RESUMO
Familial Dysautonomia (FD) is an autosomal recessive disorder caused by a splice site mutation in the gene ELP1, which disproportionally affects neurons. While classically characterized by deficits in sensory and autonomic neurons, neuronal defects in the central nervous system have also been described. Although ELP1 expression remains high in the normal developing and adult cerebellum, its role in cerebellar development is unknown. To explore the role of Elp1 in the cerebellum, we knocked out Elp1 in cerebellar granule cell progenitors (GCPs) and examined the outcome on animal behavior and cellular composition. We found that GCP-specific conditional knockout of Elp1 (Elp1cKO) resulted in ataxia by 8 weeks of age. Cellular characterization showed that the animals had smaller cerebella with fewer granule cells. This defect was already apparent as early as 7 days after birth, when Elp1cKO animals also had fewer mitotic GCPs and shorter Purkinje dendrites. Through molecular characterization, we found that loss of Elp1 was associated with an increase in apoptotic cell death and cell stress pathways in GCPs. Our study demonstrates the importance of ELP1 in the developing cerebellum, and suggests that loss of Elp1 in the GC lineage may also play a role in the progressive ataxia phenotypes of FD patients.
Assuntos
Cerebelo , Disautonomia Familiar , Camundongos Knockout , Fenótipo , Animais , Disautonomia Familiar/genética , Disautonomia Familiar/patologia , Cerebelo/metabolismo , Cerebelo/patologia , Camundongos , Modelos Animais de Doenças , Ataxia/genética , Ataxia/patologia , Ataxia/metabolismo , Células-Tronco Neurais/metabolismo , Apoptose/fisiologia , Peptídeos e Proteínas de Sinalização IntracelularRESUMO
The expansion of the neocortex, a hallmark of mammalian evolution1,2, was accompanied by an increase in cerebellar neuron numbers3. However, little is known about the evolution of the cellular programmes underlying the development of the cerebellum in mammals. In this study we generated single-nucleus RNA-sequencing data for around 400,000 cells to trace the development of the cerebellum from early neurogenesis to adulthood in human, mouse and the marsupial opossum. We established a consensus classification of the cellular diversity in the developing mammalian cerebellum and validated it by spatial mapping in the fetal human cerebellum. Our cross-species analyses revealed largely conserved developmental dynamics of cell-type generation, except for Purkinje cells, for which we observed an expansion of early-born subtypes in the human lineage. Global transcriptome profiles, conserved cell-state markers and gene-expression trajectories across neuronal differentiation show that cerebellar cell-type-defining programmes have been overall preserved for at least 160 million years. However, we also identified many orthologous genes that gained or lost expression in cerebellar neural cell types in one of the species or evolved new expression trajectories during neuronal differentiation, indicating widespread gene repurposing at the cell-type level. In sum, our study unveils shared and lineage-specific gene-expression programmes governing the development of cerebellar cells and expands our understanding of mammalian brain evolution.
Assuntos
Cerebelo , Evolução Molecular , Mamíferos , Neurogênese , Animais , Humanos , Camundongos , Linhagem da Célula/genética , Cerebelo/citologia , Cerebelo/embriologia , Cerebelo/crescimento & desenvolvimento , Feto/citologia , Feto/embriologia , Regulação da Expressão Gênica no Desenvolvimento , Neurogênese/genética , Neurônios/citologia , Neurônios/metabolismo , Gambás/embriologia , Gambás/crescimento & desenvolvimento , Células de Purkinje/citologia , Células de Purkinje/metabolismo , Análise da Expressão Gênica de Célula Única , Especificidade da Espécie , Transcriptoma , Mamíferos/embriologia , Mamíferos/crescimento & desenvolvimentoRESUMO
The vertebrate brain emerged more than ~500 million years ago in common evolutionary ancestors. To systematically trace its cellular and molecular origins, we established a spatially resolved cell type atlas of the entire brain of the sea lamprey-a jawless species whose phylogenetic position affords the reconstruction of ancestral vertebrate traits-based on extensive single-cell RNA-seq and in situ sequencing data. Comparisons of this atlas to neural data from the mouse and other jawed vertebrates unveiled various shared features that enabled the reconstruction of cell types, tissue structures and gene expression programs of the ancestral vertebrate brain. However, our analyses also revealed key tissues and cell types that arose later in evolution. For example, the ancestral brain was probably devoid of cerebellar cell types and oligodendrocytes (myelinating cells); our data suggest that the latter emerged from astrocyte-like evolutionary precursors in the jawed vertebrate lineage. Altogether, our work illuminates the cellular and molecular architecture of the ancestral vertebrate brain and provides a foundation for exploring its diversification during evolution.
Assuntos
Petromyzon , Vertebrados , Animais , Camundongos , Filogenia , Vertebrados/genética , Petromyzon/genética , Cabeça , EncéfaloRESUMO
BACKGROUND: Distinguishing the cellular origins of childhood brain tumors is key for understanding tumor initiation and identifying lineage-restricted, tumor-specific therapeutic targets. Previous strategies to map the cell-of-origin typically involved comparing human tumors to murine embryonal tissues, which is potentially limited due to species-specific differences. The aim of this study was to unravel the cellular origins of the 3 most common pediatric brain tumors, ependymoma, pilocytic astrocytoma, and medulloblastoma, using a developing human cerebellar atlas. METHODS: We used a single-nucleus atlas of the normal developing human cerebellum consisting of 176 645 cells as a reference for an in-depth comparison to 4416 bulk and single-cell transcriptome tumor datasets, using gene set variation analysis, correlation, and single-cell matching techniques. RESULTS: We find that the astroglial cerebellar lineage is potentially the origin for posterior fossa ependymomas. We propose that infratentorial pilocytic astrocytomas originate from the oligodendrocyte lineage and MHC II genes are specifically enriched in these tumors. We confirm that SHH and Group 3/4 medulloblastomas originate from the granule cell and unipolar brush cell lineages. Radiation-induced gliomas stem from cerebellar glial lineages and demonstrate distinct origins from the primary medulloblastoma. We identify tumor genes that are expressed in the cerebellar lineage of origin, and genes that are tumor specific; both gene sets represent promising therapeutic targets for future study. CONCLUSION: Based on our results, individual cells within a tumor may resemble different cell types along a restricted developmental lineage. Therefore, we suggest that tumors can arise from multiple cellular states along the cerebellar "lineage of origin."
Assuntos
Astrocitoma , Neoplasias Encefálicas , Neoplasias Cerebelares , Ependimoma , Glioma , Meduloblastoma , Criança , Humanos , Animais , Camundongos , Meduloblastoma/genética , Meduloblastoma/patologia , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Glioma/patologia , Astrocitoma/genética , Ependimoma/genética , Ependimoma/patologia , Cerebelo/patologia , Neoplasias Cerebelares/genética , Neoplasias Cerebelares/patologiaRESUMO
The testis produces gametes through spermatogenesis and evolves rapidly at both the morphological and molecular level in mammals1-6, probably owing to the evolutionary pressure on males to be reproductively successful7. However, the molecular evolution of individual spermatogenic cell types across mammals remains largely uncharacterized. Here we report evolutionary analyses of single-nucleus transcriptome data for testes from 11 species that cover the three main mammalian lineages (eutherians, marsupials and monotremes) and birds (the evolutionary outgroup), and include seven primates. We find that the rapid evolution of the testis was driven by accelerated fixation rates of gene expression changes, amino acid substitutions and new genes in late spermatogenic stages, probably facilitated by reduced pleiotropic constraints, haploid selection and transcriptionally permissive chromatin. We identify temporal expression changes of individual genes across species and conserved expression programs controlling ancestral spermatogenic processes. Genes predominantly expressed in spermatogonia (germ cells fuelling spermatogenesis) and Sertoli (somatic support) cells accumulated on X chromosomes during evolution, presumably owing to male-beneficial selective forces. Further work identified transcriptomal differences between X- and Y-bearing spermatids and uncovered that meiotic sex-chromosome inactivation (MSCI) also occurs in monotremes and hence is common to mammalian sex-chromosome systems. Thus, the mechanism of meiotic silencing of unsynapsed chromatin, which underlies MSCI, is an ancestral mammalian feature. Our study illuminates the molecular evolution of spermatogenesis and associated selective forces, and provides a resource for investigating the biology of the testis across mammals.
Assuntos
Evolução Molecular , Mamíferos , Espermatogênese , Testículo , Animais , Masculino , Cromatina/genética , Mamíferos/genética , Meiose/genética , Espermatogênese/genética , Testículo/citologia , Transcriptoma , Análise de Célula Única , Aves/genética , Primatas/genética , Regulação da Expressão Gênica , Espermatogônias/citologia , Células de Sertoli/citologia , Cromossomo X/genética , Cromossomo Y/genética , Mecanismo Genético de Compensação de Dose , Inativação GênicaRESUMO
Transcription factor 4 (TCF4) is a basic helix-loop-helix transcription factor essential for neurocognitive development. The aberrations in TCF4 are associated with neurodevelopmental disorders including schizophrenia, intellectual disability, and Pitt-Hopkins syndrome, an autism-spectrum disorder characterized by developmental delay. Several disease-associated missense mutations in TCF4 have been shown to interfere with TCF4 function, but for many mutations, the impact remains undefined. Here, we tested the effects of 12 functionally uncharacterized disease-associated missense mutations and variations in TCF4 using transient expression in mammalian cells, confocal imaging, in vitro DNA-binding assays, and reporter assays. We show that Pitt-Hopkins syndrome-associated missense mutations within the basic helix-loop-helix domain of TCF4 and a Rett-like syndrome-associated mutation in a transcription activation domain result in altered DNA-binding and transcriptional activity of the protein. Some of the missense variations found in schizophrenia patients slightly increase TCF4 transcriptional activity, whereas no effects were detected for missense mutations linked to mild intellectual disability. We in addition find that the outcomes of several disease-related mutations are affected by cell type, TCF4 isoform, and dimerization partner, suggesting that the effects of TCF4 mutations are context-dependent. Together with previous work, this study provides a basis for the interpretation of the functional consequences of TCF4 missense variants.
Assuntos
Fácies , Hiperventilação , Deficiência Intelectual , Mutação de Sentido Incorreto , Esquizofrenia , Fator de Transcrição 4 , Transcrição Gênica , Substituição de Aminoácidos , Animais , Células HEK293 , Sequências Hélice-Alça-Hélice , Humanos , Hiperventilação/genética , Hiperventilação/metabolismo , Deficiência Intelectual/genética , Deficiência Intelectual/metabolismo , Ratos , Ratos Sprague-Dawley , Esquizofrenia/genética , Esquizofrenia/metabolismo , Fator de Transcrição 4/química , Fator de Transcrição 4/genética , Fator de Transcrição 4/metabolismoRESUMO
Huntington's disease (HD) is an inherited neurodegenerative disorder with onset of characteristic motor symptoms at midlife, preceded by subtle cognitive and behavioral disturbances. Transcriptional dysregulation emerges early in the disease course and is considered central to HD pathogenesis. Using wild-type (wt) and HD knock-in mouse striatal cell lines we observed a HD genotype-dependent reduction in the protein levels of transcription factor 4 (TCF4), a member of the basic helix-loop-helix (bHLH) family with critical roles in brain development and function. We characterized mouse Tcf4 gene structure and expression of alternative mRNAs and protein isoforms in cell-based models of HD, and in four different brain regions of male transgenic HD mice (R6/1) from young to mature adulthood. The largest decrease in the levels of TCF4 at mRNA and specific protein isoforms were detected in the R6/1 mouse hippocampus. Translating this finding to human disease, we found reduced expression of long TCF4 isoforms in the postmortem hippocampal CA1 area and in the cerebral cortex of HD patients. Additionally, TCF4 protein isoforms showed differential synergism with the proneural transcription factor ASCL1 in activating reporter gene transcription in hippocampal and cortical cultured neurons. Induction of neuronal activity increased these synergistic effects in hippocampal but not in cortical neurons, suggesting brain region-dependent differences in TCF4 functions. Collectively, this study demonstrates isoform-specific changes in TCF4 expression in HD that could contribute to the progressive impairment of transcriptional regulation and neuronal function in this disease.
Assuntos
Doença de Huntington , Adulto , Animais , Modelos Animais de Doenças , Hipocampo , Humanos , Doença de Huntington/genética , Masculino , Camundongos , Camundongos Transgênicos , Neurônios , Isoformas de Proteínas , Fator de Transcrição 4/genéticaRESUMO
Organ development is orchestrated by cell- and time-specific gene regulatory networks. In this study, we investigated the regulatory basis of mouse cerebellum development from early neurogenesis to adulthood. By acquiring snATAC-seq (single-nucleus assay for transposase accessible chromatin using sequencing) profiles for ~90,000 cells spanning 11 stages, we mapped cerebellar cell types and identified candidate cis-regulatory elements (CREs). We detected extensive spatiotemporal heterogeneity among progenitor cells and a gradual divergence in the regulatory programs of cerebellar neurons during differentiation. Comparisons to vertebrate genomes and snATAC-seq profiles for â¼20,000 cerebellar cells from the marsupial opossum revealed a shared decrease in CRE conservation during development and differentiation as well as differences in constraint between cell types. Our work delineates the developmental and evolutionary dynamics of gene regulation in cerebellar cells and provides insights into mammalian organ development.
Assuntos
Evolução Biológica , Cerebelo/citologia , Cerebelo/crescimento & desenvolvimento , Neurônios/fisiologia , Elementos Reguladores de Transcrição , Animais , Cerebelo/embriologia , Cromatina/genética , Cromatina/metabolismo , DNA Intergênico , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Redes Reguladoras de Genes , Masculino , Camundongos , Células-Tronco Neurais/citologia , Células-Tronco Neurais/fisiologia , Neurogênese , Gambás/genéticaRESUMO
The CTG trinucleotide repeat (TNR) expansion in Transcription factor 4 (TCF4) intron 3 is the main cause of Fuchs' endothelial corneal dystrophy (FECD) and may confer an increased risk of developing bipolar disorder (BD). Usage of alternative 5' exons for transcribing the human TCF4 gene results in numerous TCF4 transcripts which encode for at least 18 N-terminally different protein isoforms that vary in their function and transactivation capability. Here we studied the TCF4 region containing the CTG TNR and characterized the transcription initiation sites of the nearby downstream 5' exons 4a, 4b and 4c. We demonstrate that these exons are linked to alternative promoters and show that the CTG TNR expansion decreases the activity of the nearby downstream TCF4 promoters in primary cultured neurons. We confirm this finding using two RNA sequencing (RNA-seq) datasets of corneal endothelium from FECD patients with expanded CTG TNR in the TCF4 gene. Furthermore, we report an increase in the expression of various other TCF4 transcripts in FECD, possibly indicating a compensatory mechanism. We conclude that the CTG TNR affects TCF4 expression in a transcript-specific manner both in neurons and in the cornea.
Assuntos
Distrofia Endotelial de Fuchs/genética , Fator de Transcrição 4/genética , Processamento Alternativo , Humanos , Regiões Promotoras Genéticas , Fator de Transcrição 4/metabolismo , Expansão das Repetições de TrinucleotídeosRESUMO
Mammalian transcription factor 4 (TCF4) has been linked to schizophrenia and intellectual disabilities, such as Pitt-Hopkins syndrome (PTHS). Here, we show that similarly to mammalian TCF4, fruit fly orthologue Daughterless (Da) is expressed widely in the Drosophila brain. Furthermore, silencing of da, using several central nervous system-specific Gal4 driver lines, impairs appetitive associative learning of the larvae and leads to decreased levels of the synaptic proteins Synapsin (Syn) and Discs large 1 (Dlg1), suggesting the involvement of Da in memory formation. Here, we demonstrate that Syn and dlg1 are direct target genes of Da in adult Drosophila heads, as Da binds to the regulatory regions of these genes and the modulation of Da levels alter the levels of Syn and dlg1 mRNA. Silencing of da also affects negative geotaxis of the adult flies, suggesting the impairment of locomotor function. Overall, our findings suggest that Da regulates Drosophila larval memory and adult negative geotaxis, possibly via its synaptic target genes Syn and dlg1 These behavioural phenotypes can be further used as a PTHS model to screen for therapeutics.This article has an associated First Person interview with the first author of the paper.
Assuntos
Aprendizagem por Associação , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Comportamento Animal , Encéfalo/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Hiperventilação/metabolismo , Deficiência Intelectual/metabolismo , Neurônios/metabolismo , Proteoma , Sinapses/metabolismo , Animais , Animais Geneticamente Modificados , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Encéfalo/embriologia , Encéfalo/fisiopatologia , Modelos Animais de Doenças , Proteínas de Drosophila/genética , Drosophila melanogaster/embriologia , Drosophila melanogaster/genética , Fácies , Regulação da Expressão Gênica no Desenvolvimento , Células HEK293 , Humanos , Hiperventilação/genética , Hiperventilação/fisiopatologia , Deficiência Intelectual/genética , Deficiência Intelectual/fisiopatologia , Larva/genética , Larva/metabolismo , Atividade Motora , Transdução de Sinais , Sinapses/genética , Sinapsinas/genética , Sinapsinas/metabolismo , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismoRESUMO
Neuralized functions as a positive regulator of the Notch pathway by promoting ubiquitination of Notch ligands via its E3 ligase activity, resulting in their efficient endocytosis and signaling. Using a yeast two-hybrid screen, we have identified a cGMP-hydrolysing phosphodiesterase, PDE9A, as a novel interactor and substrate of Neuralized E3 ubiquitin protein ligase 1 (NEURL1). We confirmed this interaction with co-immunoprecipitation experiments and show that both Neuralized Homology Repeat domains of NEURL1 can interact with PDE9A. We also demonstrate that NEURL1 can promote polyubiquitination of PDE9A that leads to its proteasome-mediated degradation mainly via lysine residue K27 of ubiquitin. Our results suggest that NEURL1 acts as a novel regulator of protein levels of PDE9A.
Assuntos
3',5'-AMP Cíclico Fosfodiesterases/metabolismo , GMP Cíclico/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , 3',5'-AMP Cíclico Fosfodiesterases/química , 3',5'-AMP Cíclico Fosfodiesterases/genética , Animais , Domínio Catalítico , Feminino , Células HEK293 , Humanos , Imunoprecipitação/métodos , Masculino , Neurônios/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise , Ratos , Ratos Sprague-Dawley , Transfecção , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/genética , Ubiquitinação/genéticaRESUMO
Transcription factor 4 (TCF4 also known as ITF2 or E2-2) is a basic helix-loop-helix (bHLH) protein associated with Pitt-Hopkins syndrome, intellectual disability, and schizophrenia (SCZ). Here, we show that TCF4-dependent transcription in cortical neurons cultured from embryonic rats of both sexes is induced by neuronal activity via soluble adenylyl cyclase and protein kinase A (PKA) signaling. PKA phosphorylates TCF4 directly and a PKA phosphorylation site in TCF4 is necessary for its transcriptional activity in cultured neurons and in the developing brain in vivo We also demonstrate that Gadd45g (growth arrest and DNA damage inducible gamma) is a direct target of neuronal-activity-induced, TCF4-dependent transcriptional regulation and that TCF4 missense variations identified in SCZ patients alter the transcriptional activity of TCF4 in neurons. This study identifies a new role for TCF4 as a neuronal-activity-regulated transcription factor, offering a novel perspective on the association of TCF4 with cognitive disorders.SIGNIFICANCE STATEMENT The importance of the basic helix-loop-helix transcription factor transcription factor 4 (TCF4) in the nervous system is underlined by its association with common and rare cognitive disorders. In the current study, we show that TCF4-controlled transcription in primary cortical neurons is induced by neuronal activity and protein kinase A. Our results support the hypotheses that dysregulation of neuronal-activity-dependent signaling plays a significant part in the etiology of neuropsychiatric and neurodevelopmental disorders.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Proteínas de Ligação a DNA/metabolismo , Deficiência Intelectual/metabolismo , Neurônios/metabolismo , Esquizofrenia/metabolismo , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Animais , Células Cultivadas , Córtex Cerebral/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/genética , Proteínas de Ligação a DNA/genética , Feminino , Células HEK293 , Hipocampo/metabolismo , Humanos , Deficiência Intelectual/genética , Masculino , Ratos , Ratos Sprague-Dawley , Esquizofrenia/genética , Fator de Transcrição 4 , Fatores de Transcrição/genéticaRESUMO
Mutations in TCF4 (basic helix-loop-helix transcription factor 4), a gene with complex organization and multiple transcription initiation sites, are usually associated with Pitt-Hopkins syndrome (PTHS). However, a translocation encompassing the 5' end of TCF4 and several point mutations have been linked to non-syndromic intellectual disability (NSID). Here we describe a family with autosomal dominantly inherited NSID in seven relatives with a partial deletion of TCF4, disrupting the 5' end of the gene, predicted to result in the reduction of the number of mRNAs that can be produced by alternative transcription initiation. Functional studies indicate that it leads to reduced levels of transcripts coding for TCF4 protein isoforms with a nuclear localization signal, which may be relevant to the phenotype. The findings in our family support the notion that the position of the mutation in TCF4 is relevant to the phenotype, with those mutations in the 5' region, cassette exons and regions not affecting the important functional domains being linked to NSID rather than PTHS. We suggest that screening for mutations in TCF4 could be considered in the investigation of NSID.
Assuntos
Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Deficiência Intelectual/genética , Fatores de Transcrição/genética , Transcrição Gênica , Regiões 5' não Traduzidas , Éxons/genética , Fácies , Feminino , Humanos , Hiperventilação/genética , Hiperventilação/patologia , Deficiência Intelectual/patologia , Masculino , Linhagem , Fenótipo , Mutação Puntual/genética , Fator de Transcrição 4RESUMO
To study nucleolar involvement in brain development, the nuclear and nucleolar proteomes from the rat cerebral cortex at postnatal day 7 were analyzed using LC-MS/iTRAQ methodology. Data of the analysis are available via ProteomeXchange with identifier PXD002188. Among 504 candidate nucleolar proteins, the overrepresented gene ontology terms included such cellular compartmentcategories as "nucleolus", "ribosome" and "chromatin". Consistent with such classification, the most overrepresented functional gene ontology terms were related to RNA metabolism/ribosomal biogenesis, translation, and chromatin organization. Sixteen putative nucleolar proteins were associated with neurodevelopmental phenotypes in humans. Microcephaly and/or cognitive impairment were the most common phenotypic manifestations. Although several such proteins have links to ribosomal biogenesis and/or genomic stability/chromatin structure (e.g. EMG1, RPL10, DKC1, EIF4A3, FLNA, SMC1, ATRX, MCM4, NSD1, LMNA, or CUL4B), others including ADAR, LARP7, GTF2I, or TCF4 have no such connections known. Although neither the Alazami syndrome-associated LARP7nor the Pitt-Hopkins syndrome-associated TCF4 were reported in nucleoli of non-neural cells, in neurons, their nucleolar localization was confirmed by immunostaining. In cultured rat hippocampal neurons, knockdown of LARP7 reduced both perikaryal ribosome content and general protein synthesis. Similar anti-ribosomal/anti-translation effects were observed after knockdown of the ribosomal biogenesis factor EMG1 whose deficiency underlies Bowen-Conradi syndrome. Finally, moderate reduction of ribosome content and general protein synthesis followed overexpression of two Pitt-Hopkins syndrome mutant variants of TCF4. Therefore, dysregulation of ribosomal biogenesis and/or other functions of the nucleolus may disrupt neurodevelopment resulting in such phenotypes as microcephaly and/or cognitive impairment.
Assuntos
Nucléolo Celular/metabolismo , Córtex Cerebral/crescimento & desenvolvimento , Proteínas Nucleares/isolamento & purificação , Proteômica/métodos , Animais , Animais Recém-Nascidos , Células Cultivadas , Córtex Cerebral/metabolismo , Feminino , Humanos , Modelos Animais , Mapas de Interação de Proteínas , Ratos , Ratos Sprague-Dawley , Ribossomos/metabolismoRESUMO
Pitt-Hopkins syndrome (PTHS) is caused by haploinsufficiency of Transcription factor 4 (TCF4), one of the three human class I basic helix-loop-helix transcription factors called E-proteins. Drosophila has a single E-protein, Daughterless (Da), homologous to all three mammalian counterparts. Here we show that human TCF4 can rescue Da deficiency during fruit fly nervous system development. Overexpression of Da or TCF4 specifically in adult flies significantly decreases their survival rates, indicating that these factors are crucial even after development has been completed. We generated da transgenic fruit fly strains with corresponding missense mutations R578H, R580W, R582P and A614V found in TCF4 of PTHS patients and studied the impact of these mutations in vivo. Overexpression of wild type Da as well as human TCF4 in progenitor tissues induced ectopic sensory bristles and the rough eye phenotype. By contrast, overexpression of Da(R580W) and Da(R582P) that disrupt DNA binding reduced the number of bristles and induced the rough eye phenotype with partial lack of pigmentation, indicating that these act dominant negatively. Compared to the wild type, Da(R578H) and Da(A614V) were less potent in induction of ectopic bristles and the rough eye phenotype, respectively, suggesting that these are hypomorphic. All studied PTHS-associated mutations that we introduced into Da led to similar effects in vivo as the same mutations in TCF4 in vitro. Consequently, our Drosophila models of PTHS are applicable for further studies aiming to unravel the molecular mechanisms of this disorder.
RESUMO
Technological advantages in sequencing and proteomics have revealed the remarkable diversity of alternative protein isoforms. Typically, the localization and functions of these isoforms are unknown and cannot be predicted. Also the localization signals leading to particular subnuclear compartments have not been identified and thus, predicting alternative functions due to alternative subnuclear localization is limited only to very few subnuclear compartments. Knowledge of the localization and function of alternative protein isoforms allows for a greater understanding of cellular complexity. In this article, we characterize a short and well-defined signal targeting the bovine papillomavirus type 1 E8/E2 protein to the nuclear matrix. The targeting signal comprises the peptide coded by E8 ORF, which is spliced together with part of the E2 ORF to generate the E8/E2 mRNA. Localization to the nuclear matrix correlates well with the transcription repression activities of E8/E2; a single point mutation directs the E8/E2 protein into the nucleoplasm, and transcription repression activity is lost. Our data prove that adding as few as Ë10 amino acids by alternative transcription/alternative splicing drastically alters the function and subnuclear localization of proteins. To our knowledge, E8 is the shortest known nuclear matrix targeting signal.
Assuntos
Papillomavirus Bovino 1/genética , Proteínas de Ligação a DNA/genética , Genoma Viral , Matriz Nuclear/genética , Proteínas Oncogênicas Virais/genética , Proteínas Virais/genética , Animais , Células CHO , Células COS , Núcleo Celular/metabolismo , Chlorocebus aethiops , Cricetulus , Proteínas de Ligação a DNA/metabolismo , Repressão Epigenética , Matriz Nuclear/metabolismo , Proteínas Oncogênicas Virais/metabolismo , Regiões Promotoras Genéticas , Transcrição Gênica , Proteínas Virais/metabolismoRESUMO
Huntington disease (HD) is a fatal autosomal dominant neurodegenerative disorder caused by an increased number of CAG repeats in the HTT gene coding for huntingtin. Decreased neurotrophic support and increased mitochondrial and excitotoxic stress have been reported in HD striatal and cortical neurons. The members of the class O forkhead (FOXO) transcription factor family, including FOXO3a, act as sensor proteins that are activated upon decreased survival signals and/or increased cellular stress. Using immunocytochemical screening in mouse striatal Hdh(7/7) (wild type), Hdh(7/109) (heterozygous for HD mutation), and Hdh(109/109) (homozygous for HD mutation) cells, we identified FOXO3a as a differentially regulated transcription factor in HD. We report increased nuclear FOXO3a levels in mutant Hdh cells. Additionally, we show that treatment with mitochondrial toxin 3-nitropropionic acid results in enhanced nuclear localization of FOXO3a in wild type Hdh(7/7) cells and in rat primary cortical neurons. Furthermore, mRNA levels of Foxo3a are increased in mutant Hdh cells compared with wild type cells and in 3-nitropropionic acid-treated primary neurons compared with untreated neurons. A similar increase was observed in the cortex of R6/2 mice and HD patient post-mortem caudate tissue compared with controls. Using chromatin immunoprecipitation and reporter assays, we demonstrate that FOXO3a regulates its own transcription by binding to the conserved response element in Foxo3a promoter. Altogether, the findings of this study suggest that FOXO3a levels are increased in HD cells as a result of overactive positive feedback loop.
Assuntos
Retroalimentação Fisiológica , Fatores de Transcrição Forkhead/metabolismo , Doença de Huntington/metabolismo , Proteínas da Membrana Plasmática de Transporte de Serotonina/metabolismo , Animais , Western Blotting , Células Cultivadas , Corpo Estriado/citologia , Corpo Estriado/metabolismo , Feminino , Proteína Forkhead Box O3 , Fatores de Transcrição Forkhead/genética , Expressão Gênica , Células HEK293 , Humanos , Doença de Huntington/genética , Imuno-Histoquímica , Masculino , Camundongos Transgênicos , Microscopia Confocal , Mutação , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Nitrocompostos/farmacologia , Regiões Promotoras Genéticas/genética , Propionatos/farmacologia , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas da Membrana Plasmática de Transporte de Serotonina/genéticaRESUMO
BACKGROUND: Brain derived neurotrophic factor (BDNF) belongs to a family of structurally related proteins called neurotrophins that have been shown to regulate survival and growth of neurons in the developing central and peripheral nervous system and also to take part in synaptic plasticity related processes in adulthood. Since BDNF is associated with several nervous system disorders it would be beneficial to have cellular reporter system for studying its expression regulation. METHODS: Using modified bacterial artificial chromosome (BAC), we generated several transgenic cell lines expressing humanised Renilla luciferase (hRluc)-EGFP fusion reporter gene under the control of rat BDNF gene regulatory sequences (rBDNF-hRluc-EGFP) in HeLa background. To see if the hRluc-EGFP reporter was regulated in response to known regulators of BDNF expression we treated cell lines with substances known to regulate BDNF and also overexpressed transcription factors known to regulate BDNF gene in established cell lines. RESULTS: rBDNF-hRluc-EGFP cell lines had high transgene copy numbers when assayed with qPCR and FISH analysis showed that transgene was maintained episomally in all cell lines. Luciferase activity in transgenic cell lines was induced in response to ionomycin-mediated rise of intracellular calcium levels, treatment with HDAC inhibitors and by over-expression of transcription factors known to increase BDNF expression, indicating that transcription of the transgenic reporter is regulated similarly to the endogenous BDNF gene. CONCLUSIONS: Generated rBDNF-hRluc-EGFP BAC cell lines respond to known modulators of BDNF expression and could be used for screening of compounds/small molecules or transcription factors altering BDNF expression.
Assuntos
Bioensaio/métodos , Fator Neurotrófico Derivado do Encéfalo/genética , Cromossomos Artificiais Bacterianos/genética , Desenho de Fármacos , Inibidores de Histona Desacetilases/farmacologia , Ativação Transcricional/efeitos dos fármacos , Ativação Transcricional/genética , Células HeLa , HumanosRESUMO
Transcription factor TCF4 (alias ITF2, SEF2 or E2-2) is a broadly expressed basic helix-loop-helix (bHLH) protein that functions as a homo- or heterodimer. Missense, nonsense, frame-shift and splice-site mutations as well as translocations and large deletions encompassing TCF4 gene cause Pitt-Hopkins syndrome (PTHS), a rare developmental disorder characterized by severe motor and mental retardation, typical facial features and breathing anomalies. Irrespective of the mutation, TCF4 haploinsufficiency has been proposed as an underlying mechanism for PTHS. We have recently demonstrated that human TCF4 gene is transcribed using numerous 5' exons. Here, we re-evaluated the impact of all the published PTHS-associated mutations, taking into account the diversity of TCF4 isoforms, and assessed how the reading frame elongating and missense mutations affect TCF4 functions. Our analysis revealed that not all deletions and truncating mutations in TCF4 result in complete loss-of-function and the impact of reading frame elongating and missense mutations ranges from subtle deficiencies to dominant-negative effects. We show that (i) missense mutations in TCF4 bHLH domain and the reading frame elongating mutation damage DNA-binding and transactivation ability in a manner dependent on dimer context (homodimer versus heterodimer with ASCL1 or NEUROD2); (ii) the elongating mutation and the missense mutation at the dimer interface of the HLH domain destabilize the protein; and (iii) missense mutations outside of the bHLH domain cause no major functional deficiencies. We conclude that different PTHS-associated mutations impair the functions of TCF4 by diverse mechanisms and to a varying extent, possibly contributing to the phenotypic variability of PTHS patients.