Anatomically and Functionally Distinct Lung Mesenchymal Populations Marked by Lgr5 and Lgr6

The diversity of mesenchymal cell types in the lung that influence epithelial homeostasis and regeneration is poorly defined. We used genetic lineage tracing, single-cell RNA sequencing, and organoid culture approaches to show that Lgr5 and Lgr6, well-known markers of stem cells in epithelial tissues, are markers of mesenchymal cells in the adult lung. Lgr6 + cells comprise a subpopulation of smooth muscle cells surrounding airway epithelia and promote airway differentiation of epithelial progenitors via Wnt-Fgf10 cooperation. Genetic ablation of Lgr6 + cells impairs airway injury repair in vivo. Distinct Lgr5 + cells are located in alveolar compartments and are sufficient to promote alveolar differentiation of epithelial progenitors through Wnt activation. Modulating Wnt activity altered differentiation outcomes specified by mesenchymal cells. This identification of region- and lineage-specific crosstalk between epithelium and their neighboring mesenchymal partners provides new understanding of how different cell types are maintained in the adult lung. Keywords: mesenchymal cells; bronchiolar epithelium; alveolar epithelium; lung stem cells; lung; differentiation; niche; Wnt signalin

\u3cem\u3eLkb1\u3c/em\u3e Inactivation Drives Lung Cancer Lineage Switching Governed by Polycomb Repressive Complex 2

Adenosquamous lung tumours, which are extremely poor prognosis, may result from cellular plasticity. Here, we demonstrate lineage switching of KRAS+ lung adenocarcinomas (ADC) to squamous cell carcinoma (SCC) through deletion of Lkb1 (Stk11) in autochthonous and transplant models. Chromatin analysis reveals loss of H3K27me3 and gain of H3K27ac and H3K4me3 at squamous lineage genes, including Sox2, ΔNp63 and Ngfr. SCC lesions have higher levels of the H3K27 methyltransferase EZH2 than the ADC lesions, but there is a clear lack of the essential Polycomb Repressive Complex 2 (PRC2) subunit EED in the SCC lesions. The pattern of high EZH2, but low H3K27me3 mark, is also prevalent in human lung SCC and SCC regions within ADSCC tumours. Using FACS-isolated populations, we demonstrate that bronchioalveolar stem cells and club cells are the likely cells-of-origin for SCC transitioned tumours. These findings shed light on the epigenetics and cellular origins of lineage-specific lung tumours

A telomere-dedicated RPA complex is essential for the replication of duplex telomeric DNA

Telomeres are the ends of eukaryotic chromosomes. They play a critical role in genome maintenance since impairment of telomeric homeostasis inevitably mediates genomic catastrophe, leading to cell death or, more dangerously, cancerous transformations. It is therefore imperative for every eukaryotic cell to maintain and preserve telomere integrity. In budding yeast, a telomeric dedicated RPA complex (the t-RPA complex), formed by the three essential proteins Cdc13, Stn1 and Ten1 sits at the central node of telomere homeostasis. According to the current model, the trimer, binding tightly to the telomeric single stranded repeats, ensures the elongation of the telomeres by recruiting the cellular retro- transcriptase telomerase. On the other hand, it also supposedly involved in protecting telomeres from uncontrolled resection through a still poorly understood mechanism. This dissertation describes my efforts in using the homology between the t-RPA and the original RPA complex, to decipher the overall structure of the t-RPA complex: first pinpointing the molecular details of the Ten1-Stn1 interaction and then identifying a domain of the t-RPA complex that specifically evolved to perform a new telomeric-specific function. Subsequently, the attentive review of a key temperature sensitive allele of Cdc13 together with a number of published observations, casted significant doubts on the current interpretation of the role of the t-RPA complex at telomeres, forcing the conception of a new model for telomere maintenance where the essential function of the t-RPA complex is not the protection of chromosome ends, but is actually the efficient replication of the telomere as a telomeric specific complex that becomes part of the replisome as the replication fork advances through the duplex telomeric DNA. The last sections of this manuscript describe the experimental approaches used successfully test the new hypothesis, leading to the confirmation of the t-RPA complex as a telomere dedicated replicative factor and fundamentally changing the paradigm of telomere homeostasi

Structure Prediction-Driven Genetics in Saccharomyces cerevisiae Identifies an Interface Between the t-RPA Proteins Stn1 and Ten1

In Saccharomyces cerevisiae, Cdc13, Stn1, and Ten1 are essential for both chromosome capping and telomere length homeostasis. These three proteins have been proposed to perform their roles at chromosome termini as a telomere-dedicated t-RPA complex, on the basis of several parallels with the conventional RPA complex. In this study, we have used several approaches to test whether a predicted α-helix in the N-terminal domain of the S. cerevisiae Stn1 protein is required for formation of the proposed t-RPA complex, in a manner analogous to the comparable helix in Rpa2. Analysis of a panel of Rpa2–OBStn1 chimeras indicates that whether a chimeric protein contains the Rpa2 or Stn1 version of this α-helix dictates its ability to function in place of Rpa2 or Stn1, respectively. In addition, mutations introduced into a hydrophobic surface of the predicted Stn1 α-helix eliminated association with Ten1. Strikingly, allele-specific suppression of a stn1 mutation in this helix (stn1–L164D) by a ten1 mutation (ten1–D138Y) resulted in a restored Stn1–Ten1 interaction, supporting the identification of a Stn1–Ten1 interface. We conclude that Stn1 interacts with Ten1 through an α-helix, in a manner analogous to the interaction between the comparable subunits of the RPA complex

pH-gated succinate secretion regulates muscle remodeling in response to exercise

In response to skeletal muscle contraction during exercise, paracrine factors coordinate tissue remodeling, which underlies this healthy adaptation. Here we describe a pH-sensing metabolite signal that initiates muscle remodeling through exercise. In mice and humans, exercising skeletal muscle releases the mitochondrial metabolite succinate into the local interstitium and circulation. Selective secretion of succinate is facilitated by its transient protonation, which occurs upon muscle cell acidification. In the protonated monocarboxylic form, succinate is rendered a transport substrate for monocarboxylate transporter 1, which facilitates pH-gated release. Upon secretion, succinate signals via its cognate receptor SUCNR1 in non-myofibrillar cells in muscle tissue to control muscle-remodeling transcriptional programs. This succinate-SUCNR1 signaling is required for paracrine regulation of muscle innervation, muscle matrix remodeling, and muscle strength in response to exercise training. In sum, we define a bioenergetic sensor in muscle that utilizes intracellular pH and succinate to coordinate tissue adaptation to exercise

BRG1 Loss Predisposes Lung Cancers to Replicative Stress and ATR Dependency

Inactivation of SMARCA4/BRG1, the core ATPase subunit of mammalian SWI/SNF complexes, occurs at very high frequencies in non-small cell lung cancers (NSCLC). There are no targeted therapies for this subset of lung cancers, nor is it known how mutations in BRG1 contribute to lung cancer progression. Using a combination of gain- and loss-of-function approaches, we demonstrate that deletion of BRG1 in lung cancer leads to activation of replication stress responses. Single-molecule assessment of replication fork dynamics in BRG1-deficient cells revealed increased origin firing mediated by the prelicensing protein, CDC6. Quantitative mass spectrometry and coimmunoprecipitation assays showed that BRG1-containing SWI/SNF complexes interact with RPA complexes. Finally, BRG1-deficient lung cancers were sensitive to pharmacologic inhibition of ATR. These findings provide novel mechanistic insight into BRG1-mutant lung cancers and suggest that their dependency on ATR can be leveraged therapeutically and potentially expanded to BRG1-mutant cancers in other tissues

Lkb1 inactivation drives lung cancer lineage switching governed by Polycomb Repressive Complex 2

Adenosquamous lung tumours, which are extremely poor prognosis, may result from cellular plasticity. Here, we demonstrate lineage switching of KRAS+ lung adenocarcinomas (ADC) to squamous cell carcinoma (SCC) through deletion of Lkb1 (Stk11) in autochthonous and transplant models. Chromatin analysis reveals loss of H3K27me3 and gain of H3K27ac and H3K4me3 at squamous lineage genes, including Sox2, ΔNp63 and Ngfr. SCC lesions have higher levels of the H3K27 methyltransferase EZH2 than the ADC lesions, but there is a clear lack of the essential Polycomb Repressive Complex 2 (PRC2) subunit EED in the SCC lesions. The pattern of high EZH2, but low H3K27me3 mark, is also prevalent in human lung SCC and SCC regions within ADSCC tumours. Using FACS-isolated populations, we demonstrate that bronchioalveolar stem cells and club cells are the likely cells-of-origin for SCC transitioned tumours. These findings shed light on the epigenetics and cellular origins of lineage-specific lung tumours