Lorraine Pillus

The emerging class of multi-functional proteins known as moonlighters challenges the “one protein, one function” mentality by demonstrating crosstalk between biological pathways that were previously thought to be functionally discrete. Here, we present new links between amino acid metabolism and chromatin regulation, two biological pathways that are critical for cellular and organismal homeostasis. We discovered that the threonine biosynthetic pathway is required for the transcriptional silencing of ribosomal DNA (rDNA) in Saccharomyces cerevisiae. The enzymes in the pathway promote rDNA silencing through distinct mechanisms as a subset of silencing phenotypes was rescued with exogenous threonine. In addition, we found that a key pathway enzyme, homoserine dehydrogenase, promotes DNA repair through a mechanism involving the MRX complex, a major player in DNA double strand break repair. These data further the understanding of enzymes with non-canonical roles, here demonstrated within the threonine biosynthetic pathway, and provide insight into their roles as potential anti-fungal pharmaceutical targets.

Synthetic biology enables the design of gene networks to confer specific biological functions, yet it remains a challenge to rationally engineer a biological trait as complex as longevity. A naturally occurring toggle switch underlies fate decisions toward either nucleolar or mitochondrial decline during the aging of yeast cells. We rewired this endogenous toggle to engineer an autonomous genetic clock that generates sustained oscillations between the nucleolar and mitochondrial aging processes in individual cells. These oscillations increased cellular life span through the delay of the commitment to aging that resulted from either the loss of chromatin silencing or the depletion of heme. Our results establish a connection between gene network architecture and cellular longevity that could lead to rationally designed gene circuits that slow aging.

Covalent modifications of chromatin regulate genomic structure and accessibility in diverse biological processes such as transcriptional regulation, cell cycle progression, and DNA damage repair. Many histone modifications have been characterized, yet understanding the interactions between these and their combinatorial effects remains an active area of investigation, including dissecting functional interactions between enzymes mediating these modifications. In budding yeast, the histone acetyltransferase Gcn5 interacts with Rts1, a regulatory subunit of protein phosphatase 2A (PP2A). Implicated in the interaction is the potential for the dynamic phosphorylation of conserved residues on histone H2B and the Cse4 centromere-specific histone H3 variant. To probe these dynamics, we sought to identify kinases which contribute to the phosphorylated state. In a directed screen beginning with in silico analysis of the 127 members of yeast kinome, we have now identified 16 kinases with genetic interactions with GCN5 and specifically found distinct roles for the Hog1 stress-activated protein kinase. Deletion of HOG1 (hog1Δ) rescues gcn5Δ sensitivity to the microtubule poison nocodazole and the lethality of the gcn5Δ rts1Δ double mutant. The Hog1–Gcn5 interaction requires the conserved H2B-T91 residue, which is phosphorylated in vertebrate species. Furthermore, deletion of HOG1 decreases aneuploidy and apoptotic populations in gcn5Δ cells. Together, these results introduce Hog1 as a kinase that functionally opposes Gcn5 and Rts1 in the context of the spindle assembly checkpoint and suggest further kinases may also influence GCN5's functions.

Chromatin instability and protein homeostasis (proteostasis) stress are two well-established hallmarks of aging, which have been considered largely independent of each other. Using microfluidics and single-cell imaging approaches, we observed that, during the replicative aging of Saccharomyces cerevisiae, a challenge to proteostasis occurs specifically in the fraction of cells with decreased stability within the ribosomal DNA (rDNA). A screen of 170 yeast RNA-binding proteins identified ribosomal RNA (rRNA)-binding proteins as the most enriched group that aggregate upon a decrease in rDNA stability induced by inhibition of a conserved lysine deacetylase Sir2. Further, loss of rDNA stability induces age-dependent aggregation of rRNA-binding proteins through aberrant overproduction of rRNAs. These aggregates contribute to age-induced proteostasis decline and limit cellular lifespan. Our findings reveal a mechanism underlying the interconnection between chromatin instability and proteostasis stress and highlight the importance of cell-to-cell variability in aging processes.

Programmed aging in yeast cells: Following the fate of individual yeast cells has revealed aging to be more of a programmable decision process rather than a simple accumulation of deleterious events. Li et al. combined single-cell studies and mathematical modeling to show that yeast cells showed two different forms of aging: one with less ribosomal DNA silencing, in which nucleoli were degraded, and another with less heme accumulation and hemedependent transcription, in which mitochondria were more affected. Overexpression of the lysine deacetylase Sir2, which contributes to ribosomal DNA silencing, led to a third cell-aging fate in which the average life span was extended. If other cells age in similar ways, then this study may provide new ways to consider dynamics of aging and strategies to extend the health span. Science this issue p. 325

Cellular aging is a complex process that involves many interwoven molecular processes. Studies in model organisms have identified many individual genes and factors that have profound effects on lifespan. However, how these genes and factors interact and function collectively to drive the aging process remains unclear. We investigated single-cell aging dynamics throughout the replicative lifespans of S. cerevisiae, and found that isogenic cells diverge towards two aging paths, with distinct phenotypic changes and death forms. We further identified specific molecular pathways driving each aging fate and revealed that these pathways interact and operate dynamically to enable an early-life switch that governs the aging fate decision and the progression towards death. Our work uncovers the interconnected molecular pathways that drives the aging process and opens up the possibility of designing interventions that simultaneously target multiple network nodes, instead of single genes, to more effectively extend the healthspan.

Multiple interdependent mechanisms ensure faithful segregation of chromosomes during cell division. Among these, the spindle assembly checkpoint monitors attachment of spindle microtubules to the centromere of each chromosome whereas the tension-sensing checkpoint monitors the opposing forces between sister chromatid centromeres for proper biorientation. We report here a new function for the deeply conserved Gcn5 acetyltransferase in the centromeric localization of Rts1, a key player in the tension-sensing checkpoint. Rts1 is a regulatory component of Protein Phopshatase 2A, a near universal phosphatase complex, which is recruited to centromeres by the Shugoshin (Sgo) checkpoint component under low-tension conditions to maintain sister chromatid cohesion. We report that loss of Gcn5 disrupts centromeric localization of Rts1. Increased RTS1 dosage robustly suppresses gcn5∆ cell cycle and chromosome segregation defects, including restoration of Rts1 to centromeres. Sgo1’s Rts1-binding function also plays a key role in RTS1 dosage suppression of gcn5∆ phenotypes. Notably, we have identified residues of the centromere histone H3 variant Cse4 that function in these chromosome segregation-related roles of RTS1. Together, these findings expand understanding of the mechanistic roles of Gcn5 and Cse4 in chromosome segregation.

Significance: Aging is an inevitable consequence of living, and with it comes increased morbidity and mortality. Novel approaches to mitigating age-related chronic diseases demand a better understanding of the biology of aging. Studies in model organisms have identified many conserved molecular factors that influence aging. The emerging challenge is to understand how these factors interact and change dynamically to drive aging. Using multidisciplinary technologies, we have revealed a sirtuin-dependent intermittent pattern of chromatin silencing during yeast aging that is crucial for longevity. Our findings highlight the important role of silencing dynamics in aging, which deserves careful consideration when designing schemes to delay or reverse aging by modulating sirtuins and silencing.

Enhancer of Polycomb (EPC) was first identified for its contributions to development in Drosophila and was soon-thereafter purified as a subunit of the NuA4/TIP60 acetyltransferase complex. Since then, EPC has often been left in the shadows as an essential, yet non-catalytic subunit of NuA4/TIP60; however, its deep conservation and disease association make clear that it warrants additional attention. In fact, recent studies in yeast demonstrated that its Enhancer of Polycomb, Epl1, was just as important for gene expression and acetylation as is the catalytic subunit of NuA4. Despite its conservation, studies of EPC have often remained siloed between organisms. Here, our goal is to provide a cohesive view of the current state of the EPC literature as it stands among the major model organisms in which it has been studied. EPC is involved in multiple processes, beginning with its cardinal role in regulating global and targeted histone acetylation. EPC also frequently serves as an important interaction partner in these basic cellular functions, as well as in multicellular development, such as in hematopoiesis and skeletal muscle differentiation, and in human disease. Taken together, a unifying theme from these studies highlights EPC as a critical genomic regulator.