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1.
Mol Cell ; 48(2): 195-206, 2012 Oct 26.
Artigo em Inglês | MEDLINE | ID: mdl-22959275

RESUMO

LIN28 is a conserved RNA-binding protein implicated in pluripotency, reprogramming, and oncogenesis. It was previously shown to act primarily by blocking let-7 microRNA (miRNA) biogenesis, but here we elucidate distinct roles of LIN28 regulation via its direct messenger RNA (mRNA) targets. Through crosslinking and immunoprecipitation coupled with high-throughput sequencing (CLIP-seq) in human embryonic stem cells and somatic cells expressing exogenous LIN28, we have defined discrete LIN28-binding sites in a quarter of human transcripts. These sites revealed that LIN28 binds to GGAGA sequences enriched within loop structures in mRNAs, reminiscent of its interaction with let-7 miRNA precursors. Among LIN28 mRNA targets, we found evidence for LIN28 autoregulation and also direct but differing effects on the protein abundance of splicing regulators in somatic and pluripotent stem cells. Splicing-sensitive microarrays demonstrated that exogenous LIN28 expression causes widespread downstream alternative splicing changes. These findings identify important regulatory functions of LIN28 via direct mRNA interactions.


Assuntos
Processamento Alternativo/genética , RNA Mensageiro , Proteínas de Ligação a RNA , Sítios de Ligação/genética , Células-Tronco Embrionárias , Regulação da Expressão Gênica no Desenvolvimento , Células HEK293 , Humanos , Motivos de Nucleotídeos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo
2.
Nature ; 503(7477): 525-529, 2013 Nov 28.
Artigo em Inglês | MEDLINE | ID: mdl-24153179

RESUMO

Identifying cellular and molecular differences between human and non-human primates (NHPs) is essential to the basic understanding of the evolution and diversity of our own species. Until now, preserved tissues have been the main source for most comparative studies between humans, chimpanzees (Pan troglodytes) and bonobos (Pan paniscus). However, these tissue samples do not fairly represent the distinctive traits of live cell behaviour and are not amenable to genetic manipulation. We propose that induced pluripotent stem (iPS) cells could be a unique biological resource to determine relevant phenotypical differences between human and NHPs, and that those differences could have potential adaptation and speciation value. Here we describe the generation and initial characterization of iPS cells from chimpanzees and bonobos as new tools to explore factors that may have contributed to great ape evolution. Comparative gene expression analysis of human and NHP iPS cells revealed differences in the regulation of long interspersed element-1 (L1, also known as LINE-1) transposons. A force of change in mammalian evolution, L1 elements are retrotransposons that have remained active during primate evolution. Decreased levels of L1-restricting factors APOBEC3B (also known as A3B) and PIWIL2 (ref. 7) in NHP iPS cells correlated with increased L1 mobility and endogenous L1 messenger RNA levels. Moreover, results from the manipulation of A3B and PIWIL2 levels in iPS cells supported a causal inverse relationship between levels of these proteins and L1 retrotransposition. Finally, we found increased copy numbers of species-specific L1 elements in the genome of chimpanzees compared to humans, supporting the idea that increased L1 mobility in NHPs is not limited to iPS cells in culture and may have also occurred in the germ line or embryonic cells developmentally upstream to germline specification during primate evolution. We propose that differences in L1 mobility may have differentially shaped the genomes of humans and NHPs and could have continuing adaptive significance.


Assuntos
Elementos Nucleotídeos Longos e Dispersos/genética , Pan paniscus/genética , Pan troglodytes/genética , Células-Tronco Pluripotentes/metabolismo , Animais , Proteínas Argonautas/metabolismo , Linhagem Celular , Forma Celular , Citidina Desaminase/metabolismo , Evolução Molecular , Genoma Humano/genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Cariotipagem , Camundongos Nus , Antígenos de Histocompatibilidade Menor , Pan paniscus/metabolismo , Pan troglodytes/metabolismo , Células-Tronco Pluripotentes/citologia , RNA Mensageiro/análise , RNA Mensageiro/genética , Análise de Sequência de RNA , Especificidade da Espécie
3.
Development ; 140(22): 4633-44, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-24154525

RESUMO

The neurogenic potential of the subgranular zone (SGZ) of the hippocampal dentate gyrus is likely to be regulated by molecular cues arising from its complex heterogeneous cellular environment. Through transcriptome analysis using laser microdissection coupled with DNA microarrays, in combination with analysis of genome-wide in situ hybridization data, we identified 363 genes selectively enriched in adult mouse SGZ. These genes reflect expression in the different constituent cell types, including progenitor and dividing cells, immature granule cells, astrocytes, oligodendrocytes and GABAergic interneurons. Similar transcriptional profiling in the rhesus monkey dentate gyrus across postnatal development identified a highly overlapping set of SGZ-enriched genes, which can be divided based on temporal profiles to reflect maturation of glia versus granule neurons. Furthermore, we identified a neurogenesis-related gene network with decreasing postnatal expression that is highly correlated with the declining number of proliferating cells in dentate gyrus over postnatal development. Many of the genes in this network showed similar postnatal downregulation in mouse, suggesting a conservation of molecular mechanisms underlying developmental and adult neurogenesis in rodents and primates. Conditional deletion of Sox4 and Sox11, encoding two neurogenesis-related transcription factors central in this network, produces a mouse with no hippocampus, confirming the crucial role for these genes in regulating hippocampal neurogenesis.


Assuntos
Perfilação da Expressão Gênica , Hipocampo/metabolismo , Macaca mulatta/genética , Neurogênese/genética , Animais , Animais Recém-Nascidos , Biomarcadores/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Redes Reguladoras de Genes , Genoma/genética , Hipocampo/citologia , Interneurônios/citologia , Interneurônios/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Família Multigênica , Oligodendroglia/citologia , Oligodendroglia/metabolismo , Fatores de Transcrição SOXC/genética , Fatores de Transcrição SOXC/metabolismo , Análise Espaço-Temporal , Transcrição Gênica
4.
Hum Mol Genet ; 21(17): 3825-34, 2012 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-22661500

RESUMO

Cockayne syndrome (CS) is a human premature aging disorder associated with neurological and developmental abnormalities, caused by mutations mainly in the CS group B gene (ERCC6). At the molecular level, CS is characterized by a deficiency in the transcription-couple DNA repair pathway. To understand the role of this molecular pathway in a pluripotent cell and the impact of CSB mutation during human cellular development, we generated induced pluripotent stem cells (iPSCs) from CSB skin fibroblasts (CSB-iPSC). Here, we showed that the lack of functional CSB does not represent a barrier to genetic reprogramming. However, iPSCs derived from CSB patient's fibroblasts exhibited elevated cell death rate and higher reactive oxygen species (ROS) production. Moreover, these cellular phenotypes were accompanied by an up-regulation of TXNIP and TP53 transcriptional expression. Our findings suggest that CSB modulates cell viability in pluripotent stem cells, regulating the expression of TP53 and TXNIP and ROS production.


Assuntos
Senilidade Prematura/patologia , Síndrome de Cockayne/patologia , Células-Tronco Pluripotentes Induzidas/patologia , Estresse Oxidativo , Morte Celular/genética , Hipóxia Celular/genética , Sobrevivência Celular/genética , Células Clonais , Síndrome de Cockayne/genética , Dano ao DNA/genética , Regulação da Expressão Gênica , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Modelos Biológicos , Espécies Reativas de Oxigênio/metabolismo
5.
Dev Growth Differ ; 54(7): 673-85, 2012 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-22913491

RESUMO

Salamanders possess an extraordinary capacity for tissue and organ regeneration when compared to mammals. In our effort to characterize the unique transcriptional fingerprint emerging during the early phase of salamander limb regeneration, we identified transcriptional activation of some germline-specific genes within the Mexican axolotl (Ambystoma mexicanum) that is indicative of cellular reprogramming of differentiated cells into a germline-like state. In this work, we focus on one of these genes, the long interspersed nucleotide element-1 (LINE-1) retrotransposon, which is usually active in germ cells and silent in most of the somatic tissues in other organisms. LINE-1 was found to be dramatically upregulated during regeneration. In addition, higher genomic LINE-1 content was also detected in the limb regenerate when compared to that before amputation indicating that LINE-1 retrotransposition is indeed active during regeneration. Active LINE-1 retrotransposition has been suggested to have a potentially deleterious impact on genomic integrity. Silencing of activated LINE-1 by small RNAs has been reported to be part of the machinery aiming to maintain genomic integrity. Indeed, we were able to identify putative LINE-1-related piRNAs in the limb blastema. Transposable element-related piRNAs have been identified frequently in the germline in other organisms. Thus, we present here a scenario in which a unique germline-like state is established during axolotl limb regeneration, and the re-activation of LINE-1 may serve as a marker for cellular dedifferentiation in the early-stage of limb regeneration.


Assuntos
Regulação da Expressão Gênica/fisiologia , Membro Posterior , Elementos Nucleotídeos Longos e Dispersos/fisiologia , Regeneração/fisiologia , Ambystoma mexicanum , Animais
6.
Nat Struct Mol Biol ; 27(10): 989-1000, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32807991

RESUMO

The molecular functions of the majority of RNA-binding proteins (RBPs) remain unclear, highlighting a major bottleneck to a full understanding of gene expression regulation. Here, we develop a plasmid resource of 690 human RBPs that we subject to luciferase-based 3'-untranslated-region tethered function assays to pinpoint RBPs that regulate RNA stability or translation. Enhanced UV-cross-linking and immunoprecipitation of these RBPs identifies thousands of endogenous mRNA targets that respond to changes in RBP level, recapitulating effects observed in tethered function assays. Among these RBPs, the ubiquitin-associated protein 2-like (UBAP2L) protein interacts with RNA via its RGG domain and cross-links to mRNA and rRNA. Fusion of UBAP2L to RNA-targeting CRISPR-Cas9 demonstrates programmable translational enhancement. Polysome profiling indicates that UBAP2L promotes translation of target mRNAs, particularly global regulators of translation. Our tethering survey allows rapid assignment of the molecular activity of proteins, such as UBAP2L, to specific steps of mRNA metabolism.


Assuntos
Proteínas de Transporte/metabolismo , Biossíntese de Proteínas , Estabilidade de RNA , Proteínas de Ligação a RNA/metabolismo , Regiões 3' não Traduzidas , Sítios de Ligação , Sistemas CRISPR-Cas , Proteínas de Transporte/química , Proteínas de Transporte/genética , Linhagem Celular , Humanos , Luciferases/genética , Luciferases/metabolismo , Fases de Leitura Aberta , Polirribossomos/genética , Polirribossomos/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Raios Ultravioleta
7.
Gene Expr Patterns ; 8(1): 36-46, 2007 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-17920339

RESUMO

Erythrocyte tropomodulin (E-Tmod, Tmod1) is a tropomyosin-binding protein that caps the slow-growing end of actin filaments. In erythrocytes, it may favor the formation of short actin protofilaments needed for elastic cell deformation. Previously we created a knockout mouse model in which lacZ was knocked-in downstream of the E1 promoter to report the expression of full length E-Tmod. Here we utilize E-Tmod(+/lacZ) mice to study E-Tmod expression patterns in the CNS. X-gal staining and in situ hybridization of adults revealed its restricted expression in the olfactory bulb, hippocampus, cerebral cortex, basal ganglia, nuclei of brain stem and cerebellum. In neonates, signals in the cortex and caudate putamen increased from days 15 to 40. Immunohistochemistry also revealed that signals for beta-galactosidase coincided with that of NeuN, a post-mitotic nuclear marker for neurons, but not that for GFAP+ astrocytes or APC+ oligodendrocytes, suggesting E-Tmod/lacZ-positive cells in the CNS were neurons. Large neurons, e.g., mitral cells in olfactory bulb and mossy cells in hilus of the dentate gyrus are among those that expressed very high levels of E-Tmod in the CNS.


Assuntos
Encéfalo/citologia , Neurônios/química , Tropomodulina/análise , Fatores Etários , Animais , Animais Recém-Nascidos , Química Encefálica , Sistema Nervoso Central/química , Eritrócitos , Expressão Gênica , Óperon Lac , Camundongos , Regiões Promotoras Genéticas , Tropomodulina/genética
8.
Stem Cell Reports ; 8(4): 1101-1111, 2017 04 11.
Artigo em Inglês | MEDLINE | ID: mdl-28410643

RESUMO

Reprogramming somatic cells to induced pluripotent stem cells (iPSCs) offers the possibility of studying the molecular mechanisms underlying human diseases in cell types difficult to extract from living patients, such as neurons and cardiomyocytes. To date, studies have been published that use small panels of iPSC-derived cell lines to study monogenic diseases. However, to study complex diseases, where the genetic variation underlying the disorder is unknown, a sizable number of patient-specific iPSC lines and controls need to be generated. Currently the methods for deriving and characterizing iPSCs are time consuming, expensive, and, in some cases, descriptive but not quantitative. Here we set out to develop a set of simple methods that reduce cost and increase throughput in the characterization of iPSC lines. Specifically, we outline methods for high-throughput quantification of surface markers, gene expression analysis of in vitro differentiation potential, and evaluation of karyotype with markedly reduced cost.


Assuntos
Variação Genética , Ensaios de Triagem em Larga Escala/métodos , Células-Tronco Pluripotentes Induzidas/metabolismo , Cariotipagem/métodos , Miócitos Cardíacos/metabolismo , Neurônios/metabolismo , Biomarcadores/metabolismo , Diferenciação Celular , Linhagem Celular , Reprogramação Celular/genética , Análise Custo-Benefício , Genótipo , Ensaios de Triagem em Larga Escala/economia , Ensaios de Triagem em Larga Escala/instrumentação , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Cariotipagem/economia , Miócitos Cardíacos/citologia , Neurônios/citologia , Fenótipo
9.
Neuron ; 92(6): 1337-1351, 2016 Dec 21.
Artigo em Inglês | MEDLINE | ID: mdl-27939580

RESUMO

A critical feature of neural networks is that they balance excitation and inhibition to prevent pathological dysfunction. How this is achieved is largely unknown, although deficits in the balance contribute to many neurological disorders. We show here that a microRNA (miR-101) is a key orchestrator of this essential feature, shaping the developing network to constrain excitation in the adult. Transient early blockade of miR-101 induces long-lasting hyper-excitability and persistent memory deficits. Using target site blockers in vivo, we identify multiple developmental programs regulated in parallel by miR-101 to achieve balanced networks. Repression of one target, NKCC1, initiates the switch in γ-aminobutyric acid (GABA) signaling, limits early spontaneous activity, and constrains dendritic growth. Kif1a and Ank2 are targeted to prevent excessive synapse formation. Simultaneous de-repression of these three targets completely phenocopies major dysfunctions produced by miR-101 blockade. Our results provide new mechanistic insight into brain development and suggest novel candidates for therapeutic intervention.


Assuntos
Encéfalo/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/genética , MicroRNAs/genética , Animais , Anquirinas/genética , Anquirinas/metabolismo , Comportamento Animal , Encéfalo/crescimento & desenvolvimento , Dendritos , Cinesinas/genética , Cinesinas/metabolismo , Camundongos , Rede Nervosa/crescimento & desenvolvimento , Rede Nervosa/metabolismo , Vias Neurais/crescimento & desenvolvimento , Vias Neurais/metabolismo , Técnicas de Patch-Clamp , Reação em Cadeia da Polimerase , Análise de Sequência de RNA , Membro 2 da Família 12 de Carreador de Soluto/genética , Membro 2 da Família 12 de Carreador de Soluto/metabolismo , Ácido gama-Aminobutírico/metabolismo
10.
Cell Metab ; 16(6): 833-45, 2012 Dec 05.
Artigo em Inglês | MEDLINE | ID: mdl-23217262

RESUMO

In the mouse liver, circadian transcriptional rhythms are necessary for metabolic homeostasis. Whether dynamic epigenomic modifications are associated with transcript oscillations has not been systematically investigated. We found that several antisense RNA, lincRNA, and microRNA transcripts also showed circadian oscillations in adult mouse livers. Robust transcript oscillations often correlated with rhythmic histone modifications in promoters, gene bodies, or enhancers, although promoter DNA methylation levels were relatively stable. Such integrative analyses identified oscillating expression of an antisense transcript (asPer2) to the gene encoding the circadian oscillator component Per2. Robust transcript oscillations often accompanied rhythms in multiple histone modifications and recruitment of multiple chromatin-associated clock components. Coupling of cycling histone modifications with nearby oscillating transcripts thus established a temporal relationship between enhancers, genes, and transcripts on a genome-wide scale in a mammalian liver. The results offer a framework for understanding the dynamics of metabolism, circadian clock, and chromatin modifications involved in metabolic homeostasis.


Assuntos
Ritmo Circadiano/genética , Epigenômica , Fígado/metabolismo , RNA/metabolismo , Transcrição Gênica/genética , Animais , Metilação de DNA , Loci Gênicos , Histonas/genética , Histonas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/metabolismo , Proteínas Circadianas Period/genética , Proteínas Circadianas Period/metabolismo , Regiões Promotoras Genéticas , RNA Antissenso/metabolismo , RNA Longo não Codificante/metabolismo
11.
PLoS One ; 5(11): e13829, 2010 Nov 04.
Artigo em Inglês | MEDLINE | ID: mdl-21079806

RESUMO

Two-photon scanning microscopy has advanced our understanding of neural signaling in non-mammalian species and mammals. Various developments are needed to perform two-photon scanning microscopy over prolonged periods in non-human primates performing a behavioral task. In striate cortex in two macaque monkeys, cortical neurons were transfected with a genetically encoded fluorescent calcium sensor, memTNXL, using AAV1 as a viral vector. By constructing an extremely rigid and stable apparatus holding both the two-photon scanning microscope and the monkey's head, single neurons were imaged at high magnification for prolonged periods with minimal motion artifacts for up to ten months. Structural images of single neurons were obtained at high magnification. Changes in calcium during visual stimulation were measured as the monkeys performed a fixation task. Overall, functional responses and orientation tuning curves were obtained in 18.8% of the 234 labeled and imaged neurons. This demonstrated that the two-photon scanning microscopy can be successfully obtained in behaving primates.


Assuntos
Cálcio/metabolismo , Proteínas Luminescentes/metabolismo , Neurônios/metabolismo , Córtex Visual/metabolismo , Animais , Comportamento Animal/fisiologia , Técnicas Biossensoriais/métodos , Dependovirus/genética , Transferência Ressonante de Energia de Fluorescência/métodos , Vetores Genéticos/genética , Proteínas Luminescentes/genética , Macaca mulatta , Microscopia de Fluorescência por Excitação Multifotônica/métodos , Reprodutibilidade dos Testes , Transfecção , Córtex Visual/citologia
12.
Artigo em Inglês | MEDLINE | ID: mdl-19949461

RESUMO

Short cell-type specific promoter sequences are important for targeted gene therapy and studies of brain circuitry. We report on the ability of short promoter sequences to drive fluorescent protein expression in specific types of mammalian cortical inhibitory neurons using adeno-associated virus (AAV) and lentivirus (LV) vectors. We tested many gene regulatory sequences derived from fugu (Takifugu rubripes), mouse, human, and synthetic composite regulatory elements. All fugu compact promoters expressed in mouse cortex, with only the somatostatin (SST) and the neuropeptide Y (NPY) promoters largely restricting expression to GABAergic neurons. However these promoters did not control expression in inhibitory cells in a subtype specific manner. We also tested mammalian promoter sequences derived from genes putatively coexpressed or coregulated within three major inhibitory interneuron classes (PV, SST, VIP). In contrast to the fugu promoters, many of the mammalian sequences failed to express, and only the promoter from gene A930038C07Rik conferred restricted expression, although as in the case of the fugu sequences, this too was not inhibitory neuron subtype specific. Lastly and more promisingly, a synthetic sequence consisting of a composite regulatory element assembled with PAX6 E1.1 binding sites, NRSE and a minimal CMV promoter showed markedly restricted expression to a small subset of mostly inhibitory neurons, but whose commonalities are unknown.

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