Activity-dependent transcriptional responses shape cortical function. However, we lack a comprehensive understanding of the diversity of these responses across the full range of cortical cell types, and how these changes contribute to neuronal plasticity and disease. Here we applied high-throughput single-cell RNA-sequencing to investigate the breadth of transcriptional changes that occur across cell types in mouse visual cortex following exposure to light. We identified significant and divergent transcriptional responses to stimulation in each of the 30 cell types characterized, revealing 611 stimulus-responsive genes. Excitatory pyramidal neurons exhibit inter- and intra-laminar heterogeneity in the induction of stimulus responsive genes. Non-neuronal cells demonstrated clear transcriptional responses that may regulate experience-dependent changes in neurovascular coupling and myelination. Together, these results reveal the dynamic landscape of stimulus-dependent transcriptional changes that occur across cell types in visual cortex, which are likely critical for cortical function and may be sites of de-regulation in developmental brain disorders.

Insulin expressing cells that have been differentiated from human pluripotent stem cells in vitro lack the glucose responsiveness characteristic of mature β-cells. β-cell maturation in mice was studied to find genetic markers that enable screens for factors that induce bona fide β-cells in vitro. We find that functional β-cell maturation is marked by an increase in the glucose threshold for insulin secretion and by expression of the gene urocortin 3.

Short peptide tags S6 and A1, each 12 residues in length, were identified from a phage-displayed peptide library as efficient substrates for site-specific protein labeling catalyzed by Sfp and AcpS phosphopantetheinyl transferases (PPTases), respectively. S6 and A1 tags were selected for useful levels of orthogonality in reactivities with the PPTases: the catalytic efficiency, kcat/Km of Sfp-catalyzed S6 serine phosphopantetheinylation was 442-fold greater than that for AcpS. Conversely, the kcat/Km of AcpS-catalyzed A1 labeling was 30-fold higher than that for Sfp-catalyzed A1 labeling. S6 and A1 peptide tags can be fused to N- or C-termini of proteins for orthogonal labeling of target proteins in cell lysates or on live cell surfaces. The development of the orthogonal S6 and A1 tags represents a significant enhancement of PPTase-catalyzed protein labeling, allowing tandem or iterative covalent attachment of small molecules of diverse structures to the target proteins with high efficiency and specificity.

SUMMARY In mammals during the early postnatal period the environment plays a critical role in promoting the final steps in the neuronal development. While epigenetic factors are thought to contribute to this process, the underlying molecular mechanisms remain poorly understood. Here we show that in the brain during early life the DNA methyltransferase DNMT3A transiently binds across transcribed regions of lowly expressed genes, and its binding specifies the pattern of DNA methylation at CA sequences (mCA) within these genes. We find that DNMT3A occupancy and mCA deposition within the transcribed regions of genes is negatively regulated by gene transcription and may be modified by early-life experience. Once deposited, mCA is bound by the methyl-DNA-binding protein MECP2 and functions in a rheostat-like manner to fine-tune the cell type-specific transcription of genes that are critical for brain function.

Significance Human pluripotent stem cells (hPSCs) can be produced from any person and have the potential to differentiate into any cell type in the body. This study focuses on the generation of insulin-expressing cells from hPSCs and compares their gene expression, as assayed by transcriptional gene products, to that of insulin-expressing β cells from human fetal and adult samples. We employ a new method to isolate and profile insulin-expressing cells and conclude that several different hPSC lines generate very similar insulin-expressing cells, cells whose transcripts resemble fetal rather than adult β cells. This study advances the possibility of directing the differentiation of stem cells into functional β cells by comparing and cataloging differences between hPSC-derived insulin-expressing cells and human β cells.

Kutznerides, actinomycete-derived cyclic depsipetides, consist of six nonproteinogenic residues, including a highly oxygenated tricyclic hexahydropyrroloindole, a chlorinated piperazic acid, 2-(1-methylcyclopropyl)-glycine, a ␤-branched-hydroxy acid, and 3-hydroxy glutamic acid, for which biosynthetic logic has not been elucidated. Herein we describe the biosynthetic gene cluster for the kutzneride family, identified by degenerate primer PCR for halogenating enzymes postulated to be involved in biosyntheses of these unusual monomers. The 56-kb gene cluster encodes a series of six nonribosomal peptide synthetase (NRPS) modules distributed over three proteins and a variety of tailoring enzymes, including both mononuclear nonheme iron and two flavindependent halogenases, and an array of oxygen transfer catalysts. The sequence and organization of NRPS genes support incorporation of the unusual monomer units into the densely functionalized scaffold of kutznerides. Our work provides insight into the formation of this intriguing class of compounds and provides a foundation for elucidating the timing and mechanisms of their biosynthesis.chlorination ͉ halogenases ͉ nonribosomal peptide biosynthesis K utznerides are antifungal and antimicrobial cyclic hexadepsipeptides isolated from the soil actinomycete Kutzneria sp. 744 (1). Structural elucidation of these metabolites revealed nine related compounds composed of five unusual nonproteinogenic amino acids and one hydroxy acid ( Fig. 1) but differing in the extent of substitution and stereochemistry of constituent residues (2). All kutznerides contain 2-(1-methylcyclopropyl)-D-glycine (MecPGly) connected to the ␣-hydroxyl moiety of either (S)-2-hydroxy-3-methylbutyric or (S)-2-hydroxy-3,3-dimethylbutyric acid. The hydroxy acid residue is followed by a piperazic acid moiety, found in four distinct forms: as piperazic acid in kutznerides 1, 3, 5, and 7; as dehydropiperazate in kutznerides 4 and 9; as ␥-chloro-piperazate in kutznerides 2 and 8; and as ␥-hydroxy-dehydropiperazate in kutzneride 6. Furthermore, kutznerides contain O-methyl-L-serine and either the threo or erythro isomer of 3-hydroxy-D-glutamate. Finally, an unusual tricyclic dihalogenated (2S,3aR,8aS)-6,7-dichloro-3a-hydroxy-hexahydropyrrolo[2,3-b]indole-2-carboxylic acid (PIC) is conserved in all structurally characterized kutznerides. The structural subunits of kutznerides suggest unusual enzymatic mechanisms involved in their biosynthesis.Recently, our laboratory has elucidated the enzymatic logic of carbon-chlorine bond formation during the biosynthesis of several chlorinated secondary metabolites. Among these, dichlorination of the pyrrole moiety in the biosynthesis of pyoluteorin (3) and chlorination of tryptophan in rebeccamycin biosynthesis (4) are carried out by the flavin-dependent halogenases PltA and RebH, respectively. The work of van Pee and coworkers (5) on the pyrrolnitrin halogenase PrnA demonstrated the necessity of FADH 2 , chloride, and oxygen for catalytic activity of flavoprotein halogenase...

The advent of endothermy is a defining feature of mammalian and avian evolution, achieved through continuous fine-tuned homeostatic regulation of body temperature and metabolism 1,2 . However, when challenged by food deprivation or harsh environmental conditions, many mammalian species initiate adaptive energy-conserving survival strategies, including torpor and hibernation, during which their body temperature decreases far below its homeostatic setpoint [3][4][5] . How homeothermic mammals initiate and regulate these extraordinary hypothermic states remains largely unknown. Here, we discover that entry into mouse torpor, a fasting-induced state with greatly decreased metabolic rate and body temperature as low as 20°C 6 , is regulated by neurons in the medial and lateral preoptic area of the hypothalamus. We show that re-stimulation of neurons activated during a previous bout of torpor is sufficient to initiate key features of torpor, even in animals that are not calorically restricted. Among these neurons we identify a population of glutamatergic Adcyap1+ cells whose activity accurately determines when animals naturally initiate and exit torpor, and whose inhibition disrupts the natural process of torpor entry, maintenance and arousal. Taken together, we discover a specific neuronal population in the mouse *

Neuronal development requires a complex choreography of transcriptional decisions to obtain specific cellular identities. Realizing the ultimate goal of identifying genome-wide signatures that define and drive specific neuronal fates has been hampered by enormous complexity in both time and space during development. Here, we have paired high-throughput purification of pyramidal neuron subclasses with deep profiling of spatiotemporal transcriptional dynamics during corticogenesis to resolve lineage choice decisions. We identified numerous features ranging from spatial and temporal usage of alternative mRNA isoforms and promoters to a host of mRNA genes modulated during fate specification. Notably, we uncovered numerous long non-coding RNAs with restricted temporal and cell type specific expression. To facilitate future exploration, we provide an interactive online database to enable multidimensional data mining and dissemination. This multi-faceted study generates a powerful resource and informs understanding of the transcriptional regulation underlying pyramidal neuron diversity in the neocortex.