RESUMEN
Endogenous retroviruses (ERVs) make up a large fraction of mammalian genomes and are thought to contribute to human disease, including brain disorders. In the brain, aberrant activation of ERVs is a potential trigger for an inflammatory response, but mechanistic insight into this phenomenon remains lacking. Using CRISPR/Cas9-based gene disruption of the epigenetic co-repressor protein Trim28, we found a dynamic H3K9me3-dependent regulation of ERVs in proliferating neural progenitor cells (NPCs), but not in adult neurons. In vivo deletion of Trim28 in cortical NPCs during mouse brain development resulted in viable offspring expressing high levels of ERVs in excitatory neurons in the adult brain. Neuronal ERV expression was linked to activated microglia and the presence of ERV-derived proteins in aggregate-like structures. This study demonstrates that brain development is a critical period for the silencing of ERVs and provides causal in vivo evidence demonstrating that transcriptional activation of ERV in neurons results in an inflammatory response.
Asunto(s)
Encéfalo/crecimiento & desarrollo , Encefalitis/genética , Retrovirus Endógenos/genética , Eliminación de Gen , Proteína 28 que Contiene Motivos Tripartito/genética , Animales , Encéfalo/inmunología , Encéfalo/virología , Sistemas CRISPR-Cas , Células Cultivadas , Encefalitis/inmunología , Encefalitis/virología , Retrovirus Endógenos/inmunología , Epigénesis Genética , Regulación de la Expresión Génica , Histonas/metabolismo , Ratones , Activación TranscripcionalRESUMEN
The human silencing hub (HUSH) complex binds to transcripts of LINE-1 retrotransposons (L1s) and other genomic repeats, recruiting MORC2 and other effectors to remodel chromatin. How HUSH and MORC2 operate alongside DNA methylation, a central epigenetic regulator of repeat transcription, remains largely unknown. Here we interrogate this relationship in human neural progenitor cells (hNPCs), a somatic model of brain development that tolerates removal of DNA methyltransferase DNMT1. Upon loss of MORC2 or HUSH subunit TASOR in hNPCs, L1s remain silenced by robust promoter methylation. However, genome demethylation and activation of evolutionarily-young L1s attracts MORC2 binding, and simultaneous depletion of DNMT1 and MORC2 causes massive accumulation of L1 transcripts. We identify the same mechanistic hierarchy at pericentromeric α-satellites and clustered protocadherin genes, repetitive elements important for chromosome structure and neurodevelopment respectively. Our data delineate the epigenetic control of repeats in somatic cells, with implications for understanding the vital functions of HUSH-MORC2 in hypomethylated contexts throughout human development.
Asunto(s)
ADN (Citosina-5-)-Metiltransferasa 1 , Metilación de ADN , Elementos de Nucleótido Esparcido Largo , Células-Madre Neurales , Humanos , ADN (Citosina-5-)-Metiltransferasa 1/metabolismo , ADN (Citosina-5-)-Metiltransferasa 1/genética , Células-Madre Neurales/metabolismo , Elementos de Nucleótido Esparcido Largo/genética , Epigénesis Genética , Regiones Promotoras Genéticas , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Proteínas Co-Represoras/metabolismo , Proteínas Co-Represoras/genética , Silenciador del Gen , Proteínas Represoras/metabolismo , Proteínas Represoras/genética , Proteínas del Tejido NerviosoRESUMEN
Mutations in GBA1 cause Gaucher disease and are the most important genetic risk factor for Parkinson's disease. However, analysis of transcription at this locus is complicated by its highly homologous pseudogene, GBAP1. We show that >50% of short RNA-sequencing reads mapping to GBA1 also map to GBAP1. Thus, we used long-read RNA sequencing in the human brain, which allowed us to accurately quantify expression from both GBA1 and GBAP1. We discovered significant differences in expression compared to short-read data and identify currently unannotated transcripts of both GBA1 and GBAP1. These included protein-coding transcripts from both genes that were translated in human brain, but without the known lysosomal function-yet accounting for almost a third of transcription. Analyzing brain-specific cell types using long-read and single-nucleus RNA sequencing revealed region-specific variations in transcript expression. Overall, these findings suggest nonlysosomal roles for GBA1 and GBAP1 with implications for our understanding of the role of GBA1 in health and disease.
Asunto(s)
Glucosilceramidasa , Seudogenes , Humanos , Glucosilceramidasa/genética , Glucosilceramidasa/metabolismo , Seudogenes/genética , Encéfalo/metabolismo , Anotación de Secuencia Molecular , Enfermedad de Parkinson/genética , Enfermedad de Parkinson/metabolismo , Enfermedad de Gaucher/genética , Análisis de Secuencia de ARN/métodosRESUMEN
Traumatic brain injury (TBI) is a leading cause of chronic brain impairment and results in a robust, but poorly understood, neuroinflammatory response that contributes to the long-term pathology. We used single-nuclei RNA sequencing (snRNA-seq) to study transcriptomic changes in different cell populations in human brain tissue obtained acutely after severe, life-threatening TBI. This revealed a unique transcriptional response in oligodendrocyte precursors and mature oligodendrocytes, including the activation of a robust innate immune response, indicating an important role for oligodendroglia in the initiation of neuroinflammation. The activation of an innate immune response correlated with transcriptional upregulation of endogenous retroviruses in oligodendroglia. This observation was causally linked in vitro using human glial progenitors, implicating these ancient viral sequences in human neuroinflammation. In summary, this work provides insight into the initiating events of the neuroinflammatory response in TBI, which has therapeutic implications.
Asunto(s)
Lesiones Traumáticas del Encéfalo , Lesiones Encefálicas , Retrovirus Endógenos , Humanos , Animales , Ratones , Retrovirus Endógenos/genética , Enfermedades Neuroinflamatorias , Transcriptoma/genética , Lesiones Traumáticas del Encéfalo/patología , Lesiones Encefálicas/patología , Oligodendroglía/patología , Inflamación/genética , Inflamación/patología , Ratones Endogámicos C57BLRESUMEN
The genetic mechanisms underlying the expansion in size and complexity of the human brain remain poorly understood. Long interspersed nuclear element-1 (L1) retrotransposons are a source of divergent genetic information in hominoid genomes, but their importance in physiological functions and their contribution to human brain evolution are largely unknown. Using multiomics profiling, we here demonstrate that L1 promoters are dynamically active in the developing and the adult human brain. L1s generate hundreds of developmentally regulated and cell type-specific transcripts, many that are co-opted as chimeric transcripts or regulatory RNAs. One L1-derived long noncoding RNA, LINC01876, is a human-specific transcript expressed exclusively during brain development. CRISPR interference silencing of LINC01876 results in reduced size of cerebral organoids and premature differentiation of neural progenitors, implicating L1s in human-specific developmental processes. In summary, our results demonstrate that L1-derived transcripts provide a previously undescribed layer of primate- and human-specific transcriptome complexity that contributes to the functional diversification of the human brain.
Asunto(s)
Retroelementos , Transcriptoma , Animales , Humanos , Retroelementos/genética , Elementos de Nucleótido Esparcido Largo/genética , Neuronas , Primates/genéticaRESUMEN
The human forebrain has expanded in size and complexity compared to chimpanzees despite limited changes in protein-coding genes, suggesting that gene expression regulation is an important driver of brain evolution. Here, we identify a KRAB-ZFP transcription factor, ZNF558, that is expressed in human but not chimpanzee forebrain neural progenitor cells. ZNF558 evolved as a suppressor of LINE-1 transposons but has been co-opted to regulate a single target, the mitophagy gene SPATA18. ZNF558 plays a role in mitochondrial homeostasis, and loss-of-function experiments in cerebral organoids suggests that ZNF558 influences developmental timing during early human brain development. Expression of ZNF558 is controlled by the size of a variable number tandem repeat that is longer in chimpanzees compared to humans, and variable in the human population. Thus, this work provides mechanistic insight into how a cis-acting structural variation establishes a regulatory network that affects human brain evolution.
Asunto(s)
Redes Reguladoras de Genes , Organoides , Encéfalo/metabolismo , Proteínas de Unión al ADN , Regulación de la Expresión Génica , Humanos , Organoides/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) can be differentiated into many different cell types of the central nervous system. One challenge when using pluripotent stem cells is to develop robust and efficient differentiation protocols that result in homogenous cultures of the desired cell type. Here, we have utilized the SMAD-inhibitors SB431542 and Noggin in a fully defined monolayer culture model to differentiate human pluripotent cells into homogenous forebrain neural progenitors. Temporal fate analysis revealed that this protocol results in forebrain-patterned neural progenitor cells that start to express early neuronal markers after two weeks of differentiation, allowing for the analysis of gene expression changes during neurogenesis. Using this system, we were able to identify many previously uncharacterized long intergenic non-coding RNAs that display dynamic expression during human forebrain neurogenesis.
RESUMEN
Neuroplasticity forms the basis for neuronal circuit complexity and differences between otherwise similar circuits. We show that the microphthalmia-associated transcription factor (Mitf) plays a central role in intrinsic plasticity of olfactory bulb (OB) projection neurons. Mitral and tufted (M/T) neurons from Mitf mutant mice are hyperexcitable, have a reduced A-type potassium current (IA) and exhibit reduced expression of Kcnd3, which encodes a potassium voltage-gated channel subunit (Kv4.3) important for generating the IA Furthermore, expression of the Mitf and Kcnd3 genes is activity dependent in OB projection neurons and the MITF protein activates expression from Kcnd3 regulatory elements. Moreover, Mitf mutant mice have changes in olfactory habituation and have increased habituation for an odorant following long-term exposure, indicating that regulation of Kcnd3 is pivotal for long-term olfactory adaptation. Our findings show that Mitf acts as a direct regulator of intrinsic homeostatic feedback and links neuronal activity, transcriptional changes and neuronal function.
Asunto(s)
Factor de Transcripción Asociado a Microftalmía , Bulbo Olfatorio , Animales , Ratones , Factor de Transcripción Asociado a Microftalmía/genética , Neuronas , Odorantes , OlfatoRESUMEN
Although mast cell distribution has been described in both human and canine hearts, cardiac mast cells in mice have yet to be categorically localized. We therefore sought to describe mast cell distribution within the mouse heart and characterize their dependence on the Microphthalmia-associated transcription factor (Mitf). Cardiac mast cells were visualized using Toluidine Blue and avidin staining, and their distribution within the heart described. Cardiac mast cells were most prevalent in the epicardium (50%) or myocardium (45%). Less frequently, mast cells were noted in the endocardium (5%). Within the myocardium, 31% of the mast cells had perivascular location. By studying two different Mitf mutant strains, Mitfmi-vga9 and MitfMi-wh, we demonstrated that these mutations led to near-complete deficiency of cardiac mast cells. Accordingly, expression of the mMCP-4 and mMCP-5 genes was lost and chymase enzyme activity was severely reduced. Additionally, hearts from mice heterozygous for these Mitf mutations contained significantly fewer mast cells compared to wild-type mice. Our results demonstrated that the distribution of cardiac mast cells in mice is different from humans and dogs. Cardiac mast cells are dependent on Mitf expression, with loss-of-function mutation in the Mitf gene leading to near-complete lack of cardiac mast cells. Loss of a single Mitf allele is sufficient for relative mast cell deficiency.
Asunto(s)
Regulación de la Expresión Génica/inmunología , Mastocitos/inmunología , Factor de Transcripción Asociado a Microftalmía/inmunología , Miocardio/inmunología , Pericardio/inmunología , Animales , Perros , Humanos , Mastocitos/citología , Ratones , Ratones Transgénicos , Factor de Transcripción Asociado a Microftalmía/genética , Proteínas Quimioatrayentes de Monocitos/genética , Proteínas Quimioatrayentes de Monocitos/inmunología , Mutación , Serina Endopeptidasas/genética , Serina Endopeptidasas/inmunologíaRESUMEN
SUMMARY: Melanocytes are pigment producing cells derived from the neural crest. They are primarily found in the skin and hair follicles, but can also be found in other tissues including the eye, ear and heart. Here, we describe the distribution of pigmented cells in C57BL/6J mouse meninges, the membranes that envelope the brain. These cells contain melanosomes of all four stages of development and they depend on Microphthalmia associated transcription factor (MITF), the master regulator of melanocyte development, suggesting that they are bona-fide melanocytes. The location of these pigmented cells is consistent with the location of meningeal melanomas in humans and animal models. SIGNIFICANCE: Here, we document and define pigmented cells in the meninges of the mouse brain and confirm that they are melanocytes. This is important for understanding the role of this cell type and for understanding primary meningeal melanoma, a rare disease that likely arises from normal meningeal melanocytes.