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1.
Genome Res ; 27(11): 1783-1794, 2017 11.
Artigo em Inglês | MEDLINE | ID: mdl-29030469

RESUMO

The stochastic dynamics and regulatory mechanisms that govern differentiation of individual human neural precursor cells (NPC) into mature neurons are currently not fully understood. Here, we used single-cell RNA-sequencing (scRNA-seq) of developing neurons to dissect/identify NPC subtypes and critical developmental stages of alternative lineage specifications. This study comprises an unsupervised, high-resolution strategy for identifying cell developmental bifurcations, tracking the stochastic transcript kinetics of the subpopulations, elucidating regulatory networks, and finding key regulators. Our data revealed the bifurcation and developmental tracks of the two NPC subpopulations, and we captured an early (24 h) transition phase that leads to alternative neuronal specifications. The consequent up-regulation and down-regulation of stage- and subpopulation-specific gene groups during the course of maturation revealed biological insights with regard to key regulatory transcription factors and lincRNAs that control cellular programs in the identified neuronal subpopulations.


Assuntos
Perfilação da Expressão Gênica/métodos , Redes Reguladoras de Genes , Células-Tronco Neurais/citologia , Análise de Sequência de RNA/métodos , Análise de Célula Única/métodos , Diferenciação Celular , Linhagem da Célula , Células Cultivadas , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Neurogênese , RNA Longo não Codificante/genética , Fatores de Transcrição/genética
2.
EMBO J ; 33(11): 1271-83, 2014 Jun 02.
Artigo em Inglês | MEDLINE | ID: mdl-24802670

RESUMO

Several transcription factors (TFs) have been implicated in neuroectoderm (NE) development, and recently, the TF PAX6 was shown to be critical for human NE specification. However, microRNA networks regulating human NE development have been poorly documented. We hypothesized that microRNAs activated by PAX6 should promote NE development. Using a genomics approach, we identified PAX6 binding sites and active enhancers genome-wide in an in vitro model of human NE development that was based on neural differentiation of human embryonic stem cells (hESC). PAX6 binding to active enhancers was found in the proximity of several microRNAs, including hsa-miR-135b. MiR-135b was activated during NE development, and ectopic expression of miR-135b in hESC promoted differentiation toward NE. MiR-135b promotes neural conversion by targeting components of the TGF-ß and BMP signaling pathways, thereby inhibiting differentiation into alternate developmental lineages. Our results demonstrate a novel TF-miRNA module that is activated during human neuroectoderm development and promotes the irreversible fate specification of human pluripotent cells toward the neural lineage.


Assuntos
Proteínas Morfogenéticas Ósseas/metabolismo , Proteínas do Olho/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/metabolismo , MicroRNAs/genética , Fatores de Transcrição Box Pareados/metabolismo , Proteínas Repressoras/metabolismo , Transdução de Sinais , Fator de Crescimento Transformador beta/metabolismo , Sítios de Ligação , Proteínas Morfogenéticas Ósseas/genética , Diferenciação Celular , Linhagem Celular , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Proteínas do Olho/genética , Perfilação da Expressão Gênica , Proteínas de Homeodomínio/genética , Humanos , MicroRNAs/metabolismo , Modelos Moleculares , Mutação , Placa Neural , Fator de Transcrição PAX6 , Fatores de Transcrição Box Pareados/genética , Proteínas Repressoras/genética , Análise de Sequência de DNA , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Fator de Crescimento Transformador beta/genética
3.
Stem Cells ; 34(1): 124-34, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26507573

RESUMO

The transcription factor REST is a key suppressor of neuronal genes in non-neuronal tissues. REST has been shown to suppress proneuronal microRNAs in neural progenitors indicating that REST-mediated neurogenic suppression may act in part via microRNAs. We used neural differentiation of Rest-null mouse ESC to identify dozens of microRNAs regulated by REST during neural development. One of the identified microRNAs, miR-375, was upregulated during human spinal motor neuron development. We found that miR-375 facilitates spinal motor neurogenesis by targeting the cyclin kinase CCND2 and the transcription factor PAX6. Additionally, miR-375 inhibits the tumor suppressor p53 and protects neurons from apoptosis in response to DNA damage. Interestingly, motor neurons derived from a spinal muscular atrophy patient displayed depressed miR-375 expression and elevated p53 protein levels. Importantly, SMA motor neurons were significantly more susceptible to DNA damage induced apoptosis suggesting that miR-375 may play a protective role in motor neurons.


Assuntos
MicroRNAs/genética , Neurônios Motores/metabolismo , Neurônios Motores/patologia , Degeneração Neural/patologia , Animais , Apoptose/genética , Sequência de Bases , Humanos , Camundongos , MicroRNAs/metabolismo , Dados de Sequência Molecular , Atrofia Muscular Espinal/genética , Degeneração Neural/genética , Neurogênese/genética , Transdução de Sinais/genética , Proteína Supressora de Tumor p53/metabolismo
4.
Genome Res ; 22(1): 9-24, 2012 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-22090374

RESUMO

Cell-type diversity is governed in part by differential gene expression programs mediated by transcription factor (TF) binding. However, there are few systematic studies of the genomic binding of different types of TFs across a wide range of human cell types, especially in relation to gene expression. In the ENCODE Project, we have identified the genomic binding locations across 11 different human cell types of CTCF, RNA Pol II (RNAPII), and MYC, three TFs with diverse roles. Our data and analysis revealed how these factors bind in relation to genomic features and shape gene expression and cell-type specificity. CTCF bound predominantly in intergenic regions while RNAPII and MYC preferentially bound to core promoter regions. CTCF sites were relatively invariant across diverse cell types, while MYC showed the greatest cell-type specificity. MYC and RNAPII co-localized at many of their binding sites and putative target genes. Cell-type specific binding sites, in particular for MYC and RNAPII, were associated with cell-type specific functions. Patterns of binding in relation to gene features were generally conserved across different cell types. RNAPII occupancy was higher over exons than adjacent introns, likely reflecting a link between transcriptional elongation and splicing. TF binding was positively correlated with the expression levels of their putative target genes, but combinatorial binding, in particular of MYC and RNAPII, was even more strongly associated with higher gene expression. These data illuminate how combinatorial binding of transcription factors in diverse cell types is associated with gene expression and cell-type specific biology.


Assuntos
Regulação da Expressão Gênica/fisiologia , Genoma Humano/fisiologia , RNA Polimerase II/metabolismo , Elementos de Resposta/fisiologia , Fatores de Transcrição/metabolismo , Transcrição Gênica/fisiologia , Estudo de Associação Genômica Ampla/métodos , Células HeLa , Células Hep G2 , Humanos , Células K562 , Especificidade de Órgãos/fisiologia , Splicing de RNA/fisiologia
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.
PLoS Genet ; 8(4): e1002624, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22496669

RESUMO

Increasing numbers of human diseases are being linked to genetic variants, but our understanding of the mechanistic links leading from DNA sequence to disease phenotype is limited. The majority of disease-causing nucleotide variants fall within the non-protein-coding portion of the genome, making it likely that they act by altering gene regulatory sequences. We hypothesised that SNPs within the binding sites of the transcriptional repressor REST alter the degree of repression of target genes. Given that changes in the effective concentration of REST contribute to several pathologies-various cancers, Huntington's disease, cardiac hypertrophy, vascular smooth muscle proliferation-these SNPs should alter disease-susceptibility in carriers. We devised a strategy to identify SNPs that affect the recruitment of REST to target genes through the alteration of its DNA recognition element, the RE1. A multi-step screen combining genetic, genomic, and experimental filters yielded 56 polymorphic RE1 sequences with robust and statistically significant differences of affinity between alleles. These SNPs have a considerable effect on the the functional recruitment of REST to DNA in a range of in vitro, reporter gene, and in vivo analyses. Furthermore, we observe allele-specific biases in deeply sequenced chromatin immunoprecipitation data, consistent with predicted differenes in RE1 affinity. Amongst the targets of polymorphic RE1 elements are important disease genes including NPPA, PTPRT, and CDH4. Thus, considerable genetic variation exists in the DNA motifs that connect gene regulatory networks. Recently available ChIP-seq data allow the annotation of human genetic polymorphisms with regulatory information to generate prior hypotheses about their disease-causing mechanism.


Assuntos
Sítios de Ligação/genética , Doença , Motivos de Nucleotídeos/genética , Sequências Reguladoras de Ácido Nucleico/genética , Proteínas Repressoras , Linhagem Celular , Proteínas de Ligação a DNA/genética , Doença/genética , Redes Reguladoras de Genes , Genoma Humano , Humanos , Análise de Sequência com Séries de Oligonucleotídeos , Fenótipo , Polimorfismo de Nucleotídeo Único/genética , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Fatores de Transcrição/genética
7.
Angew Chem Int Ed Engl ; 54(8): 2442-6, 2015 Feb 16.
Artigo em Inglês | MEDLINE | ID: mdl-25565332

RESUMO

To address existing limitations in live neuron imaging, we have developed NeuO, a novel cell-permeable fluorescent probe with an unprecedented ability to label and image live neurons selectively over other cells in the brain. NeuO enables robust live neuron imaging and isolation in vivo and in vitro across species; its versatility and ease of use sets the basis for its development in a myriad of neuronal targeting applications.


Assuntos
Corantes Fluorescentes/metabolismo , Neurônios/metabolismo , Animais , Compostos de Boro/química , Compostos de Boro/metabolismo , Células Cultivadas , Corantes Fluorescentes/química , Microscopia Confocal , Microscopia de Vídeo , Neurônios/citologia , Ratos , Coloração e Rotulagem
8.
Genome Biol ; 25(1): 140, 2024 05 28.
Artigo em Inglês | MEDLINE | ID: mdl-38807229

RESUMO

RNA-binding proteins (RBPs) regulate key aspects of RNA processing including alternative splicing, mRNA degradation and localization by physically binding RNA molecules. Current methods to map these interactions, such as CLIP, rely on purifying single proteins at a time. Our new method, ePRINT, maps RBP-RNA interaction networks on a global scale without purifying individual RBPs. ePRINT uses exoribonuclease XRN1 to precisely map the 5' end of the RBP binding site and uncovers direct and indirect targets of an RBP of interest. Importantly, ePRINT can also uncover RBPs that are differentially activated between cell fate transitions, including neural progenitor differentiation into neurons.


Assuntos
Proteínas de Ligação a RNA , Proteínas de Ligação a RNA/metabolismo , Sítios de Ligação , Exorribonucleases/metabolismo , Humanos , RNA/metabolismo , Animais , Ligação Proteica
9.
Nucleic Acids Res ; 39(9): 3558-73, 2011 May.
Artigo em Inglês | MEDLINE | ID: mdl-21247883

RESUMO

The E2F family of transcription factors has important roles in cell cycle progression. E2F4 is an E2F family member that has been proposed to be primarily a repressor of transcription, but the scope of its binding activity and functions in transcriptional regulation is not fully known. We used ChIP sequencing (ChIP-seq) to identify around 16,000 E2F4 binding sites which potentially regulate 7346 downstream target genes with wide-ranging functions in DNA repair, cell cycle regulation, apoptosis, and other processes. While half of all E2F4 binding sites (56%) occurred near transcription start sites (TSSs), ∼20% of sites occurred more than 20 kb away from any annotated TSS. These distal sites showed histone modifications suggesting that E2F4 may function as a long-range regulator, which we confirmed by functional experimental assays on a subset. Overexpression of E2F4 and its transcriptional cofactors of the retinoblastoma (Rb) family and its binding partner DP-1 revealed that E2F4 acts as an activator as well as a repressor. E2F4 binding sites also occurred near regulatory elements for miRNAs such as let-7a and mir-17, suggestive of regulation of miRNAs by E2F4. Taken together, our genome-wide analysis provided evidence of versatile roles of E2F4 and insights into its functions.


Assuntos
Fator de Transcrição E2F4/metabolismo , Proteínas Repressoras/metabolismo , Ativação Transcricional , Sítios de Ligação , Técnicas de Cultura de Células , Linhagem Celular , Imunoprecipitação da Cromatina , Elementos Facilitadores Genéticos , Perfilação da Expressão Gênica , Genômica , Humanos , MicroRNAs/metabolismo , Anotação de Sequência Molecular , Regiões Promotoras Genéticas , Análise de Sequência de DNA
10.
Stem Cell Reports ; 17(7): 1650-1665, 2022 07 12.
Artigo em Inglês | MEDLINE | ID: mdl-35750046

RESUMO

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the loss of motor neurons (MNs). There are no effective treatments and patients usually die within 2-5 years of diagnosis. Emerging commonalities between familial and sporadic cases of this complex multifactorial disorder include disruption to RNA processing and cytoplasmic inclusion bodies containing TDP-43 and/or FUS protein aggregates. Both TDP-43 and FUS have been implicated in RNA processing functions, including microRNA biogenesis, transcription, and splicing. In this study, we explore the misexpression of microRNAs in an iPSC-based disease model of FUS ALS. We identify the downregulation of miR-139, an MN-enriched microRNA, in FUS and sporadic ALS MN. We discover that miR-139 downregulation leads to the activation of canonical WNT signaling and demonstrate that the WNT transcriptional mediator ß-catenin is a major driver of MN degeneration in ALS. Our results highlight the importance of homeostatic RNA networks in ALS.


Assuntos
Esclerose Lateral Amiotrófica , MicroRNAs , Doenças Neurodegenerativas , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/metabolismo , Proteínas de Ligação a DNA/metabolismo , Humanos , MicroRNAs/genética , MicroRNAs/metabolismo , Neurônios Motores/metabolismo , Mutação , Doenças Neurodegenerativas/metabolismo , Proteína FUS de Ligação a RNA/genética , Proteína FUS de Ligação a RNA/metabolismo , Regulação para Cima/genética , beta Catenina/genética , beta Catenina/metabolismo
11.
PLoS Biol ; 6(3): e65, 2008 Mar 18.
Artigo em Inglês | MEDLINE | ID: mdl-18351804

RESUMO

The eukaryotic genome is packaged as chromatin with nucleosomes comprising its basic structural unit, but the detailed structure of chromatin and its dynamic remodeling in terms of individual nucleosome positions has not been completely defined experimentally for any genome. We used ultra-high-throughput sequencing to map the remodeling of individual nucleosomes throughout the yeast genome before and after a physiological perturbation that causes genome-wide transcriptional changes. Nearly 80% of the genome is covered by positioned nucleosomes occurring in a limited number of stereotypical patterns in relation to transcribed regions and transcription factor binding sites. Chromatin remodeling in response to physiological perturbation was typically associated with the eviction, appearance, or repositioning of one or two nucleosomes in the promoter, rather than broader region-wide changes. Dynamic nucleosome remodeling tends to increase the accessibility of binding sites for transcription factors that mediate transcriptional changes. However, specific nucleosomal rearrangements were also evident at promoters even when there was no apparent transcriptional change, indicating that there is no simple, globally applicable relationship between chromatin remodeling and transcriptional activity. Our study provides a detailed, high-resolution, dynamic map of single-nucleosome remodeling across the yeast genome and its relation to global transcriptional changes.


Assuntos
Montagem e Desmontagem da Cromatina , Células Eucarióticas/metabolismo , Genoma/genética , Nucleossomos/genética , Nucleossomos/metabolismo , Saccharomyces cerevisiae/genética , Transcrição Gênica , Sequência de Bases , Sítios de Ligação , Modelos Genéticos , Dados de Sequência Molecular , Fases de Leitura Aberta/genética , Regiões Promotoras Genéticas/genética , Análise de Sequência de DNA , TATA Box , Fatores de Transcrição/metabolismo , Sítio de Iniciação de Transcrição
12.
Stem Cell Reports ; 16(12): 3020-3035, 2021 12 14.
Artigo em Inglês | MEDLINE | ID: mdl-34767750

RESUMO

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative condition characterized by the loss of motor neurons. We utilized single-cell transcriptomics to uncover dysfunctional pathways in degenerating motor neurons differentiated from SOD1 E100G ALS patient-derived induced pluripotent stem cells (iPSCs) and respective isogenic controls. Differential gene expression and network analysis identified activation of developmental pathways and core transcriptional factors driving the ALS motor neuron gene dysregulation. Specifically, we identified activation of SMAD2, a downstream mediator of the transforming growth factor ß (TGF-ß) signaling pathway as a key driver of SOD1 iPSC-derived motor neuron degeneration. Importantly, our analysis indicates that activation of TGFß signaling may be a common mechanism shared between SOD1, FUS, C9ORF72, VCP, and sporadic ALS motor neurons. Our results demonstrate the utility of single-cell transcriptomics in mapping disease-relevant gene regulatory networks driving neurodegeneration in ALS motor neurons. We find that ALS-associated mutant SOD1 targets transcriptional networks that perturb motor neuron homeostasis.


Assuntos
Esclerose Lateral Amiotrófica/patologia , Perfilação da Expressão Gênica , Células-Tronco Pluripotentes Induzidas/patologia , Neurônios Motores/patologia , Degeneração Neural/genética , Análise de Célula Única , Superóxido Dismutase-1/metabolismo , Regulação da Expressão Gênica , Redes Reguladoras de Genes , Humanos , Interneurônios/metabolismo , Neurônios Motores/metabolismo , Degeneração Neural/patologia , Transdução de Sinais , Fator de Crescimento Transformador beta/metabolismo
13.
Mol Brain ; 14(1): 98, 2021 06 26.
Artigo em Inglês | MEDLINE | ID: mdl-34174924

RESUMO

Induced pluripotent stem cells (iPSCs) and their differentiated neurons (iPSC-neurons) are a widely used cellular model in the research of the central nervous system. However, it is unknown how well they capture age-associated processes, particularly given that pluripotent cells are only present during the earliest stages of mammalian development. Epigenetic clocks utilize coordinated age-associated changes in DNA methylation to make predictions that correlate strongly with chronological age. It has been shown that the induction of pluripotency rejuvenates predicted epigenetic age. As existing clocks are not optimized for the study of brain development, we developed the fetal brain clock (FBC), a bespoke epigenetic clock trained in human prenatal brain samples in order to investigate more precisely the epigenetic age of iPSCs and iPSC-neurons. The FBC was tested in two independent validation cohorts across a total of 194 samples, confirming that the FBC outperforms other established epigenetic clocks in fetal brain cohorts. We applied the FBC to DNA methylation data from iPSCs and embryonic stem cells and their derived neuronal precursor cells and neurons, finding that these cell types are epigenetically characterized as having an early fetal age. Furthermore, while differentiation from iPSCs to neurons significantly increases epigenetic age, iPSC-neurons are still predicted as being fetal. Together our findings reiterate the need to better understand the limitations of existing epigenetic clocks for answering biological research questions and highlight a limitation of iPSC-neurons as a cellular model of age-related diseases.


Assuntos
Relógios Biológicos/genética , Encéfalo/embriologia , Senescência Celular , Epigênese Genética , Feto/citologia , Células-Tronco Pluripotentes Induzidas/citologia , Modelos Biológicos , Neurônios/citologia , Senescência Celular/genética , Metilação de DNA/genética , Bases de Dados Genéticas , Feminino , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Neurônios/metabolismo , Gravidez , Reprodutibilidade dos Testes
14.
Stem Cell Reports ; 8(4): 856-869, 2017 04 11.
Artigo em Inglês | MEDLINE | ID: mdl-28366453

RESUMO

Although mutations in several genes with diverse functions have been known to cause amyotrophic lateral sclerosis (ALS), it is unknown to what extent causal mutations impinge on common pathways that drive motor neuron (MN)-specific neurodegeneration. In this study, we combined induced pluripotent stem cells-based disease modeling with genome engineering and deep RNA sequencing to identify pathways dysregulated by mutant SOD1 in human MNs. Gene expression profiling and pathway analysis followed by pharmacological screening identified activated ERK and JNK signaling as key drivers of neurodegeneration in mutant SOD1 MNs. The AP1 complex member JUN, an ERK/JNK downstream target, was observed to be highly expressed in MNs compared with non-MNs, providing a mechanistic insight into the specific degeneration of MNs. Importantly, investigations of mutant FUS MNs identified activated p38 and ERK, indicating that network perturbations induced by ALS-causing mutations converge partly on a few specific pathways that are drug responsive and provide immense therapeutic potential.


Assuntos
Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/patologia , Células-Tronco Pluripotentes Induzidas/patologia , Sistema de Sinalização das MAP Quinases , Neurônios Motores/patologia , Superóxido Dismutase-1/genética , Fator de Transcrição AP-1/metabolismo , Esclerose Lateral Amiotrófica/metabolismo , Células Cultivadas , Engenharia Genética , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Neurônios Motores/metabolismo , Mutação Puntual , Proteínas Proto-Oncogênicas c-jun/metabolismo , Superóxido Dismutase-1/metabolismo
15.
Mol Cell Biol ; 37(16)2017 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-28584195

RESUMO

Sox2 is known to be important for neuron formation, but the precise mechanism through which it activates a neurogenic program and how this differs from its well-established function in self-renewal of stem cells remain elusive. In this study, we identified a highly conserved cyclin-dependent kinase (Cdk) phosphorylation site on serine 39 (S39) in Sox2. In neural stem cells (NSCs), phosphorylation of S39 enhances the ability of Sox2 to negatively regulate neuronal differentiation, while loss of phosphorylation is necessary for chromatin retention of a truncated form of Sox2 generated during neurogenesis. We further demonstrated that nonphosphorylated cleaved Sox2 specifically induces the expression of proneural genes and promotes neurogenic commitment in vivo Our present study sheds light on how the level of Cdk kinase activity directly regulates Sox2 to tip the balance between self-renewal and differentiation in NSCs.


Assuntos
Quinases Ciclina-Dependentes/metabolismo , Neurogênese , Fosfosserina/metabolismo , Fatores de Transcrição SOXB1/metabolismo , Sequência de Aminoácidos , Animais , Diferenciação Celular , DNA/metabolismo , Regulação da Expressão Gênica , Camundongos , Modelos Biológicos , Proteínas Mutantes/metabolismo , Células NIH 3T3 , Células-Tronco Neurais/metabolismo , Neurogênese/genética , Neurônios/citologia , Neurônios/metabolismo , Fosforilação , Ligação Proteica , Estabilidade Proteica , Fatores de Transcrição SOXB1/química , Serina Proteases/metabolismo
16.
Structure ; 12(11): 1989-99, 2004 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-15530363

RESUMO

Accurate prediction of location of cavities and surface grooves in proteins is important, as these are potential sites for ligand binding. Several currently available programs for cavity detection are unable to detect cavities near the surface or surface grooves. In the present study, an optimized molecular dynamics based procedure is described for detection and quantification of interior cavities as well as surface pockets. This is based on the observation that the mobility of water in such pockets is significantly lower than that of bulk water. The algorithm efficiently detects surface grooves that are sites of protein-ligand and protein-protein interaction. The algorithm was also used to substantially improve the performance of an automated docking procedure for docking monomers of nonobligate protein-protein complexes. In addition, it was applied to predict key residues involved in the binding of the E. coli toxin CcdB with its inhibitor. Predictions were subsequently validated by mutagenesis experiments.


Assuntos
Proteínas de Escherichia coli/metabolismo , Algoritmos , Sítios de Ligação , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Ligantes , Modelos Moleculares , Mutagênese , Ligação Proteica
17.
Genome Res ; 19(8): 1325-37, 2009 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-19546172

RESUMO

We investigated functional epigenetic changes that occur in primary human T lymphocytes during entry into the cell cycle and mapped these at the single-nucleosome level by ChIP-chip on tiling arrays for chromosomes 1 and 6. We show that nucleosome loss and flanking active histone marks define active transcriptional start sites (TSSs). Moreover, these signatures are already set at many inducible genes in quiescent cells prior to cell stimulation. In contrast, there is a dearth of the inactive histone mark H3K9me3 at the TSS, and under-representation of H3K9me2 and H3K9me3 defines the body of active genes. At the DNA level, cytosine methylation (meC) is enriched for nucleosomes that remain at the TSS, whereas in general there is a dearth of meC at TSSs. Furthermore, a drop in meC also marks 3' transcription termination, and a peak of meC occurs at stop codons. This mimics the 3' nucleosomal distribution in yeast, which we show does not occur in human T cells.


Assuntos
Epigênese Genética , Fase G1/fisiologia , Fase de Repouso do Ciclo Celular/fisiologia , Linfócitos T/metabolismo , Células Cultivadas , Imunoprecipitação da Cromatina , Ilhas de CpG/genética , Metilação de DNA , Fase G1/genética , Perfilação da Expressão Gênica , Estudo de Associação Genômica Ampla , Histonas/metabolismo , Humanos , Lisina/metabolismo , Metilação , Nucleossomos/genética , Nucleossomos/metabolismo , Regiões Promotoras Genéticas/genética , Ligação Proteica , Fase de Repouso do Ciclo Celular/genética , Linfócitos T/citologia , Sítio de Iniciação de Transcrição , Transcrição Gênica
18.
Genome Res ; 17(6): 910-6, 2007 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-17568006

RESUMO

Identifying the genome-wide binding sites of transcription factors is important in deciphering transcriptional regulatory networks. ChIP-chip (Chromatin immunoprecipitation combined with microarrays) has been widely used to map transcription factor binding sites in the human genome. However, whole genome ChIP-chip analysis is still technically challenging in vertebrates. We recently developed STAGE as an unbiased method for identifying transcription factor binding sites in the genome. STAGE is conceptually based on SAGE, except that the input is ChIP-enriched DNA. In this study, we implemented an improved sequencing strategy and analysis methods and applied STAGE to map the genomic binding profile of the transcription factor STAT1 after interferon treatment. STAT1 is mainly responsible for mediating the cellular responses to interferons, such as cell proliferation, apoptosis, immune surveillance, and immune responses. We present novel algorithms for STAGE tag analysis to identify enriched loci with high specificity, as verified by quantitative ChIP. STAGE identified several previously unknown STAT1 target genes, many of which are involved in mediating the response to interferon-gamma signaling. STAGE is thus a viable method for identifying the chromosomal targets of transcription factors and generating meaningful biological hypotheses that further our understanding of transcriptional regulatory networks.


Assuntos
Mapeamento Cromossômico , Genoma Humano , Elementos de Resposta , Fator de Transcrição STAT1/genética , Sitios de Sequências Rotuladas , Transdução de Sinais/genética , Algoritmos , Imunoprecipitação da Cromatina , Células HeLa , Humanos , Interferon gama/farmacologia , Fator de Transcrição STAT1/metabolismo , Análise de Sequência de DNA , Transcrição Gênica/efeitos dos fármacos , Transcrição Gênica/genética
19.
Nat Methods ; 2(1): 47-53, 2005 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-15782160

RESUMO

Identifying the chromosomal targets of transcription factors is important for reconstructing the transcriptional regulatory networks underlying global gene expression programs. We have developed an unbiased genomic method called sequence tag analysis of genomic enrichment (STAGE) to identify the direct binding targets of transcription factors in vivo. STAGE is based on high-throughput sequencing of concatemerized tags derived from target DNA enriched by chromatin immunoprecipitation. We first used STAGE in yeast to confirm that RNA polymerase III genes are the most prominent targets of the TATA-box binding protein. We optimized the STAGE protocol and developed analysis methods to allow the identification of transcription factor targets in human cells. We used STAGE to identify several previously unknown binding targets of human transcription factor E2F4 that we independently validated by promoter-specific PCR and microarray hybridization. STAGE provides a means of identifying the chromosomal targets of DNA-associated proteins in any sequenced genome.


Assuntos
DNA/metabolismo , Técnicas Genéticas , Genoma , Células Cultivadas , Imunoprecipitação da Cromatina , Primers do DNA/química , Proteínas de Ligação a DNA/metabolismo , Fator de Transcrição E2F4 , Humanos , Imunoprecipitação , Hibridização de Ácido Nucleico , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas , Ligação Proteica , Fatores de Transcrição/metabolismo , Transcrição Gênica
20.
J Biol Chem ; 277(35): 31345-53, 2002 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-12070144

RESUMO

Accurate identification of cavities is important in the study of protein structure, stability, design, and ligand binding. Identification and quantitation of cavities is a nontrivial problem because most cavities are connected to the protein exterior. We describe a computational procedure for quantitating cavity volumes and apply this to derive an estimate of the hydrophobic driving force in protein folding. A grid-based Monte Carlo procedure is used to position water molecules on the surface of a protein. A Voronoi procedure is used to identify and quantitate empty space within the solvated protein. Additional cavities not detected by other existing procedures can be identified. Most of these are close to surface concavities. Residue volumes for both the interior and the surface residues as well as cavity volumes are in good agreement with volumes calculated from fully hydrated protein structures obtained from molecular dynamic simulations. We show that the loss of stability because of cavity-creating mutations correlates better with cavity volumes determined by this procedure than with cavity volumes determined by other methods. Available structural and thermodynamic data for a number of cavity-containing mutants were analyzed to obtain estimates of 26.1 cal x mol(-1) x A(-3) and 18.5 cal x mol(-1) x A(-2) for the relative contributions of cavity formation and the hydrophobic effect to the observed stability changes. The present estimate for the hydrophobic driving force is at the lower end of estimates derived from model compound studies and considerably lower than previous estimates of approximately 50 cal x mol(-1) x A(-2) derived from protein mutational data. In the absence of structural rearrangement, on average, deletion of a single methylene group is expected to result in losses in stability of 0.41 and 0.70 kcal x mol(-1) resulting from decrease in hydrophobicity and packing, respectively.


Assuntos
Dobramento de Proteína , Proteínas/química , Proteínas/metabolismo , Ribonucleases/química , Proteínas de Bactérias , Cinética , Modelos Moleculares , Conformação Proteica
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