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1.
Proc Natl Acad Sci U S A ; 113(12): E1691-700, 2016 Mar 22.
Artigo em Inglês | MEDLINE | ID: mdl-26957603

RESUMO

The linear distribution of genes across chromosomes and the spatial localization of genes within the nucleus are related to their transcriptional regulation. The mechanistic consequences of linear gene order, and how it may relate to the functional output of genome organization, remain to be fully resolved, however. Here we tested the relationship between linear and 3D organization of gene regulation during myogenesis. Our analysis has identified a subset of topologically associated domains (TADs) that are significantly enriched for muscle-specific genes. These lineage-enriched TADs demonstrate an expression-dependent pattern of nuclear organization that influences the positioning of adjacent nonenriched TADs. Therefore, lineage-enriched TADs inform cell-specific genome organization during myogenesis. The reduction of allelic spatial distance of one of these domains, which contains Myogenin, correlates with reduced transcriptional variability, identifying a potential role for lineage-specific nuclear topology. Using a fusion-based strategy to decouple mitosis and myotube formation, we demonstrate that the cell-specific topology of syncytial nuclei is dependent on cell division. We propose that the effects of linear and spatial organization of gene loci on gene regulation are linked through TAD architecture, and that mitosis is critical for establishing nuclear topologies during cellular differentiation.


Assuntos
Linhagem da Célula/genética , Regulação da Expressão Gênica no Desenvolvimento , Desenvolvimento Muscular/genética , Alelos , Mapeamento Cromossômico , Fibroblastos , Genes Reporter , Proteínas de Fluorescência Verde/genética , Humanos , Imageamento Tridimensional , Hibridização in Situ Fluorescente , Proteína MyoD/genética , Miogenina/genética , Estrutura Terciária de Proteína , Transcrição Gênica , Transdução Genética
2.
Nucleic Acids Res ; 44(7): 3082-94, 2016 Apr 20.
Artigo em Inglês | MEDLINE | ID: mdl-26673704

RESUMO

Higher order chromatin structure establishes domains that organize the genome and coordinate gene expression. However, the molecular mechanisms controlling transcription of individual loci within a topological domain (TAD) are not fully understood. The cystic fibrosis transmembrane conductance regulator (CFTR) gene provides a paradigm for investigating these mechanisms.CFTR occupies a TAD bordered by CTCF/cohesin binding sites within which are cell-type-selective cis-regulatory elements for the locus. We showed previously that intronic and extragenic enhancers, when occupied by specific transcription factors, are recruited to the CFTR promoter by a looping mechanism to drive gene expression. Here we use a combination of CRISPR/Cas9 editing of cis-regulatory elements and siRNA-mediated depletion of architectural proteins to determine the relative contribution of structural elements and enhancers to the higher order structure and expression of the CFTR locus. We found the boundaries of the CFTRTAD are conserved among diverse cell types and are dependent on CTCF and cohesin complex. Removal of an upstream CTCF-binding insulator alters the interaction profile, but has little effect on CFTR expression. Within the TAD, intronic enhancers recruit cell-type selective transcription factors and deletion of a pivotal enhancer element dramatically decreases CFTR expression, but has minor effect on its 3D structure.


Assuntos
Cromatina/química , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Elementos Facilitadores Genéticos , Regulação da Expressão Gênica , Elementos Isolantes , Fator de Ligação a CCCTC , Células CACO-2 , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Células Cultivadas , Proteínas Cromossômicas não Histona/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Loci Gênicos , Humanos , Proteínas Repressoras/metabolismo , Coesinas
3.
Nucleic Acids Res ; 42(15): 9612-22, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-25081205

RESUMO

Higher order chromatin structures across the genome are maintained in part by the architectural proteins CCCTC binding factor (CTCF) and the cohesin complex, which co-localize at many sites across the genome. Here, we examine the role of these proteins in mediating chromatin structure at the cystic fibrosis transmembrane conductance regulator (CFTR) gene. CFTR encompasses nearly 200 kb flanked by CTCF-binding enhancer-blocking insulator elements and is regulated by cell-type-specific intronic enhancers, which loop to the promoter in the active locus. SiRNA-mediated depletion of CTCF or the cohesin component, RAD21, showed that these two factors have distinct roles in regulating the higher order organization of CFTR. CTCF mediates the interactions between CTCF/cohesin binding sites, some of which have enhancer-blocking insulator activity. Cohesin shares this tethering role, but in addition stabilizes interactions between the promoter and cis-acting intronic elements including enhancers, which are also dependent on the forkhead box A1/A2 (FOXA1/A2) transcription factors (TFs). Disruption of the three-dimensional structure of the CFTR gene by depletion of CTCF or RAD21 increases gene expression, which is accompanied by alterations in histone modifications and TF occupancy across the locus, and causes internalization of the gene from the nuclear periphery.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Cromatina/química , Proteínas Cromossômicas não Histona/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulação da Expressão Gênica , Proteínas Repressoras/metabolismo , Sítios de Ligação , Fator de Ligação a CCCTC , Células CACO-2 , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/fisiologia , Linhagem Celular , Proteínas Cromossômicas não Histona/antagonistas & inibidores , Proteínas Cromossômicas não Histona/fisiologia , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Loci Gênicos , Fator 3-alfa Nuclear de Hepatócito/metabolismo , Fator 3-beta Nuclear de Hepatócito/metabolismo , Humanos , Proteínas Repressoras/antagonistas & inibidores , Proteínas Repressoras/fisiologia , Coesinas
4.
Sci Rep ; 9(1): 8011, 2019 05 29.
Artigo em Inglês | MEDLINE | ID: mdl-31142749

RESUMO

The three-dimensional organization of the genome in mammalian interphase nuclei is intrinsically linked to the regulation of gene expression. Whole chromosome territories and their encoded gene loci occupy preferential positions within the nucleus that changes according to the expression profile of a given cell lineage or stage. To further illuminate the relationship between chromosome organization, epigenetic environment, and gene expression, here we examine the functional organization of chromosome X and corresponding X-linked genes in a variety of healthy human and disease state X diploid (XX) cells. We observe high frequencies of homologous chromosome X colocalization (or coalescence), typically associated with initiation of X-chromosome inactivation, occurring in XX cells outside of early embryogenesis. Moreover, during chromosome X coalescence significant changes in Xist, H3K27me3, and X-linked gene expression occur, suggesting the potential exchange of gene regulatory information between the active and inactive X chromosomes. We also observe significant differences in chromosome X coalescence in disease-implicated lymphocytes isolated from systemic lupus erythematosus (SLE) patients compared to healthy controls. These results demonstrate that X chromosomes can functionally interact outside of embryogenesis when X inactivation is initiated and suggest a potential gene regulatory mechanism aberration underlying the increased frequency of autoimmunity in XX individuals.


Assuntos
Mecanismo Genético de Compensação de Dose/genética , Lúpus Eritematoso Sistêmico/genética , RNA Longo não Codificante/genética , Cromossomo X/genética , Animais , Núcleo Celular/genética , Diploide , Desenvolvimento Embrionário/genética , Feminino , Regulação da Expressão Gênica no Desenvolvimento/genética , Genes Ligados ao Cromossomo X , Humanos , Lúpus Eritematoso Sistêmico/patologia , Masculino , Inativação do Cromossomo X/genética
6.
Blood Press Monit ; 23(3): 148-152, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29677012

RESUMO

This study aims to evaluate the relationship between mean outdoor temperature and mean daily blood pressure (BP) and heart rate (HR) among six, large, geographically and climatically diverse US cities. We collected BP and HR data from Higi stations, located in a wide range of neighborhood grocery stores and retail pharmacies, from six US cities (Houston, Los Angeles, Miami, Boise, Chicago, and New York City). Outdoor daily temperature data were collected from the National Centers for Environmental Information's database. Pearson's correlation was used to assess the linear relationship between mean daily outdoor temperature and mean daily BP and HR for each city from May 2016 through April 2017. A total of 2 140 626 BP and HR readings were recorded in the six study cities. Mean outdoor temperature was inversely correlated with both mean daily average systolic (r=-0.69, P<0.0001) and diastolic (r=-0.71; P<0.0001) BPs, but not HR (r<0.0001, P=0.48). We also found that temperature change had a larger impact on BP in equatorial climates such as Miami compared with colder and more temperature variable cities like Chicago and Boise. Previous studies have found that BP varies seasonally, but few have looked at the impact of daily temperature on both BP and HR changes. Our study is one of the largest and most climatically diverse populations ever looking at this relationship. Our results suggest that temperature, and perhaps geography, should play a role in tailoring individualized evaluation and treatment for hypertensive diseases.


Assuntos
Pressão Sanguínea , Clima , Bases de Dados Factuais , Temperatura , População Urbana , Feminino , Humanos , Masculino , Estados Unidos
7.
Mol Cell Biol ; 35(5): 884-98, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25535332

RESUMO

Nuclear lamins play important roles in the organization and structure of the nucleus; however, the specific mechanisms linking lamin structure to nuclear functions are poorly defined. We demonstrate that reducing nuclear lamin B1 expression by short hairpin RNA-mediated silencing in cancer cell lines to approximately 50% of normal levels causes a delay in the cell cycle and accumulation of cells in early S phase. The S phase delay appears to be due to the stalling and collapse of replication forks. The double-strand DNA breaks resulting from replication fork collapse were inefficiently repaired, causing persistent DNA damage signaling and the assembly of extensive repair foci on chromatin. The expression of multiple factors involved in DNA replication and repair by both nonhomologous end joining and homologous repair is misregulated when lamin B1 levels are reduced. We further demonstrate that lamin B1 interacts directly with the promoters of some genes associated with DNA damage response and repair, including BRCA1 and RAD51. Taken together, the results suggest that the maintenance of lamin B1 levels is required for DNA replication and repair through regulation of the expression of key factors involved in these essential nuclear functions.


Assuntos
Cromatina/química , Regulação Neoplásica da Expressão Gênica , Regulação da Expressão Gênica , Lamina Tipo B/metabolismo , Apoptose , Proteína BRCA1/metabolismo , Ciclo Celular , Linhagem Celular Tumoral , Quebras de DNA de Cadeia Dupla , Dano ao DNA , Reparo do DNA , Replicação do DNA , Inativação Gênica , Humanos , Interferência de RNA , Rad51 Recombinase/metabolismo , Fase S
8.
Nat Cell Biol ; 12(10): 929-31, 2010 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-20885421

RESUMO

Transcriptional noise has an important role in generating diversity in cellular populations that are seemingly identical. As this noise stems from the inherent stochasticity of gene expression, it has been unclear whether it is directly controlled. Dig1, a regulator of the budding yeast mating pathway, is now shown to prevent transcriptional noise by regulating the spatial organization of downstream gene targets.


Assuntos
Regulação da Expressão Gênica , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Transcrição Gênica/genética , Fatores de Transcrição/metabolismo
9.
Science ; 321(5889): 686-91, 2008 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-18669861

RESUMO

The transition from naïve to activated T cells is marked by alternative splicing of pre-mRNA encoding the transmembrane phosphatase CD45. Using a short hairpin RNA interference screen, we identified heterogeneous ribonucleoprotein L-like (hnRNPLL) as a critical inducible regulator of CD45 alternative splicing. HnRNPLL was up-regulated in stimulated T cells, bound CD45 transcripts, and was both necessary and sufficient for CD45 alternative splicing. Depletion or overexpression of hnRNPLL in B and T cell lines and primary T cells resulted in reciprocal alteration of CD45RA and RO expression. Exon array analysis suggested that hnRNPLL acts as a global regulator of alternative splicing in activated T cells. Induction of hnRNPLL during hematopoietic cell activation and differentiation may allow cells to rapidly shift their transcriptomes to favor proliferation and inhibit cell death.


Assuntos
Processamento Alternativo , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Antígenos Comuns de Leucócito/genética , Ativação Linfocitária , Linfócitos T/imunologia , Linfócitos T/metabolismo , Linfócitos B/imunologia , Linfócitos B/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Ribonucleoproteínas Nucleares Heterogêneas/genética , Humanos , Lentivirus/genética , Lentivirus/fisiologia , Antígenos Comuns de Leucócito/química , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Interferência de RNA , Fator de Transcrição STAT5/genética , Acetato de Tetradecanoilforbol/farmacologia , Transcrição Gênica , Transdução Genética , Regulação para Cima
10.
Mol Cell Biol ; 28(17): 5209-22, 2008 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-18591248

RESUMO

ORAI1 is a pore subunit of the store-operated Ca(2+) release-activated Ca(2+) (CRAC) channel. To examine the physiological consequences of ORAI1 deficiency, we generated mice with targeted disruption of the Orai1 gene. The results of immunohistochemical analysis showed that ORAI1 is expressed in lymphocytes, skin, and muscle of wild-type mice and is not expressed in Orai1(-/-) mice. Orai1(-/-) mice with the inbred C57BL/6 background showed perinatal lethality, which was overcome by crossing them to outbred ICR mice. Orai1(-/-) mice were small in size, with eyelid irritation and sporadic hair loss resembling the cyclical alopecia observed in mice with keratinocyte-specific deletion of the Cnb1 gene. T and B cells developed normally in Orai1(-/-) mice, but B cells showed a substantial decrease in Ca(2+) influx and cell proliferation in response to B-cell receptor stimulation. Naïve and differentiated Orai1(-/-) T cells showed substantial reductions in store-operated Ca(2+) entry, CRAC currents, and cytokine production. These features are consistent with the severe combined immunodeficiency and mild extraimmunological symptoms observed in a patient with a missense mutation in human ORAI1 and distinguish the ORAI1-null mice described here from a previously reported Orai1 gene-trap mutant mouse which may be a hypomorph rather than a true null.


Assuntos
Linfócitos B/patologia , Canais de Cálcio/deficiência , Cabelo/patologia , Linfócitos T/patologia , Animais , Linfócitos B/imunologia , Linfócitos B/metabolismo , Canais de Cálcio/metabolismo , Sinalização do Cálcio , Diferenciação Celular , Proliferação de Células , Citocinas/biossíntese , Epiderme/patologia , Fibroblastos/citologia , Fibroblastos/metabolismo , Cabelo/imunologia , Ativação do Canal Iônico , Subpopulações de Linfócitos/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos ICR , Camundongos Knockout , Proteína ORAI1 , Proteína ORAI2 , Fenótipo , Linfócitos T/imunologia , Linfócitos T/metabolismo
11.
J Biol Chem ; 282(22): 16232-43, 2007 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-17293345

RESUMO

Stimulation of immune cells triggers Ca2+ entry through store-operated Ca2+ release-activated Ca2+ channels, promoting nuclear translocation of the transcription factor NFAT. Through genome-wide RNA interference screens in Drosophila, we and others identified olf186-F (Drosophila Orai, dOrai) and dStim as critical components of store-operated Ca2+ entry and showed that dOrai and its human homologue Orai1 are pore subunits of the Ca2+ release-activated Ca2+ channel. Here we report that Orai1 is predominantly responsible for store-operated Ca2+ influx in human embryonic kidney 293 cells and human T cells and fibroblasts, although its paralogue Orai3 can partly compensate in the absence of functional Orai1. All three mammalian Orai are widely expressed at the mRNA level, and all three are incorporated into the plasma membrane. In human embryonic kidney 293 cells, Orai1 is glycosylated at an asparagine residue in the predicted second extracellular loop, but mutation of the residue does not compromise function. STIM1 and Orai1 colocalize after store depletion, but Orai1 does not associate detectably with STIM1 in glycerol gradient centrifugation or coimmunoprecipitation experiments. Glutamine substitutions in two conserved glutamate residues, located within predicted transmembrane helices of Drosophila Orai and human Orai1, greatly diminish store-operated Ca2+ influx, and primary T cells ectopically expressing mutant E106Q and E190Q Orai1 proteins show reduced proliferation and cytokine secretion. Together, these data establish Orai1 as a predominant mediator of store-operated calcium entry, proliferation, and cytokine production in T cells.


Assuntos
Canais de Cálcio/metabolismo , Sinalização do Cálcio/fisiologia , Membrana Celular/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Processamento de Proteína Pós-Traducional/fisiologia , Linfócitos T/metabolismo , Substituição de Aminoácidos , Animais , Canais de Cálcio/genética , Linhagem Celular , Membrana Celular/genética , Proliferação de Células , Citocinas/biossíntese , Drosophila , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Glicosilação , Humanos , Proteínas de Membrana/genética , Mutação de Sentido Incorreto , Proteínas de Neoplasias/genética , Proteína ORAI1 , Estrutura Secundária de Proteína , Homologia de Sequência de Aminoácidos , Molécula 1 de Interação Estromal , Linfócitos T/citologia
12.
Biochem Biophys Res Commun ; 348(2): 662-8, 2006 Sep 22.
Artigo em Inglês | MEDLINE | ID: mdl-16890206

RESUMO

Ribosomal protein S1 is shown to interact with the non-coding RNA DsrA and with rpoS mRNA. DsrA is a non-coding RNA that is important in controlling expression of the rpoS gene product in Escherichia coli. Photochemical crosslinking, quadrupole-time of flight tandem mass spectrometry, and peptide sequencing have identified an interaction between DsrA and S1 in the 30S ribosomal subunit. Purified S1 binds both DsrA (K(obs) approximately 6 x 10(6) M(-1)) and rpoS mRNA (K(obs) approximately 3 x 10(7) M(-1)). Ribonuclease probing experiments indicate that S1 binding has a weak but detectable effect on the secondary structure of DsrA or rpoS mRNA.


Assuntos
Proteínas de Bactérias/genética , Proteínas de Escherichia coli/metabolismo , RNA Mensageiro/metabolismo , RNA não Traduzido/metabolismo , Proteínas Ribossômicas/metabolismo , Fator sigma/genética , Sequência de Aminoácidos , Sequência de Bases , Ensaio de Desvio de Mobilidade Eletroforética , Espectrometria de Massas , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Pequeno RNA não Traduzido , Ribonuclease Pancreático/metabolismo
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