p53 Regulates the Extent of Fibroblast Proliferation and Fibrosis in Left Ventricle Pressure Overload.

BACKGROUND: Cardiomyopathy is characterized by the pathological accumulation of resident cardiac fibroblasts that deposit ECM (extracellular matrix) and generate a fibrotic scar. However, the mechanisms that control the timing and extent of cardiac fibroblast proliferation and ECM production are not known, hampering the development of antifibrotic strategies to prevent heart failure. METHODS: We used the Tcf21 (transcription factor 21)MerCreMer mouse line for fibroblast-specific lineage tracing and p53 (tumor protein p53) gene deletion. We characterized cardiac physiology and used single-cell RNA-sequencing and in vitro studies to investigate the p53-dependent mechanisms regulating cardiac fibroblast cell cycle and fibrosis in left ventricular pressure overload induced by transaortic constriction. RESULTS: Cardiac fibroblast proliferation occurs primarily between days 7 and 14 following transaortic constriction in mice, correlating with alterations in p53-dependent gene expression. p53 deletion in fibroblasts led to a striking accumulation of Tcf21-lineage cardiac fibroblasts within the normal proliferative window and precipitated a robust fibrotic response to left ventricular pressure overload. However, excessive interstitial and perivascular fibrosis does not develop until after cardiac fibroblasts exit the cell cycle. Single-cell RNA sequencing revealed p53 null fibroblasts unexpectedly express lower levels of genes encoding important ECM proteins while they exhibit an inappropriately proliferative phenotype. in vitro studies establish a role for p53 in suppressing the proliferative fibroblast phenotype, which facilitates the expression and secretion of ECM proteins. Importantly, Cdkn2a (cyclin-dependent kinase inhibitor 2a) expression and the p16Ink4a-retinoblastoma cell cycle control pathway is induced in p53 null cardiac fibroblasts, which may eventually contribute to cell cycle exit and fulminant scar formation. CONCLUSIONS: This study reveals a mechanism regulating cardiac fibroblast accumulation and ECM secretion, orchestrated in part by p53-dependent cell cycle control that governs the timing and extent of fibrosis in left ventricular pressure overload.

A riboswitch separated from its ribosome-binding site still regulates translation.

Riboswitches regulate downstream gene expression by binding cellular metabolites. Regulation of translation initiation by riboswitches is posited to occur by metabolite-mediated sequestration of the Shine-Dalgarno sequence (SDS), causing bypass by the ribosome. Recently, we solved a co-crystal structure of a prequeuosine1-sensing riboswitch from Carnobacterium antarcticum that binds two metabolites in a single pocket. The structure revealed that the second nucleotide within the gene-regulatory SDS, G34, engages in a crystal contact, obscuring the molecular basis of gene regulation. Here, we report a co-crystal structure wherein C10 pairs with G34. However, molecular dynamics simulations reveal quick dissolution of the pair, which fails to reform. Functional and chemical probing assays inside live bacterial cells corroborate the dispensability of the C10-G34 pair in gene regulation, leading to the hypothesis that the compact pseudoknot fold is sufficient for translation attenuation. Remarkably, the C. antarcticum aptamer retained significant gene-regulatory activity when uncoupled from the SDS using unstructured spacers up to 10 nucleotides away from the riboswitch-akin to steric-blocking employed by sRNAs. Accordingly, our work reveals that the RNA fold regulates translation without SDS sequestration, expanding known riboswitch-mediated gene-regulatory mechanisms. The results infer that riboswitches exist wherein the SDS is not embedded inside a stable fold.

Analysis of a preQ1-I riboswitch in effector-free and bound states reveals a metabolite-programmed nucleobase-stacking spine that controls gene regulation.

Riboswitches are structured RNA motifs that recognize metabolites to alter the conformations of downstream sequences, leading to gene regulation. To investigate this molecular framework, we determined crystal structures of a preQ1-I riboswitch in effector-free and bound states at 2.00 Å and 2.65 Å-resolution. Both pseudoknots exhibited the elusive L2 loop, which displayed distinct conformations. Conversely, the Shine-Dalgarno sequence (SDS) in the S2 helix of each structure remained unbroken. The expectation that the effector-free state should expose the SDS prompted us to conduct solution experiments to delineate environmental changes to specific nucleobases in response to preQ1. We then used nudged elastic band computational methods to derive conformational-change pathways linking the crystallographically-determined effector-free and bound-state structures. Pathways featured: (i) unstacking and unpairing of L2 and S2 nucleobases without preQ1-exposing the SDS for translation and (ii) stacking and pairing L2 and S2 nucleobases with preQ1-sequestering the SDS. Our results reveal how preQ1 binding reorganizes L2 into a nucleobase-stacking spine that sequesters the SDS, linking effector recognition to biological function. The generality of stacking spines as conduits for effector-dependent, interdomain communication is discussed in light of their existence in adenine riboswitches, as well as the turnip yellow mosaic virus ribosome sensor.

Temporal Single Cell Analysis of Leukemia Microenvironment Identifies Taurine-Taurine Transporter Axis as a Key Regulator of Myeloid Leukemia.

Signals from the microenvironment are known to be critical for development, sustaining adult stem cells, and for oncogenic progression. While candidate niche-driven signals that can promote cancer progression have been identified1-6, concerted efforts to comprehensively map microenvironmental ligands for cancer stem cell specific surface receptors have been lacking. Here, we use temporal single cell RNA-sequencing to identify molecular cues from the bone marrow stromal niche that engage leukemia stem cells (LSC) during oncogenic progression. We integrate these data with our RNA-seq analysis of human LSCs from distinct aggressive myeloid cancer subtypes and our CRISPR based in vivo LSC dependency map7 to develop a temporal receptor-ligand interactome essential for disease progression. These analyses identify the taurine transporter (TauT)-taurine axis as a critical dependency of myeloid malignancies. We show that taurine production is restricted to the osteolineage population during cancer initiation and expansion. Inhibiting taurine synthesis in osteolineage cells impairs LSC growth and survival. Our experiments with the TauT genetic loss of function murine model indicate that its loss significantly impairs the progression of aggressive myeloid leukemias in vivo by downregulating glycolysis. Further, TauT inhibition using a small molecule strongly impairs the growth and survival of patient derived myeloid leukemia cells. Finally, we show that TauT inhibition can synergize with the clinically approved oxidative phosphorylation inhibitor venetoclax8, 9 to block the growth of primary human leukemia cells. Given that aggressive myeloid leukemias continue to be refractory to current therapies and have poor prognosis, our work indicates targeting the taurine transporter may be of therapeutic significance. Collectively, our data establishes a temporal landscape of stromal signals during cancer progression and identifies taurine-taurine transporter signaling as an important new regulator of myeloid malignancies.

Characterizing Neonatal Heart Maturation, Regeneration, and Scar Resolution Using Spatial Transcriptomics.

The neonatal mammalian heart exhibits a remarkable regenerative potential, which includes fibrotic scar resolution and the generation of new cardiomyocytes. To investigate the mechanisms facilitating heart repair after apical resection in neonatal mice, we conducted bulk and spatial transcriptomic analyses at regenerative and non-regenerative timepoints. Importantly, spatial transcriptomics provided near single-cell resolution, revealing distinct domains of atrial and ventricular myocardium that exhibit dynamic phenotypic alterations during postnatal heart maturation. Spatial transcriptomics also defined the cardiac scar, which transitions from a proliferative to secretory phenotype as the heart loses regenerative potential. The resolving scar is characterized by spatially and temporally restricted programs of inflammation, epicardium expansion and extracellular matrix production, metabolic reprogramming, lipogenic scar extrusion, and cardiomyocyte restoration. Finally, this study revealed the emergence of a regenerative border zone defined by immature cardiomyocyte markers and the robust expression of Sprr1a. Taken together, our study defines the spatially and temporally restricted gene programs that underlie neonatal heart regeneration and provides insight into cardio-restorative mechanisms supporting scar resolution.

Coordination of endothelial cell positioning and fate specification by the epicardium.

The organization of an integrated coronary vasculature requires the specification of immature endothelial cells (ECs) into arterial and venous fates based on their localization within the heart. It remains unclear how spatial information controls EC identity and behavior. Here we use single-cell RNA sequencing at key developmental timepoints to interrogate cellular contributions to coronary vessel patterning and maturation. We perform transcriptional profiling to define a heterogenous population of epicardium-derived cells (EPDCs) that express unique chemokine signatures. We identify a population of Slit2+ EPDCs that emerge following epithelial-to-mesenchymal transition (EMT), which we term vascular guidepost cells. We show that the expression of guidepost-derived chemokines such as Slit2 are induced in epicardial cells undergoing EMT, while mesothelium-derived chemokines are silenced. We demonstrate that epicardium-specific deletion of myocardin-related transcription factors in mouse embryos disrupts the expression of key guidance cues and alters EPDC-EC signaling, leading to the persistence of an immature angiogenic EC identity and inappropriate accumulation of ECs on the epicardial surface. Our study suggests that EC pathfinding and fate specification is controlled by a common mechanism and guided by paracrine signaling from EPDCs linking epicardial EMT to EC localization and fate specification in the developing heart.