Stem cells tightly regulate dead cell clearance to maintain tissue fitness.

Billions of cells are eliminated daily from our bodies1-4. Although macrophages and dendritic cells are dedicated to migrating and engulfing dying cells and debris, many epithelial and mesenchymal tissue cells can digest nearby apoptotic corpses1-4. How these non-motile, non-professional phagocytes sense and eliminate dying cells while maintaining their normal tissue functions is unclear. Here we explore the mechanisms that underlie their multifunctionality by exploiting the cyclical bouts of tissue regeneration and degeneration during hair cycling. We show that hair follicle stem cells transiently unleash phagocytosis at the correct time and place through local molecular triggers that depend on both lipids released by neighbouring apoptotic corpses and retinoids released by healthy counterparts. We trace the heart of this dual ligand requirement to RARγ-RXRα, whose activation enables tight regulation of apoptotic cell clearance genes and provides an effective, tunable mechanism to offset phagocytic duties against the primary stem cell function of preserving tissue integrity during homeostasis. Finally, we provide functional evidence that hair follicle stem cell-mediated phagocytosis is not simply redundant with professional phagocytes but rather has clear benefits to tissue fitness. Our findings have broad implications for other non-motile tissue stem or progenitor cells that encounter cell death in an immune-privileged niche.

Stem cells tightly regulate dead cell clearance to maintain tissue fitness.

Macrophages and dendritic cells have long been appreciated for their ability to migrate to and engulf dying cells and debris, including some of the billions of cells that are naturally eliminated from our body daily. However, a substantial number of these dying cells are cleared by 'non-professional phagocytes', local epithelial cells that are critical to organismal fitness. How non-professional phagocytes sense and digest nearby apoptotic corpses while still performing their normal tissue functions is unclear. Here, we explore the molecular mechanisms underlying their multifunctionality. Exploiting the cyclical bouts of tissue regeneration and degeneration during the hair cycle, we show that stem cells can transiently become non-professional phagocytes when confronted with dying cells. Adoption of this phagocytic state requires both local lipids produced by apoptotic corpses to activate RXRα, and tissue-specific retinoids for RARγ activation. This dual factor dependency enables tight regulation of the genes requisite to activate phagocytic apoptotic clearance. The tunable phagocytic program we describe here offers an effective mechanism to offset phagocytic duties against the primary stem cell function of replenishing differentiated cells to preserve tissue integrity during homeostasis. Our findings have broad implications for other non-motile stem or progenitor cells which experience cell death in an immune-privileged niche.

Stress-induced growth rate reduction restricts metabolic resource utilization to modulate osmo-adaptation time.

A near-constant feature of stress responses is a downregulation or arrest of the cell cycle, resulting in transient growth slowdown. To investigate the role of growth slowdown in the hyperosmotic shock response of S. cerevisiae, we perturbed the G1/S checkpoint protein Sic1 to enable osmo-stress response activation with diminished growth slowdown. We document that in this mutant, adaptation to stress is accelerated rather than delayed. This accelerated recovery of the mutant proceeds by liquidation of internal glycogen stores, which are then shunted into the osmo-shock response. Therefore, osmo-adaptation in wild-type cells is delayed because growth slowdown prevents full accessibility to cellular glycogen stores. However, faster adaptation comes at the cost of acute sensitivity to subsequent osmo-stresses. We suggest that stress-induced growth slowdown acts as an arbiter to regulate the resources devoted to osmo-shock, balancing short-term adaptation with long-term robustness.

Orthogonal control of mean and variability of endogenous genes in a human cell line.

Stochastic fluctuations at the transcriptional level contribute to isogenic cell-to-cell heterogeneity in mammalian cell populations. However, we still have no clear understanding of the repercussions of this heterogeneity, given the lack of tools to independently control mean expression and variability of a gene. Here, we engineer a synthetic circuit to modulate mean expression and heterogeneity of transgenes and endogenous human genes. The circuit, a Tunable Noise Rheostat (TuNR), consists of a transcriptional cascade of two inducible transcriptional activators, where the output mean and variance can be modulated by two orthogonal small molecule inputs. In this fashion, different combinations of the inputs can achieve the same mean but with different population variability. With TuNR, we achieve low basal expression, over 1000-fold expression of a transgene product, and up to 7-fold induction of the endogenous gene NGFR. Importantly, for the same mean expression level, we are able to establish varying degrees of heterogeneity in expression within an isogenic population, thereby decoupling gene expression noise from its mean. TuNR is therefore a modular tool that can be used in mammalian cells to enable direct interrogation of the implications of cell-to-cell variability.

Assembly of Genetic Circuits with the Mammalian ToolKit.

The ability to rapidly assemble and prototype cellular circuits is vital for biological research and its applications in biotechnology and medicine. The Mammalian ToolKit (MTK) is a Golden Gate-based cloning toolkit for fast, reproducible and versatile assembly of large DNA vectors and their implementation in mammalian models. The MTK consists of a curated library of characterized, modular parts that can be assembled into transcriptional units and further weaved into complex circuits. These circuits are easily repurposed and introduced in mammalian cells by different methods.

A Toolkit for Rapid Modular Construction of Biological Circuits in Mammalian Cells.

The ability to rapidly assemble and prototype cellular circuits is vital for biological research and its applications in biotechnology and medicine. Current methods for the assembly of mammalian DNA circuits are laborious, slow, and expensive. Here we present the Mammalian ToolKit (MTK), a Golden Gate-based cloning toolkit for fast, reproducible, and versatile assembly of large DNA vectors and their implementation in mammalian models. The MTK consists of a curated library of characterized, modular parts that can be assembled into transcriptional units and further weaved into complex circuits. We showcase the capabilities of the MTK by using it to generate single-integration landing pads, create and deliver libraries of protein variants and sgRNAs, and iterate through dCas9-based prototype circuits. As a biological proof of concept, we demonstrate how the MTK can speed the generation of noninfectious viral circuits to enable rapid testing of pharmacological inhibitors of emerging viruses that pose a major threat to human health.

Determination of ubiquitin fitness landscapes under different chemical stresses in a classroom setting.

Ubiquitin is essential for eukaryotic life and varies in only 3 amino acid positions between yeast and humans. However, recent deep sequencing studies indicate that ubiquitin is highly tolerant to single mutations. We hypothesized that this tolerance would be reduced by chemically induced physiologic perturbations. To test this hypothesis, a class of first year UCSF graduate students employed deep mutational scanning to determine the fitness landscape of all possible single residue mutations in the presence of five different small molecule perturbations. These perturbations uncover 'shared sensitized positions' localized to areas around the hydrophobic patch and the C-terminus. In addition, we identified perturbation specific effects such as a sensitization of His68 in HU and a tolerance to mutation at Lys63 in DTT. Our data show how chemical stresses can reduce buffering effects in the ubiquitin proteasome system. Finally, this study demonstrates the potential of lab-based interdisciplinary graduate curriculum.