RESUMEN
The olfactory epithelium is one of the few regions of the nervous system that sustains neurogenesis throughout life. Its experimental accessibility makes it especially tractable for studying molecular mechanisms that drive neural regeneration after injury-induced cell death. In this study, we used single cell sequencing to identify major regulatory players in determining olfactory epithelial stem cell fate after acute injury. We combined gene expression and accessible chromatin profiles of individual lineage traced olfactory stem cells to predict transcription factor activity specific to different lineages and stages of recovery. We further identified a discrete stem cell state that appears poised for activation, characterized by accessible chromatin around wound response and lineage specific genes prior to their later expression in response to injury. Together these results provide evidence that a subset of quiescent olfactory epithelial stem cells are epigenetically primed to support injury-induced regeneration.
RESUMEN
X-linked myotubular myopathy (XLMTM) is a severe congenital disease characterized by profound muscle weakness, respiratory failure, and early death. No approved therapy for XLMTM is currently available. Adeno-associated virus (AAV)-mediated gene replacement therapy has shown promise as an investigational therapeutic strategy. We aimed to characterize the transcriptomic changes in muscle biopsies of individuals with XLMTM who received resamirigene bilparvovec (AT132; rAAV8-Des-hMTM1) in the ASPIRO clinical trial and to identify potential biomarkers that correlate with therapeutic outcome. We leveraged RNA-sequencing data from the muscle biopsies of 15 study participants and applied differential expression analysis, gene co-expression analysis, and machine learning to characterize the transcriptomic changes at baseline (pre-dose) and at 24 and 48 weeks after resamirigene bilparvovec dosing. As expected, MTM1 expression levels were significantly increased after dosing (p < 0.0001). Differential expression analysis identified upregulated genes after dosing that were enriched in several pathways, including lipid metabolism and inflammatory response pathways, and downregulated genes were enriched in cell-cell adhesion and muscle development pathways. Genes involved in inflammatory and immune pathways were differentially expressed between participants exhibiting ventilator support reduction of either greater or less than 6 h/day after gene therapy compared to pre-dosing. Co-expression analysis identified similarly regulated genes, which were grouped into modules. Finally, the machine learning model identified five genes, including MTM1, as potential RNA biomarkers to monitor the progress of AAV gene replacement therapy. These findings further extend our understanding of AAV-mediated gene therapy in individuals with XLMTM at the transcriptomic level.
Asunto(s)
Miopatías Estructurales Congénitas , Transcriptoma , Humanos , Biomarcadores/metabolismo , Perfilación de la Expresión Génica , Terapia Genética , Músculo Esquelético/metabolismo , Miopatías Estructurales Congénitas/genética , Miopatías Estructurales Congénitas/terapia , Miopatías Estructurales Congénitas/patología , Proteínas Tirosina Fosfatasas no Receptoras/genética , Proteínas Tirosina Fosfatasas no Receptoras/metabolismo , ARN/metabolismo , Transcriptoma/genéticaRESUMEN
The assay for transposase-accessible chromatin using sequencing (ATAC-seq) allows the study of epigenetic regulation of gene expression by assessing chromatin configuration for an entire genome. Despite its popularity, there have been limited studies investigating the analytical challenges related to ATAC-seq data, with most studies leveraging tools developed for bulk transcriptome sequencing. Here, we show that GC-content effects are omnipresent in ATAC-seq datasets. Since the GC-content effects are sample specific, they can bias downstream analyses such as clustering and differential accessibility analysis. We introduce a normalization method based on smooth-quantile normalization within GC-content bins and evaluate it together with 11 different normalization procedures on 8 public ATAC-seq datasets. Accounting for GC-content effects in the normalization is crucial for common downstream ATAC-seq data analyses, improving accuracy and interpretability. Through case studies, we show that exploratory data analysis is essential to guide the choice of an appropriate normalization method for a given dataset.
Asunto(s)
Benchmarking , Secuenciación de Inmunoprecipitación de Cromatina , Epigénesis Genética , Análisis de Secuencia de ADN/métodos , Secuenciación de Nucleótidos de Alto RendimientoRESUMEN
Therapeutic exon skipping as a treatment for Duchenne muscular dystrophy (DMD) has largely concentrated on the delivery of antisense oligomers to treat out-of-frame exon deletions. Here we report on the preclinical development of an adeno-associated virus (AAV)-encapsidated viral vector containing four copies of the noncoding U7 small nuclear RNA (U7snRNA), each targeted to either the splice donor or the splice acceptor sites of DMD exon 2. We have previously shown that delivery of this vector (scAAV9.U7.ACCA) to the Dup2 mouse model results in expression of full-length dystrophin from wild-type DMD mRNA, as well as an internal ribosome entry site (IRES)-driven isoform translated only in the absence of exon 2 (deletion exon 2 [Del2] mRNA). Here we present the data from a rigorous dose escalation toxicity study in nonhuman primates, encompassing two doses (3 × 1013 and 8 × 1013 vg/kg) and two time points (3 and 6 months postinjection). No evidence for significant toxicity was seen by biochemical, histopathologic, or clinical measures, providing evidence for safety that led to initiation of a first-in-human clinical trial.
Asunto(s)
Distrofina , Distrofia Muscular de Duchenne , Animales , Distrofina/genética , Exones , Ratones , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/terapia , Primates , ARN Nuclear PequeñoRESUMEN
Altered olfactory function is a common symptom of COVID-19, but its etiology is unknown. A key question is whether SARS-CoV-2 (CoV-2) - the causal agent in COVID-19 - affects olfaction directly, by infecting olfactory sensory neurons or their targets in the olfactory bulb, or indirectly, through perturbation of supporting cells. Here we identify cell types in the olfactory epithelium and olfactory bulb that express SARS-CoV-2 cell entry molecules. Bulk sequencing demonstrated that mouse, non-human primate and human olfactory mucosa expresses two key genes involved in CoV-2 entry, ACE2 and TMPRSS2. However, single cell sequencing revealed that ACE2 is expressed in support cells, stem cells, and perivascular cells, rather than in neurons. Immunostaining confirmed these results and revealed pervasive expression of ACE2 protein in dorsally-located olfactory epithelial sustentacular cells and olfactory bulb pericytes in the mouse. These findings suggest that CoV-2 infection of non-neuronal cell types leads to anosmia and related disturbances in odor perception in COVID-19 patients.
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Infecciones por Coronavirus/patología , Trastornos del Olfato/virología , Peptidil-Dipeptidasa A/metabolismo , Neumonía Viral/patología , Serina Endopeptidasas/metabolismo , Olfato/fisiología , Enzima Convertidora de Angiotensina 2 , Animales , Betacoronavirus/fisiología , COVID-19 , Callithrix , Humanos , Macaca , Ratones , Trastornos del Olfato/genética , Mucosa Olfatoria/citología , Mucosa Olfatoria/metabolismo , Neuronas Receptoras Olfatorias/metabolismo , Pandemias , Peptidil-Dipeptidasa A/genética , SARS-CoV-2 , Serina Endopeptidasas/genética , Olfato/genética , Internalización del VirusRESUMEN
Covalent nucleotide modifications in noncoding RNAs affect a plethora of biological processes, and new functions continue to be discovered even for well-known modifying enzymes. To systematically compare the functions of a large set of noncoding RNA modifications in gene regulation, we carried out ribosome profiling in budding yeast to characterize 57 nonessential genes involved in tRNA modification. Deletion mutants exhibited a range of translational phenotypes, with enzymes known to modify anticodons, or non-tRNA substrates such as rRNA, exhibiting the most dramatic translational perturbations. Our data build on prior reports documenting translational upregulation of the nutrient-responsive transcription factor Gcn4 in response to numerous tRNA perturbations, and identify many additional translationally regulated mRNAs throughout the yeast genome. Our data also uncover unexpected roles for tRNA-modifying enzymes in regulation of TY retroelements, and in rRNA 2'-O-methylation. This dataset should provide a rich resource for discovery of additional links between tRNA modifications and gene regulation.
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ARN de Hongos/metabolismo , ARN de Transferencia/metabolismo , Ribosomas/enzimología , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimología , Transcriptoma , ARNt Metiltransferasas/metabolismo , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/biosíntesis , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Perfilación de la Expresión Génica/métodos , Regulación Fúngica de la Expresión Génica , Genotipo , Metilación , Mutación , Fenotipo , ARN de Hongos/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Ribosómico/genética , ARN Ribosómico/metabolismo , ARN de Transferencia/genética , ARN no Traducido/genética , ARN no Traducido/metabolismo , Retroelementos , Ribosomas/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/biosíntesis , Proteínas de Saccharomyces cerevisiae/genética , Secuencias Repetidas Terminales , ARNt Metiltransferasas/genéticaRESUMEN
The repeating subunit of chromatin, the nucleosome, includes two copies of each of the four core histones, and several recent studies have reported that asymmetrically-modified nucleosomes occur at regulatory elements in vivo. To probe the mechanisms by which histone modifications are read out, we designed an obligate pair of H3 heterodimers, termed H3X and H3Y, which we extensively validated genetically and biochemically. Comparing the effects of asymmetric histone tail point mutants with those of symmetric double mutants revealed that a single methylated H3K36 per nucleosome was sufficient to silence cryptic transcription in vivo. We also demonstrate the utility of this system for analysis of histone modification crosstalk, using mass spectrometry to separately identify modifications on each H3 molecule within asymmetric nucleosomes. The ability to generate asymmetric nucleosomes in vivo and in vitro provides a powerful and generalizable tool to probe the mechanisms by which H3 tails are read out by effector proteins in the cell.
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Histonas/análisis , Nucleosomas/química , Proteínas de Saccharomyces cerevisiae/análisis , Saccharomyces cerevisiae/química , Histonas/genética , Espectrometría de Masas/métodos , Procesamiento Proteico-Postraduccional , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crecimiento & desarrollo , Proteínas de Saccharomyces cerevisiae/genética , Biología Sintética/métodosRESUMEN
Long-term memory requires activity-dependent synthesis of plasticity-related proteins (PRPs) to strengthen synaptic efficacy and consequently consolidate memory. Cytoplasmic polyadenylation element binding protein (CPEB)3 is a sequence-specific RNA-binding protein that regulates translation of several PRP RNAs in neurons. To understand whether CPEB3 plays a part in learning and memory, we generated CPEB3 knock-out (KO) mice and found that the null mice exhibited enhanced hippocampus-dependent, short-term fear memory in the contextual fear conditioning test and long-term spatial memory in the Morris water maze. The basal synaptic transmission of Schaffer collateral-CA1 neurons was normal but long-term depression evoked by paired-pulse low-frequency stimulation was modestly facilitated in the juvenile KO mice. Molecular and cellular characterizations revealed several molecules in regulating plasticity of glutamatergic synapses are translationally elevated in the CPEB3 KO neurons, including the scaffolding protein PSD95 and the NMDA receptors along with the known CPEB3 target, GluA1. Together, CPEB3 functions as a negative regulator to confine the strength of glutamatergic synapses by downregulating the expression of multiple PRPs and plays a role underlying certain forms of hippocampus-dependent memories.
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Guanilato-Quinasas/metabolismo , Hipocampo/fisiología , Proteínas de la Membrana/metabolismo , Memoria a Corto Plazo , Proteínas de Unión al ARN/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Animales , Condicionamiento Clásico , Homólogo 4 de la Proteína Discs Large , Miedo , Guanilato-Quinasas/genética , Hipocampo/citología , Hipocampo/metabolismo , Depresión Sináptica a Largo Plazo , Aprendizaje por Laberinto , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neuronas/metabolismo , Neuronas/fisiología , Proteínas de Unión al ARN/genética , Receptores AMPA/genética , Receptores AMPA/metabolismo , Receptores de N-Metil-D-Aspartato/genética , Sinapsis/fisiología , Transmisión SinápticaRESUMEN
The recently identified GGGGCC repeat expansion in the noncoding region of C9ORF72 is the most common pathogenic mutation in patients with frontotemporal dementia (FTD) or amyotrophic lateral sclerosis (ALS). We generated a human neuronal model and investigated the pathological phenotypes of human neurons containing GGGGCC repeat expansions. Skin biopsies were obtained from two subjects who had >1,000 GGGGCC repeats in C9ORF72 and their respective fibroblasts were used to generate multiple induced pluripotent stem cell (iPSC) lines. After extensive characterization, two iPSC lines from each subject were selected, differentiated into postmitotic neurons, and compared with control neurons to identify disease-relevant phenotypes. Expanded GGGGCC repeats exhibit instability during reprogramming and neuronal differentiation of iPSCs. RNA foci containing GGGGCC repeats were present in some iPSCs, iPSC-derived human neurons and primary fibroblasts. The percentage of cells with foci and the number of foci per cell appeared to be determined not simply by repeat length but also by other factors. These RNA foci do not seem to sequester several major RNA-binding proteins. Moreover, repeat-associated non-ATG (RAN) translation products were detected in human neurons with GGGGCC repeat expansions and these neurons showed significantly elevated p62 levels and increased sensitivity to cellular stress induced by autophagy inhibitors. Our findings demonstrate that key neuropathological features of FTD/ALS with GGGGCC repeat expansions can be recapitulated in iPSC-derived human neurons and also suggest that compromised autophagy function may represent a novel underlying pathogenic mechanism.
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Esclerosis Amiotrófica Lateral/genética , Expansión de las Repeticiones de ADN/genética , Demencia Frontotemporal/genética , Mutación/genética , Neuronas/metabolismo , Proteínas/genética , Esclerosis Amiotrófica Lateral/metabolismo , Animales , Proteína C9orf72 , Diferenciación Celular/genética , Diferenciación Celular/fisiología , Expansión de las Repeticiones de ADN/fisiología , Demencia Frontotemporal/metabolismo , Genotipo , Humanos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Ratones , Neuronas/citología , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismoRESUMEN
Regulated RNA translation is critical to provide proteins needed to maintain persistent modification of synaptic strength, which underlies the molecular basis of long-term memory (LTM). Cytoplasmic polyadenylation element-binding proteins (CPEBs) are sequence-specific RNA-binding proteins and regulate translation in various tissues. All four CPEBs in vertebrates are expressed in the brain, including the hippocampal neurons, suggesting their potential roles in translation-dependent plasticity and memory. Although CPEB1 and CPEB3 have been shown to control specific kinds of hippocampus-related LTM, the role of CPEB2 and CPEB4 in learning and memory remains elusive. Thus, we generated CPEB4 knockout (KO) mice and analyzed them using several behavioral tests. No difference was found in the anxiety level, motor coordination, hippocampus-dependent learning and memory between the KO mice and their wild-type (WT) littermates. Electrophysiological recordings of multiple forms of synaptic plasticity in the Schaffer collateral pathway-CA1 neurons also showed normal responses in the KO hippocampal slices. Morphological analyses revealed that the CPEB4-lacking pyramidal neurons possessed slightly elongated dendritic spines. Unlike its related family members, CPEB1 and CPEB3, CPEB4 seems to be dispensable for hippocampus-dependent plasticity, learning and memory.
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Conducta Animal/fisiología , Región CA1 Hipocampal , Memoria/fisiología , Plasticidad Neuronal/genética , Proteínas de Unión al ARN/genética , Animales , Aprendizaje/fisiología , Ratones , Ratones Noqueados , Neuronas/metabolismo , Proteínas de Unión al ARN/metabolismoRESUMEN
Cognitive abilities and disorders unique to humans are thought to result from adaptively driven changes in brain transcriptomes, but little is known about the role of cis-regulatory changes affecting transcription start sites (TSS). Here, we mapped in human, chimpanzee, and macaque prefrontal cortex the genome-wide distribution of histone H3 trimethylated at lysine 4 (H3K4me3), an epigenetic mark sharply regulated at TSS, and identified 471 sequences with human-specific enrichment or depletion. Among these were 33 loci selectively methylated in neuronal but not non-neuronal chromatin from children and adults, including TSS at DPP10 (2q14.1), CNTN4 and CHL1 (3p26.3), and other neuropsychiatric susceptibility genes. Regulatory sequences at DPP10 and additional loci carried a strong footprint of hominid adaptation, including elevated nucleotide substitution rates and regulatory motifs absent in other primates (including archaic hominins), with evidence for selective pressures during more recent evolution and adaptive fixations in modern populations. Chromosome conformation capture at two neurodevelopmental disease loci, 2q14.1 and 16p11.2, revealed higher order chromatin structures resulting in physical contact of multiple human-specific H3K4me3 peaks spaced 0.5-1 Mb apart, in conjunction with a novel cis-bound antisense RNA linked to Polycomb repressor proteins and downregulated DPP10 expression. Therefore, coordinated epigenetic regulation via newly derived TSS chromatin could play an important role in the emergence of human-specific gene expression networks in brain that contribute to cognitive functions and neurological disease susceptibility in modern day humans.