Publication highlights

This work reveals that amino acids, rather than fructose 1,6-bisphosphate, are the relevant cellular regulators of pyruvate kinase M2 (PKM2), a critical node in cancer metabolism. It further elucidates the molecular mechanism of PKM2 regulation by amino acids with a new algorithm that predicts allosteric pathways in proteins, a major and difficult problem in structural biology.

Intestinal homeostasis requires a continuous dialogue between commensal bacteria and intestinal immune cells. Natural Killer T (NKT) cells are a population of CD1d-restricted lipid-reactive lymphocytes contributing to the regulation of mucosal immunity, but the mechanisms underlying this are poorly understood. Here we show that lipid presentation by CD1d+ intestinal dendritic cells and macrophages controls NKT cell function and activation which in turn regulates commensal bacteria and immune cell populations in the gut. These results reveal an NKT cell-dendritic cell crosstalk as a key mechanism for the regulation of intestinal homeostasis.

This study defined the mechanism leading to critically short telomeres in the absence of RTEL1 and showed that telomerase, which extends telomeres in normal cells, is pathological when forks encounter an obstacle within the telomere. We showed that replication forks stall and reverse at persistent t-loops, which creates a pseudo-telomere substrate that is inappropriately stabilised by telomerase. Removing telomerase or blocking replication fork reversal rescued telomere dysfunction in Rtel1 deficient cells. We proposed that when persistent t-loops stall the replisome, telomerase inhibits fork restart, triggering the excision of the t-loop by SLX1/4 and loss of a substantial part of the telomere.

This study was the first to demonstrate that RAD51 paralogues bind to and structurally remodel the pre-synaptic RAD-51-ssDNA filament to a stabilised, “open”, and flexible conformation, which facilitates strand exchange with the template duplex. We showed that RAD51 paralogues act by binding the end of the presynaptic filament, which induces a conformational change that stabilises RAD-51 bound to ssDNA and primes the filament for strand exchange. These observations established for the first time the underlying mechanism of HR stimulation by Rad51 paralogues and revealed a new paradigm for the action of HR mediator proteins.

Evidence suggested that the telomere adopts a lasso-like t-loop configuration, which safeguards chromosome ends from being recognised as DNA double strand breaks. However, the regulation and physiological importance of t-loops in end-protection was uncertain. This study uncovered a phospho-switch in TRF2 that coordinates the timely assembly and disassembly of t-loops during the cell cycle, which protects telomeres from replication stress and an unscheduled DNA damage response. These results were the first to definitively establish the t-loop as a physiologically important structure required to suppress checkpoint activation at telomere ends.

The prevailing view of neural induction in vertebrate embryos had been that cells are initially induced with anterior (forebrain) identity and then caudalising signals convert a proportion to posterior fates (spinal cord). Using chromatin accessibility, to define how cells adopt region-specific neural fates, combined with genetic and biochemical perturbations, we found that contrary to the established model, cells commit to a regional identity before acquiring neural identity. These findings prompt a revision to textbook models of neural induction. The study illustrates our adoption of new genomic methods (ATACseq) to address long-standing questions, and our capacity to productively collaborate with computational biologists.

Here, we described a cryo-EM structure of a retroviral intasome in a functional complex with a nucleosome. The structure revealed a multivalent interface of the viral integration machinery and chromatin, involving both gyres of nucleosomal DNA and histones. Whilst the histone octamer remains intact, the DNA is lifted from its surface to allow for strand transfer at highly preferred integration sites. These data provided a unique snapshot of an enzyme recognizing and acting upon nucleosomal DNA. The structure was the first to illustrate nucleosome flexibility facilitating a biological process and, as such, had far-reaching implications for chromosome biology.

Oesophageal adenocarcinoma shows high genetic heterogeneity making the identification of cancer drivers challenging. We developed a machine learning algorithm to identify cancer drivers in 261 oesophageal adenocarcinomas. Although most predicted drivers were rare or patient-specific, they all perturbed well-known cancer pathways. Using the recurrence of the same pathway perturbations rather than individual genes, we stratified patients into six groups different for their clinical features. We validated experimentally the contribution of these genes to disease progression and revealed acquired dependencies exploitable in therapy. This study described a new way to identify cancer drivers that we have recently further developed for application in precision oncology.

The MCM replicative helicase is loaded onto duplex DNA as a double hexamer. Here we use time-resolved cryo-EM to show that ORC binds to its high affinity binding site to load the first MCM hexamer. ORC then releases this site and it, or another ORC molecule then binds the B2 element, which contains a degenerate ORC binding site. This binding is stabilised by a novel interaction between the Orc6 subunit of ORC and the N-terminus of the MCM hexamer. ORC then recruits and loads the second hexamer by the same mechanism as the first hexamer. We employed newly developed in silico reconstitution approaches to describe the full context of the helicase loading reaction, studied on a near-native, chromatinised origin of replication. This study radically changes our approach to investigating chromosome replication with cryo-EM.

This paper provided the first view of how the inactive MCM double hexamer is converted to two active CMG helicases. We showed MCM remains bound to ADP after loading; firing factors trigger ADP-ATP exchange; ATP rebinding causes double hexamer splitting, initial DNA melting and CMG formation. Active helicases then translocate N-terminus first.

Metastatic melanoma is a lethal disease, in part because of rapid acquisition of resistance to therapy. Using genetically engineered mouse models, we demonstrate that the activating RAC1 P29S mutation, present in up to 5% of melanoma patients, cooperates with BRAF as a driver of melanoma initiation and promotes BRAF inhibitor resistance. The critical RAC1 effector pathway in melanoma is shown to be the transcription factor complex SRF/MRTF, which initiates a switch to a mesenchymal-like state characterized by therapy resistance. Therapeutic targeting of SRF/MRTF may have potential to reverse BRAF inhibitor resistance in melanoma patients bearing the oncogenic RAC1 P29S mutation

KRAS is the most commonly mutated oncogene in human lung cancer, but direct targeting of RAS proteins has proved difficult. A recently developed inhibitor of G12C mutant KRAS protein inhibits lung cancer progression in mouse models but does not provide durable regressions. By studying signalling pathways required for survival of KRAS mutant cells, we demonstrate a strong and selective potentiation of the effects of G12C KRAS inhibitors when mTOR and/or IGF1R are also inhibited. Using mutant specific G12C KRAS inhibitors rather than MEK inhibitors in these combinations is associated with greater specificity and lower toxicity. We propose that adding IGF1R and mTOR inhibitors will increase the impact of G12C KRAS inhibitors in clinical trials.

Protein aggregation drives neuronal death in Parkinson’s disease, although how transition of monomeric protein structures to aggregated forms causes toxicity is unknown. We demonstrate that aggregation of the protein α-synuclein generates beta sheet-rich oligomers, which localise to the mitochondrial inner membrane, where they impair complex I-dependent respiration, induce oxidation of ATP synthase and cause mitochondrial lipid peroxidation. These oxidation events result in opening of the permeability transition pore, triggering mitochondrial swelling, and ultimately cell death. This work highlights how structural conversion of a protein changes its physiological interaction with proteins and lipids, and induces pathology in human cell models of disease.

A key requirement for patterning networks is that the scale of pattern be appropriately matched to the size of the system to be patterned. Through a combination of theory and experiment, we show that failure of the PAR network to scale with cell size restricts stable cell polarity to a specific size range and imposes a minimum cell size threshold for polarity. Experimental alteration of cell size indicates that embryos are sensitive to this size threshold. We thus propose a general strategy by which cells can use intrinsic length scales of patterning networks to enable size-dependent decision making.

This paper is a continuation of our major research theme on how dividing stem cells in the CNS are able to resist environmental stresses that shut down proliferation in most other developing tissues. It reports the first identification, in any species, of lipid droplets as protectors of stem cells. We discovered that hypoxia induces lipid droplets in the neural stem cell niche and that these protect the neural stem cells themselves from damaging polyunsaturated fatty acid (PUFA) peroxidation reactions. This study laid the foundation for our current mechanistic studies into the antioxidant functions of lipid droplets during development and tumorigenesis.

This reports the first identification, in any species, of the microbiome as a key mediator of developmental stress-induced longevity. We found that mild oxidative stress during development robustly increases lifespan via the selective elimination of Acetobacter from the microbiome. This study also highlights that targeted remodelling of the early-life microbiome can provide an efficient strategy for extending healthspan and lifespan.