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The chromodomain helicase DNA-binding protein CHD8 is among the most frequently found de-novo mutations in autism (1–3). Unlike most other autism-risk genes, CHD8 mutations appear to be fully penetrant (4). Despite its prominent disease involvement, little known about its molecular function. Based on sequence homology, CHD8 is believed to be a chromatin regulator, but mechanisms for its genomic targeting and its role on chromatin are unclear. Here, we developed a human cell model carrying conditional CHD8 loss-of-function alleles. Full knockout CHD8 was required for the viability of undifferentiated human embryonic stem (ES) cells, whereas postmitotic neurons survived following CHD8 depletion. However, chromatin accessibility maps and transcriptional profiling revealed that CHD8 is a potent general chromatin activator, enhancing transcription of its direct target genes, including a large group of autism genes. CHD8’s genomic binding sites in human neurons were significantly enriched for ELK1 (ETS) motifs. Moreover, positive CHD8-dependent chromatin remodeling was enhanced at ELK1 motif-containing CHD8 binding sites. ELK1 was the most prominent ETS factor expressed in human neurons and was necessary for CHD8 to target the sites that contained the ELK1 motif, demonstrating a cooperative interaction between ELK1 and CHD8 on chromatin. We also observed potential role of CHD8 in ELK1 localization on nuclear compartments in a transcription-stage-dependent manner. Finally, inhibition of ELK1 activity or ELK1 knockdown that enhances the neurogenesis from embryonic stem cells (ES) was dependent on the presence of CHD8. In summary, our results establish that CHD8 is a strong activator of chromatin accessibility and transcription in neurons and reveals a role in regulating many high-risk autism genes. Additionally, we show there is molecular and functional interdependence of CHD8 and ELK1 in chromatin binding of CHD8, nuclear interaction of ELK1, and neurogenesis enhancement. These data imply the involvement of the MAPK/ERK pathway effector ELK1 in pathogenesis of autism caused by CHD8 mutations (5).

Summary The differentiation of pluripotent stem cells can be accomplished by sequential activation of signaling pathways or through transcription factor programming. Multistep differentiation imitates embryonic development to obtain authentic cell types, but it suffers from asynchronous differentiation with variable efficiency. Transcription factor programming induces synchronous and efficient differentiation with higher reproducibility but may not always yield authentic cell types. We systematically explored the generation of dopaminergic induced neuronal cells from mouse and human pluripotent stem cells. We found that the proneural factor Ascl1 in combination with mesencephalic factors Lmx1a and Nurr1 induce peripheral dopaminergic neurons. Co-delivery of additional midbrain transcription factors En1, FoxA2, and Pitx3 resulted in facile and robust generation of functional dopaminergic neurons of midbrain character. Our results suggest that more complex combinations of transcription factors may be needed for proper regional specification of induced neuronal cells generated by direct lineage induction., Graphical abstract, Highlights • Ascl1 alone can induce tyrosine hydroxylase (TH)-positive ES-iN cells • Ascl1 alone, or with Nurr1 and Lmx1a, induce peripheral TH-positive cells • WNT1 and neurotrophic factors increase TH-positive iN cells in culture • A 6-factor combination induces TH-positive dopamine iN cells of central identity, Dopaminergic neurons can be induced from pluripotent cells using overexpression of transcription factors. Existing transcription factor combinations result in cells of mixed regional identity. Here, Wernig and colleagues report that with extracellular signals, a six-factor combination delivered via piggyBac transposons is capable of inducing dopamine neurons of central identity from mouse and human embryonic stem cells.

The eight-carbon fatty acid octanoic acid (OA) is an important platform chemical and precursor of many industrially relevant products. Its microbial biosynthesis is regarded as a promising alternative to current unsustainable production methods. In Saccharomyces cerevisiae, the production of OA had been previously achieved by rational engineering of the fatty acid synthase. For the supply of the precursor molecule acetyl-CoA and of the redox cofactor NADPH, the native pyruvate dehydrogenase bypass had been harnessed, or the cells had been additionally provided with a pathway involving a heterologous ATP-citrate lyase. Here, we redirected the flux of glucose towards the oxidative branch of the pentose phosphate pathway and overexpressed a heterologous phosphoketolase/phosphotransacetylase shunt to improve the supply of NADPH and acetyl-CoA in a strain background with abolished OA degradation. We show that these modifications lead to an increased yield of OA during the consumption of glucose by more than 60% compared to the parental strain. Furthermore, we investigated different genetic engineering targets to identify potential factors that limit the OA production in yeast. Toxicity assays performed with the engineered strains suggest that the inhibitory effects of OA on cell growth likely impose an upper limit to attainable OA yields.

Most fungal fatty acid synthases assemble from two multidomain subunits, α and β, into a heterododecameric FAS complex. It has been recently shown that the complex assembly occurs in a cotranslational manner and is initiated by an interaction between the termini of α and β subunits. This initial engagement of subunits may be the rate-limiting phase of the assembly and subject to cellular regulation. Therefore, we hypothesized that bypassing this step by genetically fusing the subunits could be beneficial for biotechnological production of fatty acids. To test the concept, we expressed fused FAS subunits engineered for production of octanoic acid in Saccharomyces cerevisiae. Collectively, our data indicate that FAS activity is a limiting factor of fatty acid production and that FAS fusion proteins show a superior performance compared to their split counterparts. This strategy is likely a generalizable approach to optimize the production of fatty acids and derived compounds in microbial chassis organisms.

The on-target pioneer factors Ascl1 and Myod1 are sequence-related but induce two developmentally unrelated lineages—that is, neuronal and muscle identities, respectively. It is unclear how these two basic helix–loop–helix (bHLH) factors mediate such fundamentally different outcomes. The chromatin binding of Ascl1 and Myod1 was surprisingly similar in fibroblasts, yet their transcriptional outputs were drastically different. We found that quantitative binding differences explained differential chromatin remodelling and gene activation. Although strong Ascl1 binding was exclusively associated with bHLH motifs, strong Myod1-binding sites were co-enriched with non-bHLH motifs, possibly explaining why Ascl1 is less context dependent. Finally, we observed that promiscuous binding of Myod1 to neuronal targets results in neuronal reprogramming when the muscle program is inhibited by Myt1l. Our findings suggest that chromatin access of on-target pioneer factors is primarily driven by the protein–DNA interaction, unlike ordinary context-dependent transcription factors, and that promiscuous transcription factor binding requires specific silencing mechanisms to ensure lineage fidelity. Lee, Mall et al. explore why the sequence-related transcription factors Myod1 and Ascl1 lead to different reprogramming outcomes when expressed in fibroblasts, despite binding similar targets.

Summary Osteoporosis and osteoporotic fractures lead to decreased life quality and high healthcare costs. Current treatments prevent losses in bone mass and fractures to some extent but have side effects. Therefore, better therapies are needed. This study investigated whether the transcription factor Jun has a specific pro-osteogenic potency and whether modulating Jun could serve as a novel treatment for osteoporosis-associated fractures. We demonstrate that ectopically transplanted whole bones and distinct osteoprogenitors increase bone formation. Perinatal Jun induction disturbs growth plate architecture, causing a striking phenotype with shortened and thickened bones. Molecularly, Jun induces hedgehog signaling in skeletal stem cells. Therapeutically, Jun accelerates bone growth and healing in a drilling-defect model. Altogether, these results demonstrate that Jun drives bone formation by expanding osteoprogenitor populations and forcing them into the bone fate, providing a rationale for future clinical applications., Graphical Abstract, Highlights • Jun drives the formation of bone at the expense of cartilage and stroma • Jun disturbs the proper differentiation of the developing growth plate • Jun stimulates hedgehog signaling in skeletal stem cells • Jun accelerates fracture healing in a drilling-defect model, Wernig and colleagues demonstrate the striking potency of the transcription factor Jun to expand bone precursors, drive bone formation, and accelerate fracture healing. Boosting the regenerative potential of dormant bone precursor cells, they show the potential of stem cell therapy for widespread degenerative disease.

To explore how the immune system controls clearance of SARS-CoV-2, we used a single-cell, mass cytometry-based proteomics platform to profile the immune systems of 21 patients who had recovered from SARS-CoV-2 infection without need for admission to an intensive care unit or for mechanical ventilation. We focused on receptors involved in interactions between immune cells and virus-infected cells. We found that the diversity of receptor repertoires on natural killer (NK) cells was negatively correlated with the viral clearance rate. In addition, NK subsets expressing the receptor DNAM1 were increased in patients who more rapidly recovered from infection. Ex vivo functional studies revealed that NK subpopulations with high DNAM1 expression had cytolytic activities in response to target cell stimulation. We also found that SARS-CoV-2 infection induced the expression of CD155 and nectin-4, ligands of DNAM1 and its paired coinhibitory receptor TIGIT, which counterbalanced the cytolytic activities of NK cells. Collectively, our results link the cytolytic immune responses of NK cells to the clearance of SARS-CoV-2 and show that the DNAM1 pathway modulates host-pathogen interactions during SARS-CoV-2 infection.

Standard methods for the creation of neuronal cells via direct induction from primary tissue use perinatal fibroblasts, which hinders the important study of patient specific genetic lesions such as those underlying neuropsychiatric disorders. To address this we developed a novel method for the direct induction of neuronal cells (induced neuronal cells, iN cells) from adult human fibroblast cells. Reprogramming fibroblasts into iN cells via recombinant virus resulted in cells that stain for markers such as MAP2 and PSA-NCAM and exhibit electrophysiological properties such as action potentials and voltage dependent sodium- and potassium currents that reveal a neuronal phenotype. Transcriptome and chromatin analysis using RNA-Seq, microRNA-Seq and ATAC-Seq, respectively, further confirm neuronal character. 22q11.2 Deletion-Syndrome (22q11DS) is caused by a large 3 million base-pair heterozygous deletion on human chromosome 22 and is strongly associated with neurodevelopmental, neuropsychiatric phenotypes such as schizophrenia and autism. We leverage the direct-iN cell model for the study of genetic neurodevelopmental conditions by presenting gene-by-gene as well as networkwide effects of the 22q11DS deletion on gene expression in human neuronal cells, on several levels of functional genomics analysis. Some of the genes within the 22q11DS deletion boundary exhibit unexpected cell-type-specific changes in transcript levels, and genome-wide we can detect dysregulation of calcium channel subunit genes and other genes known to be involved in autism or schizophrenia, such as NRXN1, as well synaptic pathways. This genome-wide effect on gene expression can also be observed at the microRNA and chromatin levels, showing that the iN cells have indeed converted to a neuronal phenotype at several regulatory levels: chromatin, protein-coding RNAs and microRNAs, revealing relevant disease pathways and genes. We present this model of inducing neurons from fibroblasts as a useful general resource to study the genetic and molecular basis of normal and abnormal brain development and brain function.

Significance In the skin, tissue injury results in fibrosis in the form of a scar composed of dense extracellular matrix deposited by fibroblasts. Therapies that promote tissue regeneration rather than fibrosis remain elusive because principles of fibroblast programming and response to injury remain incompletely understood. Here, we present a multimodal -omics platform for the study of cell populations in complex tissue, which has allowed us to characterize wound healing fibroblasts across both time and space. We identify functionally distinct fibroblast subpopulations and track cell fate during the response to wounding. We demonstrate that populations of fibroblasts migrate, proliferate, and differentiate in an adaptive response to disruption of their environment. These results illustrate fundamental principles underlying the cellular response to tissue injury., In the skin, tissue injury results in fibrosis in the form of scars composed of dense extracellular matrix deposited by fibroblasts. The therapeutic goal of regenerative wound healing has remained elusive, in part because principles of fibroblast programming and adaptive response to injury remain incompletely understood. Here, we present a multimodal -omics platform for the comprehensive study of cell populations in complex tissue, which has allowed us to characterize the cells involved in wound healing across both time and space. We employ a stented wound model that recapitulates human tissue repair kinetics and multiple Rainbow transgenic lines to precisely track fibroblast fate during the physiologic response to skin injury. Through integrated analysis of single cell chromatin landscapes and gene expression states, coupled with spatial transcriptomic profiling, we are able to impute fibroblast epigenomes with temporospatial resolution. This has allowed us to reveal potential mechanisms controlling fibroblast fate during migration, proliferation, and differentiation following skin injury, and thereby reexamine the canonical phases of wound healing. These findings have broad implications for the study of tissue repair in complex organ systems.

Pathologic skin scarring presents a vast economic and medical burden. Unfortunately, the molecular mechanisms underlying scar formation remain to be elucidated. We used a hypertrophic scarring (HTS) mouse model in which Jun is overexpressed globally or specifically in α-smooth muscle or collagen type I–expressing cells to cause excessive extracellular matrix deposition by skin fibroblasts in the skin after wounding. Jun overexpression triggered dermal fibrosis by modulating distinct fibroblast subpopulations within the wound, enhancing reticular fibroblast numbers, and decreasing lipofibroblasts. Analysis of human scars further revealed that JUN is highly expressed across the wide spectrum of scars, including HTS and keloids. CRISPR-Cas9–mediated JUN deletion in human HTS fibroblasts combined with epigenomic and transcriptomic analysis of both human and mouse HTS fibroblasts revealed that JUN initiates fibrosis by regulating CD36. Blocking CD36 with salvianolic acid B or CD36 knockout model counteracted JUN-mediated fibrosis efficacy in both human fibroblasts and mouse wounds. In summary, JUN is a critical regulator of pathological skin scarring, and targeting its downstream effector CD36 may represent a therapeutic strategy against scarring.

Age-associated chronic inflammation (inflammageing) is a central hallmark of ageing1, but its influence on specific cells remains largely unknown. Fibroblasts are present in most tissues and contribute to wound healing2,3. They are also the most widely used cell type for reprogramming to induced pluripotent stem (iPS) cells, a process that has implications for regenerative medicine and rejuvenation strategies4. Here we show that fibroblast cultures from old mice secrete inflammatory cytokines and exhibit increased variability in the efficiency of iPS cell reprogramming between mice. Variability between individuals is emerging as a feature of old age5–8, but the underlying mechanisms remain unknown. To identify drivers of this variability, we performed multi-omics profiling of fibroblast cultures from young and old mice that have different reprogramming efficiencies. This approach revealed that fibroblast cultures from old mice contain ‘activated fibroblasts’ that secrete inflammatory cytokines, and that the proportion of activated fibroblasts in a culture correlates with the reprogramming efficiency of that culture. Experiments in which conditioned medium was swapped between cultures showed that extrinsic factors secreted by activated fibroblasts underlie part of the variability between mice in reprogramming efficiency, and we have identified inflammatory cytokines, including TNF, as key contributors. Notably, old mice also exhibited variability in wound healing rate in vivo. Single-cell RNA-sequencing analysis identified distinct subpopulations of fibroblasts with different cytokine expression and signalling in the wounds of old mice with slow versus fast healing rates. Hence, a shift in fibroblast composition, and the ratio of inflammatory cytokines that they secrete, may drive the variability between mice in reprogramming in vitro and influence wound healing rate in vivo. This variability may reflect distinct stochastic ageing trajectories between individuals, and could help in developing personalized strategies to improve iPS cell generation and wound healing in elderly individuals. Fibroblasts from old mice are heterogeneous, which affects the ability of these fibroblasts to reprogram into induced pluripotent stem cells in vitro and influences wound healing rate in vivo.

In blood, apolipoprotein E (ApoE) is a component of circulating lipoproteins and mediates the clearance of these lipoproteins from blood by binding to ApoE receptors. Humans express three genetic ApoE variants, ApoE2, ApoE3, and ApoE4, which exhibit distinct ApoE receptor-binding properties and differentially affect Alzheimer's disease (AD), such that ApoE2 protects against, and ApoE4 predisposes to AD. In brain, ApoE-containing lipoproteins are secreted by activated astrocytes and microglia, but their functions and role in AD pathogenesis are largely unknown. Ample evidence suggests that ApoE4 induces microglial dysregulation and impedes Aβ clearance in AD, but the direct neuronal effects of ApoE variants are poorly studied. Extending previous studies, we here demonstrate that the three ApoE variants differentially activate multiple neuronal signaling pathways and regulate synaptogenesis. Specifically, using human neurons (male embryonic stem cell-derived) cultured in the absence of glia to exclude indirect glial mechanisms, we show that ApoE broadly stimulates signal transduction cascades. Among others, such stimulation enhances APP synthesis and synapse formation with an ApoE4>ApoE3>ApoE2 potency rank order, paralleling the relative risk for AD conferred by these ApoE variants. Unlike the previously described induction of APP transcription, however, ApoE-induced synaptogenesis involves CREB activation rather than cFos activation. We thus propose that in brain, ApoE acts as a glia-secreted signal that activates neuronal signaling pathways. The parallel potency rank order of ApoE4>ApoE3>ApoE2 in AD risk and neuronal signaling suggests that ApoE4 may in an apparent paradox promote AD pathogenesis by causing a chronic increase in signaling, possibly via enhancing APP expression. SIGNIFICANCE STATEMENT Humans express three genetic variants of apolipoprotein E (ApoE), ApoE2, ApoE3, and ApoE4. ApoE4 constitutes the most important genetic risk factor for Alzheimer's disease (AD), whereas ApoE2 protects against AD. Significant evidence suggests that ApoE4 impairs microglial function and impedes astrocytic Aβ clearance in brain, but the direct neuronal effects of ApoE are poorly understood, and the differences between ApoE variants in these effects are unclear. Here, we report that ApoE acts on neurons as a glia-secreted signaling molecule that, among others, enhances synapse formation. In activating neuronal signaling, the three ApoE variants exhibit a differential potency of ApoE4>ApoE3>ApoE2, which mirrors their relative effects on AD risk, suggesting that differential signaling by ApoE variants may contribute to AD pathogenesis.

Exposure to early life stress (ELS) during childhood or prenatally increases the risk of future psychiatric disorders. The effect of stress exposure during the neonatal period is less well understood. In preterm infants, exposure to invasive procedures is associated with altered brain development and future stress responses suggesting that the neonatal period could be a key time for the programming of mental health. Previous studies suggest that ELS affects the hypothalamic epigenome, making it a good candidate to mediate these effects. In this study, we used a mouse model of early life stress (modified maternal separation; MMS). We hypothesised MMS would affect the hypothalamic transcriptome and DNA methylome, and impact on adult behaviour. MMS involved repeated stimulation of pups for 1.5 h/day, whilst separated from their mother, from postnatal day (P) 4–6. 3’mRNA sequencing and DNA methylation immunoprecipitation (meDIP) sequencing were performed on hypothalamic tissue at P6. Behaviour was assessed with the elevated plus, open field mazes and in-cage monitoring at 3–4 months of age. MMS was only associated with subtle changes in gene expression, but there were widespread alterations in DNA methylation. Notably, differentially methylated regions were enriched for synapse-associated loci. MMS resulted in hyperactivity in the elevated plus and open field mazes, but in-cage monitoring revealed that this was not representative of habitual hyperactivity. ELS has marked effects on DNA methylation in the hypothalamus in early life and results in stress-specific hyperactivity in young adulthood. These results have implications for the understanding of ELS-mediated effects on brain development. Supplementary Information The online version contains supplementary material available at 10.1186/s13072-021-00405-8.

Heterozygous NRXN1 deletions constitute the most prevalent currently known single-gene mutation predisposing to schizophrenia. Previous studies showed that engineered heterozygous NRXN1 deletions impaired neurotransmitter release in human neurons, suggesting a synaptic pathophysiological mechanism. Utilizing this observation for drug discovery, however, requires confidence in its robustness and validity. Here, we describe a multi-center effort to test the generality of this pivotal observation, using independent analyses at two laboratories of patient-derived and newly engineered human neurons with heterozygous NRXN1 deletions. We show that in neurons that were trans-differentiated from induced pluripotent stem cells derived from three NRXN1-deletion patients, the same impairment in neurotransmitter release was observed as in engineered NRXN1-deficient neurons. This impairment manifested as a decrease in spontaneous synaptic events and in evoked synaptic responses, and an alteration in synaptic paired-pulse depression. Nrxn1-deficient mouse neurons generated from embryonic stem cells by the same method as human neurons did not exhibit impaired neurotransmitter release, suggesting a human-specific phenotype. NRXN1 deletions produced a reproducible increase in the levels of CASK, an intracellular NRXN1-binding protein, and were associated with characteristic gene expression changes. Thus, heterozygous NRXN1 deletions robustly impair synaptic function in human neurons regardless of genetic background, enabling future drug discovery efforts.

In the skin, tissue injury results in fibrosis in the form of scars composed of dense extracellular matrix deposited by fibroblasts. The therapeutic goal of regenerative wound healing has remained elusive in part because principles of fibroblast programming and adaptive response to injury remain incompletely understood. Here, we present a multimodal -omics platform for the comprehensive study of cell populations in complex tissue, which has allowed us to characterize the cells involved in wound healing across both time and space. We employ a stented wound model that recapitulates human tissue repair kinetics and multiple Rainbow transgenic lines to precisely track fibroblast fate during the physiologic response to injury. Through integrated analysis of single cell chromatin landscapes and gene expression states, coupled with spatial transcriptomic profiling, we are able to impute fibroblast epigenomes with temporospatial resolution. This has allowed us to define the mechanisms controlling cell fate during migration, proliferation, and differentiation following tissue injury and thereby reexamine the canonical phases of wound healing. These findings have broad implications for the study of tissue repair in complex organ systems.

SUMMARY Transcriptional silencing of the FMR1 gene in fragile X syndrome (FXS) leads to the loss of the RNA-binding protein FMRP. In addition to regulating mRNA translation and protein synthesis, emerging evidence suggests that FMRP acts to coordinate proliferation and differentiation during early neural development. However, whether loss of FMRP-mediated translational control is related to impaired cell fate specification in the developing human brain remains unknown. Here, we use human patient induced pluripotent stem cell (iPSC)-derived neural progenitor cells and organoids to model neurogenesis in FXS. We developed a high-throughput, in vitro assay that allows for the simultaneous quantification of protein synthesis and proliferation within defined neural subpopulations. We demonstrate that abnormal protein synthesis in FXS is coupled to altered cellular decisions to favor proliferative over neurogenic cell fates during early development. Furthermore, pharmacologic inhibition of elevated phosphoinositide 3-kinase (PI3K) signaling corrects both excess protein synthesis and cell proliferation in a subset of patient neural cells., In brief Raj et al. developed a multiparametric assay to measure cellular and molecular phenotypes during neurogenesis in fragile X syndrome iPSC-derived neural cells. Relative to controls, FXS patient cultures have more proliferative cells with increased protein synthesis. Defects in cell fate acquisition can be normalized by inhibiting overactive PI3K signaling., Graphical Abstract

Assessment of neuroactive effects of chemicals in cell-based assays remains challenging as complex functional tissue is required for biologically relevant readouts. Recent in vitro models using rodent primary neural cultures grown on multielectrode arrays allow quantitative measurements of neural network activity suitable for neurotoxicity screening. However, robust systems for testing effects on network function in human neural models are still lacking. The increasing number of differentiation protocols for generating neurons from human-induced pluripotent stem cells (hiPSCs) holds great potential to overcome the unavailability of human primary tissue and expedite cell-based assays. Yet, the variability in neuronal activity, prolonged ontogeny and rather immature stage of most neuronal cells derived by standard differentiation techniques greatly limit their utility for screening neurotoxic effects on human neural networks. Here, we used excitatory and inhibitory neurons, separately generated by direct reprogramming from hiPSCs, together with primary human astrocytes to establish highly functional cultures with defined cell ratios. Such neuron/glia cocultures exhibited pronounced neuronal activity and robust formation of synchronized network activity on multielectrode arrays, albeit with noticeable delay compared with primary rat cortical cultures. We further investigated acute changes of network activity in human neuron/glia cocultures and rat primary cortical cultures in response to compounds with known adverse neuroactive effects, including gamma amino butyric acid receptor antagonists and multiple pesticides. Importantly, we observed largely corresponding concentration-dependent effects on multiple neural network activity metrics using both neural culture types. These results demonstrate the utility of directly converted neuronal cells from hiPSCs for functional neurotoxicity screening of environmental chemicals.

Generation of neurons of vast neurotransmitter, morphological and hodological subtype diversity involves early spatial and temporal patterning of the neuronal precursors by morphogenic gradients and combinatorial expression of homeotic transcription factors. Proneural basic-helix-loop-helix (bHLH) transcription factors play a key role in this process. However, it remains unknown how bHLH factors such as NGN2 and Ascl1, which are primarily proneural, contribute to subtype specification in a cell context-dependent manner. Here, we investigate the context-dependent molecular function of a pro-neural bHLH transcription factor NGN2; a factor which we previously showed to efficiently induce the neuronal fate in human embryonic stem (hES) cells. We tested its molecular function and lineage-inducing capacity in context of different chromatin environments and in combination with other transcription factors with spatially restricted expression patterns and evaluated NGN2 genomic localization, chromatin dynamics and transcriptional consequences using multi-omics methods. This work revealed that the combination of NGN2 and EMX1, a forebrain-restricted homeobox transcription factor, yields a highly homogeneous glutamatergic neuronal forebrain subpopulation without partial cholinergic and monoaminergic gene programs seen in NGN2 only cells. In contrast, modifying the chromatin environment that NGN2 is exposed to, strongly regulates the regional identity of resulting neurons that persists throughout reprogramming but does not influence neurotransmitter subtype specification. This shows how nature co-opts combinatorial strategies of using different chromatin landscapes and/or homeodomain transcription factors with spatially restricted expression to generate a vast variety of neuronal subtypes with a few transcription factors. Our work thus provides improved strategies to generate a plethora of defined and subtype specific neuronal cell populations from pluripotent stem cells for therapeutic or disease modeling purposes.

Terminally hydroxylated fatty acids or dicarboxylic acids are industrially relevant compounds with broad applications. Here, we present the proof of principle for the de novo biosynthesis of 8-hydroxyoctanoic acid from glucose and ethanol in the yeast Saccharomyces cerevisiae. Toxicity tests with medium-chain length ω-hydroxy fatty acids and dicarboxylic acids revealed little or no growth impairments on yeast cultures even at higher concentrations. The ability of various heterologous cytochrome P450 enzymes in combination with their cognate reductases for ω-hydroxylation of externally fed octanoic acid were compared. Finally, the most efficient P450 enzyme system was expressed in a yeast strain, whose fatty acid synthase was engineered for octanoic acid production, resulting in de novo biosynthesis of 8-hydroxyoctanoic acid up to 3 ​mg/l. Accumulation of octanoic acid revealed that cytochromes P450 activities were limiting 8-hydroxyoctanoic acid synthesis. The hydroxylation of both externally added and intracellularly produced octanoic acid was strongly dependent on the carbon source used, with ethanol being preferred. We further identified the availability of heme, a cofactor needed for P450 activity, as a limiting factor of 8-hydroxyoctanoic acid biosynthesis., Highlights • Low toxic effects of medium-chain ω-hydroxy fatty acids on yeast cells . • Systematic comparison of cytochrome P450 enzyme activities on octanoic acid . • De novo biosynthesis of 8-hydroxyoctanoic acid . • Improvement of cytochrome P450 activity with ethanol or by addition of hemin .

Hematopoietic stem cell transplantation (HSCT) is a curative therapy for blood and immune diseases with potential for many settings beyond current standard-of-care. Broad HSCT application is currently precluded largely due to morbidity and mortality associated with genotoxic irradiation or chemotherapy conditioning. Here we show that a single dose of a CD117-antibody-drug-conjugate (CD117-ADC) to saporin leads to > 99% depletion of host HSCs, enabling rapid and efficient donor hematopoietic cell engraftment. Importantly, CD117-ADC selectively targets hematopoietic stem cells yet does not cause clinically significant side-effects. Blood counts and immune cell function are preserved following CD117-ADC treatment, with effective responses by recipients to both viral and fungal challenges. These results suggest that CD117-ADC-mediated HSCT pre-treatment could serve as a non-myeloablative conditioning strategy for the treatment of a wide range of non-malignant and malignant diseases, and might be especially suited to gene therapy and gene editing settings in which preservation of immunity is desired., Hematopoietic stem cell (HSC) transplantation is a desirable treatment for many non-malignant and malignant diseases, but its use requires preconditioning of recipients with irradiation or chemotherapy that often induces high toxicity. Here the authors show that antibody-drug-conjugate to CD117, a HSC marker, allows specific and efficient preconditioning for HSC therapy.

Early life stress during childhood is associated with a number of psychiatric disorders that manifest across the life course. Preterm birth is a profound stressor, and an important cause of cognitive impairment, as well as neurodevelopmental and psychiatric disorders. However, the mechanisms that link events during the early neonatal period with later functional problems are poorly understood. We developed a novel mouse model of early life stress (modified maternal separation; MMS) with specific relevance to preterm birth (PTB) and hypothesised it would affect the hypothalamic transcriptome and DNA methylome and impact on behaviour in adulthood. MMS consisted of repeatedly stimulating pups for 1.5 hours/day, whilst separated from their mother, from postnatal day (P)4-6. 3’ RNA sequencing and DNA methylation immunoprecipitation (meDIP) sequencing was performed on the hypothalamus at P6. Behaviour was assessed with the elevated plus and open field mazes, and in-cage monitoring at 3-4 months of age. Although MMS was only associated with subtle changes in gene expression there were widespread alterations in DNA methylation. Notably, differentially methylated regions were enriched for synapse-associated loci. MMS also resulted in hyperactivity in the elevated plus and open field mazes, but in-cage monitoring revealed that this was not representative of habitual hyperactivity. In conclusion we describe a novel model of early life stress with relevance to PTB, with marked effects on DNA methylation in the hypothalamus and with stress-specific hyperactivity in young adulthood. We suggest that these results have implications for the understanding of early life stress mediated effects on brain development.

© 2020 Elsevier Inc. The cyclin-dependent kinase 1 (Cdk1) drives cell division. To uncover additional functions of Cdk1, we generated knockin mice expressing an analog-sensitive version of Cdk1 in place of wild-type Cdk1. In our study, we focused on embryonic stem cells (ESCs), because this cell type displays particularly high Cdk1 activity. We found that in ESCs, a large fraction of Cdk1 substrates is localized on chromatin. Cdk1 phosphorylates many proteins involved in epigenetic regulation, including writers and erasers of all major histone marks. Consistent with these findings, inhibition of Cdk1 altered histone-modification status of ESCs. High levels of Cdk1 in ESCs phosphorylate and partially inactivate Dot1l, the H3K79 methyltransferase responsible for placing activating marks on gene bodies. Decrease of Cdk1 activity during ESC differentiation de-represses Dot1l, thereby allowing coordinated expression of differentiation genes. These analyses indicate that Cdk1 functions to maintain the epigenetic identity of ESCs.

Cells achieve highly efficient and accurate communication through cellular projections such as neurites and filopodia, yet there is a lack of genetically encoded tools that can selectively manipulate their composition and dynamics. Here, we present a versatile optogenetic toolbox of artificial multi-headed myosin motors that can move bidirectionally within long cellular extensions and allow for the selective transport of GFP-tagged cargo with light. Utilizing these engineered motors, we could transport bulky transmembrane receptors and organelles as well as actin remodellers to control the dynamics of both filopodia and neurites. Using an optimized in vivo imaging scheme, we further demonstrate that, upon limb amputation in axolotls, a complex array of filopodial extensions is formed. We selectively modulated these filopodial extensions and showed that they re-establish a Sonic Hedgehog signalling gradient during regeneration. Considering the ubiquitous existence of actin-based extensions, this toolbox shows the potential to manipulate cellular communication with unprecedented accuracy. Zhang et al. design optogenetically controlled artificial transport vehicles that can be activated reversibly to manipulate cargo transport, impede neurite development and functionally characterize filopodial networks in axolotls.

Human cell models for disease based on induced pluripotent stem (iPS) cells have proven to be powerful new assets for investigating disease mechanisms. New insights have been obtained studying single mutations using isogenic controls generated by gene targeting. Modeling complex, multigenetic traits using patient-derived iPS cells is much more challenging due to line-to-line variability and technical limitations of scaling to dozens or more patients. Induced neuronal (iN) cells reprogrammed directly from dermal fibroblasts or urinary epithelia could be obtained from many donors, but such donor cells are heterogeneous, show interindividual variability, and must be extensively expanded, which can introduce random mutations. Moreover, derivation of dermal fibroblasts requires invasive biopsies. Here we show that human adult peripheral blood mononuclear cells, as well as defined purified T lymphocytes, can be directly converted into fully functional iN cells, demonstrating that terminally differentiated human cells can be efficiently transdifferentiated into a distantly related lineage. T cell-derived iN cells, generated by nonintegrating gene delivery, showed stereotypical neuronal morphologies and expressed multiple pan-neuronal markers, fired action potentials, and were able to form functional synapses. These cells were stable in the absence of exogenous reprogramming factors. Small molecule addition and optimized culture systems have yielded conversion efficiencies of up to 6.2%, resulting in the generation of >50,000 iN cells from 1 mL of peripheral blood in a single step without the need for initial expansion. Thus, our method allows the generation of sufficient neurons for experimental interrogation from a defined, homogeneous, and readily accessible donor cell population.

Summary RTN4-binding proteins were widely studied as “NoGo” receptors, but their physiological interactors and roles remain elusive. Similarly, BAI adhesion-GPCRs were associated with numerous activities, but their ligands and functions remain unclear. Using unbiased approaches, we observed an unexpected convergence: RTN4 receptors are high-affinity ligands for BAI adhesion-GPCRs. A single thrombospondin type 1-repeat (TSR) domain of BAIs binds to the leucine-rich repeat domain of all three RTN4-receptor isoforms with nanomolar affinity. In the 1.65 A crystal structure of the BAI1/RTN4-receptor complex, C-mannosylation of tryptophan and O-fucosylation of threonine in the BAI TSR-domains creates a RTN4-receptor/BAI interface shaped by unusual glycoconjugates that enables high-affinity interactions. In human neurons, RTN4 receptors regulate dendritic arborization, axonal elongation, and synapse formation by differential binding to glial versus neuronal BAIs, thereby controlling neural network activity. Thus, BAI binding to RTN4/NoGo receptors represents a receptor-ligand axis that, enabled by rare post-translational modifications, controls development of synaptic circuits.

Approaches to differentiating pluripotent stem cells (PSCs) into neurons currently face two major challenges-(i) generated cells are immature, with limited functional properties; and (ii) cultures exhibit heterogeneous neuronal subtypes and maturation stages. Using lineage-determining transcription factors, we previously developed a single-step method to generate glutamatergic neurons from human PSCs. Here, we show that transient expression of the transcription factors Ascl1 and Dlx2 (AD) induces the generation of exclusively GABAergic neurons from human PSCs with a high degree of synaptic maturation. These AD-induced neuronal (iN) cells represent largely nonoverlapping populations of GABAergic neurons that express various subtype-specific markers. We further used AD-iN cells to establish that human collybistin, the loss of gene function of which causes severe encephalopathy, is required for inhibitory synaptic function. The generation of defined populations of functionally mature human GABAergic neurons represents an important step toward enabling the study of diseases affecting inhibitory synaptic transmission.

A sustainable production of pharmaceuticals, chemicals, and biofuels is indispensable for an environmentally friendly future. Many of these compounds are based on fatty acids and their derivatives, generically referred to as oleochemicals. As an alternative to both petrochemistry and extraction from oil plants, modern approaches focus on engineering microbes for production of oleochemicals from biomass. This review describes strategies developed in Saccharomyces cerevisiae, a eukaryotic host in which substantial advances in the production of oleochemicals have been very recently achieved through a combination of metabolic engineering and optimized fermentation regimes. The survey of the available literature shows that model-based pathway optimization holds promise to enable an economically feasible production of oleochemicals in the near future.

Visual Abstract, Bipolar disorder (BP) and schizophrenia (SCZ) are major psychiatric disorders, but the molecular mechanisms underlying the complicated pathologies of these disorders remain unclear. It is difficult to establish adequate in vitro models for pathological analysis because of the heterogeneity of these disorders. In the present study, to recapitulate the pathologies of these disorders in vitro, we established in vitro models by differentiating mature neurons from human induced pluripotent stem cells (hiPSCs) derived from BP and SCZ patient with contributive copy number variations, as follows: two BP patients with PCDH15 deletion and one SCZ patient with RELN deletion. Glutamatergic neurons and GABAergic neurons were induced from hiPSCs under optimized conditions. Both types of induced neurons from both hiPSCs exhibited similar phenotypes of MAP2 (microtubule-associated protein 2)-positive dendrite shortening and decreasing synapse numbers. Additionally, we analyzed isogenic PCDH15- or RELN-deleted cells. The dendrite and synapse phenotypes of isogenic neurons were partially similar to those of patient-derived neurons. These results suggest that the observed phenotypes are general phenotypes of psychiatric disorders, and our in vitro models using hiPSC-based technology may be suitable for analysis of the pathologies of psychiatric disorders.

Summary Human pluripotent stem cells can be rapidly converted into functional neurons by ectopic expression of proneural transcription factors. Here we show that directly reprogrammed neurons, despite their rapid maturation kinetics, can model teratogenic mechanisms that specifically affect early neurodevelopment. We delineated distinct phases of in vitro maturation during reprogramming of human neurons and assessed the cellular phenotypes of valproic acid (VPA), a teratogenic drug. VPA exposure caused chronic impairment of dendritic morphology and functional properties of developing neurons, but not those of mature neurons. These pathogenic effects were associated with VPA-mediated inhibition of the histone deacetylase (HDAC) and glycogen synthase kinase-3 (GSK-3) pathways, which caused transcriptional downregulation of many genes, including MARCKSL1, an actin-stabilizing protein essential for dendritic morphogenesis and synapse maturation during early neurodevelopment. Our findings identify a developmentally restricted pathogenic mechanism of VPA and establish the use of reprogrammed neurons as an effective platform for modeling teratogenic pathways.

Diffuse intrinsic pontine glioma (DIPG) is an aggressive childhood tumor of the brainstem with currently no curative treatment available. The vast majority of DIPGs carry a histone H3 mutation leading to a lysine 27-to-methionine exchange (H3K27M). We engineered human induced pluripotent stem cells (iPSCs) to carry an inducible H3.3-K27M allele in the endogenous locus and studied the effects of the mutation in different disease-relevant neural cell types. H3.3-K27M upregulated bivalent promoter-associated developmental genes, producing diverse outcomes in different cell types. While being fatal for iPSCs, H3.3-K27M increased proliferation in neural stem cells (NSCs) and to a lesser extent in oligodendrocyte progenitor cells (OPCs). Only NSCs gave rise to tumors upon induction of H3.3-K27M and TP53 inactivation in an orthotopic xenograft model recapitulating human DIPGs. In NSCs, H3.3-K27M leads to maintained expression of stemness and proliferative genes and a premature activation of OPC programs that together may cause tumor initiation.

Pelizaeus-Merzbacher disease (PMD) is an X-linked disorder caused by mutation in the proteolipid protein-1 (PLP1) gene, which encodes the proteolipid protein of myelinating oligodendroglia. PMD exhibits phenotypic variability that reflects its considerable genotypic heterogeneity, but all forms of the disease result in central hypomyelination, associated in most cases with early neurological dysfunction, progressive deterioration, and ultimately death. PMD may present as a connatal, classic and transitional forms, or as the less severe spastic paraplegia type 2 and PLP-null phenotypes. These disorders are most often associated with duplications of the PLP1 gene, but can also be caused by coding and noncoding point mutations as well as full or partial deletion of the gene. A number of genetically-distinct but phenotypically-similar disorders of hypomyelination exist which, like PMD, lack any effective therapy. Yet as relatively pure CNS hypomyelinating disorders, with limited involvement of the PNS and relatively little attendant neuronal pathology, PMD and similar hypomyelinating disorders are attractive therapeutic targets for neural stem cell and glial progenitor cell transplantation, efforts at which are now underway in a number of research centers.

Direct lineage reprogramming represents a remarkable conversion of cellular and transcriptome states. However, the intermediate stages through which individual cells progress during reprogramming are largely undefined. Here we use single-cell RNA sequencing at multiple time points to dissect direct reprogramming from mouse embryonic fibroblasts to induced neuronal cells. By deconstructing heterogeneity at each time point and ordering cells by transcriptome similarity, we find that the molecular reprogramming path is remarkably continuous. Overexpression of the proneural pioneer factor Ascl1 results in a well-defined initialization, causing cells to exit the cell cycle and re-focus gene expression through distinct neural transcription factors. The initial transcriptional response is relatively homogeneous among fibroblasts, suggesting that the early steps are not limiting for productive reprogramming. Instead, the later emergence of a competing myogenic program and variable transgene dynamics over time appear to be the major efficiency limits of direct reprogramming. Moreover, a transcriptional state, distinct from donor and target cell programs, is transiently induced in cells undergoing productive reprogramming. Our data provide a high-resolution approach for understanding transcriptome states during lineage differentiation.

Cell replacement therapy with human pluripotent stem cell-derived neurons has the potential to ameliorate neurodegenerative dysfunction and central nervous system injuries, but reprogrammed neurons are dissociated and spatially disorganized during transplantation, rendering poor cell survival, functionality and engraftment in vivo. Here, we present the design of three-dimensional (3D) microtopographic scaffolds, using tunable electrospun microfibrous polymeric substrates that promote in situ stem cell neuronal reprogramming, neural network establishment and support neuronal engraftment into the brain. Scaffold-supported, reprogrammed neuronal networks were successfully grafted into organotypic hippocampal brain slices, showing an ∼3.5-fold improvement in neurite outgrowth and increased action potential firing relative to injected isolated cells. Transplantation of scaffold-supported neuronal networks into mouse brain striatum improved survival ∼38-fold at the injection site relative to injected isolated cells, and allowed delivery of multiple neuronal subtypes. Thus, 3D microscale biomaterials represent a promising platform for the transplantation of therapeutic human neurons with broad neuro-regenerative relevance., Human pluripotent stem cell derived neurons have the potential for cell replacement therapy for brain injury and disease but problems on transplantation need to be overcome. Here, the authors use a microtopographic scaffold to graft neurons into both hippocampal organoids and the mouse brain striatum.

Direct reprogramming of fibroblasts to neurons induces widespread cellular and transcriptional reconfiguration. Here, we characterized global epigenomic changes during the direct reprogramming of mouse fibroblasts to neurons using whole-genome base-resolution DNA methylation (mC) sequencing. We found that the pioneer transcription factor Ascl1 alone is sufficient for inducing the uniquely neuronal feature of non-CG methylation (mCH), but co-expression of Brn2 and Mytl1 was required to establish a global mCH pattern reminiscent of mature cortical neurons. Ascl1 alone induced promoter CG methylation (mCG) of fibroblast specific genes, while BAM overexpression additionally targets a competing myogenic program and directs a more faithful conversion to neuronal cells. Ascl1 induces local demethylation at its binding sites. Surprisingly, co-expression with Brn2 and Mytl1 inhibited the ability of Ascl1 to induce demethylation, suggesting a contextual regulation of transcription factor - epigenome interaction. Finally, we found that de novo methylation by DNMT3A is required for efficient neuronal reprogramming.

Long noncoding RNAs (lncRNAs) have been shown to act as important cell biological regulators including cell fate decisions but are often ignored in human genetics. Combining differential lncRNA expression during neuronal lineage induction with copy number variation morbidity maps of a cohort of children with autism spectrum disorder/intellectual disability versus healthy controls revealed focal genomic mutations affecting several lncRNA candidate loci. Here we find that a t(5:12) chromosomal translocation in a family manifesting neurodevelopmental symptoms disrupts specifically lnc-NR2F1. We further show that lnc-NR2F1 is an evolutionarily conserved lncRNA functionally enhances induced neuronal cell maturation and directly occupies and regulates transcription of neuronal genes including autism-associated genes. Thus, integrating human genetics and functional testing in neuronal lineage induction is a promising approach for discovering candidate lncRNAs involved in neurodevelopmental diseases.

Graft-versus-host disease (GVHD) remains a major complication of allogeneic hematopoietic cell transplantation. Acute GVHD (aGVHD) results from direct damage by donor T cells, whereas the biology of chronic GVHD (cGVHD) with its autoimmune-like manifestations remains poorly understood, mainly because of the paucity of representative preclinical models. We examined over an extended time period 7 MHC-matched, minor antigen–mismatched mouse models for development of cGVHD. Development and manifestations of cGVHD were determined by a combination of MHC allele type and recipient strain, with BALB recipients being the most susceptible. The C57BL/6 into BALB.B combination most closely modeled the human syndrome. In this strain combination moderate aGVHD was observed and BALB.B survivors developed overt cGVHD at 6 to 12 months affecting eyes, skin, and liver. Naive CD4+ cells caused this syndrome as no significant pathology was induced by grafts composed of purified hematopoietic stem cells (HSCs) or HSC plus effector memory CD4+ or CD8+ cells. Furthermore, co-transferred naive and effector memory CD4+ T cells demonstrated differential homing patterns and locations of persistence. No clear association with donor Th17 cells and the phenotype of aGVHD or cGVHD was observed in this model. Donor CD4+ cells caused injury to medullary thymic epithelial cells, a key population responsible for negative T cell selection, suggesting that impaired thymic selection was an underlying cause of the cGVHD syndrome. In conclusion, we report for the first time that the C57BL/6 into BALB.B combination is a representative model of cGVHD that evolves from immunologic events during the early post-transplant period.

Age-associated chronic inflammation (inflammaging) has emerged as a central hallmark of aging1-3, but its impact on specific cells is still largely unknown. Fibroblasts are present in all tissues and contribute to wound healing4-6. They are also the cell type that is mostly used for induced pluripotent stem cell (iPSC) reprogramming7 – a process that has implications for regenerative medicine and rejuvenation strategies8-17. Here we show that primary fibroblasts from old mice secrete inflammatory cytokines and that there is an increased variability in reprogramming efficiency between fibroblast cultures from old individuals. Individual-to-individual variability is emerging as a key feature of old age18-21, which could reflect distinct aging trajectories, but the underlying causes remain unknown. To identify drivers of this variability, we perform a multi-omic assessment of young and old fibroblast cultures with different reprogramming efficiency. This approach, coupled with single cell transcriptomics, reveals that old fibroblast cultures are heterogeneous and show a greater proportion of ‘activated fibroblasts’ that secrete inflammatory cytokines, which correlates with reprogramming efficiency. We experimentally validate that activated fibroblasts express inflammatory cytokines in vivo and that their presence is linked to enhanced reprogramming efficiency in culture. Conditioned-media swapping experiments show that extrinsic factors secreted by activated fibroblasts are more critical than intrinsic factors for the individual-to-individual variability in reprogramming efficiency, and we identify TNFα as a key inflammatory cytokine underlying this variability. Interestingly, old mice also exhibit variability in wound healing efficiency in vivo and old wounds show an increased subpopulation of activated fibroblasts with a unique TNFα signature. Our study shows that a switch in fibroblast composition, and the ratio of inflammatory cytokines they secrete, drives variability in reprogramming in vitro and may influence wound healing in vivo. These findings could help identify personalized strategies to improve iPSC generation and wound healing in older individuals.

Long noncoding RNAs (lncRNAs) have been shown to act as important cell biological regulators including cell fate decisions but are often ignored in human genetics. Combining differential lncRNA expression during neuronal lineage induction with copy number variation morbidity maps of a cohort of children with autism spectrum disorder/intellectual disability versus healthy controls revealed focal genomic mutations affecting several lncRNA candidate loci. Here we find that a t(5:12) chromosomal translocation in a family manifesting neurodevelopmental symptoms disrupts specifically lnc-NR2F1. We further show that lnc-NR2F1 is an evolutionarily conserved lncRNA functionally enhances induced neuronal cell maturation and directly occupies and regulates transcription of neuronal genes including autism-associated genes. Thus, integrating human genetics and functional testing in neuronal lineage induction is a promising approach for discovering candidate lncRNAs involved in neurodevelopmental diseases.

Fragile X syndrome (FXS) is an X chromosome-linked disease leading to severe intellectual disabilities. FXS is caused by inactivation of the fragile X mental retardation 1 (FMR1) gene, but how FMR1 inactivation induces FXS remains unclear. Using human neurons generated from control and FXS patient-derived induced pluripotent stem (iPS) cells or from embryonic stem cells carrying conditional FMR1 mutations, we show here that loss of FMR1 function specifically abolished homeostatic synaptic plasticity without affecting basal synaptic transmission. We demonstrated that, in human neurons, homeostatic plasticity induced by synaptic silencing was mediated by retinoic acid, which regulated both excitatory and inhibitory synaptic strength. FMR1 inactivation impaired homeostatic plasticity by blocking retinoic acid-mediated regulation of synaptic strength. Repairing the genetic mutation in the FMR1 gene in an FXS patient cell line restored fragile X mental retardation protein (FMRP) expression and fully rescued synaptic retinoic acid signaling. Thus, our study reveals a robust functional impairment caused by FMR1 mutations that might contribute to neuronal dysfunction in FXS. In addition, our results suggest that FXS patient iPS cell-derived neurons might be useful for studying the mechanisms mediating functional abnormalities in FXS.

Direct reprogramming of fibroblasts to neurons induces widespread cellular and transcriptional reconfiguration. In this study, we characterized global epigenomic changes during direct reprogramming using whole-genome base-resolution DNA methylome (mC) sequencing. We found that the pioneer transcription factor Ascl1 alone is sufficient for inducing the uniquely neuronal feature of non-CG methylation (mCH), but co-expression of Brn2 and Mytl1 was required to establish a global mCH pattern reminiscent of mature cortical neurons. Ascl1 alone induced strong promoter CG methylation (mCG) of fibroblast specific genes, while BAM overexpression additionally targets a competing myogenic program and directs a more faithful conversion to neuronal cells. Ascl1 induces local demethylation at its binding sites. Surprisingly, co-expression with Brn2 and Mytl1 inhibited the ability of Ascl1 to induce demethylation, suggesting a contextual regulation of transcription factor - epigenome interaction. Finally, we found thatde novomethylation by DNMT3A is required for efficient neuronal reprogramming.

SUMMARYTissue development results from lineage-specific transcription factors (TF) programming a dynamic chromatin landscape through progressive cell fate transitions. Here, we interrogate the epigenomic landscape during epidermal differentiation and create an inference network that ranks the coordinate effects of TF-accessible regulatory element-target gene expression triplets on lineage commitment. We discover two critical transition periods: surface ectoderm initiation and keratinocyte maturation, and identify TFAP2C and p63 as lineage initiation and maturation factors, respectively. Surprisingly, we find that TFAP2C, and not p63, is sufficient to initiate surface ectoderm differentiation, with TFAP2C-initiated progenitor cells capable of maturing into functional keratinocytes. Mechanistically, TFAP2C primes the surface ectoderm chromatin landscape and induces p63 expression and binding sites, thus allowing maturation factor p63 to positively auto-regulate its expression and close a subset of the TFAP2C-initiated early program. Our work provides a general framework to infer TF networks controlling chromatin transitions that will facilitate future regenerative medicine advances.

Global transcription factor activity is measured by screening cell extracts with an oligonucleotide library.

Pluripotent stem cells, defined by an unlimited self-renewal capacity and an undifferentiated state, are best typified by embryonic stem cells. These cells have a unique cell cycle compared to somatic cells as defined by a rapid progression through the cell cycle and a minimal time spent in G1. Recent reports indicate that pluripotency and cell cycle regulation are mechanistically linked. In this review, we discuss the reciprocal co-regulation of these processes, how this co-regulation may prevent differentiation, and how cellular reprogramming can re-establish the unique cell cycle regulation in induced pluripotent stem cells.

Heterozygous mutations of the NRXN1 gene, which encodes the presynaptic cell-adhesion molecule neurexin-1, were repeatedly associated with autism and schizophrenia. However, diverse clinical presentations of NRXN1 mutations in patients raise the question whether heterozygous NRXN1 mutations alone directly impair synaptic function. To address this question under conditions that precisely control for genetic background, we generated human ES cells with different heterozygous conditional NRXN1 mutations, and analyzed two different types of isogenic control and NRXN1-mutant neurons derived from these ES cells. Both heterozygous NRXN1 mutations selectively impaired neurotransmitter release in human neurons without changing neuronal differentiation or synapse formation. Moreover, NRXN1-mutant human neurons exhibited increased levels of CASK, a critical synaptic scaffolding protein that binds to neurexin-1. Our results show that, unexpectedly, heterozygous inactivation of NRXN1 directly impairs synaptic function in human neurons, and illustrate the value of this conditional deletion approach for studying the functional effects of disease-associated mutations.