ABSTRACT
Psychedelics are a broad class of drugs defined by their ability to induce an altered state of consciousness1,2. These drugs have been used for millennia in both spiritual and medicinal contexts, and a number of recent clinical successes have spurred a renewed interest in developing psychedelic therapies3-9. Nevertheless, a unifying mechanism that can account for these shared phenomenological and therapeutic properties remains unknown. Here we demonstrate in mice that the ability to reopen the social reward learning critical period is a shared property across psychedelic drugs. Notably, the time course of critical period reopening is proportional to the duration of acute subjective effects reported in humans. Furthermore, the ability to reinstate social reward learning in adulthood is paralleled by metaplastic restoration of oxytocin-mediated long-term depression in the nucleus accumbens. Finally, identification of differentially expressed genes in the 'open state' versus the 'closed state' provides evidence that reorganization of the extracellular matrix is a common downstream mechanism underlying psychedelic drug-mediated critical period reopening. Together these results have important implications for the implementation of psychedelics in clinical practice, as well as the design of novel compounds for the treatment of neuropsychiatric disease.
Subject(s)
Critical Period, Psychological , Hallucinogens , Learning , Reward , Animals , Humans , Mice , Consciousness/drug effects , Hallucinogens/pharmacology , Hallucinogens/therapeutic use , Learning/drug effects , Time Factors , Oxytocin/metabolism , Nucleus Accumbens/drug effects , Nucleus Accumbens/metabolism , Long-Term Synaptic Depression/drug effects , Extracellular Matrix/drug effectsABSTRACT
Recent studies suggested that microglia, the primary brain immune cells, can affect circuit connectivity and neuronal function1,2. Microglia infiltrate the neuroepithelium early in embryonic development and are maintained in the brain throughout adulthood3,4. Several maternal environmental factors-such as an aberrant microbiome, immune activation and poor nutrition-can influence prenatal brain development5,6. Nevertheless, it is unknown how changes in the prenatal environment instruct the developmental trajectory of infiltrating microglia, which in turn affect brain development and function. Here we show that, after maternal immune activation (MIA) in mice, microglia from the offspring have a long-lived decrease in immune reactivity (blunting) across the developmental trajectory. The blunted immune response was accompanied by changes in chromatin accessibility and reduced transcription factor occupancy of the open chromatin. Single-cell RNA-sequencing analysis revealed that MIA does not induce a distinct subpopulation but, rather, decreases the contribution to inflammatory microglia states. Prenatal replacement of microglia from MIA offspring with physiological infiltration of naive microglia ameliorated the immune blunting and restored a decrease in presynaptic vesicle release probability onto dopamine receptor type-two medium spiny neurons, indicating that aberrantly formed microglia due to an adverse prenatal environment affect the long-term microglia reactivity and proper striatal circuit development.
Subject(s)
Inflammation , Microglia , Mothers , Neural Pathways , Prenatal Exposure Delayed Effects , Animals , Chromatin/genetics , Chromatin/metabolism , Female , Inflammation/immunology , Inflammation/pathology , Mice , Microglia/immunology , Microglia/pathology , Neostriatum/cytology , Neural Pathways/pathology , Neurons/pathology , Pregnancy , Prenatal Exposure Delayed Effects/genetics , Prenatal Exposure Delayed Effects/immunology , RNA-Seq , Receptors, Dopamine/metabolism , Single-Cell Analysis , Transcription Factors/metabolismABSTRACT
Commitment to the innate lymphoid cell (ILC) lineage is determined by Id2, a transcriptional regulator that antagonizes T and B cell-specific gene expression programs. Yet how Id2 expression is regulated in each ILC subset remains poorly understood. We identified a cis-regulatory element demarcated by a long non-coding RNA (lncRNA) that controls the function and lineage identity of group 1 ILCs, while being dispensable for early ILC development and homeostasis of ILC2s and ILC3s. The locus encoding this lncRNA, which we termed Rroid, directly interacted with the promoter of its neighboring gene, Id2, in group 1 ILCs. Moreover, the Rroid locus, but not the lncRNA itself, controlled the identity and function of ILC1s by promoting chromatin accessibility and deposition of STAT5 at the promoter of Id2 in response to interleukin (IL)-15. Thus, non-coding elements responsive to extracellular cues unique to each ILC subset represent a key regulatory layer for controlling the identity and function of ILCs.
Subject(s)
Gene Expression Regulation , Immunity, Innate/genetics , Lymphocytes/metabolism , RNA, Long Noncoding/genetics , Regulatory Sequences, Nucleic Acid , Animals , Cell Differentiation , Cell Lineage/genetics , Cell Lineage/immunology , Chromatin Assembly and Disassembly , Female , Gene Expression Profiling , Genetic Loci , Homeostasis , Inhibitor of Differentiation Protein 2/genetics , Killer Cells, Natural/cytology , Killer Cells, Natural/immunology , Killer Cells, Natural/metabolism , Lymphocyte Subsets/immunology , Lymphocyte Subsets/metabolism , Lymphocytes/immunology , Male , Mice , Promoter Regions, Genetic , STAT5 Transcription Factor/metabolism , Transcription, GeneticABSTRACT
The lacrimal gland is of central interest in ophthalmology both as the source of the aqueous component of tear fluid and as the site of autoimmune pathology in the context of Sjogren's syndrome (SjS). To provide a foundational description of mouse lacrimal gland cell types and their patterns of gene expression, we have analyzed single-cell transcriptomes from wild-type (Balb/c) mice and from two genetically based SjS models, MRL/lpr and NOD (nonobese diabetic).H2b, and defined the localization of multiple cell-type-specific protein and mRNA markers. This analysis has uncovered a previously undescribed cell type, Car6+ cells, which are located at the junction of the acini and the connecting ducts. More than a dozen secreted polypeptides that are likely to be components of tear fluid are expressed by acinar cells and show pronounced sex differences in expression. Additional examples of gene expression heterogeneity within a single cell type were identified, including a gradient of Claudin4 along the length of the ductal system and cell-to-cell heterogeneity in transcription factor expression within acinar and myoepithelial cells. The patterns of expression of channels, transporters, and pumps in acinar, Car6+, and ductal cells make strong predictions regarding the mechanisms of water and electrolyte secretion. In MRL/lpr and NOD.H2b lacrimal glands, distinctive changes in parenchymal gene expression and in immune cell subsets reveal widespread interferon responses, a T cell-dominated infiltrate in the MRL/lpr model, and a mixed B cell and T cell infiltrate in the NOD.H2b model.
Subject(s)
Lacrimal Apparatus , Sjogren's Syndrome , Female , Mice , Male , Animals , Sjogren's Syndrome/metabolism , Lacrimal Apparatus/metabolism , Mice, Inbred MRL lpr , Mice, Inbred NOD , Mice, Inbred BALB C , Disease Models, AnimalABSTRACT
The receptor tyrosine kinase RET plays a critical role in the fate specification of enteric neural crest-derived cells (ENCDCs) during enteric nervous system (ENS) development. RET loss of function (LoF) is associated with Hirschsprung disease (HSCR), which is marked by aganglionosis of the gastrointestinal (GI) tract. Although the major phenotypic consequences and the underlying transcriptional changes from Ret LoF in the developing ENS have been described, cell type- and state-specific effects are unknown. We performed single-cell RNA sequencing on an enriched population of ENCDCs from the developing GI tract of Ret null heterozygous and homozygous mice at embryonic day (E)12.5 and E14.5. We demonstrate four significant findings: 1) Ret-expressing ENCDCs are a heterogeneous population comprising ENS progenitors as well as glial- and neuronal-committed cells; 2) neurons committed to a predominantly inhibitory motor neuron developmental trajectory are not produced under Ret LoF, leaving behind a mostly excitatory motor neuron developmental program; 3) expression patterns of HSCR-associated and Ret gene regulatory network genes are impacted by Ret LoF; and 4) Ret deficiency leads to precocious differentiation and reduction in the number of proliferating ENS precursors. Our results support a model in which Ret contributes to multiple distinct cellular phenotypes during development of the ENS, including the specification of inhibitory neuron subtypes, cell cycle dynamics of ENS progenitors, and the developmental timing of neuronal and glial commitment.
Subject(s)
Enteric Nervous System , Hirschsprung Disease , Proto-Oncogene Proteins c-ret , Animals , Mice , Cell Differentiation , Cell Proliferation , Hirschsprung Disease/genetics , Neural Crest , Proto-Oncogene Proteins c-ret/geneticsABSTRACT
Haematopoiesis relies on tightly controlled gene expression patterns as development proceeds through a series of progenitors. While the regulation of hematopoietic development has been well studied, the role of noncoding elements in this critical process is a developing field. In particular, the discovery of new regulators of lymphopoiesis could have important implications for our understanding of the adaptive immune system and disease. Here we elucidate how a noncoding element is capable of regulating a broadly expressed transcription factor, Ikaros, in a lymphoid lineage-specific manner, such that it imbues Ikaros with the ability to specify the lymphoid lineage over alternate fates. Deletion of the Daedalus locus, which is proximal to Ikaros, led to a severe reduction in early lymphoid progenitors, exerting control over the earliest fate decisions during lymphoid lineage commitment. Daedalus locus deletion led to alterations in Ikaros isoform expression and a significant reduction in Ikaros protein. The Daedalus locus may function through direct DNA interaction as Hi-C analysis demonstrated an interaction between the two loci. Finally, we identify an Ikaros-regulated erythroid-lymphoid checkpoint that is governed by Daedalus in a lymphoid-lineage-specific manner. Daedalus appears to act as a gatekeeper of Ikaros's broad lineage-specifying functions, selectively stabilizing Ikaros activity in the lymphoid lineage and permitting diversion to the erythroid fate in its absence. These findings represent a key illustration of how a transcription factor with broad lineage expression must work in concert with noncoding elements to orchestrate hematopoietic lineage commitment.
Subject(s)
Hematopoiesis/genetics , Ikaros Transcription Factor/genetics , Lymphopoiesis/genetics , RNA, Untranslated/genetics , Animals , Cell Differentiation/genetics , Cell Lineage/genetics , DNA-Binding Proteins/genetics , Gene Deletion , Gene Expression Regulation, Developmental/genetics , MiceABSTRACT
Somatic LINE-1 (L1) retrotransposition has been detected in early embryos, adult brains, and the gastrointestinal (GI) tract, and many cancers, including epithelial GI tumors. We previously found numerous somatic L1 insertions in paired normal and GI cancerous tissues. Here, using a modified method of single-cell analysis for somatic L1 insertions, we studied adenocarcinomas of colon, pancreas, and stomach, and found a variable number of somatic L1 insertions in tumors of the same type from patient to patient. We detected no somatic L1 insertions in single cells of 5 of 10 tumors studied. In three tumors, aneuploid cells were detected by FACS. In one pancreatic tumor, there were many more L1 insertions in aneuploid than in euploid tumor cells. In one gastric cancer, both aneuploid and euploid cells contained large numbers of likely clonal insertions. However, in a second gastric cancer with aneuploid cells, no somatic L1 insertions were found. We suggest that when the cellular environment is favorable to retrotransposition, aneuploidy predisposes tumor cells to L1 insertions, and retrotransposition may occur at the transition from euploidy to aneuploidy. Seventeen percent of insertions were also present in normal cells, similar to findings in genomic DNA from normal tissues of GI tumor patients. We provide evidence that: 1) The number of L1 insertions in tumors of the same type is highly variable, 2) most somatic L1 insertions in GI cancer tissues are absent from normal tissues, and 3) under certain conditions, somatic L1 retrotransposition exhibits a propensity for occurring in aneuploid cells.
Subject(s)
Adenocarcinoma/genetics , Gastrointestinal Neoplasms/genetics , Long Interspersed Nucleotide Elements/genetics , Adenocarcinoma/pathology , Artifacts , Gastrointestinal Neoplasms/pathology , Humans , Single-Cell AnalysisABSTRACT
Radial glial progenitor cells (RGCs) in the dorsal telencephalon directly or indirectly produce excitatory projection neurons and macroglia of the neocortex. Recent evidence shows that the pool of RGCs is more heterogeneous than originally thought and that progenitor subpopulations can generate particular neuronal cell types. Using single-cell RNA sequencing, we have studied gene expression patterns of RGCs with different neurogenic behavior at early stages of cortical development. At this early age, some RGCs rapidly produce postmitotic neurons, whereas others self-renew and undergo neurogenic divisions at a later age. We have identified candidate genes that are differentially expressed among these early RGC subpopulations, including the transcription factor Sox9. Using in utero electroporation in embryonic mice of either sex, we demonstrate that elevated Sox9 expression in progenitors affects RGC cell cycle duration and leads to the generation of upper layer cortical neurons. Our data thus reveal molecular differences between progenitor cells with different neurogenic behavior at early stages of corticogenesis and indicates that Sox9 is critical for the maintenance of RGCs to regulate the generation of upper layer neurons.SIGNIFICANCE STATEMENT The existence of heterogeneity in the pool of RGCs and its relationship with the generation of cellular diversity in the cerebral cortex has been an interesting topic of debate for many years. Here we describe the existence of RGCs with reduced neurogenic behavior at early embryonic ages presenting a particular molecular signature. This molecular signature consists of differential expression of some genes including the transcription factor Sox9, which has been found to be a specific regulator of this subpopulation of progenitor cells. Functional experiments perturbing expression levels of Sox9 reveal its instructive role in the regulation of the neurogenic behavior of RGCs and its relationship with the generation of upper layer projection neurons at later ages.
Subject(s)
Cell Self Renewal/genetics , Ependymoglial Cells/cytology , Gene Expression Regulation, Developmental/genetics , Neocortex/cytology , Nerve Tissue Proteins/physiology , Neurogenesis/genetics , SOX9 Transcription Factor/physiology , Animals , Cell Cycle/genetics , Electroporation , Ependymoglial Cells/metabolism , Female , Genes, Reporter , Genetic Vectors/administration & dosage , Injections, Intraventricular , Mice , Mice, Inbred C57BL , Neocortex/embryology , Neocortex/growth & development , Nerve Tissue Proteins/biosynthesis , Nerve Tissue Proteins/genetics , Neuroglia/cytology , Neurons/cytology , Pregnancy , Promoter Regions, Genetic/genetics , SOX9 Transcription Factor/biosynthesis , SOX9 Transcription Factor/genetics , Single-Cell Analysis , Transcription, GeneticABSTRACT
Regulation of metabolic pathways in the immune system provides a mechanism to actively control cellular function, growth, proliferation, and survival. Here, we report that miR-181 is a nonredundant determinant of cellular metabolism and is essential for supporting the biosynthetic demands of early NKT cell development. As a result, miR-181-deficient mice showed a complete absence of mature NKT cells in the thymus and periphery. Mechanistically, miR-181 modulated expression of the phosphatase PTEN to control PI3K signaling, which was a primary stimulus for anabolic metabolism in immune cells. Thus miR-181-deficient mice also showed severe defects in lymphoid development and T cell homeostasis associated with impaired PI3K signaling. These results uncover miR-181 as essential for NKT cell development and establish this family of miRNAs as central regulators of PI3K signaling and global metabolic fitness during development and homeostasis.
Subject(s)
Lymphopoiesis/genetics , MicroRNAs/metabolism , Natural Killer T-Cells/metabolism , PTEN Phosphohydrolase/metabolism , Animals , Cell Differentiation , Chemokine CXCL12/metabolism , Down-Regulation , Homeostasis , Lymphocytes/metabolism , Mice , MicroRNAs/genetics , Phosphatidylinositol 3-Kinases/metabolism , Signal Transduction/geneticsABSTRACT
Neutrophils, eosinophils and 'classical' monocytes collectively account for about 70% of human blood leukocytes and are among the shortest-lived cells in the body. Precise regulation of the lifespan of these myeloid cells is critical to maintain protective immune responses and minimize the deleterious consequences of prolonged inflammation. However, how the lifespan of these cells is strictly controlled remains largely unknown. Here we identify a long non-coding RNA that we termed Morrbid, which tightly controls the survival of neutrophils, eosinophils and classical monocytes in response to pro-survival cytokines in mice. To control the lifespan of these cells, Morrbid regulates the transcription of the neighbouring pro-apoptotic gene, Bcl2l11 (also known as Bim), by promoting the enrichment of the PRC2 complex at the Bcl2l11 promoter to maintain this gene in a poised state. Notably, Morrbid regulates this process in cis, enabling allele-specific control of Bcl2l11 transcription. Thus, in these highly inflammatory cells, changes in Morrbid levels provide a locus-specific regulatory mechanism that allows rapid control of apoptosis in response to extracellular pro-survival signals. As MORRBID is present in humans and dysregulated in individuals with hypereosinophilic syndrome, this long non-coding RNA may represent a potential therapeutic target for inflammatory disorders characterized by aberrant short-lived myeloid cell lifespan.
Subject(s)
Bcl-2-Like Protein 11/genetics , Myeloid Cells/cytology , Myeloid Cells/metabolism , RNA, Long Noncoding/genetics , Alleles , Animals , Antigens, Ly/metabolism , Apoptosis , Bcl-2-Like Protein 11/biosynthesis , Cell Survival , Down-Regulation , Eosinophils/cytology , Eosinophils/metabolism , Female , Humans , Male , Mice , Monocytes/cytology , Monocytes/metabolism , Neutrophils/cytology , Neutrophils/metabolism , Promoter Regions, GeneticABSTRACT
Autoimmune uveoretinitis is a significant cause of visual loss, and mouse models offer unique opportunities to study its disease mechanisms. Aire-/- mice fail to express self-antigens in the thymus, exhibit reduced central tolerance, and develop a spontaneous, chronic, and progressive uveoretinitis. Using single-cell RNA sequencing (scRNA-seq), we characterized wild-type and Aire-/- retinas to define, in a comprehensive and unbiased manner, the cell populations and gene expression patterns associated with disease. Based on scRNA-seq, immunostaining, and in situ hybridization, we infer that 1) the dominant effector response in Aire-/- retinas is Th1-driven, 2) a subset of monocytes convert to either a macrophage/microglia state or a dendritic cell state, 3) the development of tertiary lymphoid structures constitutes part of the Aire-/- retinal phenotype, 4) all major resident retinal cell types respond to interferon gamma (IFNG) by changing their patterns of gene expression, and 5) Muller glia up-regulate specific genes in response to IFN gamma and may act as antigen-presenting cells.
ABSTRACT
The mammalian CNS is capable of tolerating chronic hypoxia, but cell type-specific responses to this stress have not been systematically characterized. In the Norrin KO (NdpKO ) mouse, a model of familial exudative vitreoretinopathy (FEVR), developmental hypovascularization of the retina produces chronic hypoxia of inner nuclear-layer (INL) neurons and Muller glia. We used single-cell RNA sequencing, untargeted metabolomics, and metabolite labeling from 13C-glucose to compare WT and NdpKO retinas. In NdpKO retinas, we observe gene expression responses consistent with hypoxia in Muller glia and retinal neurons, and we find a metabolic shift that combines reduced flux through the TCA cycle with increased synthesis of serine, glycine, and glutathione. We also used single-cell RNA sequencing to compare the responses of individual cell types in NdpKO retinas with those in the hypoxic cerebral cortex of mice that were housed for 1 week in a reduced oxygen environment (7.5% oxygen). In the hypoxic cerebral cortex, glial transcriptome responses most closely resemble the response of Muller glia in the NdpKO retina. In both retina and brain, vascular endothelial cells activate a previously dormant tip cell gene expression program, which likely underlies the adaptive neoangiogenic response to chronic hypoxia. These analyses of retina and brain transcriptomes at single-cell resolution reveal both shared and cell type-specific changes in gene expression in response to chronic hypoxia, implying both shared and distinct cell type-specific physiologic responses.
Subject(s)
Hypoxia/metabolism , Neuroglia/metabolism , Neurons/metabolism , Animals , Brain/metabolism , Disease Models, Animal , Endothelial Cells/metabolism , Familial Exudative Vitreoretinopathies/genetics , Familial Exudative Vitreoretinopathies/physiopathology , Female , Male , Mice , Mice, Inbred C57BL , Nerve Tissue Proteins/metabolism , Retina/metabolism , Retina/physiology , Retinal Neurons/metabolism , Retinal Vessels/metabolism , Sequence Analysis, RNA/methods , Single-Cell Analysis/methodsABSTRACT
Omics data contain signals from the molecular, physical, and kinetic inter- and intracellular interactions that control biological systems. Matrix factorization (MF) techniques can reveal low-dimensional structure from high-dimensional data that reflect these interactions. These techniques can uncover new biological knowledge from diverse high-throughput omics data in applications ranging from pathway discovery to timecourse analysis. We review exemplary applications of MF for systems-level analyses. We discuss appropriate applications of these methods, their limitations, and focus on the analysis of results to facilitate optimal biological interpretation. The inference of biologically relevant features with MF enables discovery from high-throughput data beyond the limits of current biological knowledge - answering questions from high-dimensional data that we have not yet thought to ask.
Subject(s)
Data Interpretation, Statistical , Genomics/statistics & numerical data , Proteomics/statistics & numerical data , Algorithms , Humans , Systems Biology/statistics & numerical dataABSTRACT
Genetic variation modulating risk of sporadic Parkinson disease (PD) has been primarily explored through genome-wide association studies (GWASs). However, like many other common genetic diseases, the impacted genes remain largely unknown. Here, we used single-cell RNA-seq to characterize dopaminergic (DA) neuron populations in the mouse brain at embryonic and early postnatal time points. These data facilitated unbiased identification of DA neuron subpopulations through their unique transcriptional profiles, including a postnatal neuroblast population and substantia nigra (SN) DA neurons. We use these population-specific data to develop a scoring system to prioritize candidate genes in all 49 GWAS intervals implicated in PD risk, including genes with known PD associations and many with extensive supporting literature. As proof of principle, we confirm that the nigrostriatal pathway is compromised in Cplx1-null mice. Ultimately, this systematic approach establishes biologically pertinent candidates and testable hypotheses for sporadic PD, informing a new era of PD genetic research.
Subject(s)
Dopaminergic Neurons/metabolism , Genetic Association Studies , Parkinson Disease/genetics , Parkinson Disease/pathology , Sequence Analysis, RNA , Single-Cell Analysis/methods , Animals , Cell Separation , Gene Regulatory Networks , Genetic Loci , Genetic Markers , Genome-Wide Association Study , Mice, Knockout , Substantia Nigra/pathologyABSTRACT
MOTIVATION: Dimension reduction techniques are widely used to interpret high-dimensional biological data. Features learned from these methods are used to discover both technical artifacts and novel biological phenomena. Such feature discovery is critically importent in analysis of large single-cell datasets, where lack of a ground truth limits validation and interpretation. Transfer learning (TL) can be used to relate the features learned from one source dataset to a new target dataset to perform biologically driven validation by evaluating their use in or association with additional sample annotations in that independent target dataset. RESULTS: We developed an R/Bioconductor package, projectR, to perform TL for analyses of genomics data via TL of clustering, correlation and factorization methods. We then demonstrate the utility TL for integrated data analysis with an example for spatial single-cell analysis. AVAILABILITY AND IMPLEMENTATION: projectR is available on Bioconductor and at https://github.com/genesofeve/projectR. CONTACT: gsteinobrien@jhmi.edu or ejfertig@jhmi.edu. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
Subject(s)
Genomics , Software , Cluster Analysis , Machine Learning , Single-Cell AnalysisABSTRACT
Kabuki syndrome is a Mendelian intellectual disability syndrome caused by mutations in either of two genes (KMT2D and KDM6A) involved in chromatin accessibility. We previously showed that an agent that promotes chromatin opening, the histone deacetylase inhibitor (HDACi) AR-42, ameliorates the deficiency of adult neurogenesis in the granule cell layer of the dentate gyrus and rescues hippocampal memory defects in a mouse model of Kabuki syndrome (Kmt2d+/ßGeo). Unlike a drug, a dietary intervention could be quickly transitioned to the clinic. Therefore, we have explored whether treatment with a ketogenic diet could lead to a similar rescue through increased amounts of beta-hydroxybutyrate, an endogenous HDACi. Here, we report that a ketogenic diet in Kmt2d+/ßGeo mice modulates H3ac and H3K4me3 in the granule cell layer, with concomitant rescue of both the neurogenesis defect and hippocampal memory abnormalities seen in Kmt2d+/ßGeo mice; similar effects on neurogenesis were observed on exogenous administration of beta-hydroxybutyrate. These data suggest that dietary modulation of epigenetic modifications through elevation of beta-hydroxybutyrate may provide a feasible strategy to treat the intellectual disability seen in Kabuki syndrome and related disorders.
Subject(s)
Abnormalities, Multiple/diet therapy , Diet, Ketogenic/methods , Face/abnormalities , Hematologic Diseases/diet therapy , Hippocampus/metabolism , Histones/biosynthesis , Intellectual Disability/diet therapy , Neurogenesis/physiology , Vestibular Diseases/diet therapy , 3-Hydroxybutyric Acid/metabolism , Abnormalities, Multiple/genetics , Animals , Disease Models, Animal , Hematologic Diseases/genetics , Hippocampus/cytology , Histone Demethylases/genetics , Histone-Lysine N-Methyltransferase/genetics , Intellectual Disability/genetics , Mice , Mice, Inbred C57BL , Mice, Transgenic , Myeloid-Lymphoid Leukemia Protein/genetics , Neurogenesis/genetics , Vestibular Diseases/geneticsABSTRACT
Recent genome-wide association studies (GWAS) identified numerous schizophrenia (SZ) and Alzheimer's disease (AD) associated loci, most outside protein-coding regions and hypothesized to affect gene transcription. We used a massively parallel reporter assay to screen, 1,049 SZ and 30 AD variants in 64 and nine loci, respectively for allele differences in driving reporter gene expression. A library of synthetic oligonucleotides assaying each allele five times was transfected into K562 chronic myelogenous leukemia lymphoblasts and SK-SY5Y human neuroblastoma cells. One hundred forty eight variants showed allelic differences in K562 and 53 in SK-SY5Y cells, on average 2.6 variants per locus. Nine showed significant differences in both lines, a modest overlap reflecting different regulatory landscapes of these lines that also differ significantly in chromatin marks. Eight of nine were in the same direction. We observe no preference for risk alleles to increase or decrease expression. We find a positive correlation between the number of SNPs in linkage disequilibrium and the proportion of functional SNPs supporting combinatorial effects that may lead to haplotype selection. Our results prioritize future functional follow up of disease associated SNPs to determine the driver GWAS variant(s), at each locus and enhance our understanding of gene regulation dynamics.
Subject(s)
Alzheimer Disease/genetics , Gene Expression Regulation/genetics , Schizophrenia/genetics , Alleles , Cell Line, Tumor , Gene Expression/genetics , Gene Frequency/genetics , Genetic Predisposition to Disease , Genetic Variation/genetics , Genome-Wide Association Study/methods , Haplotypes , Humans , K562 Cells , Linkage Disequilibrium , Polymorphism, Single Nucleotide/genetics , Quantitative Trait LociABSTRACT
BACKGROUND: Massively parallel reporter assays (MPRAs) have emerged as a popular means for understanding noncoding variation in a variety of conditions. While a large number of experiments have been described in the literature, analysis typically uses ad-hoc methods. There has been little attention to comparing performance of methods across datasets. RESULTS: We present the mpralm method which we show is calibrated and powerful, by analyzing its performance on multiple MPRA datasets. We show that it outperforms existing statistical methods for analysis of this data type, in the first comprehensive evaluation of statistical methods on several datasets. We investigate theoretical and real-data properties of barcode summarization methods and show an unappreciated impact of summarization method for some datasets. Finally, we use our model to conduct a power analysis for this assay and show substantial improvements in power by performing up to 6 replicates per condition, whereas sequencing depth has smaller impact; we recommend to always use at least 4 replicates. An R package is available from the Bioconductor project. CONCLUSIONS: Together, these results inform recommendations for differential analysis, general group comparisons, and power analysis and will help improve design and analysis of MPRA experiments.
Subject(s)
Genome, Human , High-Throughput Nucleotide Sequencing/methods , Humans , Linear Models , Sequence Analysis, DNA/methods , Sequence Analysis, RNA , SoftwareABSTRACT
DNA methylation was first described almost a century ago; however, the rules governing its establishment and maintenance remain elusive. Here we present data demonstrating that active transcription regulates levels of genomic methylation. We identify a novel RNA arising from the CEBPA gene locus that is critical in regulating the local DNA methylation profile. This RNA binds to DNMT1 and prevents CEBPA gene locus methylation. Deep sequencing of transcripts associated with DNMT1 combined with genome-scale methylation and expression profiling extend the generality of this finding to numerous gene loci. Collectively, these results delineate the nature of DNMT1-RNA interactions and suggest strategies for gene-selective demethylation of therapeutic targets in human diseases.
Subject(s)
CCAAT-Enhancer-Binding Proteins/genetics , DNA (Cytosine-5-)-Methyltransferases/metabolism , DNA Methylation/genetics , Gene Expression Regulation/genetics , RNA, Untranslated/metabolism , Base Sequence , Cell Line , DNA/genetics , DNA/metabolism , DNA (Cytosine-5-)-Methyltransferase 1 , Gene Expression Profiling , Genome, Human/genetics , Humans , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Untranslated/genetics , RNA-Binding Proteins/metabolism , Substrate Specificity , Transcription, Genetic/geneticsABSTRACT
Cell type-specific changes in neuronal excitability have been proposed to contribute to the selective degeneration of corticospinal neurons in amyotrophic lateral sclerosis (ALS) and to neocortical hyperexcitability, a prominent feature of both inherited and sporadic variants of the disease, but the mechanisms underlying selective loss of specific cell types in ALS are not known. We analyzed the physiological properties of distinct classes of cortical neurons in the motor cortex of hSOD1G93A mice of both sexes and found that they all exhibit increases in intrinsic excitability that depend on disease stage. Targeted recordings and in vivo calcium imaging further revealed that neurons adapt their functional properties to normalize cortical excitability as the disease progresses. Although different neuron classes all exhibited increases in intrinsic excitability, transcriptional profiling indicated that the molecular mechanisms underlying these changes are cell type specific. The increases in excitability in both excitatory and inhibitory cortical neurons show that selective dysfunction of neuronal cell types cannot account for the specific vulnerability of corticospinal motor neurons in ALS. Furthermore, the stage-dependent alterations in neuronal function highlight the ability of cortical circuits to adapt as disease progresses. These findings show that both disease stage and cell type must be considered when developing therapeutic strategies for treating ALS.SIGNIFICANCE STATEMENT It is not known why certain classes of neurons preferentially die in different neurodegenerative diseases. It has been proposed that the enhanced excitability of affected neurons is a major contributor to their selective loss. We show using a mouse model of amyotrophic lateral sclerosis (ALS), a disease in which corticospinal neurons exhibit selective vulnerability, that changes in excitability are not restricted to this neuronal class and that excitability does not increase monotonically with disease progression. Moreover, although all neuronal cell types tested exhibited abnormal functional properties, analysis of their gene expression demonstrated cell type-specific responses to the ALS-causing mutation. These findings suggest that therapies for ALS may need to be tailored for different cell types and stages of disease.