Senescent mouse cells fail to overtly regulate the HIRA histone chaperone and do not form robust Senescence Associated Heterochromatin Foci

Background

Cellular senescence is a permanent growth arrest that occurs in response to cellular stressors, such as telomere shortening or activation of oncogenes. Although the process of senescence growth arrest is somewhat conserved between mouse and human cells, there are some critical differences in the molecular pathways of senescence between these two species. Recent studies in human fibroblasts have defined a cell signaling pathway that is initiated by repression of a specific Wnt ligand, Wnt2. This, in turn, activates a histone chaperone HIRA, and culminates in formation of specialized punctate domains of facultative heterochromatin, called Senescence-Associated Heterochromatin Foci (SAHF), that are enriched in the histone variant, macroH2A. SAHF are thought to repress expression of proliferation-promoting genes, thereby contributing to senescence-associated proliferation arrest. We asked whether this Wnt2-HIRA-SAHF pathway is conserved in mouse fibroblasts.

Results

We show that mouse embryo fibroblasts (MEFs) and mouse skin fibroblasts, do not form robust punctate SAHF in response to an activated Ras oncogene or shortened telomeres. However, senescent MEFs do exhibit elevated levels of macroH2A staining throughout the nucleus as a whole. Consistent with their failure to fully activate the SAHF assembly pathway, the Wnt2-HIRA signaling axis is not overtly regulated between proliferating and senescent mouse cells.

Conclusions

In addition to the previously defined differences between mouse and human cells in the mechanisms and phenotypes associated with senescence, we conclude that senescent mouse and human fibroblasts also differ at the level of chromatin and the signaling pathways used to regulate chromatin. These differences between human and mouse senescence may contribute to the increased propensity of mouse fibroblasts (and perhaps other mouse cell types) to become immortalized and transformed, compared to human cells.     

Quantifying the importance of geographic replication and representativeness when estimating demographic rates,using a coastal species as a case study

Demographic rates are rarely estimated over an entire species range, limiting empirical tests of ecological patterns and theories, and raising questions about the representativeness of studies that use data from a small part of a range. The uncertainty that results from using demographic rates from just a few sites is especially pervasive in population projections, which are critical for a wide range of questions in ecology and conservation. We developed a simple simulation to quantify how this lack of geographic representativeness can affect inferences about the global mean and variance of growth rates, which has implications for the robust design of a wide range of population studies. Using a coastal songbird, saltmarsh sparrow Ammodramus caudacutus, as a case study, we first estimated survival, fecundity, and population growth rates at 21 sites distributed across much of their breeding range. We then subsampled this large, representative dataset according to five sampling scenarios in order to simulate a variety of geographic biases in study design. We found spatial variation in demographic rates, but no large systematic patterns. Estimating the global mean and variance of growth rates using subsets of the data suggested that at least 10–15 sites were required for reasonably unbiased estimates, highlighting how relying on demographic data from just a few sites can lead to biased results when extrapolating across a species range. Sampling at the full 21 sites, however, offered diminishing returns, raising the possibility that for some species accepting some geographical bias in sampling can still allow for robust range‐wide inferences. The subsampling approach presented here, while conceptually simple, could be used with both new and existing data to encourage efficiency in the design of long‐term or large‐scale ecological studies.     

Human Rev1 relies on insert-2 to promote selective binding and accurate replication of stabilized G-quadruplex motifs

We previously reported that human Rev1 (hRev1) bound to a parallel-stranded G-quadruplex (G4) from the c-MYC promoter with high affinity. We have extended those results to include other G4 motifs, finding that hRev1 exhibited stronger affinity for parallel-stranded G4 than either anti-parallel or hybrid folds. Amino acids in the αE helix of insert-2 were identified as being important for G4 binding. Mutating E466 and Y470 to alanine selectively perturbed G4 binding affinity. The E466K mutant restored wild-type G4 binding properties. Using a forward mutagenesis assay, we discovered that loss of hRev1 increased G4 mutation frequency >200-fold compared to the control sequence. Base substitutions and deletions occurred around and within the G4 motif. Pyridostatin (PDS) exacerbated this effect, as the mutation frequency increased >700-fold over control and deletions upstream of the G4 site more than doubled. Mutagenic replication of G4 DNA (±PDS) was partially rescued by wild-type and E466K hRev1. The E466A or Y470A mutants failed to suppress the PDS-induced increase in G4 mutation frequency. These findings have implications for the role of insert-2, a motif conserved in vertebrates but not yeast or plants, in Rev1-mediated suppression of mutagenesis during G4 replication.     

Meta-analysis identifies pleiotropic loci controlling phenotypic trade-offs in sorghum

Community association populations are composed of phenotypically and genetically diverse accessions. Once these populations are genotyped, the resulting marker data can be reused by different groups investigating the genetic basis of different traits. Because the same genotypes are observed and scored for a wide range of traits in different environments, these populations represent a unique resource to investigate pleiotropy. Here, we assembled a set of 234 separate trait datasets for the Sorghum Association Panel, a group of 406 sorghum genotypes widely employed by the sorghum genetics community. Comparison of genome-wide association studies (GWAS) conducted with two independently generated marker sets for this population demonstrate that existing genetic marker sets do not saturate the genome and likely capture only 35–43% of potentially detectable loci controlling variation for traits scored in this population. While limited evidence for pleiotropy was apparent in cross-GWAS comparisons, a multivariate adaptive shrinkage approach recovered both known pleiotropic effects of existing loci and new pleiotropic effects, particularly significant impacts of known dwarfing genes on root architecture. In addition, we identified new loci with pleiotropic effects consistent with known trade-offs in sorghum development. These results demonstrate the potential for mining existing trait datasets from widely used community association populations to enable new discoveries from existing trait datasets as new, denser genetic marker datasets are generated for existing community association populations.     

Developmental constraints on fin diversity

The fish fin is a breathtaking repository full of evolutionary diversity, novelty, and convergence. Over 500 million years, the adaptation to novel habitats has provided landscapes of fin diversity. Although comparative anatomy of evolutionarily divergent patterns over centuries has highlighted the fundamental architectures and evolutionary trends of fins, including convergent evolution, the developmental constraints on fin evolution, which bias the evolutionary trajectories of fin morphology, largely remain elusive. Here, we review the evolutionary history, developmental mechanisms, and evolutionary underpinnings of paired fins, illuminating possible developmental constraints on fin evolution. Our compilation of anatomical and genetic knowledge of fin development sheds light on the canalized and the unpredictable aspects of fin shape in evolution. Leveraged by an arsenal of genomic and genetic tools within the working arena of spectacular fin diversity, evolutionary developmental biology embarks on the establishment of conceptual framework for developmental constraints, previously enigmatic properties of evolution.     

Circadian rhythms in Neurospora crassa: Dynamics of the clock component frequency visualized using a fluorescent reporter

The frequency (frq) gene of Neurospora crassa has long been considered essential to the function of this organism’s circadian rhythm. Increasingly, deciphering the coupling of core oscillator genes such as frq to the output pathways of the circadian rhythm has become a major focus of circadian research. To address this coupling it is critical to have a reporter of circadian activity that can deliver high resolution spatial and temporal information about the dynamics of core oscillatory proteins such as FRQ. However, due to the difficulty of studying the expression of circadian rhythm genes in aerobic N. crassa cultures, little is known about the dynamics of this gene under physiologically realistic conditions. To address these issues we report a fluorescent fusion to the frq gene using a codon optimized version of the mCherry gene. To trace the expression and accumulation of FRQ–mCherryNC (FRQ–mCh) during the circadian rhythm, growing vegetative hyphae were scanned every hour under confocal microscopy (100×). Fluorescence of FRQ–mCh was detected only at the growing edge of the colony, and located in the cytoplasm and nuclei of vegetative hyphae for a distance of approximately 150–200 μm from the apices of leading hyphae. When driven by the frq promoter, apparently there was also a second FRQ entrance into the nucleus during the circadian cycle; however the second entrance had a lower accumulation level than the first entrance. Thus this fluorescent fusion protein has proven useful in tracking the spatial dynamics of the frq protein and has indicated that the dynamics of the FRQ protein’s nuclear trafficking may be more complex than previously realized.     

Molecular crowding enhances facilitated diffusion of two human DNA glycosylases

Intracellular space is at a premium due to the high concentrations of biomolecules and is expected to have a fundamental effect on how large macromolecules move in the cell. Here, we report that crowded solutions promote intramolecular DNA translocation by two human DNA repair glycosylases. The crowding effect increases both the efficiency and average distance of DNA chain translocation by hindering escape of the enzymes to bulk solution. The increased contact time with the DNA chain provides for redundant damage patrolling within individual DNA chains at the expense of slowing the overall rate of damaged base removal from a population of molecules. The significant biological implication is that a crowded cellular environment could influence the mechanism of damage recognition as much as any property of the enzyme or DNA.A significant triumph in biochemistry over the last 20 years was the ability to isolate human DNA repair enzymes and study their in vitro properties using defined DNA substrates containing damaged sites. Typically, these studies have been performed using dilute conditions, where the concentration of the enzyme, DNA and buffer components were low compared to the concentration of water. Although a wealth of insights into the thermodynamic, kinetic and structural properties of enzymes have resulted from such approaches (1–7), DNA repair enzymes act in a crowded cellular environment with quite different physical properties (8,9). Thus, an open question is how the complex intracellular milieu affects the ability of enzymes to locate and repair damage sites embedded in a large polymeric DNA substrate.The human intracellular environment has numerous physical properties that could dramatically affect enzyme activity. These include high inorganic ion and metabolite concentrations (10,11), lower dielectric properties (12–14), higher bulk viscosity (15,16), and the presence of high concentrations of macromolecules which consume available volume (‘molecular crowding’) (17,18). Indeed, the concentration of macromolecules in human cells is an astounding ∼100–300 mg/ml (9,19), which means that 10–40% of the total cellular volume is consumed by large molecules (often called the excluded volume). Taken together,­ these parameters could affect association of an enzyme with its target in complex ways. For instance, high ion concentrations are expected to shield electrostatic interactions between an enzyme and its highly charged DNA substrate (10,20,21), while a lower dielectric constant could have an opposite effect. Increases in macroscopic viscosity will slow the translational movement of macromolecules and due to entropic effects, crowded environments will push macromolecular association when the complex consumes a smaller volume than the free component species (9,22,23).Although volume exclusion largely explains the effects of crowded environments on binding equilibria, crowding has been reported to have a surprisingly small effect on the diffusion-controlled association kinetics of macromolecules (24). Indeed, it has been observed that some diffusion-controlled association reactions occur at nearly the same rates in crowded solutions and in cells as they do in dilute solution (24,25). These kinetic effects are counterintuitive, but can be understood by considering that macromolecular crowders alter the macroscopic viscosity and available volume in crowded solutions, but do not change the microscopic viscosity (26,27). Thus, over short nanometer distances, the rotational and translational diffusion of proteins is not greatly affected by crowding because the protein only feels the microscopic viscosity of the solvent that is present in the spaces between the larger crowding molecules (28). Over larger distances, hard sphere repulsion between the protein and crowding molecules increases the effective viscosity and slows translational diffusion (8,28,29). When two binding partners approach one another, they are captured within a low viscosity (high mobility) cage created by the larger crowding molecules, which increases the probability for a productive encounter event. Surprisingly, the capture of two binding partners within a high mobility cage can in some cases offset all of the negative effects of high viscosity on the overall association rate (29).The above considerations raise the interesting question of what effect molecular crowding has on enzyme association with DNA, and in particular, the property of facilitated diffusion along a DNA chain? Facilitated diffusion on the DNA chain (‘translocation’) is a distinct process that involves transient states of an enzyme and DNA that are not directly observable in equilibrium binding, steady-state or rapid kinetic measurements (1–4,30). Here, we measure the effect of inert crowding agents on the probability that the DNA repair enzymes uracil and 8-oxguanine DNA repair glycosylase will successfully translocate between two damaged sites in a DNA chain. We find that crowding increases the likelihood that each enzyme will successfully translocate between their respective target sites without dissociation to bulk solution and also increases the average translocation distance. For both enzymes, crowding biases the damage search process toward a chain tracking search mode rather than a 3D search mode. Such a crowder-induced transition in the search mode could significantly impact the effectiveness of the damage search in a crowded nuclear environment. These enzymes represent two of the largest superfamilies of glycosylases and their similar behavior in these studies suggests that the findings will be general for other related glycosylases.     

Chronic mTOR inhibition in mice with rapamycin alters T,B, myeloid,and innate lymphoid cells and gut flora and prolongs life of immune‐deficient mice

The mammalian (mechanistic) target of rapamycin (mTOR) regulates critical immune processes that remain incompletely defined. Interest in mTOR inhibitor drugs is heightened by recent demonstrations that the mTOR inhibitor rapamycin extends lifespan and healthspan in mice. Rapamycin or related analogues (rapalogues) also mitigate age‐related debilities including increasing antigen‐specific immunity, improving vaccine responses in elderly humans, and treating cancers and autoimmunity, suggesting important new clinical applications. Nonetheless, immune toxicity concerns for long‐term mTOR inhibition, particularly immunosuppression, persist. Although mTOR is pivotal to fundamental, important immune pathways, little is reported on immune effects of mTOR inhibition in lifespan or healthspan extension, or with chronic mTOR inhibitor use. We comprehensively analyzed immune effects of rapamycin as used in lifespan extension studies. Gene expression profiling found many and novel changes in genes affecting differentiation, function, homeostasis, exhaustion, cell death, and inflammation in distinct T‐ and B‐lymphocyte and myeloid cell subpopulations. Immune functions relevant to aging and inflammation, and to cancer and infections, and innate lymphoid cell effects were validated in vitro and in vivo. Rapamycin markedly prolonged lifespan and healthspan in cancer‐ and infection‐prone mice supporting disease mitigation as a mechanism for mTOR suppression‐mediated longevity extension. It modestly altered gut metagenomes, and some metagenomic effects were linked to immune outcomes. Our data show novel mTOR inhibitor immune effects meriting further studies in relation to longevity and healthspan extension.     

Asymmetric reproductive character displacement in male aggregation behaviour

Reproductive character displacement--the evolution of traits that minimize reproductive interactions between species--can promote striking divergence in male signals or female mate preferences between populations that do and do not occur with heterospecifics. However, reproductive character displacement can affect other aspects of mating behaviour. Indeed, avoidance of heterospecific interactions might contribute to spatial (or temporal) aggregation of conspecifics. We examined this possibility in two species of hybridizing spadefoot toad (genus Spea). We found that in Spea bombifrons sympatric males were more likely than allopatric males to associate with calling males. Moreover, contrary to allopatric males, sympatric S. bombifrons males preferentially associated with conspecific male calls. By contrast, Spea multiplicata showed no differences between sympatry and allopatry in likelihood to associate with calling males. Further, sympatric and allopatric males did not differ in preference for conspecifics. However, allopatric S. multiplicata were more variable than sympatric males in their responses. Thus, in S. multiplicata, character displacement may have refined pre-existing aggregation behaviour. Our results suggest that heterospecific interactions can foster aggregative behaviour that might ultimately contribute to clustering of conspecifics. Such clustering can generate spatial or temporal segregation of reproductive activities among species and ultimately promote reproductive isolation.