RESUMO
Histone deacetylases (HDACs) are enzymes that catalyze the removal of acetyl groups from the epsilon-amino groups of conserved lysine residues in the amino terminal tail of histones. In humans, there are 18 potential deacetylase enzymes that are responsible for the removal of acetyl groups and maintenance of the equilibrium of lysine acetylation on histones. Like most histone modification enzymes, accumulating evidence suggests that many, if not all, HDACs can also modify non-histone proteins. The focus of this article is to provide up-to-date, easy to follow, approaches and techniques specifically for the assay of HDAC enzymatic activities.
Assuntos
Bioensaio/métodos , Histona Desacetilases/metabolismo , Animais , Linhagem Celular , Histona Desacetilases/isolamento & purificação , Histonas/metabolismo , Humanos , Coloração e Rotulagem , Especificidade por SubstratoRESUMO
Histone deacetylases (HDACs) are enzymes that regulate the functions of histones as well as nonhistones by catalyzing the removal of acetyl groups from lysine residues. HDACs regulate many biological processes, including the cell division cycle and tumorigenesis. Although recent studies have implicated HDAC8 in tumor cell proliferation, the molecular mechanisms linking HDAC8 to cell growth remain unknown. Here, we report that the human ortholog of the yeast ever-shorter telomeres 1B (EST1B) binds HDAC8. This interaction is regulated by protein kinase A-mediated HDAC8 phosphorylation and protects human EST1B (hEST1B) from ubiquitin-mediated degradation. Phosphorylated HDAC8 preferentially recruits Hsp70 to a complex that inhibits the CHIP (C-terminal heat shock protein interacting protein) E3 ligase-mediated degradation of hEST1B. Importantly, HDAC8 regulation of hEST1B protein stability modulates total telomerase enzymatic activity. Our findings reveal a novel mechanism by which HDAC8 contributes to tumorigenesis by regulating telomerase activity.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Histona Desacetilases/metabolismo , Proteínas Repressoras/metabolismo , Telomerase/metabolismo , Ubiquitina/metabolismo , Acetilação , Sequência de Bases , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , DNA Complementar/genética , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Inativação Gênica , Proteínas de Choque Térmico HSP70/química , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico HSP90/química , Proteínas de Choque Térmico HSP90/metabolismo , Células HeLa , Histona Desacetilases/química , Histona Desacetilases/genética , Humanos , Técnicas In Vitro , Complexos Multiproteicos , Fosforilação , Ligação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Repressoras/química , Proteínas Repressoras/genética , Telomerase/química , Telomerase/genética , Técnicas do Sistema de Duplo-Híbrido , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismoAssuntos
Histona Desacetilases/isolamento & purificação , Histona Desacetilases/metabolismo , Animais , Vetores Genéticos , Células HeLa , Histona Desacetilases/genética , Humanos , Insetos , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Sitios de Sequências Rotuladas , Transfecção/métodosRESUMO
Histone deacetylases (HDACs) are enzymes that catalyze the removal of acetyl groups from lysine residues of histone and nonhistone proteins. Recent studies suggest that they are key regulators of many cellular events, including cell proliferation and cancer development. Human class I HDACs possess homology to the yeast RPD3 protein and include HDAC1, HDAC2, HDAC3, and HDAC8. While HDAC1, HDAC2, and HDAC3 have been characterized extensively, almost nothing is known about HDAC8. Here we report that HDAC8 is phosphorylated by cyclic AMP-dependent protein kinase A (PKA) in vitro and in vivo. The PKA phosphoacceptor site of HDAC8 is Ser(39), a nonconserved residue among class I HDACs. Mutation of Ser(39) to Ala enhances the deacetylase activity of HDAC8. In contrast, mutation of Ser(39) to Glu or induction of HDAC8 phosphorylation by forskolin, a potent activator of adenyl cyclase, decreases HDAC8's enzymatic activity. Remarkably, inhibition of HDAC8 activity by hyperphosphorylation leads to hyperacetylation of histones H3 and H4, suggesting that PKA-mediated phosphorylation of HDAC8 plays a central role in the overall acetylation status of histones.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Histona Desacetilases/metabolismo , Proteínas Repressoras/metabolismo , Acetilação , Sequência de Aminoácidos , Sequência de Bases , Sítios de Ligação/genética , Domínio Catalítico/genética , DNA Complementar/genética , Células HeLa , Histona Desacetilases/química , Histona Desacetilases/genética , Histonas/química , Histonas/metabolismo , Humanos , Técnicas In Vitro , Dados de Sequência Molecular , Fosforilação , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Repressoras/química , Proteínas Repressoras/genética , Serina/químicaRESUMO
Methylation of specific residues within the N-terminal histone tails plays a critical role in regulating eukaryotic gene expression. Although great advances have been made toward identifying histone methyltransferases (HMTs) and elucidating the consequences of histone methylation, little is known about the recruitment of HMTs to regulatory regions of chromatin. Here we report that the sequence-specific DNA-binding transcription factor Yin Yang 1 (YY1) binds to and recruits the histone H4 (Arg 3)-specific methyltransferase, PRMT1, to a YY1-activated promoter. Our data confirm that histone methylation does not occur randomly but rather is a targeted event and provides one mechanism by which HMTs can be recruited to chromatin to activate gene expression.