DNA and as a molecular chaperone that modulates cell differentiation and apoptosis by binding to telomere protein complexes in Ginkgo biloba. This study contributes to a significantly understanding of the unique telomere complex structure and regulatory mechanisms in Ginkgo biloba, a long-lived tree species.

RAP1-GTPase immunostaining is altered in human precancerous and cancerous cervical lesions.

Aim: This study investigated RAP1 immunostaining variation in different cell types during CC progression.Methods: Paraffin-embedded cervical tissues from 101 patients were categorized into control, pre-neoplastic and neoplastic groups. RAP1 immunolocalization, HPV detection and genotyping were performed. A semiquantitative immunoreactive score was employed to compare labeling intensity, cellular localization, nuclear labeling, percentage and distribution of reactive cells.Results: 73% (72/99) of cervical specimens were HPV+. RAP1 was localized in the nucleus and cytoplasm of all samples. Cytoplasmic RAP1 immunoscore was higher than nuclear score in all CC groups. RAP1 intensity increased with lesion severity. SCC samples exhibited predominantly intense RAP1 immunostaining.Conclusion: RAP1 is an efficient biomarker for detecting invasive CC lesions but has limited utility in distinguishing SCC grades.

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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.

Analyzing Telomeric Protein-DNA Interactions Using Single-Molecule Magnetic Tweezers.

Telomeres, the protective structures at the ends of chromosomes, are crucial for maintaining cellular longevity and genome stability. Their proper function depends on tightly regulated processes of replication, elongation, and damage response. The shelterin complex, especially Telomere Repeat-binding Factor 1 (TRF1) and TRF2, plays a pivotal role in telomere protection and has emerged as a potential anti-cancer target for drug discovery. These proteins bind to the repetitive telomeric DNA motif TTAGGG, facilitating the formation of protective structures and recruitment of other telomeric proteins. Structural methods and advanced imaging techniques have provided insights into telomeric protein-DNA interactions, but probing the dynamic processes requires single-molecule approaches. Tools like magnetic tweezers, optical tweezers, and atomic force microscopy (AFM) have been employed to study telomeric protein-DNA interactions, revealing important details such as TRF2-dependent DNA distortion and telomerase catalysis. However, the preparation of single-molecule constructs with telomeric repetitive motifs continues to be a challenging task, potentially limiting the breadth of studies utilizing single-molecule mechanical methods. To address this, we developed a method to study interactions using full-length human telomeric DNA with magnetic tweezers. This protocol describes how to express and purify TRF2, prepare telomeric DNA, set up single-molecule mechanical assays, and analyze data. This detailed guide will benefit researchers in telomere biology and telomere-targeted drug discovery.

Protection of the Telomeric Junction by the Shelterin Complex.

Shelterin serves critical roles in suppressing superfluous DNA damage repair pathways on telomeres. The junction between double-stranded telomeric tracts (dsTEL) and single-stranded telomeric overhang (ssTEL) is the most accessible region of the telomeric DNA. The shelterin complex contains dsTEL and ssTEL binding proteins and can protect this junction by bridging the ssTEL and dsTEL tracts. To test this possibility, we monitored shelterin binding to telomeric DNA substrates with varying ssTEL and dsTEL lengths and quantified its impact on telomere accessibility using single-molecule fluorescence microscopy methods in vitro. We identified the first dsTEL repeat nearest the junction as the preferred binding site for creating the shelterin bridge. Shelterin requires at least two ssTEL repeats, while the POT1 subunit of shelterin that binds to ssTEL requires longer ssTEL tracts for stable binding to telomeres and effective protection of the junction region. The ability of POT1 to protect the junction is significantly enhanced by the 5'-phosphate at the junction. Collectively, our results show that shelterin enhances the binding stability of POT1 to ssTEL and provides more effective protection compared with POT1 alone by bridging single- and double-stranded telomeric tracts.

CST3 alleviates retinal vascular leakage by regulating the Rap1 signaling pathway.

Retinal vascular leakage is a major event in several retinal diseases, including diabetic retinopathy (DR). In a previous study, we demonstrated that the aqueous humor concentration of Cystatin C (CST3), a physiological inhibitor of cysteine protease, is negatively correlated with the severity of diabetic macular edema. However, its function in the retina has not been clearly elucidated. In this study, we found a significant decrease in the aqueous humor concentration of CST3 with DR progression. Furthermore, we found that CST3 was expressed in retinal endothelial cells and that its expression was significantly downregulated in high glucose-treated human retinal microvascular endothelial cells (HRMECs) and the retinal vessels of oxygen-induced retinopathy (OIR) mice. Silencing CST3 expression resulted in decreased HRMEC migration and tubule formation ability. Exogenous addition of the CST3 protein significantly improved HRMEC migration and tubular formation. In-vivo experiments demonstrated that CST3 silencing induced retinal vascular leakage in WT mice, while its intravitreal injection significantly reduced retinal leakage in OIR mice. Mechanistically, CST3 promoted the expression of the downstream adhesion molecules, claudin5, VE-cadherin, and ZO-1, in retinal vascular cells by regulating the Rap1 signaling pathway. Therefore, this study revealed a novel mechanism by which CST3 improves retinal vascular function and provided evidence that it is a potential therapeutic target for retinal vascular leakage.

Involvement of the splicing factor SART1 in the BRCA1-dependent homologous recombination repair of DNA double-strand breaks.

Although previous studies have reported that pre-mRNA splicing factors (SFs) are involved in the repair of DNA double-strand breaks (DSBs) via homologous recombination (HR), their exact role in promoting HR remains poorly understood. Here, we showed that SART1, an SF upregulated in several types of cancer, promotes DSB end resection, an essential first step of HR. The resection-promoting function of SART1 requires phosphorylation at threonine 430 and 695 by ATM/ATR. SART1 is recruited to DSB sites in a manner dependent on transcription and its RS domain. SART1 is epistatic with BRCA1, a major HR factor, in the promotion of resection, especially transcription-associated resection in the G2 phase. SART1 and BRCA1 accumulate at DSB sites in an interdependent manner, and epistatically counteract the resection blockade posed by 53BP1 and RIF1. Furthermore, chromosome analysis demonstrated that SART1 and BRCA1 epistatically suppressed genomic alterations caused by DSB misrepair in the G2 phase. Collectively, these results indicate that SART1 and BRCA1 cooperatively facilitate resection of DSBs arising in transcriptionally active genomic regions in the G2 phase, thereby promoting faithful repair by HR, and suppressing genome instability.

The molecular mechanism for TERRA recruitment and annealing to telomeres.

Telomeric repeat containing RNA (TERRA) is a noncoding RNA that is transcribed from telomeres. Previous study showed that TERRA trans anneals by invading into the telomeric duplex to form an R-loop in mammalian cells. Here, we elucidate the molecular mechanism underlying TERRA recruitment and invasion into telomeres in the context of shelterin proteins, RAD51 and RNase H using single molecule (sm) assays. We demonstrate that TERRA trans annealing into telomeric DNA exhibits dynamic movement that is stabilized by TRF2. TERRA annealing to the telomeric duplex results in the formation of a stable triplex structure which differs from a conventional R-loop. We identified that the presence of a sub-telomeric DNA and a telomeric overhang in the form of a G-quadruplex significantly enhances TERRA annealing to telomeric duplex. We also demonstrate that RAD51-TERRA complex invades telomere duplex more efficiently than TERRA alone. Additionally, TRF2 increases TERRA affinity to telomeric duplex and protects it from RNase H digestion. In contrast, TRF1 represses TERRA annealing to telomeric duplex and fails to provide protection against RNase H digestion. Our findings provide an in-depth molecular mechanism underpinning TERRA recruitment and annealing to the telomere.

Novel truncating germline variant reinforces TINF2 as a susceptibility gene for familial non-medullary thyroid cancer.

BACKGROUND: It has long been observed that there are families in which non-medullary thyroid cancer (NMTC) occurs, but few syndromes and genes have been described to date. Proteins in the shelterin complex have been implied in cancer. Here, we have studied shelterin genes in families affected by NMTC (FNMTC). METHODS: We performed whole-exome sequencing (WES) in 10 affected individuals from four families with at least three affected members. Polymerase chain reaction (PCR) and Sanger sequencing were performed to search for variants in the TINF2 gene in 40 FNMTC families. TINF2 transcripts and loss of heterozygosity (LOH) were studied in several affected patients of one family. RESULTS: We found the c.507G>T variant in heterozygosis in the TINF2 gene in one family, co-segregating in all five affected members. This variant affects the normal splicing. LOH was not observed. CONCLUSIONS: Our results reinforce the TINF2 gene as a susceptibility cause of FNMTC suggesting the importance of location of frameshift variants in TINF2. According to our data and previous literature, TINF2 pathogenic variants appear to be a significant risk factor for the development of NMTC and/or melanoma.

Structural biology of shelterin and telomeric chromatin: the pieces and an unfinished puzzle.

The six-subunit shelterin complex binds to mammalian telomeres and protects them from triggering multiple DNA damage response pathways. The loss of this protective function by shelterin can have detrimental effects on cells. In this review, we first discuss structural studies of shelterin, detailing the contributions of each subunit and inter-subunit interactions in protecting chromosome ends. We then examine the influence of telomeric chromatin dynamics on the function of shelterin at telomeres. These studies provide valuable insights and underscore the challenges that future research must tackle to attain high-resolution structures of shelterin.

The evolving genetic landscape of telomere biology disorder dyskeratosis congenita.

Dyskeratosis congenita (DC) is a rare inherited bone marrow failure syndrome, caused by genetic mutations that principally affect telomere biology. Approximately 35% of cases remain uncharacterised at the genetic level. To explore the genetic landscape, we conducted genetic studies on a large collection of clinically diagnosed cases of DC as well as cases exhibiting features resembling DC, referred to as 'DC-like' (DCL). This led us to identify several novel pathogenic variants within known genetic loci and in the novel X-linked gene, POLA1. In addition, we have also identified several novel variants in POT1 and ZCCHC8 in multiple cases from different families expanding the allelic series of DC and DCL phenotypes. Functional characterisation of novel POLA1 and POT1 variants, revealed pathogenic effects on protein-protein interactions with primase, CTC1-STN1-TEN1 (CST) and shelterin subunit complexes, that are critical for telomere maintenance. ZCCHC8 variants demonstrated ZCCHC8 deficiency and signs of pervasive transcription, triggering inflammation in patients' blood. In conclusion, our studies expand the current genetic architecture and broaden our understanding of disease mechanisms underlying DC and DCL disorders.

Overexpression of protection of telomeres 1 (POT1), a single-stranded DNA-binding proteins in alfalfa (Medicago sativa), enhances seed vigor.

Extensive bodies of research are dedicated to the study of seed aging with a particular focus on the roles of reactive oxygen species (ROS), and the ensuing oxidative damage during storage, as a primary cause of seed vigor decreasing. ROS diffuse to the nucleus and damage the telomeres, resulting in a loss of genetic integrity. Protection of telomeres 1 (POT1) is a telomeric protein that binds to the telomere region, and plays an essential role in maintaining genomic stability in plants. In this study, there were totally four MsPOT1 genes obtained from alfalfa genome. Expression analysis of four MsPOT1 genes in germinated seed presented the different expressions. Four MsPOT1 genes displayed high expression levels at the early stage of seed germination, Among the four POT1 genes, it was found that MS. gene040108 was significantly up-regulated in the early germination stage of CK seeds, but down-regulated in aged seeds. RT-qPCR assays and RNA-seq data revealed that MsPOT1-X gene was significantly induced by seed aging treatment. Transgenic seeds overexpressing MsPOT1-X gene in Arabidopsis thaliana and Medicago trunctula exhibited enhanced seed vigor, telomere length, telomerase activity associated with reduced H2O2 content. These results would provide a new way to understand aging stress-responsive MsPOT1 genes for genetic improvement of seed vigor. Although a key gene regulating seed vigor was identified in this study, the specific mechanism of MsPOT1-X gene regulating seed vigor needs to be further explored.

Dynamics of Replication-Associated Protein Levels through the Cell Cycle.

The measurement of dynamic changes in protein level and localization throughout the cell cycle is of major relevance to studies of cellular processes tightly coordinated with the cycle, such as replication, transcription, DNA repair, and checkpoint control. Currently available methods include biochemical assays of cells in bulk following synchronization, which determine protein levels with poor temporal and no spatial resolution. Taking advantage of genetic engineering and live-cell microscopy, we performed time-lapse imaging of cells expressing fluorescently tagged proteins under the control of their endogenous regulatory elements in order to follow their levels throughout the cell cycle. We effectively discern between cell cycle phases and S subphases based on fluorescence intensity and distribution of co-expressed proliferating cell nuclear antigen (PCNA)-mCherry. This allowed us to precisely determine and compare the levels and distribution of multiple replication-associated factors, including Rap1-interacting factor 1 (RIF1), minichromosome maintenance complex component 6 (MCM6), origin recognition complex subunit 1 (ORC1, and Claspin, with high spatiotemporal resolution in HeLa Kyoto cells. Combining these data with available mass spectrometry-based measurements of protein concentrations reveals the changes in the concentration of these proteins throughout the cell cycle. Our approach provides a practical basis for a detailed interrogation of protein dynamics in the context of the cell cycle.

TRF2-RAP1 represses RAD51-dependent homology-directed telomere repair by promoting BLM-mediated D-loop unwinding and inhibiting BLM-DNA2-dependent 5'-end resection.

Inappropriate homology-directed repair (HDR) of telomeres results in catastrophic telomere loss and aberrant chromosome fusions, leading to genome instability. We have previously shown that the TRF2-RAP1 heterodimer protects telomeres from engaging in aberrant telomere HDR. Cells lacking the basic domain of TRF2 and functional RAP1 display HDR-mediated telomere clustering, resulting in the formation of ultrabright telomeres (UTs) and massive chromosome fusions. Using purified proteins, we uncover three distinct molecular pathways that the TRF2-RAP1 heterodimer utilizes to protect telomeres from engaging in aberrant HDR. We show mechanistically that TRF2-RAP1 inhibits RAD51-initiated telomeric D-loop formation. Both the TRF2 basic domain and RAP1-binding to TRF2 are required to block RAD51-mediated homology search. TRF2 recruits the BLM helicase to telomeres through its TRFH domain to promote BLM-mediated unwinding of telomere D-loops. In addition, TRF2-RAP1 inhibits BLM-DNA2-mediated 5' telomere end resection, preventing the generation of 3' single-stranded telomere overhangs necessary for RAD51-dependent HDR. Importantly, cells expressing BLM mutants unable to interact with TRF2 accumulate telomere D-loops and UTs. Our findings uncover distinct molecular mechanisms coordinated by TRF2-RAP1 to protect telomeres from engaging in aberrant HDR.

CoPixie, a novel algorithm for single-particle track colocalization, enables efficient quantification of telomerase dynamics at telomeres.

Single-particle imaging and tracking can be combined with colocalization analysis to study the dynamic interactions between macromolecules in living cells. Indeed, single-particle tracking has been extensively used to study protein-DNA interactions and dynamics. Still, unbiased identification and quantification of binding events at specific genomic loci remains challenging. Herein, we describe CoPixie, a new software that identifies colocalization events between a theoretically unlimited number of imaging channels, including single-particle movies. CoPixie is an object-based colocalization algorithm that relies on both pixel and trajectory overlap to determine colocalization between molecules. We employed CoPixie with live-cell single-molecule imaging of telomerase and telomeres, to test the model that cancer-associated POT1 mutations facilitate telomere accessibility. We show that POT1 mutants Y223C, D224N or K90E increase telomere accessibility for telomerase interaction. However, unlike the POT1-D224N mutant, the POT1-Y223C and POT1-K90E mutations also increase the duration of long-lasting telomerase interactions at telomeres. Our data reveal that telomere elongation in cells expressing cancer-associated POT1 mutants arises from the dual impact of these mutations on telomere accessibility and telomerase retention at telomeres. CoPixie can be used to explore a variety of questions involving macromolecular interactions in living cells, including between proteins and nucleic acids, from multicolor single-particle tracks.

Protein SUMOylation promotes cAMP-independent EPAC1 activation.

Protein SUMOylation is a prevalent stress-response posttranslational modification crucial for maintaining cellular homeostasis. Herein, we report that protein SUMOylation modulates cellular signaling mediated by cAMP, an ancient and universal stress-response second messenger. We identify K561 as a primary SUMOylation site in exchange protein directly activated by cAMP (EPAC1) via site-specific mapping of SUMOylation using mass spectrometry. Sequence and site-directed mutagenesis analyses reveal that a functional SUMO-interacting motif in EPAC1 is required for the binding of SUMO-conjugating enzyme UBC9, formation of EPAC1 nuclear condensate, and EPAC1 cellular SUMOylation. Heat shock-induced SUMO modification of EPAC1 promotes Rap1/2 activation in a cAMP-independent manner. Structural modeling and molecular dynamics simulation studies demonstrate that SUMO substituent on K561 of EPAC1 promotes Rap1 interaction by increasing the buried surface area between the SUMOylated receptor and its effector. Our studies identify a functional SUMOylation site in EPAC1 and unveil a novel mechanism in which SUMOylation of EPAC1 leads to its autonomous activation. The findings of SUMOylation-mediated activation of EPAC1 not only provide new insights into our understanding of cellular regulation of EPAC1 but also will open up a new field of experimentation concerning the cross-talk between cAMP/EPAC1 signaling and protein SUMOylation, two major cellular stress response pathways, during cellular homeostasis.