Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 14 de 14
Filtrar
1.
Cell ; 178(4): 850-866.e26, 2019 08 08.
Artigo em Inglês | MEDLINE | ID: mdl-31398340

RESUMO

We performed a comprehensive assessment of rare inherited variation in autism spectrum disorder (ASD) by analyzing whole-genome sequences of 2,308 individuals from families with multiple affected children. We implicate 69 genes in ASD risk, including 24 passing genome-wide Bonferroni correction and 16 new ASD risk genes, most supported by rare inherited variants, a substantial extension of previous findings. Biological pathways enriched for genes harboring inherited variants represent cytoskeletal organization and ion transport, which are distinct from pathways implicated in previous studies. Nevertheless, the de novo and inherited genes contribute to a common protein-protein interaction network. We also identified structural variants (SVs) affecting non-coding regions, implicating recurrent deletions in the promoters of DLG2 and NR3C2. Loss of nr3c2 function in zebrafish disrupts sleep and social function, overlapping with human ASD-related phenotypes. These data support the utility of studying multiplex families in ASD and are available through the Hartwell Autism Research and Technology portal.


Assuntos
Transtorno do Espectro Autista/genética , Predisposição Genética para Doença/genética , Linhagem , Mapas de Interação de Proteínas/genética , Animais , Criança , Bases de Dados Genéticas , Modelos Animais de Doenças , Feminino , Deleção de Genes , Guanilato Quinases/genética , Humanos , Padrões de Herança/genética , Aprendizado de Máquina , Masculino , Núcleo Familiar , Regiões Promotoras Genéticas/genética , Receptores de Mineralocorticoides/genética , Fatores de Risco , Proteínas Supressoras de Tumor/genética , Sequenciamento Completo do Genoma , Peixe-Zebra/genética
2.
PLoS Biol ; 14(2): e1002368, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26844991

RESUMO

Both scientists and the public would benefit from improved communication of basic scientific research and from integrating scientists into education outreach, but opportunities to support these efforts are limited. We have developed two low-cost programs--"Present Your PhD Thesis to a 12-Year-Old" and "Shadow a Scientist"--that combine training in science communication with outreach to area middle schools. We assessed the outcomes of these programs and found a 2-fold benefit: scientists improve their communication skills by explaining basic science research to a general audience, and students' enthusiasm for science and their scientific knowledge are increased. Here we present details about both programs, along with our assessment of them, and discuss the feasibility of exporting these programs to other universities.


Assuntos
Comunicação , Relações Comunidade-Instituição , Pesquisa , Estudantes , Humanos
3.
BMC Genomics ; 16: 40, 2015 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-25653011

RESUMO

BACKGROUND: The pathways regulating the transition of mammalian cells from quiescence to proliferation are mediated by multiple miRNAs. Despite significant improvements in our understanding of miRNA targeting, the majority of miRNA regulatory networks are still largely unknown and require experimental validation. RESULTS: Here we identified miR-503, miR-103, and miR-494 as negative regulators of proliferation in primary human cells. We experimentally determined their genome wide target profiles using RNA-induced silencing complex (RISC) immunoprecipitations and gene expression profiling. Analysis of the genome wide target profiles revealed evidence of extensive regulation of gene expression through non-canonical target pairing by miR-503. We identified the proto-oncogene DDHD2 as a target of miR-503 that requires pairing outside of the canonical 5' seed region of miR-503, representing a novel mode of miRNA-target pairing. Further bioinformatics analysis implicated miR-503 and DDHD2 in breast cancer tumorigenesis. CONCLUSIONS: Our results provide an extensive genome wide set of targets for miR-503, miR-103, and miR-494, and suggest that miR-503 may act as a tumor suppressor in breast cancer by its direct non-canonical targeting of DDHD2.


Assuntos
Neoplasias da Mama/genética , MicroRNAs/genética , MicroRNAs/metabolismo , Fosfolipases/metabolismo , Neoplasias da Mama/patologia , Carcinogênese , Proliferação de Células/genética , Feminino , Regulação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Genoma Humano , Humanos , Estimativa de Kaplan-Meier , Proto-Oncogene Mas
4.
RNA ; 19(7): 958-70, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23697550

RESUMO

Mobile group II introns encode reverse transcriptases (RTs) that function in intron mobility ("retrohoming") by a process that requires reverse transcription of a highly structured, 2-2.5-kb intron RNA with high processivity and fidelity. Although the latter properties are potentially useful for applications in cDNA synthesis and next-generation RNA sequencing (RNA-seq), group II intron RTs have been difficult to purify free of the intron RNA, and their utility as research tools has not been investigated systematically. Here, we developed general methods for the high-level expression and purification of group II intron-encoded RTs as fusion proteins with a rigidly linked, noncleavable solubility tag, and we applied them to group II intron RTs from bacterial thermophiles. We thus obtained thermostable group II intron RT fusion proteins that have higher processivity, fidelity, and thermostability than retroviral RTs, synthesize cDNAs at temperatures up to 81°C, and have significant advantages for qRT-PCR, capillary electrophoresis for RNA-structure mapping, and next-generation RNA sequencing. Further, we find that group II intron RTs differ from the retroviral enzymes in template switching with minimal base-pairing to the 3' ends of new RNA templates, making it possible to efficiently and seamlessly link adaptors containing PCR-primer binding sites to cDNA ends without an RNA ligase step. This novel template-switching activity enables facile and less biased cloning of nonpolyadenylated RNAs, such as miRNAs or protein-bound RNA fragments. Our findings demonstrate novel biochemical activities and inherent advantages of group II intron RTs for research, biotechnological, and diagnostic methods, with potentially wide applications.


Assuntos
DNA Complementar/biossíntese , Íntrons , DNA Polimerase Dirigida por RNA/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Análise de Sequência de RNA/métodos , Sequência de Bases , Clonagem Molecular , Sequência Conservada , DNA Complementar/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Perfilação da Expressão Gênica , Biblioteca Gênica , Geobacillus stearothermophilus/genética , Geobacillus stearothermophilus/metabolismo , Células HeLa , Humanos , Células MCF-7 , MicroRNAs/genética , MicroRNAs/metabolismo , Dados de Sequência Molecular , Fases de Leitura Aberta , Proteínas Periplásmicas de Ligação/genética , Proteínas Periplásmicas de Ligação/metabolismo , Plasmídeos/genética , Plasmídeos/metabolismo , Estabilidade Proteica , DNA Polimerase Dirigida por RNA/genética , Proteínas Recombinantes de Fusão/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Temperatura
5.
Nucleic Acids Res ; 41(4): 2239-54, 2013 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-23303785

RESUMO

The transition of mammalian cells from quiescence to proliferation is accompanied by the differential expression of several microRNAs (miRNAs) and transcription factors. However, the interplay between transcription factors and miRNAs in modulating gene regulatory networks involved in human cell proliferation is largely unknown. Here we show that the miRNA miR-22 promotes proliferation in primary human cells, and through a combination of Argonaute-2 immunoprecipitation and reporter assays, we identified multiple novel targets of miR-22, including several cell-cycle arrest genes that mediate the effects of the tumor-suppressor p53. In addition, we found that miR-22 suppresses interferon gene expression by directly targeting high mobility group box-1 and interferon regulatory factor (IRF)-5, preventing activation of IRF3 and NF-κB, which are activators of interferon genes. The expression of interferon genes is elevated in quiescent cells and their expression is inhibitory for cell proliferation. In addition, we find that miR-22 is activated by the transcription factor Myc when quiescent cells enter proliferation and that miR-22 inhibits the Myc transcriptional repressor MXD4, mediating a feed-forward loop to elevate Myc expression levels. Our results implicate miR-22 in downregulating the anti-proliferative p53 and interferon pathways and reveal a new transcription factor-miRNA network that regulates the transition of primary human cells from quiescence to proliferation.


Assuntos
Pontos de Checagem do Ciclo Celular/genética , Proliferação de Células , Redes Reguladoras de Genes , Interferons/genética , MicroRNAs/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/antagonistas & inibidores , Células Cultivadas , Regulação para Baixo , Genes cdc , Células HeLa , Humanos , Interferons/biossíntese , MicroRNAs/biossíntese , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas Repressoras/antagonistas & inibidores
6.
bioRxiv ; 2023 Sep 30.
Artigo em Inglês | MEDLINE | ID: mdl-37808727

RESUMO

The development of successful therapeutics for dementias requires an understanding of their shared and distinct molecular features in the human brain. We performed single-nuclear RNAseq and ATACseq in Alzheimer disease (AD), Frontotemporal degeneration (FTD), and Progressive Supranuclear Palsy (PSP), analyzing 40 participants, yielding over 1.4M cells from three brain regions ranging in vulnerability and pathological burden. We identify 35 shared disease-associated cell types and 14 that are disease-specific, replicating those previously identified in AD. Disease - specific cell states represent molecular features of disease-specific glial-immune mechanisms and neuronal vulnerability in each disorder, layer 4/5 intra-telencephalic neurons in AD, layer 2/3 intra-telencephalic neurons in FTD, and layer 5/6 near-projection neurons in PSP. We infer intrinsic disease-associated gene regulatory networks, which we empirically validate by chromatin footprinting. We find that causal genetic risk acts in specific neuronal and glial cells that differ across disorders, primarily non-neuronal cells in AD and specific neuronal subtypes in FTD and PSP. These data illustrate the heterogeneous spectrum of glial and neuronal composition and gene expression alterations in different dementias and identify new therapeutic targets by revealing shared and disease-specific cell states.

7.
Stem Cell Reports ; 16(10): 2548-2564, 2021 10 12.
Artigo em Inglês | MEDLINE | ID: mdl-34506726

RESUMO

The specification of inhibitory neurons has been described for the mouse and human brain, and many studies have shown that pluripotent stem cells (PSCs) can be used to create interneurons in vitro. It is unclear whether in vitro methods to produce human interneurons generate all the subtypes found in brain, and how similar in vitro and in vivo interneurons are. We applied single-nuclei and single-cell transcriptomics to model interneuron development from human cortex and interneurons derived from PSCs. We provide a direct comparison of various in vitro interneuron derivation methods to determine the homogeneity achieved. We find that PSC-derived interneurons capture stages of development prior to mid-gestation, and represent a minority of potential subtypes found in brain. Comparison with those found in fetal or adult brain highlighted decreased expression of synapse-related genes. These analyses highlight the potential to tailor the method of generation to drive formation of particular subtypes.


Assuntos
Interneurônios/metabolismo , Células-Tronco Neurais/metabolismo , Células-Tronco Pluripotentes/metabolismo , Transcriptoma , Diferenciação Celular , Técnicas de Reprogramação Celular/métodos , Humanos , Análise de Célula Única , Fatores de Transcrição/metabolismo
8.
Cell Rep ; 33(7): 108398, 2020 11 17.
Artigo em Inglês | MEDLINE | ID: mdl-33207193

RESUMO

To understand how neural-immune-associated genes and pathways contribute to neurodegenerative disease pathophysiology, we performed a systematic functional genomic analysis in purified microglia and bulk tissue from mouse and human AD, FTD, and PSP. We uncover a complex temporal trajectory of microglial-immune pathways involving the type 1 interferon response associated with tau pathology in the early stages, followed by later signatures of partial immune suppression and, subsequently, the type 2 interferon response. We find that genetic risk for dementias shows disease-specific patterns of pathway enrichment. We identify drivers of two gene co-expression modules conserved from mouse to human, representing competing arms of microglial-immune activation (NAct) and suppression (NSupp) in neurodegeneration. We validate our findings by using chemogenetics, experimental perturbation data, and single-cell sequencing in post-mortem brains. Our results refine the understanding of stage- and disease-specific microglial responses, implicate microglial viral defense pathways in dementia pathophysiology, and highlight therapeutic windows.


Assuntos
Demência/genética , Tauopatias/genética , Proteínas tau/metabolismo , Idoso , Animais , Encéfalo/metabolismo , Feminino , Demência Frontotemporal/genética , Redes Reguladoras de Genes/genética , Predisposição Genética para Doença , Genômica/métodos , Humanos , Terapia de Imunossupressão , Inflamação/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microglia/metabolismo , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/metabolismo , Cultura Primária de Células , Fatores de Risco , Tauopatias/metabolismo , Tauopatias/fisiopatologia , Proteínas tau/genética , Proteínas tau/fisiologia
10.
Nat Neurosci ; 23(4): 500-509, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32203496

RESUMO

Although the cerebral cortex is organized into six excitatory neuronal layers, it is unclear whether glial cells show distinct layering. In the present study, we developed a high-content pipeline, the large-area spatial transcriptomic (LaST) map, which can quantify single-cell gene expression in situ. Screening 46 candidate genes for astrocyte diversity across the mouse cortex, we identified superficial, mid and deep astrocyte identities in gradient layer patterns that were distinct from those of neurons. Astrocyte layer features, established in the early postnatal cortex, mostly persisted in adult mouse and human cortex. Single-cell RNA sequencing and spatial reconstruction analysis further confirmed the presence of astrocyte layers in the adult cortex. Satb2 and Reeler mutations that shifted neuronal post-mitotic development were sufficient to alter glial layering, indicating an instructive role for neuronal cues. Finally, astrocyte layer patterns diverged between mouse cortical regions. These findings indicate that excitatory neurons and astrocytes are organized into distinct lineage-associated laminae.


Assuntos
Astrócitos/citologia , Córtex Cerebral/citologia , Neurônios/citologia , Transcriptoma , Animais , Astrócitos/metabolismo , Mapeamento Encefálico , Córtex Cerebral/metabolismo , Humanos , Camundongos , Neurônios/metabolismo
11.
Nat Commun ; 11(1): 3358, 2020 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-32620757

RESUMO

Neurodevelopmental disorders have a heritable component and are associated with region specific alterations in brain anatomy. However, it is unclear how genetic risks for neurodevelopmental disorders are translated into spatially patterned brain vulnerabilities. Here, we integrated cortical neuroimaging data from patients with neurodevelopmental disorders caused by genomic copy number variations (CNVs) and gene expression data from healthy subjects. For each of the six investigated disorders, we show that spatial patterns of cortical anatomy changes in youth are correlated with cortical spatial expression of CNV genes in neurotypical adults. By transforming normative bulk-tissue cortical expression data into cell-type expression maps, we link anatomical change maps in each analysed disorder to specific cell classes as well as the CNV-region genes they express. Our findings reveal organizing principles that regulate the mapping of genetic risks onto regional brain changes in neurogenetic disorders. Our findings will enable screening for candidate molecular mechanisms from readily available neuroimaging data.


Assuntos
Córtex Cerebral/patologia , Variações do Número de Cópias de DNA , Predisposição Genética para Doença , Transtornos do Neurodesenvolvimento/genética , Adolescente , Adulto , Mapeamento Encefálico , Córtex Cerebral/citologia , Córtex Cerebral/diagnóstico por imagem , Córtex Cerebral/crescimento & desenvolvimento , Criança , Estudos de Coortes , Feminino , Perfilação da Expressão Gênica , Genoma Humano , Humanos , Imageamento por Ressonância Magnética , Masculino , Pessoa de Meia-Idade , Transtornos do Neurodesenvolvimento/diagnóstico , Transtornos do Neurodesenvolvimento/patologia , Neuroimagem , Neurônios/metabolismo , Neurônios/patologia , Oligodendroglia/metabolismo , Oligodendroglia/patologia , Análise Espacial , Adulto Jovem
12.
Neuron ; 103(5): 785-801.e8, 2019 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-31303374

RESUMO

We performed RNA sequencing on 40,000 cells to create a high-resolution single-cell gene expression atlas of developing human cortex, providing the first single-cell characterization of previously uncharacterized cell types, including human subplate neurons, comparisons with bulk tissue, and systematic analyses of technical factors. These data permit deconvolution of regulatory networks connecting regulatory elements and transcriptional drivers to single-cell gene expression programs, significantly extending our understanding of human neurogenesis, cortical evolution, and the cellular basis of neuropsychiatric disease. We tie cell-cycle progression with early cell fate decisions during neurogenesis, demonstrating that differentiation occurs on a transcriptomic continuum; rather than only expressing a few transcription factors that drive cell fates, differentiating cells express broad, mixed cell-type transcriptomes before telophase. By mapping neuropsychiatric disease genes to cell types, we implicate dysregulation of specific cell types in ASD, ID, and epilepsy. We developed CoDEx, an online portal to facilitate data access and browsing.


Assuntos
Bases de Dados Genéticas , Regulação da Expressão Gênica no Desenvolvimento , Redes Reguladoras de Genes/genética , Neocórtex/embriologia , Neurogênese/genética , Neurônios/metabolismo , Transtorno do Espectro Autista/genética , Ciclo Celular , Córtex Cerebral/citologia , Córtex Cerebral/embriologia , Córtex Cerebral/metabolismo , Células Ependimogliais/metabolismo , Epilepsia/embriologia , Epilepsia/genética , Feminino , Perfilação da Expressão Gênica , Idade Gestacional , Humanos , Deficiência Intelectual/embriologia , Deficiência Intelectual/genética , Interneurônios/metabolismo , Neocórtex/citologia , Neocórtex/metabolismo , Células-Tronco Neurais/metabolismo , Gravidez , Segundo Trimestre da Gravidez , RNA-Seq , Análise de Célula Única , Telófase/genética
13.
Neuron ; 96(3): 542-557, 2017 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-29096072

RESUMO

A comprehensive characterization of neuronal cell types, their distributions, and patterns of connectivity is critical for understanding the properties of neural circuits and how they generate behaviors. Here we review the experiences of the BRAIN Initiative Cell Census Consortium, ten pilot projects funded by the U.S. BRAIN Initiative, in developing, validating, and scaling up emerging genomic and anatomical mapping technologies for creating a complete inventory of neuronal cell types and their connections in multiple species and during development. These projects lay the foundation for a larger and longer-term effort to generate whole-brain cell atlases in species including mice and humans.


Assuntos
Atlas como Assunto , Mapeamento Encefálico/métodos , Encéfalo/citologia , Encéfalo/fisiologia , Rede Nervosa/citologia , Rede Nervosa/fisiologia , Animais , Encéfalo/anatomia & histologia , Mapeamento Encefálico/tendências , Humanos , Rede Nervosa/anatomia & histologia , Projetos Piloto
14.
PLoS One ; 10(5): e0126535, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25992613

RESUMO

miRNAs play a central role in numerous pathologies including multiple cancer types. miR-191 has predominantly been studied as an oncogene, but the role of miR-191 in the proliferation of primary cells is not well characterized, and the miR-191 targetome has not been experimentally profiled. Here we utilized RNA induced silencing complex immunoprecipitations as well as gene expression profiling to construct a genome wide miR-191 target profile. We show that miR-191 represses proliferation in primary human fibroblasts, identify multiple proto-oncogenes as novel miR-191 targets, including CDK9, NOTCH2, and RPS6KA3, and present evidence that miR-191 extensively mediates target expression through coding sequence (CDS) pairing. Our results provide a comprehensive genome wide miR-191 target profile, and demonstrate miR-191's regulation of primary human fibroblast proliferation.


Assuntos
Quinase 9 Dependente de Ciclina/genética , Fibroblastos/metabolismo , MicroRNAs/genética , Complexo de Inativação Induzido por RNA/genética , Receptor Notch2/genética , Proteínas Quinases S6 Ribossômicas 90-kDa/genética , Contagem de Células , Proliferação de Células , Quinase 9 Dependente de Ciclina/metabolismo , Fibroblastos/citologia , Regulação da Expressão Gênica , Células HEK293 , Células HeLa , Humanos , Imunoprecipitação , MicroRNAs/antagonistas & inibidores , MicroRNAs/metabolismo , Cultura Primária de Células , Proto-Oncogenes , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Complexo de Inativação Induzido por RNA/metabolismo , Receptor Notch2/metabolismo , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismo , Transdução de Sinais , Transfecção
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA