Meiotic resetting of the cellular Sod1 pool is driven by protein aggregation, degradation, and transient LUTI-mediated repression.

Gametogenesis requires packaging of the cellular components needed for the next generation. In budding yeast, this process includes degradation of many mitotically stable proteins, followed by their resynthesis. Here, we show that one such case-Superoxide dismutase 1 (Sod1), a protein that commonly aggregates in human ALS patients-is regulated by an integrated set of events, beginning with the formation of pre-meiotic Sod1 aggregates. This is followed by degradation of a subset of the prior Sod1 pool and clearance of Sod1 aggregates. As degradation progresses, Sod1 protein production is transiently blocked during mid-meiotic stages by transcription of an extended and poorly translated SOD1 mRNA isoform, SOD1LUTI. Expression of SOD1LUTI is induced by the Unfolded Protein Response, and it acts to repress canonical SOD1 mRNA expression. SOD1LUTI is no longer expressed following the meiotic divisions, enabling a resurgence of canonical mRNA and synthesis of new Sod1 protein such that gametes inherit a full complement of Sod1 protein. Failure to aggregate and degrade Sod1 results in reduced gamete fitness in the presence of oxidants, highlighting the importance of this regulation. Investigation of Sod1 during yeast gametogenesis, an unusual cellular context in which Sod1 levels are tightly regulated, could shed light on conserved aspects of its aggregation and degradation, with relevance to understanding Sod1's role in human disease.

Tunable Transcriptional Interference at the Endogenous Alcohol Dehydrogenase Gene Locus in Drosophila melanogaster.

Neighboring sequences of a gene can influence its expression. In the phenomenon known as transcriptional interference, transcription at one region in the genome can repress transcription at a nearby region in cis Transcriptional interference occurs at a number of eukaryotic loci, including the alcohol dehydrogenase (Adh) gene in Drosophila melanogasterAdh is regulated by two promoters, which are distinct in their developmental timing of activation. It has been shown using transgene insertion that when the promoter distal from the Adh start codon is deleted, transcription from the proximal promoter becomes de-regulated. As a result, the Adh proximal promoter, which is normally active only during the early larval stages, becomes abnormally activated in adults. Whether this type of regulation occurs in the endogenous Adh context, however, remains unclear. Here, we employed the CRISPR/Cas9 system to edit the endogenous Adh locus and found that removal of the distal promoter also resulted in the untimely expression of the proximal promoter-driven mRNA isoform in adults, albeit at lower levels than previously reported. Importantly, transcription from the distal promoter was sufficient to repress proximal transcription in larvae, and the degree of this repression was dependent on the degree of distal promoter activity. Finally, upregulation of the distal Adh transcript led to the enrichment of histone 3 lysine 36 trimethylation over the Adh proximal promoter. We conclude that the endogenous Adh locus is developmentally regulated by transcriptional interference in a tunable manner.

Transaldolase inhibition impairs mitochondrial respiration and induces a starvation-like longevity response in Caenorhabditis elegans.

Mitochondrial dysfunction can increase oxidative stress and extend lifespan in Caenorhabditis elegans. Homeostatic mechanisms exist to cope with disruptions to mitochondrial function that promote cellular health and organismal longevity. Previously, we determined that decreased expression of the cytosolic pentose phosphate pathway (PPP) enzyme transaldolase activates the mitochondrial unfolded protein response (UPRmt) and extends lifespan. Here we report that transaldolase (tald-1) deficiency impairs mitochondrial function in vivo, as evidenced by altered mitochondrial morphology, decreased respiration, and increased cellular H2O2 levels. Lifespan extension from knockdown of tald-1 is associated with an oxidative stress response involving p38 and c-Jun N-terminal kinase (JNK) MAPKs and a starvation-like response regulated by the transcription factor EB (TFEB) homolog HLH-30. The latter response promotes autophagy and increases expression of the flavin-containing monooxygenase 2 (fmo-2). We conclude that cytosolic redox established through the PPP is a key regulator of mitochondrial function and defines a new mechanism for mitochondrial regulation of longevity.

Recurrent Gene Duplication Diversifies Genome Defense Repertoire in Drosophila.

Transposable elements (TEs) comprise large fractions of many eukaryotic genomes and imperil host genome integrity. The host genome combats these challenges by encoding proteins that silence TE activity. Both the introduction of new TEs via horizontal transfer and TE sequence evolution requires constant innovation of host-encoded TE silencing machinery to keep pace with TEs. One form of host innovation is the adaptation of existing, single-copy host genes. Indeed, host suppressors of TE replication often harbor signatures of positive selection. Such signatures are especially evident in genes encoding the piwi-interacting-RNA pathway of gene silencing, for example, the female germline-restricted TE silencer, HP1D/Rhino Host genomes can also innovate via gene duplication and divergence. However, the importance of gene family expansions, contractions, and gene turnover to host genome defense has been largely unexplored. Here, we functionally characterize Oxpecker, a young, tandem duplicate gene of HP1D/rhino We demonstrate that Oxpecker supports female fertility in Drosophila melanogaster and silences several TE families that are incompletely silenced by HP1D/Rhino in the female germline. We further show that, like Oxpecker, at least ten additional, structurally diverse, HP1D/rhino-derived daughter and "granddaughter" genes emerged during a short 15-million year period of Drosophila evolution. These young paralogs are transcribed primarily in germline tissues, where the genetic conflict between host genomes and TEs plays out. Our findings suggest that gene family expansion is an underappreciated yet potent evolutionary mechanism of genome defense diversification.

Mitotic fidelity requires transgenerational action of a testis-restricted HP1.

Sperm-packaged DNA must undergo extensive reorganization to ensure its timely participation in embryonic mitosis. Whereas maternal control over this remodeling is well described, paternal contributions are virtually unknown. In this study, we show that Drosophila melanogaster males lacking Heterochromatin Protein 1E (HP1E) sire inviable embryos that undergo catastrophic mitosis. In these embryos, the paternal genome fails to condense and resolve into sister chromatids in synchrony with the maternal genome. This delay leads to a failure of paternal chromosomes, particularly the heterochromatin-rich sex chromosomes, to separate on the first mitotic spindle. Remarkably, HP1E is not inherited on mature sperm chromatin. Instead, HP1E primes paternal chromosomes during spermatogenesis to ensure faithful segregation post-fertilization. This transgenerational effect suggests that maternal control is necessary but not sufficient for transforming sperm DNA into a mitotically competent pronucleus. Instead, paternal action during spermiogenesis exerts post-fertilization control to ensure faithful chromosome segregation in the embryo.

Activation of the mitochondrial unfolded protein response does not predict longevity in Caenorhabditis elegans.

Recent studies have propagated the model that the mitochondrial unfolded protein response (UPR(mt)) is causal for lifespan extension from inhibition of the electron transport chain (ETC) in Caenorhabditis elegans. Here we report a genome-wide RNAi screen for negative regulators of the UPR(mt). Lifespan analysis of nineteen RNAi clones that induce the hsp-6p::gfp reporter demonstrate differential effects on longevity. Deletion of atfs-1, which is required for induction of the UPR(mt), fails to prevent lifespan extension from knockdown of two genes identified in our screen or following knockdown of the ETC gene cco-1. RNAi knockdown of atfs-1 also has no effect on lifespan extension caused by mutation of the ETC gene isp-1. Constitutive activation of the UPR(mt) by gain of function mutations in atfs-1 fails to extend lifespan. These observations identify several new factors that promote mitochondrial homoeostasis and demonstrate that the UPR(mt), as currently defined, is neither necessary nor sufficient for lifespan extension.

Molecular mechanisms underlying genotype-dependent responses to dietary restriction.

Dietary restriction (DR) increases lifespan and attenuates age-related phenotypes in many organisms; however, the effect of DR on longevity of individuals in genetically heterogeneous populations is not well characterized. Here, we describe a large-scale effort to define molecular mechanisms that underlie genotype-specific responses to DR. The effect of DR on lifespan was determined for 166 single gene deletion strains in Saccharomyces cerevisiae. Resulting changes in mean lifespan ranged from a reduction of 79% to an increase of 103%. Vacuolar pH homeostasis, superoxide dismutase activity, and mitochondrial proteostasis were found to be strong determinants of the response to DR. Proteomic analysis of cells deficient in prohibitins revealed induction of a mitochondrial unfolded protein response (mtUPR), which has not previously been described in yeast. Mitochondrial proteotoxic stress in prohibitin mutants was suppressed by DR via reduced cytoplasmic mRNA translation. A similar relationship between prohibitins, the mtUPR, and longevity was also observed in Caenorhabditis elegans. These observations define conserved molecular processes that underlie genotype-dependent effects of DR that may be important modulators of DR in higher organisms.

End-of-life cell cycle arrest contributes to stochasticity of yeast replicative aging.

There is growing evidence that stochastic events play an important role in determining individual longevity. Studies in model organisms have demonstrated that genetically identical populations maintained under apparently equivalent environmental conditions display individual variation in life span that can be modeled by the Gompertz-Makeham law of mortality. Here, we report that within genetically identical haploid and diploid wild-type populations, shorter-lived cells tend to arrest in a budded state, while cells that arrest in an unbudded state are significantly longer-lived. This relationship is particularly notable in diploid BY4743 cells, where mother cells that arrest in a budded state have a shorter mean life span (25.6 vs. 35.6) and larger coefficient of variance with respect to individual life span (0.42 vs. 0.32) than cells that arrest in an unbudded state. Mutations that cause genomic instability tend to shorten life span and increase the proportion of the population that arrest in a budded state. These observations suggest that randomly occurring damage may contribute to stochasticity during replicative aging by causing a subset of the population to terminally arrest prematurely in the S or G2 phase of the cell cycle.

pH neutralization protects against reduction in replicative lifespan following chronological aging in yeast.

Chronological and replicative aging have been studied in yeast as alternative paradigms for post-mitotic and mitotic aging, respectively. It has been known for more than a decade that cells of the S288C background aged chronologically in rich medium have reduced replicative lifespan relative to chronologically young cells. Here we report replication of this observation in the diploid BY4743 strain background. We further show that the reduction in replicative lifespan from chronological aging is accelerated when cells are chronologically aged under standard conditions in synthetic complete medium rather than rich medium. The loss of replicative potential with chronological age is attenuated by buffering the pH of the chronological aging medium to 6.0, an intervention that we have previously shown can extend chronological lifespan. These data demonstrate that extracellular acidification of the culture medium can cause intracellular damage in the chronologically aging population that is asymmetrically segregated by the mother cell to limit subsequent replicative lifespan.