ETV4 is a mechanical transducer linking cell crowding dynamics to lineage specification.

Dynamic changes in mechanical microenvironments, such as cell crowding, regulate lineage fates as well as cell proliferation. Although regulatory mechanisms for contact inhibition of proliferation have been extensively studied, it remains unclear how cell crowding induces lineage specification. Here we found that a well-known oncogene, ETS variant transcription factor 4 (ETV4), serves as a molecular transducer that links mechanical microenvironments and gene expression. In a growing epithelium of human embryonic stem cells, cell crowding dynamics is translated into ETV4 expression, serving as a pre-pattern for future lineage fates. A switch-like ETV4 inactivation by cell crowding derepresses the potential for neuroectoderm differentiation in human embryonic stem cell epithelia. Mechanistically, cell crowding inactivates the integrin-actomyosin pathway and blocks the endocytosis of fibroblast growth factor receptors (FGFRs). The disrupted FGFR endocytosis induces a marked decrease in ETV4 protein stability through ERK inactivation. Mathematical modelling demonstrates that the dynamics of cell density in a growing human embryonic stem cell epithelium precisely determines the spatiotemporal ETV4 expression pattern and, consequently, the timing and geometry of lineage development. Our findings suggest that cell crowding dynamics in a stem cell epithelium drives spatiotemporal lineage specification using ETV4 as a key mechanical transducer.

Measuring transcription factor binding and gene expression using barcoded self-reporting transposon calling cards and transcriptomes.

Calling cards technology using self-reporting transposons enables the identification of DNA-protein interactions through RNA sequencing. Although immensely powerful, current implementations of calling cards in bulk experiments on populations of cells are technically cumbersome and require many replicates to identify independent insertions into the same genomic locus. Here, we have drastically reduced the cost and labor requirements of calling card experiments in bulk populations of cells by introducing a DNA barcode into the calling card itself. An additional barcode incorporated during reverse transcription enables simultaneous transcriptome measurement in a facile and affordable protocol. We demonstrate that barcoded self-reporting transposons recover in vitro binding sites for four basic helix-loop-helix transcription factors with important roles in cell fate specification: ASCL1, MYOD1, NEUROD2 and NGN1. Further, simultaneous calling cards and transcriptional profiling during transcription factor overexpression identified both binding sites and gene expression changes for two of these factors. Lastly, we demonstrated barcoded calling cards can record binding in vivo in the mouse brain. In sum, RNA-based identification of transcription factor binding sites and gene expression through barcoded self-reporting transposon calling cards and transcriptomes is an efficient and powerful method to infer gene regulatory networks in a population of cells.

Mapping cis-regulatory elements in human neurons links psychiatric disease heritability and activity-regulated transcriptional programs.

Genome-wide association studies (GWASs) have identified hundreds of loci associated with psychiatric diseases, yet there is a lack of understanding of disease pathophysiology. Common risk variants can shed light on the underlying molecular mechanisms; however, identifying causal variants remains challenging. We map cis-regulatory elements in human neurons derived from pluripotent stem cells. This system allows us to determine enhancers that activate the transcription of neuronal activity-regulated gene programs, which are thought to be critical for synaptic plasticity and are not possible to identify from postmortem tissues. Using the activity-by-contact model, we create variant-to-gene maps to interpret the function of GWAS variants. Our work nominates a subset of variants to elucidate the molecular mechanisms involving GWAS-significant loci. It also highlights that in vitro human cellular models are a powerful platform for identifying and mechanistic studies of human trait-associated genetic variants in cell states that are inaccessible from other types of human samples.

Cellular and genetic drivers of RNA editing variation in the human brain.

Posttranscriptional adenosine-to-inosine modifications amplify the functionality of RNA molecules in the brain, yet the cellular and genetic regulation of RNA editing is poorly described. We quantify base-specific RNA editing across three major cell populations from the human prefrontal cortex: glutamatergic neurons, medial ganglionic eminence-derived GABAergic neurons, and oligodendrocytes. We identify more selective editing and hyper-editing in neurons relative to oligodendrocytes. RNA editing patterns are highly cell type-specific, with 189,229 cell type-associated sites. The cellular specificity for thousands of sites is confirmed by single nucleus RNA-sequencing. Importantly, cell type-associated sites are enriched in GTEx RNA-sequencing data, edited ~twentyfold higher than all other sites, and variation in RNA editing is largely explained by neuronal proportions in bulk brain tissue. Finally, we uncover 661,791 cis-editing quantitative trait loci across thirteen brain regions, including hundreds with cell type-associated features. These data reveal an expansive repertoire of highly regulated RNA editing sites across human brain cell types and provide a resolved atlas linking cell types to editing variation and genetic regulatory effects.

Rapid and Extraction-Free Detection of SARS-CoV-2 from Saliva by Colorimetric Reverse-Transcription Loop-Mediated Isothermal Amplification.

BACKGROUND: Rapid, reliable, and widespread testing is required to curtail the ongoing COVID-19 pandemic. Current gold-standard nucleic acid tests are hampered by supply shortages in critical reagents including nasal swabs, RNA extraction kits, personal protective equipment, instrumentation, and labor. METHODS: To overcome these challenges, we developed a rapid colorimetric assay using reverse-transcription loop-mediated isothermal amplification (RT-LAMP) optimized on human saliva samples without an RNA purification step. We describe the optimization of saliva pretreatment protocols to enable analytically sensitive viral detection by RT-LAMP. We optimized the RT-LAMP reaction conditions and implemented high-throughput unbiased methods for assay interpretation. We tested whether saliva pretreatment could also enable viral detection by conventional reverse-transcription quantitative polymerase chain reaction (RT-qPCR). Finally, we validated these assays on clinical samples. RESULTS: The optimized saliva pretreatment protocol enabled analytically sensitive extraction-free detection of SARS-CoV-2 from saliva by colorimetric RT-LAMP or RT-qPCR. In simulated samples, the optimized RT-LAMP assay had a limit of detection of 59 (95% confidence interval: 44-104) particle copies per reaction. We highlighted the flexibility of LAMP assay implementation using 3 readouts: naked-eye colorimetry, spectrophotometry, and real-time fluorescence. In a set of 30 clinical saliva samples, colorimetric RT-LAMP and RT-qPCR assays performed directly on pretreated saliva samples without RNA extraction had accuracies greater than 90%. CONCLUSIONS: Rapid and extraction-free detection of SARS-CoV-2 from saliva by colorimetric RT-LAMP is a simple, sensitive, and cost-effective approach with broad potential to expand diagnostic testing for the virus causing COVID-19.

High-throughput single-cell functional elucidation of neurodevelopmental disease-associated genes reveals convergent mechanisms altering neuronal differentiation.

The overwhelming success of exome- and genome-wide association studies in discovering thousands of disease-associated genes necessitates developing novel high-throughput functional genomics approaches to elucidate the molecular mechanisms of these genes. Here, we have coupled multiplexed repression of neurodevelopmental disease-associated genes to single-cell transcriptional profiling in differentiating human neurons to rapidly assay the functions of multiple genes in a disease-relevant context, assess potentially convergent mechanisms, and prioritize genes for specific functional assays. For a set of 13 autism spectrum disorder (ASD)-associated genes, we show that this approach generated important mechanistic insights, revealing two functionally convergent modules of ASD genes: one that delays neuron differentiation and one that accelerates it. Five genes that delay neuron differentiation (ADNP, ARID1B, ASH1L, CHD2, and DYRK1A) mechanistically converge, as they all dysregulate genes involved in cell-cycle control and progenitor cell proliferation. Live-cell imaging after individual ASD-gene repression validated this functional module, confirming that these genes reduce neural progenitor cell proliferation and neurite growth. Finally, these functionally convergent ASD gene modules predicted shared clinical phenotypes among individuals with mutations in these genes. Altogether, these results show the utility of a novel and simple approach for the rapid functional elucidation of neurodevelopmental disease-associated genes.

Self-Reporting Transposons Enable Simultaneous Readout of Gene Expression and Transcription Factor Binding in Single Cells.

Cellular heterogeneity confounds in situ assays of transcription factor (TF) binding. Single-cell RNA sequencing (scRNA-seq) deconvolves cell types from gene expression, but no technology links cell identity to TF binding sites (TFBS) in those cell types. We present self-reporting transposons (SRTs) and use them in single-cell calling cards (scCC), a novel assay for simultaneously measuring gene expression and mapping TFBS in single cells. The genomic locations of SRTs are recovered from mRNA, and SRTs deposited by exogenous, TF-transposase fusions can be used to map TFBS. We then present scCC, which map SRTs from scRNA-seq libraries, simultaneously identifying cell types and TFBS in those same cells. We benchmark multiple TFs with this technique. Next, we use scCC to discover BRD4-mediated cell-state transitions in K562 cells. Finally, we map BRD4 binding sites in the mouse cortex at single-cell resolution, establishing a new method for studying TF biology in situ.

Rapid and extraction-free detection of SARS-CoV-2 from saliva with colorimetric LAMP.

Rapid, reliable, and widespread testing is required to curtail the ongoing COVID-19 pandemic. Current gold standard nucleic acid tests are hampered by supply shortages in critical reagents including nasal swabs, RNA extraction kits, personal protective equipment (PPE), instrumentation, and labor. Here we present an approach to overcome these challenges with the development of a rapid colorimetric assay using reverse-transcription loop-mediated isothermal amplification (RT-LAMP) optimized on human saliva samples without an RNA purification step. We describe our optimizations of the LAMP reaction and saliva pretreatment protocols that enabled rapid and sensitive detection of < 102 viral genomes per reaction in contrived saliva controls. Moreover, our saliva pretreatment protocol enabled sensitive viral detection by conventional quantitative reverse transcription polymerase chain reaction (qRT-PCR) without RNA extraction. We validated the high performance of these assays on clinical samples and demonstrate a promising approach to overcome the current bottlenecks limiting widespread testing.

FOXM1 drives proximal tubule proliferation during repair from acute ischemic kidney injury.

The proximal tubule has a remarkable capacity for repair after acute injury, but the cellular lineage and molecular mechanisms underlying this repair response are incompletely understood. Here, we developed a Kim1-GFPCreERt2 knockin mouse line (Kim1-GCE) in order to perform genetic lineage tracing of dedifferentiated cells while measuring the cellular transcriptome of proximal tubule during repair. Acutely injured genetically labeled clones coexpressed KIM1, VIMENTIN, SOX9, and KI67, indicating a dedifferentiated and proliferative state. Clonal analysis revealed clonal expansion of Kim1+ cells, indicating that acutely injured, dedifferentiated proximal tubule cells, rather than fixed tubular progenitor cells, account for repair. Translational profiling during injury and repair revealed signatures of both successful and unsuccessful maladaptive repair. The transcription factor Foxm1 was induced early in injury, was required for epithelial proliferation in vitro, and was dependent on epidermal growth factor receptor (EGFR) stimulation. In conclusion, dedifferentiated proximal tubule cells effect proximal tubule repair, and we reveal an EGFR/FOXM1-dependent signaling pathway that drives proliferative repair after injury.

Resistance to autosomal dominant Alzheimer's disease in an APOE3 Christchurch homozygote: a case report.

We identified a PSEN1 (presenilin 1) mutation carrier from the world's largest autosomal dominant Alzheimer's disease kindred, who did not develop mild cognitive impairment until her seventies, three decades after the expected age of clinical onset. The individual had two copies of the APOE3 Christchurch (R136S) mutation, unusually high brain amyloid levels and limited tau and neurodegenerative measurements. Our findings have implications for the role of APOE in the pathogenesis, treatment and prevention of Alzheimer's disease.

Soluble syntaxin 3 functions as a transcriptional regulator.

Syntaxins are a conserved family of SNARE proteins and contain C-terminal transmembrane anchors required for their membrane fusion activity. Here we show that Stx3 (syntaxin 3) unexpectedly also functions as a nuclear regulator of gene expression. We found that alternative splicing creates a soluble isoform that we termed Stx3S, lacking the transmembrane anchor. Soluble Stx3S binds to the nuclear import factor RanBP5 (RAN-binding protein 5), targets to the nucleus, and interacts physically and functionally with several transcription factors, including ETV4 (ETS variant 4) and ATF2 (activating transcription factor 2). Stx3S is differentially expressed in normal human tissues, during epithelial cell polarization, and in breast cancer versus normal breast tissue. Inhibition of endogenous Stx3S expression alters the expression of cancer-associated genes and promotes cell proliferation. Similar nuclear-targeted, soluble forms of other syntaxins were identified, suggesting that nuclear signaling is a conserved, novel function common among these membrane-trafficking proteins.

MCP-1 and eotaxin-1 selectively and negatively associate with memory in MCI and Alzheimer's disease dementia phenotypes.

INTRODUCTION: MCP-1 and eotaxin-1 are encoded on chromosome 17 and have been shown to reduce hippocampal neurogenesis in mice. We investigated whether these chemokines selectively associate with memory in individuals with mild cognitive impairment (MCI) and Alzheimer's disease (AD) dementia. METHODS: MCP-1 and eotaxin-1 were assayed in controls, MCI, and AD dementia patients with varying phenotypes (n = 171). A subset of 55 individuals had magnetic resonance imaging (MRI) scans available. Composite scores for cognitive variables were created, and medial temporal lobe volumes were obtained. RESULTS: An interaction was noted between MCP-1 and eotaxin-1, such that deleterious associations with memory were seen when both chemokines were elevated. These associations remained significant after adding APOE genotype and comparison (non-chromosome 17) chemokines into the model. These chemokines predicted left medial temporal lobe volume and were not related to other cognitive domains. DISCUSSION: These results suggest a potentially selective role for MCP-1 and eotaxin-1 in memory dysfunction in the context of varied MCI and AD dementia phenotypes.

Primary Cilium-Autophagy-Nrf2 (PAN) Axis Activation Commits Human Embryonic Stem Cells to a Neuroectoderm Fate.

Under defined differentiation conditions, human embryonic stem cells (hESCs) can be directed toward a mesendoderm (ME) or neuroectoderm (NE) fate, the first decision during hESC differentiation. Coupled with lineage-specific G1 lengthening, a divergent ciliation pattern emerged within the first 24 hr of induced lineage specification, and these changes heralded a neuroectoderm decision before any neural precursor markers were expressed. By day 2, increased ciliation in NE precursors induced autophagy that resulted in the inactivation of Nrf2 and thereby relieved transcriptional activation of OCT4 and NANOG. Nrf2 binds directly to upstream regions of these pluripotency genes to promote their expression and repress NE derivation. Nrf2 suppression was sufficient to rescue poorly neurogenic iPSC lines. Only after these events had been initiated did neural precursor markers get expressed at day 4. Thus, we have identified a primary cilium-autophagy-Nrf2 (PAN) control axis coupled to cell-cycle progression that directs hESCs toward NE.

Haploinsufficiency of BAZ1B contributes to Williams syndrome through transcriptional dysregulation of neurodevelopmental pathways.

Williams syndrome (WS) is a neurodevelopmental disorder caused by a genomic deletion of ∼28 genes that results in a cognitive and behavioral profile marked by overall intellectual impairment with relative strength in expressive language and hypersocial behavior. Advancements in protocols for neuron differentiation from induced pluripotent stem cells allowed us to elucidate the molecular circuitry underpinning the ontogeny of WS. In patient-derived stem cells and neurons, we determined the expression profile of the Williams-Beuren syndrome critical region-deleted genes and the genome-wide transcriptional consequences of the hemizygous genomic microdeletion at chromosome 7q11.23. Derived neurons displayed disease-relevant hallmarks and indicated novel aberrant pathways in WS neurons including over-activated Wnt signaling accompanying an incomplete neurogenic commitment. We show that haploinsufficiency of the ATP-dependent chromatin remodeler, BAZ1B, which is deleted in WS, significantly contributes to this differentiation defect. Chromatin-immunoprecipitation (ChIP-seq) revealed BAZ1B target gene functions are enriched for neurogenesis, neuron differentiation and disease-relevant phenotypes. BAZ1B haploinsufficiency caused widespread gene expression changes in neural progenitor cells, and together with BAZ1B ChIP-seq target genes, explained 42% of the transcriptional dysregulation in WS neurons. BAZ1B contributes to regulating the balance between neural precursor self-renewal and differentiation and the differentiation defect caused by BAZ1B haploinsufficiency can be rescued by mitigating over-active Wnt signaling in neural stem cells. Altogether, these results reveal a pivotal role for BAZ1B in neurodevelopment and implicate its haploinsufficiency as a likely contributor to the neurological phenotypes in WS.

Genomic DISC1 Disruption in hiPSCs Alters Wnt Signaling and Neural Cell Fate.

Genetic and clinical association studies have identified disrupted in schizophrenia 1 (DISC1) as a candidate risk gene for major mental illness. DISC1 is interrupted by a balanced chr(1;11) translocation in a Scottish family in which the translocation predisposes to psychiatric disorders. We investigate the consequences of DISC1 interruption in human neural cells using TALENs or CRISPR-Cas9 to target the DISC1 locus. We show that disruption of DISC1 near the site of the translocation results in decreased DISC1 protein levels because of nonsense-mediated decay of long splice variants. This results in an increased level of canonical Wnt signaling in neural progenitor cells and altered expression of fate markers such as Foxg1 and Tbr2. These gene expression changes are rescued by antagonizing Wnt signaling in a critical developmental window, supporting the hypothesis that DISC1-dependent suppression of basal Wnt signaling influences the distribution of cell types generated during cortical development.

Synaptic dysregulation in a human iPS cell model of mental disorders.

Dysregulated neurodevelopment with altered structural and functional connectivity is believed to underlie many neuropsychiatric disorders, and 'a disease of synapses' is the major hypothesis for the biological basis of schizophrenia. Although this hypothesis has gained indirect support from human post-mortem brain analyses and genetic studies, little is known about the pathophysiology of synapses in patient neurons and how susceptibility genes for mental disorders could lead to synaptic deficits in humans. Genetics of most psychiatric disorders are extremely complex due to multiple susceptibility variants with low penetrance and variable phenotypes. Rare, multiply affected, large families in which a single genetic locus is probably responsible for conferring susceptibility have proven invaluable for the study of complex disorders. Here we generated induced pluripotent stem (iPS) cells from four members of a family in which a frameshift mutation of disrupted in schizophrenia 1 (DISC1) co-segregated with major psychiatric disorders and we further produced different isogenic iPS cell lines via gene editing. We showed that mutant DISC1 causes synaptic vesicle release deficits in iPS-cell-derived forebrain neurons. Mutant DISC1 depletes wild-type DISC1 protein and, furthermore, dysregulates expression of many genes related to synapses and psychiatric disorders in human forebrain neurons. Our study reveals that a psychiatric disorder relevant mutation causes synapse deficits and transcriptional dysregulation in human neurons and our findings provide new insight into the molecular and synaptic etiopathology of psychiatric disorders.

Nrf2, a regulator of the proteasome, controls self-renewal and pluripotency in human embryonic stem cells.

Nuclear factor, erythroid 2-like 2 (Nrf2) is a master transcription factor for cellular defense against endogenous and exogenous stresses by regulating expression of many antioxidant and detoxification genes. Here, we show that Nrf2 acts as a key pluripotency gene and a regulator of proteasome activity in human embryonic stem cells (hESCs). Nrf2 expression is highly enriched in hESCs and dramatically decreases upon differentiation. Nrf2 inhibition impairs both the self-renewal ability of hESCs and re-establishment of pluripotency during cellular reprogramming. Nrf2 activation can delay differentiation. During early hESC differentiation, Nrf2 closely colocalizes with OCT4 and NANOG. As an underlying mechanism, our data show that Nrf2 regulates proteasome activity in hESCs partially through proteasome maturation protein (POMP), a proteasome chaperone, which in turn controls the proliferation of self-renewing hESCs, three germ layer differentiation and cellular reprogramming. Even modest proteasome inhibition skews the balance of early differentiation toward mesendoderm at the expense of an ectodermal fate by decreasing the protein level of cyclin D1 and delaying the degradation of OCT4 and NANOG proteins. Taken together, our findings suggest a new potential link between environmental stress and stemness with Nrf2 and the proteasome coordinately positioned as key mediators.

Translational profiles of medullary myofibroblasts during kidney fibrosis.

Myofibroblasts secrete matrix during chronic injury, and their ablation ameliorates fibrosis. Development of new biomarkers and therapies for CKD will be aided by a detailed analysis of myofibroblast gene expression during the early stages of fibrosis. However, dissociating myofibroblasts from fibrotic kidney is challenging. We therefore adapted translational ribosome affinity purification (TRAP) to isolate and profile mRNA from myofibroblasts and their precursors during kidney fibrosis. We generated and characterized a transgenic mouse expressing an enhanced green fluorescent protein (eGFP)-tagged L10a ribosomal subunit protein under control of the collagen1α1 promoter. We developed a one-step procedure for isolation of polysomal RNA from collagen1α1-eGFPL10a mice subject to unilateral ureteral obstruction and analyzed and validated the resulting transcriptional profiles. Pathway analysis revealed strong gene signatures for cell proliferation, migration, and shape change. Numerous novel genes and candidate biomarkers were upregulated during fibrosis, specifically in myofibroblasts, and we validated these results by quantitative PCR, in situ, and Western blot analysis. This study provides a comprehensive analysis of early myofibroblast gene expression during kidney fibrosis and introduces a new technique for cell-specific polysomal mRNA isolation in kidney injury models that is suited for RNA-sequencing technologies.

Origin of the PSEN1 E280A mutation causing early-onset Alzheimer's disease.

BACKGROUND: A mutation in presenilin 1 (E280A) causes early-onset Alzheimer's disease. Understanding the origin of this mutation will inform medical genetics. METHODS: We sequenced the genomes of 102 individuals from Antioquia, Colombia. We applied identity-by-descent analysis to identify regions of common ancestry. We estimated the age of the E280A mutation and the local ancestry of the haplotype harboring this mutation. RESULTS: All affected individuals share a minimal haplotype of 1.8 Mb containing E280A. We estimate a time to most recent common ancestor of E280A of 10 (95% credible interval, 7.2-12.6) generations. We date the de novo mutation event to 15 (95% credible interval, 11-25) generations ago. We infer a western European geographic origin of the shared haplotype. CONCLUSIONS: The age and geographic origin of E280A are consistent with a single founder dating from the time of the Spanish Conquistadors who began colonizing Colombia during the early 16th century.

Differentiated kidney epithelial cells repair injured proximal tubule.

Whether kidney proximal tubule harbors a scattered population of epithelial stem cells is a major unsolved question. Lineage-tracing studies, histologic characterization, and ex vivo functional analysis results conflict. To address this controversy, we analyzed the lineage and clonal behavior of fully differentiated proximal tubule epithelial cells after injury. A CreER(T2) cassette was knocked into the sodium-dependent inorganic phosphate transporter SLC34a1 locus, which is expressed only in differentiated proximal tubule. Tamoxifen-dependent recombination was absolutely specific to proximal tubule. Clonal analysis after injury and repair showed that the bulk of labeled cells proliferate after injury with increased clone size after severe compared with mild injury. Injury to labeled proximal tubule epithelia induced expression of CD24, CD133, vimentin, and kidney-injury molecule-1, markers of putative epithelial stem cells in the human kidney. Similar results were observed in cultured proximal tubules, in which labeled clones proliferated and expressed dedifferentiation and injury markers. When mice with completely labeled kidneys were subject to injury and repair there was no dilution of fate marker despite substantial proliferation, indicating that unlabeled progenitors do not contribute to kidney repair. During nephrogenesis and early kidney growth, single proximal tubule clones expanded, suggesting that differentiated cells also contribute to tubule elongation. These findings provide no evidence for an intratubular stem-cell population, but rather indicate that terminally differentiated epithelia reexpress apparent stem-cell markers during injury-induced dedifferentiation and repair.