Yeast Npl3 regulates replicative senescence outside of TERRA R-loop resolution and co-transcriptional processing.

Eukaryotic cells without telomerase experience progressively shorter telomeres with each round of cell division until cell cycle arrest is initiated, leading to replicative senescence. When yeast TLC1, which encodes the RNA template of telomerase, is deleted, senescence is accompanied by increased expression of TERRA (non-coding telomere repeat-containing RNA). Deletion of Npl3, an RNA-processing protein with telomere maintenance functions, accelerates senescence in tlc1Δ cells and significantly increases TERRA levels. Using genetic approaches, we set out to determine how Npl3 is involved in regulating TERRA expression and maintaining telomere homeostasis. Even though Npl3 regulates hyperrecombination, we found that Npl3 does not help resolve RNA:DNA hybrids formed during TERRA synthesis in the same way as RNase H1 and H2. Furthermore, Rad52 is still required for cells to escape senescence by telomere recombination in the absence of Npl3. Npl3 also works separately from the THO/TREX pathway for processing nascent RNA for nuclear export. However, deleting Dot1, a histone methyltransferase involved in tethering telomeres to the nuclear periphery, rescued the accelerated senescence phenotype of npl3Δ cells. Thus, our study suggests that Npl3 plays an additional role in regulating cellular senescence outside of RNA:DNA hybrid resolution and co-transcriptional processing.

Validation of Salamander Dermal Mucus Swabs as a Novel, Nonlethal Approach for Amphibian Metabolomics and Glutathione Analysis Following Pesticide Exposure.

Evaluating biomarkers of stress in amphibians is critical to conservation, yet current techniques are often destructive and/or time-consuming, which limits ease of use. In the present study, we validate the use of dermal swabs in spotted salamanders (Ambystoma maculatum) for biochemical profiling, as well as glutathione (GSH) stress response following pesticide exposure. Thirty-three purchased spotted salamanders were acclimated to laboratory conditions at Washington College (Chestertown, MD, USA) for 4 weeks. Following acclimation, salamanders were randomly sorted into three groups for an 8-h pesticide exposure on soil: control with no pesticide, 2,4-dichlorophenoxyacetic acid (2,4-D), or chlorpyrifos. Before and after exposure, mucus samples were obtained by gently rubbing a polyester-tipped swab 50 times across the ventral and dorsal surfaces. Salamanders were humanely euthanized and dissected to remove the brain for acetylcholinesterase and liver for GSH and hepatic metabolome analyses, and a whole-body tissue homogenate was used for pesticide quantification. Levels of GSH were present in lower quantities on dermal swabs relative to liver tissues for chlorpyrifos, 2,4-D, and control treatments. However, 2,4-D exposures demonstrated a large effect size increase for GSH levels in livers (Cohen's d = 0.925, p = 0.036). Other GSH increases were statistically insignificant, and effect sizes were characterized as small for 2,4-D mucosal swabs (d = 0.36), medium for chlorpyrifos mucosal swabs (d = 0.713), and negligible for chlorpyrifos liver levels (d = 0.012). The metabolomics analyses indicated that the urea cycle, alanine, and glutamate metabolism biological pathways were perturbed by both sets of pesticide exposures. Obtaining mucus samples through dermal swabbing in amphibians is a viable technique for evaluating health in these imperiled taxa. Environ Toxicol Chem 2024;43:1126-1137. © 2024 SETAC.

TERRA and the histone methyltransferase Dot1 cooperate to regulate senescence in budding yeast.

The events underlying senescence induced by critical telomere shortening are not fully understood. Here we provide evidence that TERRA, a non-coding RNA transcribed from subtelomeres, contributes to senescence in yeast lacking telomerase (tlc1Δ). Levels of TERRA expressed from multiple telomere ends appear elevated at senescence, and expression of an artificial RNA complementary to TERRA (anti-TERRA) binds TERRA in vivo and delays senescence. Anti-TERRA acts independently from several other mechanisms known to delay senescence, including those elicited by deletions of EXO1, TEL1, SAS2, and genes encoding RNase H enzymes. Further, it acts independently of the senescence delay provided by RAD52-dependent recombination. However, anti-TERRA delays senescence in a fashion epistatic to inactivation of the conserved histone methyltransferase Dot1. Dot1 associates with TERRA, and anti-TERRA disrupts this interaction in vitro and in vivo. Surprisingly, the anti-TERRA delay is independent of the C-terminal methyltransferase domain of Dot1 and instead requires only its N-terminus, which was previously found to facilitate release of telomeres from the nuclear periphery. Together, these data suggest that TERRA and Dot1 cooperate to drive senescence.

Inactivation of yeast Isw2 chromatin remodeling enzyme mimics longevity effect of calorie restriction via induction of genotoxic stress response.

ATP-dependent chromatin remodeling is involved in all DNA transactions and is linked to numerous human diseases. We explored functions of chromatin remodelers during cellular aging. Deletion of ISW2, or mutations inactivating the Isw2 enzyme complex, extends yeast replicative lifespan. This extension by ISW2 deletion is epistatic to the longevity effect of calorie restriction (CR), and this mechanism is distinct from suppression of TOR signaling by CR. Transcriptome analysis indicates that isw2Δ partially mimics an upregulated stress response in CR cells. In particular, isw2Δ cells show an increased response to genotoxic stresses, and the DNA repair enzyme Rad51 is important for isw2Δ-mediated longevity. We show that lifespan is also extended in C. elegans by reducing levels of athp-2, a putative ortholog of Itc1/ACF1, a critical subunit of the enzyme complex. Our findings demonstrate that the ISWI class of ATP-dependent chromatin remodeling complexes plays a conserved role during aging and in CR.

Rap1 relocalization contributes to the chromatin-mediated gene expression profile and pace of cell senescence.

Cellular senescence is accompanied by dramatic changes in chromatin structure and gene expression. Using Saccharomyces cerevisiae mutants lacking telomerase (tlc1Δ) to model senescence, we found that with critical telomere shortening, the telomere-binding protein Rap1 (repressor activator protein 1) relocalizes to the upstream promoter regions of hundreds of new target genes. The set of new Rap1 targets at senescence (NRTS) is preferentially activated at senescence, and experimental manipulations of Rap1 levels indicate that it contributes directly to NRTS activation. A notable subset of NRTS includes the core histone-encoding genes; we found that Rap1 contributes to their repression and that histone protein levels decline at senescence. Rap1 and histones also display a target site-specific antagonism that leads to diminished nucleosome occupancy at the promoters of up-regulated NRTS. This antagonism apparently impacts the rate of senescence because underexpression of Rap1 or overexpression of the core histones delays senescence. Rap1 relocalization is not a simple consequence of lost telomere-binding sites, but rather depends on the Mec1 checkpoint kinase. Rap1 relocalization is thus a novel mechanism connecting DNA damage responses (DDRs) at telomeres to global changes in chromatin and gene expression while driving the pace of senescence.

Cdc13 OB2 dimerization required for productive Stn1 binding and efficient telomere maintenance.

Cdc13 is an essential yeast protein required for telomere length regulation and genome stability. It does so via its telomere-capping properties and by regulating telomerase access to the telomeres. The crystal structure of the Saccharomyces cerevisiae Cdc13 domain located between the recruitment and DNA binding domains reveals an oligonucleotide-oligosaccharide binding fold (OB2) with unusually long loops extending from the core of the protein. These loops are involved in extensive interactions between two Cdc13 OB2 folds leading to stable homodimerization. Interestingly, the functionally impaired cdc13-1 mutation inhibits OB2 dimerization. Biochemical assays indicate OB2 is not involved in telomeric DNA or Stn1 binding. However, disruption of the OB2 dimer in full-length Cdc13 affects Cdc13-Stn1 association, leading to telomere length deregulation, increased temperature sensitivity, and Stn1 binding defects. We therefore propose that dimerization of the OB2 domain of Cdc13 is required for proper Cdc13, Stn1, Ten1 (CST) assembly and productive telomere capping.

Telomere stability and carcinogenesis: an off-again, on-again relationship.

Previous studies in mice have demonstrated antagonistic effects of telomerase loss on carcinogenesis. Telomere attrition can promote genome instability, thereby stimulating initiation of early-stage cancers, but can also inhibit tumorigenesis by promoting permanent cell growth arrest or death. Human cancers likely develop in cell lineages with low levels of telomerase, leading to telomere losses in early lesions, followed by subsequent activation of telomerase. Mouse models constitutively lacking telomerase have thus not addressed how telomere losses within telomerase-proficient cells have an impact on carcinogenesis. Using a novel transgenic mouse model, Begus-Nahrmann et al. demonstrate in this issue of the JCI that transient telomere dysfunction in telomerase-proficient animals is a potent stimulus of tumor formation.

Csm4, in collaboration with Ndj1, mediates telomere-led chromosome dynamics and recombination during yeast meiosis.

Chromosome movements are a general feature of mid-prophase of meiosis. In budding yeast, meiotic chromosomes exhibit dynamic movements, led by nuclear envelope (NE)-associated telomeres, throughout the zygotene and pachytene stages. Zygotene motion underlies the global tendency for colocalization of NE-associated chromosome ends in a "bouquet." In this study, we identify Csm4 as a new molecular participant in these processes and show that, unlike the two previously identified components, Ndj1 and Mps3, Csm4 is not required for meiosis-specific telomere/NE association. Instead, it acts to couple telomere/NE ensembles to a force generation mechanism. Mutants lacking Csm4 and/or Ndj1 display the following closely related phenotypes: (i) elevated crossover (CO) frequencies and decreased CO interference without abrogation of normal pathways; (ii) delayed progression of recombination, and recombination-coupled chromosome morphogenesis, with resulting delays in the MI division; and (iii) nondisjunction of homologs at the MI division for some reason other than absence of (the obligatory) CO(s). The recombination effects are discussed in the context of a model where the underlying defect is chromosome movement, the absence of which results in persistence of inappropriate chromosome relationships that, in turn, results in the observed mutant phenotypes.

The effect of genetic background on the function of Saccharomyces cerevisiae mlh1 alleles that correspond to HNPCC missense mutations.

Germline mutations in the DNA mismatch repair (MMR) gene MLH1 are associated with a large percentage of hereditary non-polyposis colorectal cancers. There are approximately 250 known human mutations in MLH1. Of these, one-third are missense variants that are often difficult to characterize with regards to pathogenicity. We analysed 28 alleles of baker's yeast MLH1 that correspond to non-truncating human mutant alleles listed in online HNPCC databases, 13 of which had not been previously studied in functional assays. Using the highly sensitive lys2::InsE-A(14) reversion rate assay, we determined the MMR proficiency conferred by each allele in the S288c strain of Saccharomyces cerevisiae. Seven alleles conferred a null phenotype for MMR and eight others showed significant MMR defects, suggesting that all 15 are likely to be pathogenic in humans. In addition, we observed a strong correlation between these results, limited results from previous functional assays and clinical data. To test whether the potential pathogenicity of certain alleles depends on the genetic background of the host, we examined the mutation rates conferred by the mlh1 alleles in a second yeast strain, SK1, which is approximately 0.7% divergent from S288c. Many alleles displayed a difference in MMR efficiency between strain backgrounds with decreasing differences as the severity of the MMR defect increased. These findings suggest that genetic background can play an important role in determining the pathogenicity of MMR alleles and may explain cases of atypical colorectal cancer inheritance.