Autophagy is a cellular catabolic process that relies on the cooperation of autophagosomes and lysosomes. During starvation, the cell expands both compartments to enhance degradation processes. We ...found that starvation activates a transcriptional program that controls major steps of the autophagic pathway, including autophagosome formation, autophagosome-lysosome fusion, and substrate degradation. The transcription factor EB (TFEB), a master gene for lysosomal biogenesis, coordinated this program by driving expression of autophagy and lysosomal genes. Nuclear localization and activity of TFEB were regulated by serine phosphorylation mediated by the extracellular signal—regulated kinase 2, whose activity was tuned by the levels of extracellular nutrients. Thus, a mitogen-activated protein kinase—dependent mechanism regulates autophagy by controlling the biogenesis and partnership of two distinct cellular organelles.
Genome editing has attracted wide interest for the generation of cellular models of disease using human pluripotent stem cells and other cell types. CRISPR-Cas systems and TALENs can target desired ...genomic sites with high efficiency in human cells, but recent publications have led to concern about the extent to which these tools may cause off-target mutagenic effects that could potentially confound disease-modeling studies. Using CRISPR-Cas9 and TALEN targeted human pluripotent stem cell clones, we performed whole-genome sequencing at high coverage in order to assess the degree of mutagenesis across the entire genome. In both types of clones, we found that off-target mutations attributable to the nucleases were very rare. From this analysis, we suggest that, although some cell types may be at risk for off-target mutations, the incidence of such effects in human pluripotent stem cells may be sufficiently low and thus not a significant concern for disease modeling and other applications.
Display omitted
•CRISPR-Cas9 and TALENs were used to genome edit human pluripotent stem cells•Whole-genome sequencing of individual clones found very few off-target mutations
Recent work suggested that off-target mutagenesis is a problem for genome-editing technology, but Veres et al. show that, at least for human pluripotent stem cells, the frequency of such mutations appears to be low.
Functional analyses of genomic data within the context of a priori biomolecular networks can give valuable mechanistic insights. However, such analyses are not a trivial task, owing to the complexity ...of biological networks and lack of computational methods for their effective integration with experimental data.
We developed a software application suite, NetWalker, as a one-stop platform featuring a number of novel holistic (i.e. assesses the whole data distribution without requiring data cutoffs) data integration and analysis methods for network-based comparative interpretations of genome-scale data. The central analysis components, NetWalk and FunWalk, are novel random walk-based network analysis methods that provide unique analysis capabilities to assess the entire data distributions together with network connectivity to prioritize molecular and functional networks, respectively, most highlighted in the supplied data. Extensive inter-operability between the analysis components and with external applications, including R, adds to the flexibility of data analyses. Here, we present a detailed computational analysis of our microarray gene expression data from MCF7 cells treated with lethal and sublethal doses of doxorubicin.
NetWalker, a detailed step-by-step tutorial containing the analyses presented in this paper and a manual are available at the web site http://netwalkersuite.org.
•Exome sequencing on unprecedented scale has identified 400 genes associated with ASD and NDD.•Functional networks disrupted in NDD converge on distinct spatiotemporal expression patterns during ...early neurodevelopment.•There is growing evidence that genes involved in broad regulatory processes can alter synaptic function.•Signaling pathways and cytoskeleton dynamics may underlie convergent pathogenic mechanisms in ASD and NDD.
Over the last two years, remarkable gene discovery efforts have implicated disruption of pathways involving gene regulatory functions and neuronal processes in autism spectrum disorder (ASD), and more broadly defined neurodevelopmental disorders (NDDs). Functional studies in the developing brain and across cell types demonstrate that the spatiotemporal expression patterns of many of these genes coalesce on subnetworks with distinct developmental trajectories. Here, we review the convergent biological processes derived from gene discovery and functional genomics in ASD and NDD from 2018−2020. We further probe the mechanistic insights that suggest these frequently perturbed pathways are interconnected and, ultimately, converge on specific functional deficits in human neurodevelopment.
Truncating mutations of chromodomain helicase DNA-binding protein 8 ( CHD8 ), and of many other genes with diverse functions, are strong-effect risk factors for autism spectrum disorder (ASD), ...suggesting multiple mechanisms of pathogenesis. We explored the transcriptional networks that CHD8 regulates in neural progenitor cells (NPCs) by reducing its expression and then integrating transcriptome sequencing (RNA sequencing) with genome-wide CHD8 binding (ChIP sequencing). Suppressing CHD8 to levels comparable with the loss of a single allele caused altered expression of 1,756 genes, 64.9% of which were up-regulated. CHD8 showed widespread binding to chromatin, with 7,324 replicated sites that marked 5,658 genes. Integration of these data suggests that a limited array of direct regulatory effects of CHD8 produced a much larger network of secondary expression changes. Genes indirectly down-regulated (i.e., without CHD8-binding sites) reflect pathways involved in brain development, including synapse formation, neuron differentiation, cell adhesion, and axon guidance, whereas CHD8-bound genes are strongly associated with chromatin modification and transcriptional regulation. Genes associated with ASD were strongly enriched among indirectly down-regulated loci ( P < 10 ⁻⁸) and CHD8-bound genes ( P = 0.0043), which align with previously identified coexpression modules during fetal development. We also find an intriguing enrichment of cancer-related gene sets among CHD8-bound genes ( P < 10 ⁻¹⁰). In vivo suppression of chd8 in zebrafish produced macrocephaly comparable to that of humans with inactivating mutations. These data indicate that heterozygous disruption of CHD8 precipitates a network of gene-expression changes involved in neurodevelopmental pathways in which many ASD-associated genes may converge on shared mechanisms of pathogenesis.
Significance Truncating mutation of chromodomain helicase DNA-binding protein 8 ( CHD8 ) represents one of the strongest known risk factors for autism spectrum disorder (ASD). We mimicked the effects of such heterozygous loss-of-function mutations in neural progenitor cells and integrated RNA sequencing with genome-wide delineation of CHD8 binding. Our results reveal that the molecular mechanism by which CHD8 alters neurodevelopmental pathways may involve both direct and indirect effects, the latter involving down-regulation following CHD8 suppression. We also find that chd8 suppression in zebrafish results in macrocephaly, consistent with observations in patients harboring loss-of-function mutations. We show that reduced expression of CHD8 impacts a variety of other functionally distinct ASD-associated genes, suggesting that the diverse functions of ASD risk factors may constitute multiple means of triggering a smaller number of final common pathways.
Familial dysautonomia (FD) is a rare recessive neurodevelopmental disease caused by a splice mutation in the Elongator acetyltransferase complex subunit 1 (ELP1) gene. This mutation results in a ...tissue-specific reduction of ELP1 protein, with the lowest levels in the central and peripheral nervous systems (CNS and PNS, respectively). FD patients exhibit complex neurological phenotypes due to the loss of sensory and autonomic neurons. Disease symptoms include decreased pain and temperature perception, impaired or absent myotatic reflexes, proprioceptive ataxia, and progressive retinal degeneration. While the involvement of the PNS in FD pathogenesis has been clearly recognized, the underlying mechanisms responsible for the preferential neuronal loss remain unknown. In this study, we aimed to elucidate the molecular mechanisms underlying FD by conducting a comprehensive transcriptome analysis of neuronal tissues from the phenotypic mouse model TgFD9; Elp1
. This mouse recapitulates the same tissue-specific ELP1 mis-splicing observed in patients while modeling many of the disease manifestations. Comparison of FD and control transcriptomes from dorsal root ganglion (DRG), trigeminal ganglion (TG), medulla (MED), cortex, and spinal cord (SC) showed significantly more differentially expressed genes (DEGs) in the PNS than the CNS. We then identified genes that were tightly co-expressed and functionally dependent on the level of full-length ELP1 transcript. These genes, defined as ELP1 dose-responsive genes, were combined with the DEGs to generate tissue-specific dysregulated FD signature genes and networks. Within the PNS networks, we observed direct connections between Elp1 and genes involved in tRNA synthesis and genes related to amine metabolism and synaptic signaling. Importantly, transcriptomic dysregulation in PNS tissues exhibited enrichment for neuronal subtype markers associated with peptidergic nociceptors and myelinated sensory neurons, which are known to be affected in FD. In summary, this study has identified critical tissue-specific gene networks underlying the etiology of FD and provides new insights into the molecular basis of the disease.
Abstract
Pre-mRNA splicing is a key controller of human gene expression. Disturbances in splicing due to mutation lead to dysregulated protein expression and contribute to a substantial fraction of ...human disease. Several classes of splicing modulator compounds (SMCs) have been recently identified and establish that pre-mRNA splicing represents a target for therapy. We describe herein the identification of BPN-15477, a SMC that restores correct splicing of
ELP1
exon 20. Using transcriptome sequencing from treated fibroblast cells and a machine learning approach, we identify BPN-15477 responsive sequence signatures. We then leverage this model to discover 155 human disease genes harboring ClinVar mutations predicted to alter pre-mRNA splicing as targets for BPN-15477. Splicing assays confirm successful correction of splicing defects caused by mutations in
CFTR
,
LIPA
,
MLH1
and
MAPT
. Subsequent validations in two disease-relevant cellular models demonstrate that BPN-15477 increases functional protein, confirming the clinical potential of our predictions.
Chromosome 16p11.2 reciprocal genomic disorder, resulting from recurrent copy-number variants (CNVs), involves intellectual disability, autism spectrum disorder (ASD), and schizophrenia, but the ...responsible mechanisms are not known. To systemically dissect molecular effects, we performed transcriptome profiling of 350 libraries from six tissues (cortex, cerebellum, striatum, liver, brown fat, and white fat) in mouse models harboring CNVs of the syntenic 7qF3 region, as well as cellular, transcriptional, and single-cell analyses in 54 isogenic neural stem cell, induced neuron, and cerebral organoid models of CRISPR-engineered 16p11.2 CNVs. Transcriptome-wide differentially expressed genes were largely tissue-, cell-type-, and dosage-specific, although more effects were shared between deletion and duplication and across tissue than expected by chance. The broadest effects were observed in the cerebellum (2,163 differentially expressed genes), and the greatest enrichments were associated with synaptic pathways in mouse cerebellum and human induced neurons. Pathway and co-expression analyses identified energy and RNA metabolism as shared processes and enrichment for ASD-associated, loss-of-function constraint, and fragile X messenger ribonucleoprotein target gene sets. Intriguingly, reciprocal 16p11.2 dosage changes resulted in consistent decrements in neurite and electrophysiological features, and single-cell profiling of organoids showed reciprocal alterations to the proportions of excitatory and inhibitory GABAergic neurons. Changes both in neuronal ratios and in gene expression in our organoid analyses point most directly to calretinin GABAergic inhibitory neurons and the excitatory/inhibitory balance as targets of disruption that might contribute to changes in neurodevelopmental and cognitive function in 16p11.2 carriers. Collectively, our data indicate the genomic disorder involves disruption of multiple contributing biological processes and that this disruption has relative impacts that are context specific.
Display omitted
The 16p11.2 genomic disorder involves deletion and duplication of a contiguous set of genes, resulting in neurodevelopmental and other abnormalities. On the basis of comparisons of mouse models and human neuronal cells, the effects of these lesions on genome-wide gene expression and cellular function are highly dependent on cellular context.
Cerebral organoids can be used to gain insights into cell type specific processes perturbed by genetic variants associated with neuropsychiatric disorders. However, robust and scalable phenotyping of ...organoids remains challenging. Here, we perform RNA sequencing on 71 samples comprising 1,420 cerebral organoids from 25 donors, and describe a framework (Orgo-Seq) to integrate bulk RNA and single-cell RNA sequence data. We apply Orgo-Seq to 16p11.2 deletions and 15q11-13 duplications, two loci associated with autism spectrum disorder, to identify immature neurons and intermediate progenitor cells as critical cell types for 16p11.2 deletions. We further applied Orgo-Seq to identify cell type-specific driver genes. Our work presents a quantitative phenotyping framework to integrate multi-transcriptomic datasets for the identification of cell types and cell type-specific co-expressed driver genes associated with neuropsychiatric disorders.
PDF
