Houseley Group

Although lifespan has long been the focus of ageing research, preventing functional decline late in life is a more pressing societal need. Here, we investigate the basis of senescence and declining fitness during replicative ageing in budding yeast, and describe a metabolic perturbation that preserves late-life fitness even on an unrestricted glucose diet. We show that senescence can be prevented by constitutive activation of AMPK, though only for approximately half the ageing population, and use genetic and functional assays to link this heterogeneous response with differences in cytosolic acetyl coenzyme A (Acetyl-CoA) metabolism. In one class of ageing cell, AMPK activity maintains fitness late in life through pathways that transport cytosolic Acetyl-CoA into mitochondria, but AMPK also inhibits fatty acid synthesis which leads to lipid starvation in the other class of ageing cell. Therefore, AMPK activity has both positive and negative effects, but we show that constitutive AMPK activity uncoupled from fatty acid synthesis inhibition (the A2A mutant) suppresses senescence and maintains fitness in both classes of ageing cell. Our findings support a model in which lipid starvation and excess Acetyl-CoA availability are major drivers of senescence in replicatively aged wild-type yeast. This work shows that ageing is not intrinsically associated with declining fitness, at least in yeast, and that re-engineering highly conserved metabolic pathways allows fitness to be preserved very late in life.

Replication stress poses a major threat to genome integrity, yet how higher-order chromatin organization contributes to replication fork protection remains unclear. Here we show that replication stress induces the formation of transient chromatin loops that enclose de novo heterochromatin-enriched stalled replication forks. Stressed forks preferentially stall at convergent CTCF motifs, triggering stress-dependent CTCF enrichment that constrains loop extrusion and stabilizes these structures. Loop stabilization requires both CTCF anchoring and G9a-dependent heterochromatin (trimethylation of Lys9 of histone H3 (H3K9me3)) deposition on nascent DNA within the loop body. These loops function as protective scaffolds that shield stalled and reversed forks from degradation by multiple nucleases. By contrast, combined loss of stress-induced heterochromatin and CTCF enrichment destabilizes the loop scaffold, exposing multiple entry points for nucleolytic attack and resulting in extensive nascent-strand degradation through mechanisms distinct from classical fork-reversal-dependent pathways. This protective architecture is similarly critical in BRCA2-deficient cells, in which replication-stress-associated loops predominantly safeguard replication initiation zones, while nascent DNA outside these loops undergoes massive degradation and remains highly susceptible to mutations. Our study elucidates the fundamental role of replication-stress-induced three-dimensional genome reorganization in preserving replication fork stability, thereby mitigating mutagenesis and genomic instability.

TrAEL-seq is a robust method for profiling DNA replication genome-wide that works in unsynchronized cells and does not require drugs or nucleotide analogues. Here, we provide an updated method for TrAEL-seq that improves sample quality and includes multiplexing of up to six samples which dramatically improves throughput, and we validate TrAEL-seq in multiple mammalian cell lines. The updated protocol is straightforward and robust yet provides excellent resolution comparable to OK-seq in mammalian cell samples. High resolution replication profiles can be obtained across large panels of samples and in dynamic systems, for example during the progressive onset of oncogene induced senescence. In addition to mapping zones where replication initiates and terminates, TrAEL-seq is sensitive to replication fork speed, revealing effects of both transcription and proximity to replication Initiation Zones on fork progression. Although forks move more slowly through transcribed regions, this does not have a significant impact on the broader dynamics of replication fork progression, and instead replication forks accelerate across the first ∼1 Mb of travel irrespective of local transcriptional activity. We propose that this is a consequence of fewer replication forks being active later in S-phase when these distal regions replicate and there being less competition for replication factors.