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1.
PLoS One ; 6(12): e28049, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-22162999

RESUMO

BACKGROUND: Scaffold attachment factor A (SAF-A) participates in the regulation of gene expression by organizing chromatin into transcriptionally active domains and by interacting directly with RNA polymerase II. METHODOLOGY: Here we use co-localization, co-immunoprecipitation (co-IP) and in situ proximity ligation assay (PLA) to identify Brahma Related Gene 1 (BRG1), the ATP-driven motor of the human SWI-SNF chromatin remodeling complex, as another SAF-A interaction partner in mouse embryonic stem (mES) cells. We also employ RNA interference to investigate functional aspects of the SAF-A/BRG1 interaction. PRINCIPAL FINDINGS: We find that endogenous SAF-A protein interacts with endogenous BRG1 protein in mES cells, and that the interaction does not solely depend on the presence of mRNA. Moreover the interaction remains intact when cells are induced to differentiate. Functional analyses reveal that dual depletion of SAF-A and BRG1 abolishes global transcription by RNA polymerase II, while the nucleolar RNA polymerase I transcription machinery remains unaffected. CONCLUSIONS: We demonstrate that SAF-A interacts with BRG1 and that both components are required for RNA Polymerase II Mediated Transcription.


Assuntos
DNA Helicases/metabolismo , Proteínas de Ligação a DNA/química , Ribonucleoproteínas Nucleares Heterogêneas Grupo U/metabolismo , Proteínas Nucleares/metabolismo , RNA Polimerase II/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Animais , Diferenciação Celular , Linhagem Celular , RNA Polimerases Dirigidas por DNA/metabolismo , Células-Tronco Embrionárias/citologia , Humanos , Camundongos , Microscopia Confocal/métodos , Modelos Biológicos , Ligação Proteica , RNA Mensageiro/metabolismo , Transcrição Gênica
2.
Nephrol Dial Transplant ; 26(7): 2377-81, 2011 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-21459783

RESUMO

BACKGROUND: Increasing numbers of living donor kidney transplantations calls for better knowledge about long-term donor outcomes and risks. METHODS: To explore long-term kidney donor outcomes and risks, we conducted a cross sectional retrospective study. To this end, we analysed renal function using measured glomerular filtration rate (mGFR) and estimated glomerular filtration rate (eGFR) as well as microalbuminuria, blood pressure (BP), body mass index, haemoglobin, albumin and parathyroid hormone in kidney donors nephrectomized between 1965 and 2005. RESULTS: A total number of 573 kidney donors agreed to undergo medical follow-up examinations. The mean age (standard deviation) at donation was 47 (11) years and the mean time since donation was 14 (9) years. Both mean mGFR [68 (15) mL/min/1.73 m(2) body surface; P = 0.028] and mean eGFR [71 (16) mL/min/1.73 m(2) body surface; P < 0.001], based on modified diet renal dysfunction and iohexol or Cr-EDTA clearance, respectively, were found to decrease with age and to increase with time since donation. Special multivariable regression analyses reveal that for 30-year old donors, the median eGFR typically increases during the first 17 years, then remains constant for ~8 years and slowly declines thereafter. For 50-year-old donors, the median eGFR is expected to increase during the first 15 years or so and then to enter a phase of slight progressive decline. In total, 23% (126/546) of the donors were on antihypertensive medication. An additional 22% (117/543) of the donors were found to suffer from hitherto undiagnosed hypertension (BP >140/90 mm Hg). CONCLUSION: Renal function of the remaining kidney in living donors is expected to improve for many years but will show signs of slight deterioration in the longer run.


Assuntos
Transplante de Rim , Rim/fisiopatologia , Doadores Vivos , Nefrectomia , Complicações Pós-Operatórias , Adulto , Idoso , Idoso de 80 Anos ou mais , Albuminúria , Índice de Massa Corporal , Estudos de Coortes , Creatinina/metabolismo , Estudos Transversais , Feminino , Seguimentos , Taxa de Filtração Glomerular , Humanos , Testes de Função Renal , Masculino , Pessoa de Meia-Idade , Prognóstico , Estudos Retrospectivos , Fatores de Risco , Coleta de Tecidos e Órgãos , Adulto Jovem
3.
Cell Reprogram ; 13(1): 13-27, 2011 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-21235343

RESUMO

Methodologies to reprogram somatic cells into patient-specific pluripotent cells, which could potentially be used in personalized drug discovery and cell replacement therapies, are currently under development. Oct4 activation is essential for successful reprogramming and pluripotency of embryonic stem (ES) cells, albeit molecular details of Oct4 activation are not completely understood. Here we report that endogenous SAF-A is involved in regulation of Oct4 expression, binds the Oct4 proximal promoter in ES cells, and dissociates from the promoter upon early differentiation induced by LIF withdrawal. Depletion of SAF-A decreases Oct4 expression even in the presence of LIF, and results in an increase of the mesodermal marker Brachyury. The overexpression of wild-type human SAF-A rescues the mouse knock-down phenotype and results in increased Oct4 level. We also demonstrate that endogenous SAF-A interacts with the C-terminal domain (CTD) of endogenous RNA polymerase II and that the interaction is independent of CTD phosphorylation and mRNA. Moreover, we show that SAF-A exist in complexes with transcription factors Sox2 and Oct4 as well as STAT3 in ES cells. The number of endogenous SAF-A:Oct4 and SAF-A:Sox2 complexes decreases upon LIF depletion. These discoveries allow us to propose a model for activation of Oct4 transcription.


Assuntos
Células-Tronco Embrionárias/fisiologia , Regulação da Expressão Gênica , Ribonucleoproteínas Nucleares Heterogêneas Grupo U/metabolismo , Fator 3 de Transcrição de Octâmero/metabolismo , Regiões Promotoras Genéticas , Transcrição Gênica , Animais , Linhagem Celular , Células-Tronco Embrionárias/citologia , Ribonucleoproteínas Nucleares Heterogêneas Grupo U/genética , Humanos , Camundongos , Fator 3 de Transcrição de Octâmero/genética , RNA Polimerase II/metabolismo , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo , Fator de Transcrição STAT3/metabolismo
4.
PLoS One ; 5(10): e13678, 2010 Oct 27.
Artigo em Inglês | MEDLINE | ID: mdl-21048921

RESUMO

BACKGROUND: Reprogramming of somatic cells for derivation of either embryonic stem (ES) cells, by somatic cell nuclear transfer (SCNT), or ES-like cells, by induced pluripotent stem (iPS) cell procedure, provides potential routes toward non-immunogenic cell replacement therapies. Nucleolar proteins serve as markers for activation of embryonic genes, whose expression is crucial for successful reprogramming. Although Nucleolin (Ncl) is one of the most abundant nucleolar proteins, its interaction partners in ES cells have remained unidentified. METHODOLOGY: Here we explored novel Ncl-interacting proteins using in situ proximity ligation assay (PLA), colocalization and immunoprecipitation (IP) in ES cells. PRINCIPAL FINDINGS: We found that phosphorylated Ncl (Ncl-P) interacted with translationally controlled tumor protein (Tpt1) in murine ES cells. The Ncl-P/Tpt1 complex peaked during mitosis and was reduced upon retinoic acid induced differentiation, signifying a role in cell proliferation. In addition, we showed that Ncl-P interacted with the transcription factor Oct4 during interphase in human as well as murine ES cells, indicating of a role in transcription. The Ncl-P/Oct4 complex peaked during early stages of spontaneous human ES cell differentiation and may thus be involved in the initial differentiation event(s) of mammalian development. CONCLUSIONS: Here we described two novel protein-protein interactions in ES cells, which give us further insight into the complex network of interacting proteins in pluripotent cells.


Assuntos
Células-Tronco Embrionárias/metabolismo , Interfase , Mitose , Fator 3 de Transcrição de Octâmero/metabolismo , Fosfoproteínas/metabolismo , Biossíntese de Proteínas , Proteínas de Ligação a RNA/metabolismo , Sequência de Aminoácidos , Animais , Western Blotting , Eletroforese em Gel de Poliacrilamida , Células-Tronco Embrionárias/citologia , Humanos , Imunoprecipitação , Espectrometria de Massas/métodos , Camundongos , Dados de Sequência Molecular , Fosfoproteínas/química , Fosforilação , Ligação Proteica , Proteínas de Ligação a RNA/química , Proteína Tumoral 1 Controlada por Tradução , Nucleolina
5.
Proc Natl Acad Sci U S A ; 107(37): 16072-7, 2010 Sep 14.
Artigo em Inglês | MEDLINE | ID: mdl-20798345

RESUMO

The human mitochondrial transcription machinery generates the primers required for initiation of leading-strand DNA replication. According to one model, the 3' end of the primer is defined by transcription termination at conserved sequence block II (CSB II) in the mitochondrial DNA control region. We here demonstrate that this site-specific termination event is caused by G-quadruplex structures formed in nascent RNA upon transcription of CSB II. We also demonstrate that a poly-dT stretch downstream of CSB II has a modest stimulatory effect on the termination efficiency. The mechanism is reminiscent of Rho-independent transcription termination in prokaryotes, with the exception that a G-quadruplex structure replaces the hairpin loop formed in bacterial mRNA during transcription of terminator sequences.


Assuntos
Primers do DNA/genética , Quadruplex G , Mitocôndrias/química , RNA/química , Regiões Terminadoras Genéticas , Transcrição Gênica , Sequência Conservada , Mitocôndrias/genética , Dados de Sequência Molecular
6.
Cell Cycle ; 9(11): 2160-9, 2010 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-20505363

RESUMO

Somatic cell nuclear transfers and the generation of induced pluripotent stem cells provide potential routes towards non-immunogenic cell replacement therapies. Translationally controlled tumor protein (Tpt1) was recently suggested to regulate cellular pluripotency. Here we explore functions of Tpt1 in mouse embryonic stem (ES) cells. We find that Tpt1 is present in the nucleus and cytoplasm of ES cells, and that specifically nuclear Tpt1 decreases upon cell differentiation. We also find that endogenous Tpt1 forms a complex with endogenous nucleophosmin/nucleoplasmin family member 1 (Npm1) in a cell cycle dependent manner. The Tpt1-Npm1 complex peaks sharply during mitosis and is independent of phosphorylation by Polo-like kinase. Differentiation by retinoic acid decreases Tpt1-Npm1 complex levels. Moreover, Tpt1 knock-down or over-expression reduces proliferation whereas Npm1 over-expression increases proliferation in ES cells. Cells depleted for both Tpt1 and Npm1 exhibit significantly reduced proliferation compared to cells depleted for Tpt1 alone, whereas cells over-expressing both Tpt1 and Npm1 show normal proliferation. Our findings reveal a role for the Tpt1-Npm1 complex in cell proliferation and identify the Tpt1-Npm1 complex as a potential biomarker for mitotic ES cells.


Assuntos
Biomarcadores Tumorais/metabolismo , Células-Tronco Embrionárias/metabolismo , Mitose , Proteínas Nucleares/metabolismo , Animais , Biomarcadores/metabolismo , Biomarcadores Tumorais/antagonistas & inibidores , Biomarcadores Tumorais/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Proliferação de Células , Células-Tronco Embrionárias/citologia , Camundongos , Nucleofosmina , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Proteína Tumoral 1 Controlada por Tradução , Quinase 1 Polo-Like
7.
FASEB J ; 23(8): 2587-94, 2009 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-19329760

RESUMO

The 6-protein complex shelterin protects the telomeres of human chromosomes. The recent discovery that telomeres are important for epigenetic gene regulation and vertebrate embryonic development calls for the establishment of model organisms to study shelterin and telomere function under normal developmental conditions. Here, we report the sequences of the shelterin-encoding genes in Xenopus laevis and its close relation Xenopus tropicalis. In vitro expression and biochemical characterization of the Xenopus shelterin proteins TRF1, TRF2, POT1, TIN2, RAP1, TPP1, and the shelterin accessory factor PINX1 indicate that all main functions of their human orthologs are conserved in Xenopus. The XlTRF1 and XtTRF1 proteins bind double-stranded telomeric DNA sequence specifically and interact with XlTIN2 and XtTIN2, respectively. Similarly, the XlTRF2 and XtTRF2 proteins bind double-stranded telomeric DNA and interact with XlRAP1 and XtRAP1, respectively, whereas the XlPOT1 and XtPOT1 proteins bind single-stranded telomeric DNA. Real-time PCR further reveals the gene expression profiles for telomerase and the shelterin genes during embryogenesis. Notably, the composition of shelterin and the formation of its subcomplexes appear to be temporally regulated during embryonic development. Moreover, unexpectedly high telomerase and shelterin gene expression during early embryogenesis may reflect a telomere length-resetting mechanism, similar to that reported for induced pluripotent stem cells and for animals cloned through somatic nuclear transfer.


Assuntos
Proteínas de Ligação a Telômeros/metabolismo , Telômero/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus/crescimento & desenvolvimento , Xenopus/metabolismo , Animais , Sequência de Bases , DNA/genética , DNA/metabolismo , Primers do DNA/genética , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Modelos Biológicos , Dados de Sequência Molecular , Óvulo/metabolismo , Complexo Shelterina , Telômero/genética , Proteínas de Ligação a Telômeros/química , Proteínas de Ligação a Telômeros/genética , Proteína 1 de Ligação a Repetições Teloméricas/genética , Proteína 1 de Ligação a Repetições Teloméricas/metabolismo , Proteína 2 de Ligação a Repetições Teloméricas/genética , Proteína 2 de Ligação a Repetições Teloméricas/metabolismo , Xenopus/genética , Proteínas de Xenopus/química , Proteínas de Xenopus/genética , Xenopus laevis/genética , Xenopus laevis/crescimento & desenvolvimento , Xenopus laevis/metabolismo
8.
Nat Struct Mol Biol ; 15(6): 598-604, 2008 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-18488043

RESUMO

The telomeric G-overhangs of the ciliate Stylonychia lemnae fold into a G-quadruplex DNA structure in vivo. Telomeric G-quadruplex formation requires the presence of two telomere end binding proteins, TEBPalpha and TEBPbeta, and is regulated in a cell-cycle dependent manner. Unfolding of this structure in S phase is dependent on the phosphorylation of TEBPbeta. Here we show that TEBPbeta phosphorylation is necessary but not sufficient for a G-quadruplex unfolding rate compatible with telomere synthesis. The telomerase seems to be actively involved in telomeric G-quadruplex DNA structure unfolding in vivo. Significantly, the telomerase is recruited to telomeres by phosphorylated TEBPbeta, and hence telomerase recruitment is cell-cycle regulated through phosphorylation. These observations allow us to propose a model for the regulation of G-quadruplex unfolding and telomere synthesis during the cell cycle.


Assuntos
Cilióforos , Quadruplex G , Telomerase/metabolismo , Proteínas de Ligação a Telômeros/metabolismo , Animais , Ciclo Celular , Conformação de Ácido Nucleico , Fosforilação , Transporte Proteico , Telômero
9.
J Biol Chem ; 283(5): 2693-701, 2008 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-17977837

RESUMO

Whereas mammalian cells harbor two double strand telomeric repeat binding factors, TRF1 and TRF2, the fission yeast Schizosaccharomyces pombe has been thought to harbor solely the TRF1/TRF2 ortholog Taz1p to perform comparable functions. Here we report the identification of telomeric repeat binding factor 1 (Tbf1), a second TRF1/TRF2 ortholog in S. pombe. Like the Taz1p, the identified Tbf1p shares amino acid sequence similarity, as well as structural and functional characteristics, with the mammalian TRF1 and TRF2 proteins. This family of proteins shares a common architecture with two separate structural domains. An N-terminal domain is necessary and sufficient for the formation of homodimers, and a C-terminal MYB/homeodomain mediates sequence specific recognition of double-stranded telomeric DNA. The identified Tbf1p binds S. pombe telomeric DNA with high sequence specificity in vitro. Targeted deletion of the tbf1 gene reveals that it is essential for survival, and overexpression of the tbf1 gene leads to telomere elongation in vivo, which is dependent upon the MYB domain. These data suggest that fission yeast, like mammals, have two factors that bind double-stranded telomeric DNA and perform distinct roles in telomere length regulation.


Assuntos
Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/metabolismo , Proteína 1 de Ligação a Repetições Teloméricas/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Sítios de Ligação/genética , Primers do DNA/genética , DNA Fúngico/genética , DNA Fúngico/metabolismo , Marcação de Genes , Genes Fúngicos , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Proteínas de Saccharomyces cerevisiae/genética , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/química , Proteínas de Schizosaccharomyces pombe/genética , Homologia de Sequência de Aminoácidos , Telômero/metabolismo , Proteína 1 de Ligação a Repetições Teloméricas/química , Proteína 1 de Ligação a Repetições Teloméricas/genética
10.
Nat Struct Mol Biol ; 12(10): 847-54, 2005 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-16142245

RESUMO

Telomere end-binding proteins (TEBPs) bind to the guanine-rich overhang (G-overhang) of telomeres. Although the DNA binding properties of TEBPs have been investigated in vitro, little is known about their functions in vivo. Here we use RNA interference to explore in vivo functions of two ciliate TEBPs, TEBPalpha and TEBPbeta. Silencing the expression of genes encoding both TEBPs shows that they cooperate to control the formation of an antiparallel guanine quadruplex (G-quadruplex) DNA structure at telomeres in vivo. This function seems to depend on the role of TEBPalpha in attaching telomeres in the nucleus and in recruiting TEBPbeta to these sites. In vitro DNA binding and footprinting studies confirm the in vivo observations and highlight the role of the C terminus of TEBPbeta in G-quadruplex formation. We have also found that G-quadruplex formation in vivo is regulated by the cell cycle-dependent phosphorylation of TEBPbeta.


Assuntos
Cilióforos/genética , DNA/química , DNA/metabolismo , Guanina/química , Proteínas de Protozoários/metabolismo , Proteínas de Ligação a Telômeros/metabolismo , Telômero/metabolismo , Animais , Núcleo Celular/genética , Núcleo Celular/metabolismo , Cilióforos/metabolismo , Quadruplex G , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Proteínas de Protozoários/genética , Interferência de RNA , Telômero/química , Proteínas de Ligação a Telômeros/análise , Proteínas de Ligação a Telômeros/genética
11.
Trends Biochem Sci ; 28(12): 632-8, 2003 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-14659694

RESUMO

Recent data indicates that controlled in vivo synthesis of telomeric DNA is a 'ménage à trois', in which sister chromatid termini paired by telomere end-binding protein constitute a core substrate for a telomerase dimer. Such an arrangement could serve to fine tune synthesis of telomeric DNA and might ensure similar telomerase processivity for paired sister chromatid termini, possibly to secure the maintenance of individual telomere lengths in daughter cells. It also suggests that the bona fide end-capping protein, which is likely to regulate telomerase by restricting access to chromosome termini, has escaped detection in man.


Assuntos
DNA/metabolismo , Telomerase/metabolismo , Sequência de Aminoácidos , Animais , DNA/genética , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Molecular , Oxytricha/enzimologia , Ligação Proteica , Alinhamento de Sequência , Especificidade por Substrato , Telômero/genética , Telômero/metabolismo , Proteínas de Ligação a Telômeros/química , Proteínas de Ligação a Telômeros/genética , Proteínas de Ligação a Telômeros/metabolismo
12.
EMBO Rep ; 3(12): 1139-45, 2002 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-12475927

RESUMO

Telomeres are protein-DNA complexes that cap chromosome ends and protect them from being recognized and processed as DNA breaks. Loss of capping function results in genetic instability and loss of cellular viability. The emerging view is that maintenance of an appropriate telomere structure is essential for function. Structural information on telomeric proteins that bind to double and single-stranded telomeric DNA shows that, despite a lack of extensive amino-acid sequence conservation, telomeric DNA recognition occurs via conserved DNA-binding domains. Furthermore, telomeric proteins have multidomain structures and hence are conformationally flexible. A possibility is that telomeric proteins take up different conformations when bound to different partners, providing a simple mechanism for modulating telomere architecture.


Assuntos
DNA/metabolismo , Proteínas de Homeodomínio/metabolismo , Proteínas Oncogênicas v-myb/metabolismo , Telômero/química , Telômero/metabolismo , Animais , Proteínas de Ligação a DNA/metabolismo , Oxytricha/química , Oxytricha/metabolismo
13.
Biochem Biophys Res Commun ; 290(1): 11-5, 2002 Jan 11.
Artigo em Inglês | MEDLINE | ID: mdl-11779125

RESUMO

The purpose of the study was to elucidate how DNA tetraplex (also referred to as G-quadruplex)-forming oligonucleotides mediate suppression of the human c-myc gene at the level of transcription initiation. A 22-base-long oligonucleotide, which is rich in guanines and folds into an intrastrand DNA tetraplex under physiological conditions, was administered to a Burkitt's lymphoma cell line overexpressing a (8:14) translocated c-myc allele. Administration of the oligonucleotide at nanomolar concentrations to the surrounding medium resulted in efficient cellular uptake, and was accompanied by a substantial concentration- and conformation-dependent decrease in growth rate. We discuss how c-myc transcription is initiated at the molecular level and speculate that the oligonucleotide exerts a dual effect on c-myc expression in vivo.


Assuntos
Linfoma de Burkitt/metabolismo , Genes myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Transcrição Gênica , Alelos , Antineoplásicos/farmacologia , Linfoma de Burkitt/genética , Divisão Celular/efeitos dos fármacos , Cromatina/metabolismo , DNA , Relação Dose-Resposta a Droga , Quadruplex G , Terapia Genética , Humanos , Microscopia de Fluorescência , Modelos Biológicos , Oligonucleotídeos/metabolismo , Regiões Promotoras Genéticas , Conformação Proteica , Telômero/metabolismo , Fatores de Tempo , Translocação Genética , Células Tumorais Cultivadas
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