The rockefeller university

Bacterial genomes contain large reservoirs of biosynthetic gene clusters (BGCs) that are predicted to encode unexplored natural products. Heterologous expression of previously unstudied BGCs should facilitate the discovery of additional therapeutically relevant bioactive molecules from bacterial culture collections, but the large-scale manipulation of BGCs remains cumbersome. Here, we describe a method to parallelize the identification, mobilization and heterologous expression of BGCs. Our solution simultaneously captures large numbers of BGCs by cloning the genomes of a strain collection in a large-insert library and uses the CONKAT-seq (co-occurrence network analysis of targeted sequences) sequencing pipeline to efficiently localize clones carrying intact BGCs which represent candidates for heterologous expression. Our discovery of several natural products, including an antibiotic that is active against multi-drug resistant Staphylococcus aureus, demonstrates the potential of leveraging economies of scale with this approach to systematically interrogate cryptic BGCs contained in strain collections.

Piezo proteins are mechanosensitive ion channels that can locally curve the membrane into a dome shape [Y. R. Guo, R. MacKinnon, eLife 6, e33660 (2017)]. The curved shape of the Piezo dome is expected to deform the surrounding lipid bilayer membrane into a membrane footprint, which may serve to amplify Piezo's sensitivity to applied forces [C. A. Haselwandter, R. MacKinnon, eLife 7, e41968 (2018)]. If Piezo proteins are embedded in lipid bilayer vesicles, the membrane shape deformations induced by the Piezo dome depend on the vesicle size. We employ here membrane elasticity theory to predict, with no free parameters, the shape of such Piezo vesicles outside the Piezo dome, and show that the predicted vesicle shapes agree quantitatively with the corresponding measured vesicle shapes obtained through cryoelectron tomography, for a range of vesicle sizes [W. Helfrich, Z. Naturforsch. C 28, 693-703 (1973)]. On this basis, we explore the coupling between Piezo and membrane shape and demonstrate that the features of the Piezo dome affecting Piezo's membrane footprint approximately follow a spherical cap geometry. Our work puts into place the foundation for deducing key elastic properties of the Piezo dome from membrane shape measurements and provides a general framework for quantifying how proteins deform bilayer membranes.

Integrin receptors are established drug targets, but many of the drugs that have been developed act as partial agonists, inducing the receptor into a high-affinity, ligand-binding state. Lin et al. discovered a general mechanism to circumvent this problem-stabilizing a key water molecule that prevents receptor activation. Their findings are likely to impact future therapeutic development.

Chemical synapses between axons and dendrites mediate neuronal intercellular communication. Here, we describe a synapse between axons and primary cilia: the axo-ciliary synapse. Using enhanced focused ion beam-scanning electron microscopy on samples with optimally preserved ultrastructure, we discovered synapses between brainstem serotonergic axons and the primary cilia of hippocampal CA1 pyramidal neurons. Functionally, these cilia are enriched in a ciliary-restricted serotonin receptor, the 5-hydroxytryptamine receptor 6 (5-HTR6). Using a cilia-targeted serotonin sensor, we show that opto-and chemogenetic stimulation of serotonergic axons releases serotonin onto cilia. Ciliary 5-HTR6 stimulation activates a non-canonical G(alpha q/11)-RhoA pathway, which modulates nuclear actin and increases histone acetylation and chromatin accessibility. Ablation of this pathway reduces chromatin accessibility in CA1 pyramidal neurons. As a signaling apparatus with proximity to the nucleus, axo-ciliary synapses short circuit neurotransmission to alter the postsynaptic neuron's epigenetic state.

Motoneurons and motoneuron-like pancreatic b cells arise from radial glia and ductal cells, respectively, both tube-lining progenitors that share molecular regulators. To uncover programs underlying motoneuron forma-tion, we studied a similar, cell-division-independent transformation of the C. elegans tube-lining Y cell into the PDA motoneuron. We find that lin-12/Notch acts through ngn-1/Ngn and its regulator hlh-16/Olig to control transformation timing. lin-12 loss blocks transformation, while lin-12(gf) promotes precocious PDA forma-tion. Early basal expression of ngn-1/Ngn and hlh-16/Olig depends on sem-4/Sall and egl-5/Hox. Later, coin-cident with Y cell morphological changes, ngn-1/Ngn expression is upregulated in a sem-4/Sall and egl-5/ Hox-dependent but hlh-16/Olig-independent manner. Subsequently, Y cell retrograde extension forms an anchored process priming PDA axon extension. Extension requires ngn-1-dependent expression of the cyto-skeleton organizers UNC-119, UNC-44/ANK, and UNC-33/CRMP, which also activate PDA terminal-gene expression. Our findings uncover cell-division-independent regulatory events leading to motoneuron gener-ation, suggesting a conserved pathway for epithelial-to-motoneuron/motoneuron-like cell differentiation.

How intestinal microbes regulate metabolic syndrome is incompletely understood. We show that intestinal micro -biota protects against development of obesity, metabolic syndrome, and pre-diabetic phenotypes by inducing commensal-specific Th17 cells. High-fat, high-sugar diet promoted metabolic disease by depleting Th17-inducing microbes, and recovery of commensal Th17 cells restored protection. Microbiota-induced Th17 cells afforded protection by regulating lipid absorption across intestinal epithelium in an IL-17-dependent manner. Diet-induced loss of protective Th17 cells was mediated by the presence of sugar. Eliminating sugar from high-fat diets protected mice from obesity and metabolic syndrome in a manner dependent on commensal -spe-cific Th17 cells. Sugar and ILC3 promoted outgrowth of Faecalibaculum rodentium that displaced Th17-inducing microbiota. These results define dietary and microbiota factors posing risk for metabolic syndrome. They also define a microbiota-dependent mechanism for immuno-pathogenicity of dietary sugar and highlight an elaborate interaction between diet, microbiota, and intestinal immunity in regulation of metabolic disorders.

The corticotropin-releasing hormone receptor 2 (CRHR2) gene encodes CRHR2, contributing to the hypothalamic-pituitary-adrenal stress response and to hyperglycemia and insulin resistance. CRHR2-/- mice are hypersensitive to stress, and the CRHR2 locus has been linked to type 2 diabetes and depression. While CRHR2 variants confer risk for mood disorders, MDD, and type 2 diabetes, they have not been investigated in familial T2D and MDD. In 212 Italian families with type 2 diabetes and depression, we tested 17 CRHR2 single nucleotide polymorphisms (SNPs), using two-point parametric-linkage and linkage-disequilibrium (i.e., association) analysis (models: dominant-complete-penetrance-D1, dominant-incomplete-penetrance-D2, recessive-complete-penetrance-R1, recessive-incomplete-penetrance-R2). We detected novel linkage/linkage-disequilibrium/association to/with depression (3 SNPs/D1, 2 SNPs/D2, 3 SNPs/R1, 3 SNPs/R2) and type 2 diabetes (3 SNPs/D1, 2 SNPs/D2, 2 SNPs/R1, 1 SNP/R2). All detected risk variants are novel. Two depression-risk variants within one linkage-disequilibrium block replicate each other. Two independent novel SNPs were comorbid while the most significant conferred either depression- or type 2 diabetes-risk. Although the families were primarily ascertained for type 2 diabetes, depression-risk variants showed higher significance than type 2 diabetes-risk variants, implying CRHR2 has a stronger role in depression-risk than type 2 diabetes-risk. In silico analysis predicted variants' dysfunction. CRHR2 is for the first time linked to/in linkage-disequilibrium/association with depression-type 2 diabetes comorbidity and may underlie the shared genetic pathogenesis via pleiotropy.

Background Many short-read genome assemblies have been found to be incomplete and contain mis-assemblies. The Vertebrate Genomes Project has been producing new reference genome assemblies with an emphasis on being as complete and error-free as possible, which requires utilizing long reads, long-range scaffolding data, new assembly algorithms, and manual curation. A more thorough evaluation of the recent references relative to prior assemblies can provide a detailed overview of the types and magnitude of improvements. Results Here we evaluate new vertebrate genome references relative to the previous assemblies for the same species and, in two cases, the same individuals, including a mammal (platypus), two birds (zebra finch, Anna's hummingbird), and a fish (climbing perch). We find that up to 11% of genomic sequence is entirely missing in the previous assemblies. In the Vertebrate Genomes Project zebra finch assembly, we identify eight new GC- and repeat-rich micro-chromosomes with high gene density. The impact of missing sequences is biased towards GC-rich 5 '-proximal promoters and 5 ' exon regions of protein-coding genes and long non-coding RNAs. Between 26 and 60% of genes include structural or sequence errors that could lead to misunderstanding of their function when using the previous genome assemblies. Conclusions Our findings reveal novel regulatory landscapes and protein coding sequences that have been greatly underestimated in previous assemblies and are now present in the Vertebrate Genomes Project reference genomes.

The ETO-family transcriptional corepressors, including ETO, ETO2, and MTGR1, are all involved in leukemia-causing chromosomal translocations. In every case, an ETO-family corepressor acquires a DNA-binding domain (DBD) to form a typical transcription factor-the DBD binds to DNA, while the ETO moiety manifests transcriptional activity. A directly comparative study of these "homologous " fusion transcription factors may clarify their similarities and differences in regulating transcription and leukemogenesis. Here, we performed a side-by-side comparison between AML1-ETO and ETO2-GLIS2, the most common fusion proteins in M2-and M7-subtypes of acute myeloid leukemia, respectively, by inducible expression of them in U937 leukemia cells. We found that, although AML1-ETO and ETO2-GLIS2 can use their own DBDs to bind DNA, they share a large proportion of genome-wide binding regions dependent on other cooperative transcription factors, including the ETS-, bZIP- and bHLH-family proteins. AML1-ETO acts as either transcriptional repressor or activator, whereas ETO2-GLIS2 mainly acts as activator. The repressor-versus-activator functions of AML1-ETO might be determined by the abundance of cooperative transcription factors/cofactors on the target genes. Importantly, AML1-ETO and ETO2-GLIS2 differentially regulate key transcription factors in myeloid differentiation including PU.1 and C/EBP beta. Consequently, AML1-ETO inhibits, but ETO2-GLIS2 facilitates, myeloid differentiation of U937 cells. This function of ETO2-GLIS2 is reminiscent of a similar effect of MLL-AF9 as previously reported. Taken together, this directly comparative study between AML1-ETO and ETO2-GLIS2 in the same cellular context provides insights into context-dependent transcription regulatory mechanisms that may underlie how these seemingly "homologous " fusion transcription factors exert distinct functions to drive different subtypes of leukemia.