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1.
Genome Res ; 29(4): 554-563, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30709850

RESUMO

Most mammalian RNA polymerase II initiation events occur at CpG islands, which are rich in CpGs and devoid of DNA methylation. Despite their relevance for gene regulation, it is unknown to what extent the CpG dinucleotide itself actually contributes to promoter activity. To address this question, we determined the transcriptional activity of a large number of chromosomally integrated promoter constructs and monitored binding of transcription factors assumed to play a role in CpG island activity. This revealed that CpG density significantly improves motif-based prediction of transcription factor binding. Our experiments also show that high CpG density alone is insufficient for transcriptional activity, yet results in increased transcriptional output when combined with particular transcription factor motifs. However, this CpG contribution to promoter activity is independent of DNA methyltransferase activity. Together, this refines our understanding of mammalian promoter regulation as it shows that high CpG density within CpG islands directly contributes to an environment permissive for full transcriptional activity.


Assuntos
Ilhas de CpG , Metilação de DNA , Regiões Promotoras Genéticas , Ativação Transcricional , Animais , Linhagem Celular , Células Cultivadas , Camundongos , Ligação Proteica , Fatores de Transcrição/metabolismo
2.
Nature ; 520(7546): 243-7, 2015 Apr 09.
Artigo em Inglês | MEDLINE | ID: mdl-25607372

RESUMO

DNA methylation is an epigenetic modification associated with transcriptional repression of promoters and is essential for mammalian development. Establishment of DNA methylation is mediated by the de novo DNA methyltransferases DNMT3A and DNMT3B, whereas DNMT1 ensures maintenance of methylation through replication. Absence of these enzymes is lethal, and somatic mutations in these genes have been associated with several human diseases. How genomic DNA methylation patterns are regulated remains poorly understood, as the mechanisms that guide recruitment and activity of DNMTs in vivo are largely unknown. To gain insights into this matter we determined genomic binding and site-specific activity of the mammalian de novo DNA methyltransferases DNMT3A and DNMT3B. We show that both enzymes localize to methylated, CpG-dense regions in mouse stem cells, yet are excluded from active promoters and enhancers. By specifically measuring sites of de novo methylation, we observe that enzymatic activity reflects binding. De novo methylation increases with CpG density, yet is excluded from nucleosomes. Notably, we observed selective binding of DNMT3B to the bodies of transcribed genes, which leads to their preferential methylation. This targeting to transcribed sequences requires SETD2-mediated methylation of lysine 36 on histone H3 and a functional PWWP domain of DNMT3B. Together these findings reveal how sequence and chromatin cues guide de novo methyltransferase activity to ensure methylome integrity.


Assuntos
DNA (Citosina-5-)-Metiltransferases/metabolismo , Metilação de DNA/genética , Epigênese Genética/genética , Genoma/genética , Animais , Linhagem Celular , Cromatina/química , Cromatina/genética , Cromatina/metabolismo , Ilhas de CpG/genética , DNA (Citosina-5-)-Metiltransferases/química , DNA Metiltransferase 3A , Células-Tronco Embrionárias/enzimologia , Células-Tronco Embrionárias/metabolismo , Elementos Facilitadores Genéticos/genética , Genômica , Histona-Lisina N-Metiltransferase/deficiência , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/química , Histonas/metabolismo , Lisina/metabolismo , Camundongos , Regiões Promotoras Genéticas/genética , Ligação Proteica , Estrutura Terciária de Proteína , Transporte Proteico , Transcrição Gênica/genética , DNA Metiltransferase 3B
3.
Nature ; 480(7378): 490-5, 2011 Dec 14.
Artigo em Inglês | MEDLINE | ID: mdl-22170606

RESUMO

Methylation of cytosines is an essential epigenetic modification in mammalian genomes, yet the rules that govern methylation patterns remain largely elusive. To gain insights into this process, we generated base-pair-resolution mouse methylomes in stem cells and neuronal progenitors. Advanced quantitative analysis identified low-methylated regions (LMRs) with an average methylation of 30%. These represent CpG-poor distal regulatory regions as evidenced by location, DNase I hypersensitivity, presence of enhancer chromatin marks and enhancer activity in reporter assays. LMRs are occupied by DNA-binding factors and their binding is necessary and sufficient to create LMRs. A comparison of neuronal and stem-cell methylomes confirms this dependency, as cell-type-specific LMRs are occupied by cell-type-specific transcription factors. This study provides methylome references for the mouse and shows that DNA-binding factors locally influence DNA methylation, enabling the identification of active regulatory regions.


Assuntos
Citosina/metabolismo , Metilação de DNA , Proteínas de Ligação a DNA/metabolismo , Epigenômica , Animais , Diferenciação Celular , Ilhas de CpG , Células-Tronco Embrionárias/citologia , Camundongos , Neurônios/citologia , Regiões Promotoras Genéticas/genética , Ligação Proteica , Células-Tronco/citologia , Fatores de Transcrição/metabolismo
4.
Proc Natl Acad Sci U S A ; 109(16): E934-43, 2012 Apr 17.
Artigo em Inglês | MEDLINE | ID: mdl-22474351

RESUMO

Topoisomerases are essential for DNA replication in dividing cells, but their genomic targets and function in postmitotic cells remain poorly understood. Here we show that a switch in the expression from Topoisomerases IIα (Top2α) to IIß (Top2ß) occurs during neuronal differentiation in vitro and in vivo. Genome-scale location analysis in stem cell-derived postmitotic neurons reveals Top2ß binding to chromosomal sites that are methylated at lysine 4 of histone H3, a feature of regulatory regions. Indeed Top2ß-bound sites are preferentially promoters and become targets during the transition from neuronal progenitors to neurons, at a time when cells exit the cell cycle. Absence of Top2ß protein or its activity leads to changes in transcription and chromatin accessibility at many target genes. Top2ß deficiency does not impair stem cell properties and early steps of neuronal differentiation but causes premature death of postmitotic neurons. This neuronal degeneration is caused by up-regulation of Ngfr p75, a gene bound and repressed by Top2ß. These findings suggest a chromatin-based targeting of Top2ß to regulatory regions in the genome to govern the transcriptional program associated with neuronal differentiation and longevity.


Assuntos
Cromatina/genética , DNA Topoisomerases Tipo II/genética , Proteínas de Ligação a DNA/genética , Neurônios/metabolismo , Animais , Western Blotting , Diferenciação Celular/genética , Sobrevivência Celular/genética , Células Cultivadas , DNA Topoisomerases Tipo II/metabolismo , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/metabolismo , Dicetopiperazinas , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/efeitos dos fármacos , Células-Tronco Embrionárias/metabolismo , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Imunoprecipitação , Masculino , Camundongos , Camundongos da Linhagem 129 , Camundongos Knockout , Neurônios/citologia , Neurônios/efeitos dos fármacos , Análise de Sequência com Séries de Oligonucleotídeos , Piperazinas/farmacologia , Ligação Proteica , Interferência de RNA , Receptor de Fator de Crescimento Neural/genética , Receptor de Fator de Crescimento Neural/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Tempo , Inibidores da Topoisomerase II/farmacologia
5.
Nat Genet ; 56(6): 1203-1212, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38816647

RESUMO

Catalytic activity of the imitation switch (ISWI) family of remodelers is critical for nucleosomal organization and DNA binding of certain transcription factors, including the insulator protein CTCF. Here we define the contribution of individual subcomplexes by deriving a panel of isogenic mouse stem cell lines, each lacking one of six ISWI accessory subunits. Individual deletions of subunits of either CERF, RSF, ACF, WICH or NoRC subcomplexes only moderately affect the chromatin landscape, while removal of the NURF-specific subunit BPTF leads to a strong reduction in chromatin accessibility and SNF2H ATPase localization around CTCF sites. This affects adjacent nucleosome occupancy and CTCF binding. At a group of sites with reduced chromatin accessibility, CTCF binding persists but cohesin occupancy is reduced, resulting in decreased insulation. These results suggest that CTCF binding can be separated from its function as an insulator in nuclear organization and identify a specific role for NURF in mediating SNF2H localization and chromatin opening at bound CTCF sites.


Assuntos
Adenosina Trifosfatases , Fator de Ligação a CCCTC , Cromatina , Proteínas Repressoras , Fatores de Transcrição , Fator de Ligação a CCCTC/metabolismo , Fator de Ligação a CCCTC/genética , Animais , Camundongos , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Proteínas Repressoras/metabolismo , Proteínas Repressoras/genética , Cromatina/metabolismo , Cromatina/genética , Adenosina Trifosfatases/metabolismo , Adenosina Trifosfatases/genética , Ligação Proteica , Linhagem Celular , Proteínas Cromossômicas não Histona/metabolismo , Proteínas Cromossômicas não Histona/genética , Nucleossomos/metabolismo , Nucleossomos/genética , Subunidades Proteicas/metabolismo , Subunidades Proteicas/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Sítios de Ligação
6.
Nat Genet ; 54(12): 1895-1906, 2022 12.
Artigo em Inglês | MEDLINE | ID: mdl-36471082

RESUMO

Cytosine methylation efficiently silences CpG-rich regulatory regions of genes and repeats in mammalian genomes. To what extent this entails direct inhibition of transcription factor (TF) binding versus indirect inhibition via recruitment of methyl-CpG-binding domain (MBD) proteins is unclear. Here we show that combinatorial genetic deletions of all four proteins with functional MBDs in mouse embryonic stem cells, derived neurons or a human cell line do not reactivate genes or repeats with methylated promoters. These do, however, become activated by methylation-restricted TFs if DNA methylation is removed. We identify several causal TFs in neurons, including ONECUT1, which is methylation sensitive only at a motif variant. Rampantly upregulated retrotransposons in methylation-free neurons feature a CRE motif, which activates them in the absence of DNA methylation via methylation-sensitive binding of CREB1. Our study reveals methylation-sensitive TFs in vivo and argues that direct inhibition, rather than indirect repression by the tested MBD proteins, is the prevailing mechanism of methylation-mediated repression at regulatory regions and repeats.


Assuntos
Metilação de DNA , Fatores de Transcrição , Animais , Humanos , Camundongos , Metilação de DNA/genética , Fator 6 Nuclear de Hepatócito , Mamíferos , Fatores de Transcrição/genética
7.
EMBO J ; 26(24): 4974-84, 2007 Dec 12.
Artigo em Inglês | MEDLINE | ID: mdl-18007591

RESUMO

Post-translational modifications of histones are involved in transcript initiation and elongation. Methylation of lysine 36 of histone H3 (H3K36me) resides promoter distal at transcribed regions in Saccharomyces cerevisiae and is thought to prevent spurious initiation through recruitment of histone-deacetylase activity. Here, we report surprising complexity in distribution, regulation and readout of H3K36me in Drosophila involving two histone methyltransferases (HMTases). Dimethylation of H3K36 peaks adjacent to promoters and requires dMes-4, whereas trimethylation accumulates toward the 3' end of genes and relies on dHypb. Reduction of H3K36me3 is lethal in Drosophila larvae and leads to elevated levels of acetylation, specifically at lysine 16 of histone H4 (H4K16ac). In contrast, reduction of both di- and trimethylation decreases lysine 16 acetylation. Thus di- and trimethylation of H3K36 have opposite effects on H4K16 acetylation, which we propose enable dynamic changes in chromatin compaction during transcript elongation.


Assuntos
Drosophila melanogaster/metabolismo , Histonas/metabolismo , Lisina/metabolismo , Processamento de Proteína Pós-Traducional , Transcrição Gênica , Acetilação , Animais , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Regulação da Expressão Gênica , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Humanos , Metilação , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Regiões Promotoras Genéticas , Interferência de RNA
8.
Mol Cell Biol ; 26(15): 5784-96, 2006 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-16847331

RESUMO

Inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene is linked to the development of tumors of the eyes, kidneys, and central nervous system. VHL encodes two gene products, pVHL30 and pVHL19, of which one, pVHL30, associates functionally with microtubules (MTs) to regulate their stability. Here we report that pVHL30 is a novel substrate of glycogen synthase kinase 3 (GSK3) in vitro and in vivo. Phosphorylation of pVHL on serine 68 (S68) by GSK3 requires a priming phosphorylation event at serine 72 (S72) mediated in vitro by casein kinase I. Functional analysis of pVHL species carrying nonphosphorylatable or phosphomimicking mutations at S68 and/or S72 reveals a central role for these phosphorylation events in the regulation of pVHL's MT stabilization (but not binding) activity. Taken together, our results identify pVHL as a novel priming-dependent substrate of GSK3 and suggest a dual-kinase mechanism in the control of pVHL's MT stabilization function. Since GSK3 is a component of multiple signaling pathways that are altered in human cancer, our results further imply that normal operation of the GSK3-pVHL axis may be a critical aspect of pVHL's tumor suppressor mechanism through the regulation of MT dynamics.


Assuntos
Quinase 3 da Glicogênio Sintase/metabolismo , Fragmentos de Peptídeos/metabolismo , Isoformas de Proteínas/metabolismo , Proteína Supressora de Tumor Von Hippel-Lindau/metabolismo , Sequência de Aminoácidos , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Linhagem Celular , Quinase 3 da Glicogênio Sintase/genética , Humanos , Camundongos , Microtúbulos/metabolismo , Dados de Sequência Molecular , Mutação , Fragmentos de Peptídeos/genética , Fosforilação , Ligação Proteica , Isoformas de Proteínas/genética , Alinhamento de Sequência , Serina/metabolismo , Transdução de Sinais/fisiologia , Proteína Supressora de Tumor Von Hippel-Lindau/genética
9.
Mol Cell Biol ; 26(16): 5994-6004, 2006 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-16880511

RESUMO

Ubiquitin-mediated degradation of the cyclin-dependent kinase inhibitor p27 provides a powerful route for enforcing normal progression through the mammalian cell cycle. According to a current model, the ubiquitination of p27 during S-phase progression is mediated by SCF(Skp2) E3 ligase that captures Thr187-phosphorylated p27 by means of the F-box protein Skp2, which in turn couples the bound substrate via Skp1 to a catalytic core complex composed of Cul1 and the Rbx/Roc RING finger protein. Here we identify Skp2 as a component of an Skp1-cullin-F-box complex that is based on a Cul1-Ro52 RING finger B-box coiled-coil motif family protein catalytic core. Ro52-containing complexes display E3 ligase activity and promote the ubiquitination of Thr187-phosphorylated p27 in a RING-dependent manner in vitro. The knockdown of Ro52 expression in human cells with small interfering RNAs causes the accumulation of p27 and the failure of cells to enter S phase. Importantly, these effects are abrogated by the simultaneous removal of p27. Taken together, these data suggest a key role for Ro52 RING finger protein in the regulation of p27 degradation and S-phase progression in mammalian cells and provide evidence for the existence of a Cul1-based catalytic core that utilizes Ro52 RING protein to promote ubiquitination.


Assuntos
Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Processamento de Proteína Pós-Traducional , Ribonucleoproteínas/química , Ribonucleoproteínas/metabolismo , Fase S , Motivos de Aminoácidos , Proteínas de Ciclo Celular/metabolismo , Proteínas Culina/metabolismo , Regulação para Baixo/genética , Células HeLa , Humanos , Fosfotreonina/metabolismo , Ligação Proteica , Ribonucleoproteínas/isolamento & purificação , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Proteínas Contendo Repetições de beta-Transducina/metabolismo
10.
Mol Cell Biol ; 25(12): 5052-60, 2005 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-15923622

RESUMO

Inactivation of the HRPT2 tumor suppressor gene is associated with the pathogenesis of the hereditary hyperparathyroidism-jaw tumor syndrome and malignancy in sporadic parathyroid tumors. The cellular function of the HPRT2 gene product, parafibromin, has not been defined yet. Here we show that parafibromin physically interacts with human orthologs of yeast Paf1 complex components, including PAF1, LEO1, and CTR9, that are involved in transcription elongation and 3' end processing. It also associates with modified forms of the large subunit of RNA polymerase II, in particular those phosphorylated on serine 5 or 2 within the carboxy-terminal domain, that are important for the coordinate recruitment of transcription elongation and RNA processing machineries during the transcription cycle. These interactions depend on a C-terminal domain of parafibromin, which is deleted in ca. 80% of clinically relevant mutations. Finally, RNAi-induced downregulation of parafibromin promotes entry into S phase, implying a role for parafibromin as an inhibitor of cell cycle progression. Taken together, these findings link the tumor suppressor parafibromin to the transcription elongation and RNA processing pathway as a PAF1 complex- and RNA polymerase II-bound protein. Dysfunction of this pathway may be a general phenomenon in the majority of cases of hereditary parathyroid cancer.


Assuntos
Regulação da Expressão Gênica , Proteínas Nucleares/metabolismo , Isoformas de Proteínas/metabolismo , RNA Polimerase II/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Animais , Ciclo Celular/fisiologia , Linhagem Celular , Humanos , Hiperparatireoidismo/genética , Hiperparatireoidismo/metabolismo , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Complexos Multiproteicos , Proteínas Nucleares/genética , Isoformas de Proteínas/genética , Interferência de RNA , RNA Polimerase II/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição , Proteínas Supressoras de Tumor/genética
11.
Nat Genet ; 43(11): 1091-7, 2011 Oct 02.
Artigo em Inglês | MEDLINE | ID: mdl-21964573

RESUMO

Cytosine methylation is a repressive, epigenetically propagated DNA modification. Although patterns of DNA methylation seem tightly regulated in mammals, it is unclear how these are specified and to what extent this process entails genetic or epigenetic regulation. To dissect the role of the underlying DNA sequence, we sequentially inserted over 50 different DNA elements into the same genomic locus in mouse stem cells. Promoter sequences of approximately 1,000 bp autonomously recapitulated correct DNA methylation in pluripotent cells. Moreover, they supported proper de novo methylation during differentiation. Truncation analysis revealed that this regulatory potential is contained within small methylation-determining regions (MDRs). MDRs can mediate both hypomethylation and de novo methylation in cis, and their activity depends on developmental state, motifs for DNA-binding factors and a critical CpG density. These results demonstrate that proximal sequence elements are both necessary and sufficient for regulating DNA methylation and reveal basic constraints of this regulation.


Assuntos
Metilação de DNA , Animais , Ilhas de CpG , Mutação , Regiões Promotoras Genéticas , Células-Tronco/metabolismo
12.
Nat Genet ; 44(1): 94-100, 2011 Dec 18.
Artigo em Inglês | MEDLINE | ID: mdl-22179133

RESUMO

Signaling mediates cellular responses to extracellular stimuli. The c-Jun NH(2)-terminal kinase (JNK) pathway exemplifies one subgroup of the mitogen-activated protein (MAP) kinases, which, besides having established functions in stress response, also contribute to development by an unknown mechanism. We show by genome-wide location analysis that JNK binds to a large set of active promoters during the differentiation of stem cells into neurons. JNK-bound promoters are enriched with binding motifs for the transcription factor NF-Y but not for AP-1. NF-Y occupies these predicted sites, and overexpression of dominant-negative NF-YA reduces the JNK presence on chromatin. We find that histone H3 Ser10 (H3S10) is a substrate for JNK, and JNK-bound promoters are enriched for H3S10 phosphorylation. Inhibition of JNK signaling in post-mitotic neurons reduces phosphorylation at H3S10 and the expression of target genes. These results establish MAP kinase binding and function on chromatin at a novel class of target genes during stem cell differentiation.


Assuntos
Diferenciação Celular , Cromatina/metabolismo , MAP Quinase Quinase 4/metabolismo , Animais , Fator de Ligação a CCAAT/metabolismo , Células-Tronco Embrionárias , Regulação da Expressão Gênica , Sistema de Sinalização das MAP Quinases/genética , Camundongos , Células-Tronco Neurais , Neurônios/fisiologia , Fosforilação , Regiões Promotoras Genéticas , Ligação Proteica , Células-Tronco , Regulação para Cima
13.
Mol Cell Biol ; 28(10): 3401-9, 2008 May.
Artigo em Inglês | MEDLINE | ID: mdl-18347056

RESUMO

In Drosophila melanogaster, dosage compensation relies on the targeting of the male-specific lethal (MSL) complex to hundreds of sites along the male X chromosome. Transcription-coupled methylation of histone H3 lysine 36 is enriched toward the 3' end of active genes, similar to the MSL proteins. Here, we have studied the link between histone H3 methylation and MSL complex targeting using RNA interference and chromatin immunoprecipitation. We show that trimethylation of histone H3 at lysine 36 (H3K36me3) relies on the histone methyltransferase Hypb and is localized promoter distal at dosage-compensated genes, similar to active genes on autosomes. However, H3K36me3 has an X-specific function, as reduction specifically decreases acetylation of histone H4 lysine 16 on the male X chromosome. This hypoacetylation is caused by compromised MSL binding and results in a failure to increase expression twofold. Thus, H3K36me3 marks the body of all active genes yet is utilized in a chromosome-specific manner to enhance histone acetylation at sites of dosage compensation.


Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Histonas/metabolismo , Fatores de Transcrição/metabolismo , Animais , Sítios de Ligação/genética , Linhagem Celular , Imunoprecipitação da Cromatina , Mecanismo Genético de Compensação de Dose , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Feminino , Histonas/química , Histonas/genética , Lisina/química , Masculino , Metilação , Modelos Biológicos , Complexos Multiproteicos , Interferência de RNA , Processos de Determinação Sexual , Fatores de Transcrição/química , Fatores de Transcrição/genética , Transcrição Gênica , Cromossomo X/genética , Cromossomo X/metabolismo
14.
Mol Cell ; 28(1): 28-40, 2007 Oct 12.
Artigo em Inglês | MEDLINE | ID: mdl-17936702

RESUMO

S6 kinase 1 (S6K1) acts to integrate nutrient and growth factor signals to promote cell growth but also cell survival as a mitochondria-tethered protein kinase that phosphorylates and inactivates the proapoptotic molecule BAD. Here we report that the prefoldin chaperone URI represents a mitochondrial substrate of S6K1. In growth factor-deprived or rapamycin-treated cells, URI forms stable complexes with protein phosphatase (PP)1gamma at mitochondria, thereby inhibiting the activity of the bound enzyme. Growth factor stimulation induces disassembly of URI/PP1gamma complexes through S6K1-mediated phosphorylation of URI at serine 371. This activates a PP1gamma-dependent negative feedback program that decreases S6K1 activity and BAD phosphorylation, thereby altering the threshold for apoptosis. These findings establish URI and PP1gamma as integral components of an S6K1-regulated mitochondrial pathway dedicated, in part, to oppose sustained S6K1 survival signaling and to ensure that the mitochondrial threshold for apoptosis is set in accord with nutrient and growth factor availability.


Assuntos
Sobrevivência Celular/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Mitocôndrias/metabolismo , Chaperonas Moleculares/metabolismo , Proteína Fosfatase 1/metabolismo , Proteínas Quinases S6 Ribossômicas/metabolismo , Transdução de Sinais/fisiologia , Sequência de Aminoácidos , Animais , Apoptose/fisiologia , Linhagem Celular , Retroalimentação Fisiológica/fisiologia , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Isoenzimas/genética , Isoenzimas/metabolismo , Chaperonas Moleculares/genética , Dados de Sequência Molecular , Complexos Multiproteicos/metabolismo , Proteína Fosfatase 1/genética , Estrutura Terciária de Proteína , RNA Interferente Pequeno/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Repressoras , Proteínas Quinases S6 Ribossômicas/genética , Alinhamento de Sequência , Serina/metabolismo , Sirolimo/metabolismo , Proteína de Morte Celular Associada a bcl/genética , Proteína de Morte Celular Associada a bcl/metabolismo
15.
Genes Dev ; 19(15): 1761-6, 2005 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-16077006

RESUMO

Deposition of variant histones provides a mechanism to reset and to potentially specify chromatin states. We determined the distribution of H3 and its variant H3.3 relative to chromatin structure and elongating polymerase. H3.3 is enriched throughout active genes similar to polymerase, yet its distribution is very distinct from that of several euchromatic histone modifications, which are highly biased toward the 5' part of active genes. Upon gene induction we observe displacement of both H3 and H3.3 followed by selective deposition of H3.3. These results support a model in which H3.3 deposition compensates for transcription-coupled nucleosomal displacement yet does not predetermine tail modifications.


Assuntos
Histonas/metabolismo , Nucleossomos/metabolismo , Regiões Promotoras Genéticas , Animais , Drosophila , Imunofluorescência , Cinética
16.
Mol Cell ; 13(1): 101-11, 2004 Jan 16.
Artigo em Inglês | MEDLINE | ID: mdl-14731398

RESUMO

The AU-rich element (ARE) in the 3' untranslated region of unstable mRNAs mediate their rapid degradation. ARE binding proteins (AUBPs) have been described that either stabilize or otherwise degrade ARE-mRNAs by recruiting the exosome, a complex of 3'-to-5' exoribonucleases. We have identified RHAU, a putative DExH RNA helicase that was isolated in association with the ARE of urokinase plasminogen activator mRNA (ARE(uPA)). RHAU physically interacts with the deadenylase PARN and the human exosome and enhances the deadenylation and decay of ARE(uPA)-mRNAs. An alternatively spliced isoform of RHAU that localized to the cytoplasm had a more pronounced effect on ARE(uPA)-mRNA destabilization than full-length RHAU. Furthermore, the ATPase activity of RHAU is essential for its mRNA-destabilizing function. ARE(uPA)-mRNA recognition by RHAU may be mediated through its RNA-dependent interaction with the AUBPs HuR and NFAR1. A model is presented to describe the action of RHAU in ARE(uPA)-directed mRNA turnover.


Assuntos
Antígenos de Superfície , Exorribonucleases/metabolismo , Fosfoproteínas , Isoformas de Proteínas/metabolismo , RNA Helicases/química , RNA Helicases/metabolismo , RNA Mensageiro/metabolismo , Regiões 3' não Traduzidas , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo , Processamento Alternativo , Motivos de Aminoácidos , Sequência de Aminoácidos , Sequência Conservada , Citoplasma/metabolismo , Regulação para Baixo , Proteínas ELAV , Proteína Semelhante a ELAV 1 , Exorribonucleases/genética , Glutationa Transferase/metabolismo , Células HeLa , Humanos , Dados de Sequência Molecular , Proteínas do Fator Nuclear 90 , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , RNA Helicases/genética , Estabilidade de RNA , Proteínas de Ligação a RNA/metabolismo , Proteínas Recombinantes/metabolismo , Deleção de Sequência , Homologia de Sequência de Aminoácidos , Ativador de Plasminogênio Tipo Uroquinase/metabolismo
17.
Science ; 302(5648): 1208-12, 2003 Nov 14.
Artigo em Inglês | MEDLINE | ID: mdl-14615539

RESUMO

Prefoldins (PFDs) are members of a recently identified, small-molecular weight protein family able to assemble into molecular chaperone complexes. Here we describe an unusually large member of this family, termed URI, that forms complexes with other small-molecular weight PFDs and with RPB5, a shared subunit of all three RNA polymerases. Functional analysis of the yeast and human orthologs of URI revealed that both are targets of nutrient signaling and participate in gene expression controlled by the TOR kinase. Thus, URI is a component of a signaling pathway that coordinates nutrient availability with gene expression.


Assuntos
Aminoácidos/metabolismo , Proteínas de Transporte/metabolismo , Regulação da Expressão Gênica , Peptídeos e Proteínas de Sinalização Intracelular , Saccharomyces cerevisiae/genética , Transdução de Sinais , Transcrição Gênica , Sequência de Aminoácidos , Proteínas de Transporte/química , Proteínas de Transporte/genética , Proteínas de Ciclo Celular , Linhagem Celular , Proteínas de Ligação a DNA/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Fatores de Transcrição GATA , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Chaperonas Moleculares , Dados de Sequência Molecular , Fosforilação , Proteínas Quinases/metabolismo , Subunidades Proteicas/metabolismo , Interferência de RNA , Proteínas Repressoras/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Sirolimo/farmacologia , Serina-Treonina Quinases TOR , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Gênica/efeitos dos fármacos , Transfecção
18.
Genes Dev ; 18(11): 1263-71, 2004 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-15175259

RESUMO

The covalent modification of nucleosomal histones has emerged as a major determinant of chromatin structure and gene activity. To understand the interplay between various histone modifications, including acetylation and methylation, we performed a genome-wide chromatin structure analysis in a higher eukaryote. We found a binary pattern of histone modifications among euchromatic genes, with active genes being hyperacetylated for H3 and H4 and hypermethylated at Lys 4 and Lys 79 of H3, and inactive genes being hypomethylated and deacetylated at the same residues. Furthermore, the degree of modification correlates with the level of transcription, and modifications are largely restricted to transcribed regions, suggesting that their regulation is tightly linked to polymerase activity.


Assuntos
Cromatina/genética , Drosophila melanogaster/genética , Perfilação da Expressão Gênica/métodos , Histonas/metabolismo , Acetilação , Animais , Cromatina/metabolismo , Replicação do DNA , Proteínas de Drosophila/genética , Células Eucarióticas/fisiologia , Genoma , Histonas/genética , Metilação , Análise de Sequência com Séries de Oligonucleotídeos , RNA/biossíntese , Transcrição Gênica
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