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In this issue of Developmental Cell, Muncie et al. describe the generation of gastrulation-like foci of cells within micropatterned colonies of pluripotent stem cells. This demonstration of mechanosensitive beta-catenin/Wnt-dependent specification of cell fate during gastrulation illustrates the insights gleaned by placing stem cells in embryo-like mechanical environments.

Polycomb repressive complex 2 (PRC2) installs and spreads repressive histone methylation marks on eukaryotic chromosomes. Because of the key roles that PRC2 plays in development and disease, how this epigenetic machinery interacts with DNA and nucleosomes is of major interest. Nonetheless, the mechanism by which PRC2 engages with native-like chromatin remains incompletely understood. In this work, we employ single-molecule force spectroscopy and molecular dynamics simulations to dissect the behavior of PRC2 on polynucleosome arrays. Our results reveal an unexpectedly diverse repertoire of PRC2 binding configurations on chromatin. Besides reproducing known binding modes in which PRC2 interacts with bare DNA, mononucleosomes, and adjacent nucleosome pairs, our data also provide direct evidence that PRC2 can bridge pairs of distal nucleosomes. In particular, the "1-3" bridging mode, in which PRC2 engages two nucleosomes separated by one spacer nucleosome, is a preferred low-energy configuration. Moreover, we show that the distribution and stability of different PRC2-chromatin interaction modes are modulated by accessory subunits, oncogenic histone mutations, and the methylation state of chromatin. Overall, these findings have implications for the mechanism by which PRC2 spreads histone modifications and compacts chromatin. The experimental and computational platforms developed here provide a framework for understanding the molecular basis of epigenetic maintenance mediated by Polycombgroup proteins.

While the static structure of the nuclear pore complex (NPC) continues to be refined with cryo-EM and x-ray crystallography, in vivo conformational changes of the NPC remain under-explored. We developed sensors that report on the orientation of NPC components by rigidly conjugating mEGFP to different NPC proteins. Our studies show conformational changes to select domains of nucleoporins (Nups) within the inner ring (Nup54, Nup58, Nup62) when transport through the NPC is perturbed and no conformational changes to Nups elsewhere in the NPC. Our results suggest that select components of the NPC are flexible and undergo conformational changes upon engaging with cargo.

Eating disorders are life-interrupting psychiatric conditions with high morbidity and mortality, yet the basic mechanisms underlying these conditions are understudied compared with other psychiatric disorders. In this opinion, we suggest that recent knowledge gleaned from genomic and neuroimaging investigations of eating disorders in humans presents a rich opportunity to sharpen animal models of eating disorders and to identify neural mechanisms that contribute to the risk and maintenance of these conditions. Our article reflects the state of the science, with a primary focus on anorexia nervosa (AN) and binge-eating behavior, and encourages further study of all conditions categorized under feeding and eating disorders.

The serum metabolome contains a plethora of biomarkers and causative agents of various diseases, some of which are endogenously produced and some that have been taken up from the environment(1). The origins of specific compounds are known, including metabolites that are highly heritable(2,3), or those that are influenced by the gut microbiome(4), by lifestyle choices such as smoking(5), or by diet(6). However, the key determinants of most metabolites are still poorly understood. Here we measured the levels of 1,251 metabolites in serum samples from a unique and deeply phenotyped healthy human cohort of 491 individuals. We applied machine-learning algorithms to predict metabolite levels in held-out individuals on the basis of host genetics, gut microbiome, clinical parameters, diet, lifestyle and anthropometric measurements, and obtained statistically significant predictions for more than 76% of the profiled metabolites. Diet and microbiome had the strongest predictive power, and each explained hundreds of metabolites-in some cases, explaining more than 50% of the observed variance. We further validated microbiome-related predictions by showing a high replication rate in two geographically independent cohorts(7,8) that were not available to us when we trained the algorithms. We used feature attribution analysis(9) to reveal specific dietary and bacterial interactions. We further demonstrate that some of these interactions might be causal, as some metabolites that we predicted to be positively associated with bread were found to increase after a randomized clinical trial of bread intervention. Overall, our results reveal potential determinants of more than 800 metabolites, paving the way towards a mechanistic understanding of alterations in metabolites under different conditions and to designing interventions for manipulating the levels of circulating metabolites. The levels of 1,251 metabolites are measured in 475 phenotyped individuals, and machine-learning algorithms reveal that diet and the microbiome are the determinants with the strongest predictive power for the levels of these metabolites.

Diseases of immunity, including autoimmune diseases such as multiple sclerosis, transplantation graft rejection, allergy, and asthma, are prevalent and increasing in prevalence. They contribute to significant morbidity and mortality; however, few if any curative therapies exist, and those that are available lack either potency or specificity. Dendritic cells (DCs) are sentinels of the immune system that connect the innate and adaptive immune system and are critical regulators of both immunity and tolerance. We posited that the tolerogenic potential of DC could be harnessed to develop more specific and potent therapies for diseases of immunity by delivering autoantigen to a sufficient number of tolerogenic DCs in situ that could then inhibit pathogenic effector T cell responses. Specifically, we hypothesized that the steroid dexamethasone covalently coupled to a peptide antigen could be processed by DCs, induce tolerogenic DCs, and attenuate antigen-specific pathogenic T cell responses. To test this hypothesis, we synthesized a series of dexamethasone-peptide immunoconjugates by standard solid-phase peptide synthesis. The antigenic portion of the immunoconjugate could be presented by DCs, and the immunoconjugate induced a tolerogenic phenotype in DCs that then inhibited antigen-specific T cell proliferation in vitro. When the immunoconjugate was administered prophylactically in the murine experimental autoimmune encephalomyelitis model of multiple sclerosis, disease was attenuated compared to dexamethasone and peptide delivered as uncoupled components. Together, this work demonstrates the utility of immunoconjugates for inducing tolerance while establishing the foundation for future studies exploring methods to enrich and target DCs for tolerogenic therapies.

The DNA polymerase (Pol) 8 of Saccharomyces cerevisiae (S.c.) is composed of the catalytic subunit Pol3 along with two regulatory subunits, Pol31 and Pol32. Pol 8 binds to proliferating cell nuclear antigen (PCNA) and functions in genome replication, repair, and recombination. Unique among DNA polymerases, the Pol3 catalytic subunit contains a 4Fe-4S cluster that may sense the cellular redox state. Here we report the 3.2-angstrom cryo-EM structure of S.c. Pol 8 in complex with primed DNA, an incoming ddTTP, and the PCNA clamp. Unexpectedly, Pol 8 binds only one subunit of the PCNA trimer. This singular yet extensive interaction holds DNA such that the 2-nm-wide DNA threads through the center of the 3-nm interior channel of the clamp without directly contacting the protein. Thus, a water-mediated clamp and DNA interface enables the PCNA clamp to "waterskate" along the duplex with minimum drag. Pol31 and Pol32 are positioned off to the side of the catalytic Pol3-PCNA-DNA axis. We show here that Pol31-Pol32 binds single stranded DNA that we propose underlies polymerase recycling during lagging strand synthesis, in analogy to Escherichia coli replicase. Interestingly, the 4Fe-4S cluster in the C-terminal CysB domain of Pol3 forms the central interface to Pol31-Pol32, and this strategic location may explain the regulation of the oxidation state on Pol 8 activity, possibly useful during cellular oxidative stress. Importantly, human cancer and other disease mutations map to nearly every domain of Pol3, suggesting that all aspects of Pol 8 replication are important to human health and disease.

Tumor-associated macrophages (TAMs) can have protumor properties, including suppressing immune responses, promoting vascularization and, consequently, augmenting tumor progression. To stop TAM-mediated immunosuppression, we use a novel treatment by injecting antibodies specific for scavenger receptor MARCO, which is expressed on a specific subpopulation of TAMs in the tumor. We now report the location of this TAM as well as the pleiotropic mechanism of action of anti-MARCO antibody treatment on tumor progression and further show that this is potentially relevant to humans. Using specific targeting, we observed decreased tumor vascularization, a switch in the metabolic program of MARCO-expressing macrophages, and activation of natural killer (NK) cell killing through TNF-related apoptosis-inducing ligand (TRAIL). This latter activity reverses the effect of melanoma cell-conditioned macrophages in blocking NK activation and synergizes with T cell-directed immunotherapy, such as antibodies to PD-1 or PD-L1, to enhance tumor killing. Our study thus reveals an approach to targeting the immunosuppressive tumor microenvironment with monoclonal antibodies to enhance NK cell activation and NK cell-mediated killing. This can complement existing T cell-directed immunotherapy, providing a promising approach to combinatorial immunotherapy for cancer.

Although the cerebellum was first described in the 17th century and its cytoarchitecture was mapped in the early 20th century, our understanding of the role of the cerebellum is rapidly changing. Initially thought to carry out simple motor control, the cerebellum is now considered to function in complex cognitive tasks. On page 1436 of this issue, Kebschull et al. (1) show that the cerebellar nuclei (CN) evolved from amniotes to humans by duplicating “subnuclei” consisting of two classes of excitatory neurons and three classes of inhibitory neurons. The excitatory cell class of the lateral nucleus that projects to the frontal cortex in mice and is affected in developmental disorders such as autism spectrum disorder (ASD) (2, 3) predominates in the greatly expanded human cerebellum. These studies thus provide molecular insights into emerging studies showing a role for the cerebellum in cognitive behaviors, including modulating dopaminergic reward circuits (4), language (5), and social behavior (6, 7).