Microbial RNAs Pressure Piezo1 to Respond.

Serotonin production by enterochromaffin cells (ECs) is microbiota-dependent, but the mechanism of this is unknown. In this issue of Cell, Sugisawa et al. demonstrate that Piezo1 in ECs senses single-strand RNA (ssRNA) from intestinal microbiota to promote serotonin production. Deletion of Piezo1 in intestinal epithelium promotes bone formation, decreases peristalsis, and protects from colitis because of decreased serotonin.

Single-Cell Profiling of Ebola Virus Disease In Vivo Reveals Viral and Host Dynamics.

Ebola virus (EBOV) causes epidemics with high mortality yet remains understudied due to the challenge of experimentation in high-containment and outbreak settings. Here, we used single-cell transcriptomics and CyTOF-based single-cell protein quantification to characterize peripheral immune cells during EBOV infection in rhesus monkeys. We obtained 100,000 transcriptomes and 15,000,000 protein profiles, finding that immature, proliferative monocyte-lineage cells with reduced antigen-presentation capacity replace conventional monocyte subsets, while lymphocytes upregulate apoptosis genes and decline in abundance. By quantifying intracellular viral RNA, we identify molecular determinants of tropism among circulating immune cells and examine temporal dynamics in viral and host gene expression. Within infected cells, EBOV downregulates STAT1 mRNA and interferon signaling, and it upregulates putative pro-viral genes (e.g., DYNLL1 and HSPA5), nominating pathways the virus manipulates for its replication. This study sheds light on EBOV tropism, replication dynamics, and elicited immune response and provides a framework for characterizing host-virus interactions under maximum containment.

ATG8-Binding UIM Proteins Define a New Class of Autophagy Adaptors and Receptors.

During autophagy, vesicle dynamics and cargo recruitment are driven by numerous adaptors and receptors that become tethered to the phagophore through interactions with lipidated ATG8/LC3 decorating the expanding membrane. Most currently described ATG8-binding proteins exploit a well-defined ATG8-interacting motif (AIM, or LC3-interacting region [LIR]) that contacts a hydrophobic patch on ATG8 known as the LIR/AIM docking site (LDS). Here we describe a new class of ATG8 interactors that exploit ubiquitin-interacting motif (UIM)-like sequences for high-affinity binding to an alternative ATG8 interaction site. Assays with candidate UIM-containing proteins together with unbiased screens identified a large collection of UIM-based ATG8 interactors in plants, yeast, and humans. Analysis of a subset also harboring ubiquitin regulatory X (UBX) domains revealed a role for UIM-directed autophagy in clearing non-functional CDC48/p97 complexes, including some impaired in human disease. With this new class of adaptors and receptors, we greatly extend the reach of selective autophagy and identify new factors regulating autophagic vesicle dynamics.

A Growth-Based Framework for Leaf Shape Development and Diversity.

How do genes modify cellular growth to create morphological diversity? We study this problem in two related plants with differently shaped leaves: Arabidopsis thaliana (simple leaf shape) and Cardamine hirsuta (complex shape with leaflets). We use live imaging, modeling, and genetics to deconstruct these organ-level differences into their cell-level constituents: growth amount, direction, and differentiation. We show that leaf shape depends on the interplay of two growth modes: a conserved organ-wide growth mode that reflects differentiation; and a local, directional mode that involves the patterning of growth foci along the leaf edge. Shape diversity results from the distinct effects of two homeobox genes on these growth modes: SHOOTMERISTEMLESS broadens organ-wide growth relative to edge-patterning, enabling leaflet emergence, while REDUCED COMPLEXITY inhibits growth locally around emerging leaflets, accentuating shape differences created by patterning. We demonstrate the predictivity of our findings by reconstructing key features of C. hirsuta leaf morphology in A. thaliana. VIDEO ABSTRACT.

Embryonic macrophages function during early life to determine invariant natural killer T cell levels at barrier surfaces.

It is increasingly recognized that immune development within mucosal tissues is under the control of environmental factors during early life. However, the cellular mechanisms that underlie such temporally and regionally restrictive governance of these processes are unclear. Here, we uncover an extrathymic pathway of immune development within the colon that is controlled by embryonic but not bone marrow-derived macrophages, which determines the ability of these organs to receive invariant natural killer T (iNKT) cells and allow them to establish local residency. Consequently, early-life perturbations of fetal-derived macrophages result in persistent decreases of mucosal iNKT cells and is associated with later-life susceptibility or resistance to iNKT cell-associated mucosal disorders. These studies uncover a host developmental program orchestrated by ontogenically distinct macrophages that is regulated by microbiota, and they reveal an important postnatal function of macrophages that emerge in fetal life.

Dietary and Microbial Oxazoles Induce Intestinal Inflammation by Modulating Aryl Hydrocarbon Receptor Responses.

Genome-wide association studies have identified risk loci associated with the development of inflammatory bowel disease, while epidemiological studies have emphasized that pathogenesis likely involves host interactions with environmental elements whose source and structure need to be defined. Here, we identify a class of compounds derived from dietary, microbial, and industrial sources that are characterized by the presence of a five-membered oxazole ring and induce CD1d-dependent intestinal inflammation. We observe that minimal oxazole structures modulate natural killer T cell-dependent inflammation by regulating lipid antigen presentation by CD1d on intestinal epithelial cells (IECs). CD1d-restricted production of interleukin 10 by IECs is limited through activity of the aryl hydrocarbon receptor (AhR) pathway in response to oxazole induction of tryptophan metabolites. As such, the depletion of the AhR in the intestinal epithelium abrogates oxazole-induced inflammation. In summary, we identify environmentally derived oxazoles as triggers of CD1d-dependent intestinal inflammatory responses that occur via activation of the AhR in the intestinal epithelium.

Low-Affinity Binding Sites and the Transcription Factor Specificity Paradox in Eukaryotes.

Eukaryotic transcription factors (TFs) from the same structural family tend to bind similar DNA sequences, despite the ability of these TFs to execute distinct functions in vivo. The cell partly resolves this specificity paradox through combinatorial strategies and the use of low-affinity binding sites, which are better able to distinguish between similar TFs. However, because these sites have low affinity, it is challenging to understand how TFs recognize them in vivo. Here, we summarize recent findings and technological advancements that allow for the quantification and mechanistic interpretation of TF recognition across a wide range of affinities. We propose a model that integrates insights from the fields of genetics and cell biology to provide further conceptual understanding of TF binding specificity. We argue that in eukaryotes, target specificity is driven by an inhomogeneous 3D nuclear distribution of TFs and by variation in DNA binding affinity such that locally elevated TF concentration allows low-affinity binding sites to be functional.

Inflammatory bowel disease.

Insights into inflammatory bowel disease (IBD) are advancing rapidly owing to immunologic investigations of a plethora of animal models of intestinal inflammation, ground-breaking advances in the interrogation of diseases that are inherited as complex genetic traits, and the development of culture-independent methods to define the composition of the intestinal microbiota. These advances are bringing a deeper understanding to the genetically determined interplay between the commensal microbiota, intestinal epithelial cells, and the immune system and the manner in which this interplay might be modified by relevant environmental factors in the pathogenesis of IBD. This review examines these interactions and, where possible, potential lessons from IBD-directed, biologic therapies that may allow for elucidation of pathways that are central to disease pathogenesis in humans.

Molecular basis of calcium signaling in lymphocytes: STIM and ORAI.

Ca(2+) entry into cells of the peripheral immune system occurs through highly Ca(2+)-selective channels known as CRAC (calcium release-activated calcium) channels. CRAC channels are a very well-characterized example of store-operated Ca(2+) channels, so designated because they open when the endoplasmic reticulum (ER) Ca(2+) store becomes depleted. Physiologically, Ca(2+) is released from the ER lumen into the cytoplasm when activated receptors couple to phospholipase C and trigger production of the second messenger inositol 1,4,5-trisphosphate (IP(3)). IP(3) binds to IP(3) receptors in the ER membrane and activates Ca(2+) release. The proteins STIM and ORAI were discovered through limited and genome-wide RNAi screens, respectively, performed in Drosophila cells and focused on identifying modulators of store-operated Ca(2+) entry. STIM1 and STIM2 sense the depletion of ER Ca(2+) stores, whereas ORAI1 is a pore subunit of the CRAC channel. In this review, we discuss selected aspects of Ca(2+) signaling in cells of the immune system, focusing on the roles of STIM and ORAI proteins in store-operated Ca(2+) entry.

Endoplasmic reticulum stress in the intestinal epithelium initiates purine metabolite synthesis and promotes Th17 cell differentiation in the gut.

Intestinal IL-17-producing T helper (Th17) cells are dependent on adherent microbes in the gut for their development. However, how microbial adherence to intestinal epithelial cells (IECs) promotes Th17 cell differentiation remains enigmatic. Here, we found that Th17 cell-inducing gut bacteria generated an unfolded protein response (UPR) in IECs. Furthermore, subtilase cytotoxin expression or genetic removal of X-box binding protein 1 (Xbp1) in IECs caused a UPR and increased Th17 cells, even in antibiotic-treated or germ-free conditions. Mechanistically, UPR activation in IECs enhanced their production of both reactive oxygen species (ROS) and purine metabolites. Treating mice with N-acetyl-cysteine or allopurinol to reduce ROS production and xanthine, respectively, decreased Th17 cells that were associated with an elevated UPR. Th17-related genes also correlated with ER stress and the UPR in humans with inflammatory bowel disease. Overall, we identify a mechanism of intestinal Th17 cell differentiation that emerges from an IEC-associated UPR.

Project DRIVE: A Compendium of Cancer Dependencies and Synthetic Lethal Relationships Uncovered by Large-Scale, Deep RNAi Screening.

Elucidation of the mutational landscape of human cancer has progressed rapidly and been accompanied by the development of therapeutics targeting mutant oncogenes. However, a comprehensive mapping of cancer dependencies has lagged behind and the discovery of therapeutic targets for counteracting tumor suppressor gene loss is needed. To identify vulnerabilities relevant to specific cancer subtypes, we conducted a large-scale RNAi screen in which viability effects of mRNA knockdown were assessed for 7,837 genes using an average of 20 shRNAs per gene in 398 cancer cell lines. We describe findings of this screen, outlining the classes of cancer dependency genes and their relationships to genetic, expression, and lineage features. In addition, we describe robust gene-interaction networks recapitulating both protein complexes and functional cooperation among complexes and pathways. This dataset along with a web portal is provided to the community to assist in the discovery and translation of new therapeutic approaches for cancer.

Control of CD1d-restricted antigen presentation and inflammation by sphingomyelin.

Invariant natural killer T (iNKT) cells recognize activating self and microbial lipids presented by CD1d. CD1d can also bind non-activating lipids, such as sphingomyelin. We hypothesized that these serve as endogenous regulators and investigated humans and mice deficient in acid sphingomyelinase (ASM), an enzyme that degrades sphingomyelin. We show that ASM absence in mice leads to diminished CD1d-restricted antigen presentation and iNKT cell selection in the thymus, resulting in decreased iNKT cell levels and resistance to iNKT cell-mediated inflammatory conditions. Defective antigen presentation and decreased iNKT cells are also observed in ASM-deficient humans with Niemann-Pick disease, and ASM activity in healthy humans correlates with iNKT cell phenotype. Pharmacological ASM administration facilitates antigen presentation and restores the levels of iNKT cells in ASM-deficient mice. Together, these results demonstrate that control of non-agonistic CD1d-associated lipids is critical for iNKT cell development and function in vivo and represents a tight link between cellular sphingolipid metabolism and immunity.

The Importance of Timing.

Building a nervous system requires a precise sequence of genetic transitions, mediated in part by the temporal and spatial regulation of transcription factors. Quan et al. add to our understanding of this regulation by describing an evolutionarily conserved post-translational mechanism that rapidly extinguishes proneural protein activity in neural precursors.

Elucidating the structure and function of the nucleus-The NIH Common Fund 4D Nucleome program.

Genomic architecture appears to play crucial roles in health and a variety of diseases. How nuclear structures reorganize over different timescales is elusive, partly because the tools needed to probe and perturb them are not as advanced as needed by the field. To fill this gap, the National Institutes of Health Common Fund started a program in 2015, called the 4D Nucleome (4DN), with the goal of developing and ultimately applying technologies to interrogate the structure and function of nuclear organization in space and time.

Deconvolving the recognition of DNA shape from sequence.

Protein-DNA binding is mediated by the recognition of the chemical signatures of the DNA bases and the 3D shape of the DNA molecule. Because DNA shape is a consequence of sequence, it is difficult to dissociate these modes of recognition. Here, we tease them apart in the context of Hox-DNA binding by mutating residues that, in a co-crystal structure, only recognize DNA shape. Complexes made with these mutants lose the preference to bind sequences with specific DNA shape features. Introducing shape-recognizing residues from one Hox protein to another swapped binding specificities in vitro and gene regulation in vivo. Statistical machine learning revealed that the accuracy of binding specificity predictions improves by adding shape features to a model that only depends on sequence, and feature selection identified shape features important for recognition. Thus, shape readout is a direct and independent component of binding site selection by Hox proteins.

Low affinity binding site clusters confer hox specificity and regulatory robustness.

In animals, Hox transcription factors define regional identity in distinct anatomical domains. How Hox genes encode this specificity is a paradox, because different Hox proteins bind with high affinity in vitro to similar DNA sequences. Here, we demonstrate that the Hox protein Ultrabithorax (Ubx) in complex with its cofactor Extradenticle (Exd) bound specifically to clusters of very low affinity sites in enhancers of the shavenbaby gene of Drosophila. These low affinity sites conferred specificity for Ubx binding in vivo, but multiple clustered sites were required for robust expression when embryos developed in variable environments. Although most individual Ubx binding sites are not evolutionarily conserved, the overall enhancer architecture-clusters of low affinity binding sites-is maintained and required for enhancer function. Natural selection therefore works at the level of the enhancer, requiring a particular density of low affinity Ubx sites to confer both specific and robust expression.

A 12.4-day periodicity in a close binary system after a supernova.

Neutron stars and stellar-mass black holes are the remnants of massive star explosions1. Most massive stars reside in close binary systems2, and the interplay between the companion star and the newly formed compact object has been theoretically explored3, but signatures for binarity or evidence for the formation of a compact object during a supernova explosion are still lacking. Here we report a stripped-envelope supernova, SN 2022jli, which shows 12.4-day periodic undulations during the declining light curve. Narrow Hα emission is detected in late-time spectra with concordant periodic velocity shifts, probably arising from hydrogen gas stripped from a companion and accreted onto the compact remnant. A new Fermi-LAT γ-ray source is temporally and positionally consistent with SN 2022jli. The observed properties of SN 2022jli, including periodic undulations in the optical light curve, coherent Hα emission shifting and evidence for association with a γ-ray source, point to the explosion of a massive star in a binary system leaving behind a bound compact remnant. Mass accretion from the companion star onto the compact object powers the light curve of the supernova and generates the γ-ray emission.

Sphingolipids from a symbiotic microbe regulate homeostasis of host intestinal natural killer T cells.

Coevolution of beneficial microorganisms with the mammalian intestine fundamentally shapes mammalian physiology. Here, we report that the intestinal microbe Bacteroides fragilis modifies the homeostasis of host invariant natural killer T (iNKT) cells by supplementing the host's endogenous lipid antigen milieu with unique inhibitory sphingolipids. The process occurs early in life and effectively impedes iNKT cell proliferation during neonatal development. Consequently, total colonic iNKT cell numbers are restricted into adulthood, and hosts are protected against experimental iNKT cell-mediated, oxazolone-induced colitis. In studies with neonatal mice lacking access to bacterial sphingolipids, we found that treatment with B. fragilis glycosphingolipids-exemplified by an isolated peak (MW = 717.6) called GSL-Bf717-reduces colonic iNKT cell numbers and confers protection against oxazolone-induced colitis in adulthood. Our results suggest that the distinctive inhibitory capacity of GSL-Bf717 and similar molecules may prove useful in the treatment of autoimmune and allergic disorders in which iNKT cell activation is destructive.

The γδ IEL effector API5 masks genetic susceptibility to Paneth cell death.

Loss of Paneth cells and their antimicrobial granules compromises the intestinal epithelial barrier and is associated with Crohn's disease, a major type of inflammatory bowel disease1-7. Non-classical lymphoid cells, broadly referred to as intraepithelial lymphocytes (IELs), intercalate the intestinal epithelium8,9. This anatomical position has implicated them as first-line defenders in resistance to infections, but their role in inflammatory disease pathogenesis requires clarification. The identification of mediators that coordinate crosstalk between specific IEL and epithelial subsets could provide insight into intestinal barrier mechanisms in health and disease. Here we show that the subset of IELs that express γ and δ T cell receptor subunits (γδ IELs) promotes the viability of Paneth cells deficient in the Crohn's disease susceptibility gene ATG16L1. Using an ex vivo lymphocyte-epithelium co-culture system, we identified apoptosis inhibitor 5 (API5) as a Paneth cell-protective factor secreted by γδ IELs. In the Atg16l1-mutant mouse model, viral infection induced a loss of Paneth cells and enhanced susceptibility to intestinal injury by inhibiting the secretion of API5 from γδ IELs. Therapeutic administration of recombinant API5 protected Paneth cells in vivo in mice and ex vivo in human organoids with the ATG16L1 risk allele. Thus, we identify API5 as a protective γδ IEL effector that masks genetic susceptibility to Paneth cell death.