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1.
Nucleic Acids Res ; 42(6): 3542-50, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24393775

RESUMO

The epigenome is defined as a type of information that can be transmitted independently of the DNA sequence, at the chromatin level, through post-translational modifications present on histone tails. Recent advances in the identification of histone 3 variants suggest a new model of information transmission through deposition of specific histone variants. To date, several non-centromeric histone 3 variants have been identified in mammals. Despite protein sequence similarity, specific deposition complexes have been characterized for both histone 3.1 (H3.1) and histone 3.3 (H3.3), whereas no deposition complex for histone 3.2 (H3.2) has been identified to date. Here, we identified human H3.2 partners by immunopurification of nuclear H3.2 complexes followed by mass spectrometry analysis. Further biochemical analyses highlighted two major complexes associated with H3.2, one containing chromatin associated factor-1 subunits and the other consisting of a subcomplex of mini chromosome maintenance helicases, together with Asf1. The purified complexes could associate with a DNA template in vitro.


Assuntos
Histonas/metabolismo , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Núcleo Celular/metabolismo , Cromatina/metabolismo , Células HeLa , Humanos , Proteínas de Manutenção de Minicromossomo/metabolismo , Chaperonas Moleculares , Fase S
2.
Mol Cell ; 25(3): 369-83, 2007 Feb 09.
Artigo em Inglês | MEDLINE | ID: mdl-17289585

RESUMO

Although the proteasome facilitates transcription from several yeast promoters, it is unclear if its role is proteolytic or which subunits are involved. We show that the proteasome regulates the HIV-1 promoter in both proteolytic and nonproteolytic modes. In the absence of transcription factor, Tat, proteasome was associated with promoter and coding regions, and its proteolytic activity regulated the level of basal transcription emanating from the promoter. Tat switched the proteasome to a nonproteolytic mode by recruiting a proteasome-associated protein, PAAF1, which favors proteasome dissociation into 19S and 20S particles. Gel filtration chromatography showed that expression of both Tat and PAAF1 enhanced the abundance of a 19S-like complex in nuclear extracts. 19S, but not 20S, subunits were strongly recruited to the promoter in the presence of Tat and PAAF1 and coactivated Tat-dependent transcription. 19S components facilitated transcriptional elongation and may be involved in clearance of paused transcriptional elongation complexes from the promoter.


Assuntos
Regulação Viral da Expressão Gênica , HIV-1/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Transcrição Gênica , Proteínas Adaptadoras de Transdução de Sinal , Proteínas de Transporte/metabolismo , Produtos do Gene tat/genética , Produtos do Gene tat/metabolismo , Repetição Terminal Longa de HIV , Células HeLa , Humanos , Regiões Promotoras Genéticas , Produtos do Gene tat do Vírus da Imunodeficiência Humana
3.
Nat Cell Biol ; 5(8): 754-61, 2003 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-12883554

RESUMO

The human immunodeficiency virus type 1 (HIV-1) encodes a potent transactivator, Tat, which functions through binding to a short leader RNA, called transactivation responsive element (TAR). Recent studies suggest that Tat activates the HIV-1 long terminal repeat (LTR), mainly by adapting co-activator complexes, such as p300, PCAF and the positive transcription elongation factor P-TEFb, to the promoter. Here, we show that the proto-oncoprotein Hdm2 interacts with Tat and mediates its ubiquitination in vitro and in vivo. In addition, Hdm2 is a positive regulator of Tat-mediated transactivation, indicating that the transcriptional properties of Tat are stimulated by ubiquitination. Fusion of ubiquitin to Tat bypasses the requirement of Hdm2 for efficient transactivation, supporting the notion that ubiquitin has a non-proteolytic function in Tat-mediated transactivation.


Assuntos
Produtos do Gene tat/metabolismo , HIV-1/genética , Proteínas Nucleares , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas/metabolismo , Ativação Transcricional , Ubiquitina/metabolismo , Linhagem Celular , Produtos do Gene tat/genética , Repetição Terminal Longa de HIV , HIV-1/metabolismo , Humanos , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-mdm2 , RNA Interferente Pequeno , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Produtos do Gene tat do Vírus da Imunodeficiência Humana
4.
EMBO J ; 21(24): 6811-9, 2002 Dec 16.
Artigo em Inglês | MEDLINE | ID: mdl-12486002

RESUMO

The HIV-1 transactivator protein, Tat, is an atypical transcriptional activator that functions through binding, not to DNA, but to a short leader RNA, TAR. Although details of its functional mechanism are still unknown, emerging findings suggest that Tat serves primarily to adapt co-activator complexes such as p300, PCAF and P-TEFb to the HIV-1 long terminal repeat. Hence, an understanding of how Tat interacts with these cofactors is crucial. It has recently been shown that acetylation at a single lysine, residue 50, regulated the association of Tat with PCAF. Here, we report that in the absence of Tat acetylation, PCAF binds to amino acids 20-40 within Tat. Interestingly, acetylation of Tat at Lys28 abrogates Tat-PCAF interaction. Acetylation at Lys50 creates a new site for binding to PCAF and dictates the formation of a ternary complex of Tat-PCAF-P-TEFb. Thus, differential lysine acetylation of Tat coordinates the interactions with its co-activators, cyclin T1 and PCAF. Our results may help in understanding the ordered recruitment of Tat co-activators to the HIV-1 promoter.


Assuntos
Acetiltransferases/metabolismo , Proteínas de Ciclo Celular/metabolismo , Ciclinas/metabolismo , Produtos do Gene tat/química , Produtos do Gene tat/metabolismo , Acetilação , Western Blotting , Cromatografia de Afinidade , Ciclina T , Glutationa Transferase/metabolismo , Repetição Terminal Longa de HIV , Células HeLa , Histona Acetiltransferases , Humanos , Lisina/química , Modelos Biológicos , Proteínas Nucleares/metabolismo , Peptídeos/química , Ligação Proteica , Proteínas Recombinantes de Fusão/metabolismo , Transativadores/metabolismo , Fatores de Transcrição , Ativação Transcricional , Fatores de Transcrição de p300-CBP
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