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Anal Biochem ; 402(1): 65-8, 2010 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-20338149


Histone acetyl transferases are important regulators of cellular homeostasis. This study describes a sensitive acetyl transferase electrophoretic mobility shift assay applicable both for kinetic analysis of acetyl transferase inhibitors and for high-throughput testing. Application of the assay for human GCN5L2 enabled dissection of inhibitor competition with respect to acetyl coenzyme A. Furthermore, we demonstrated that the assay can detect time-dependent inhibition of human GCN5L2 by reactive inhibitors.

Ensaio de Desvio de Mobilidade Eletroforética/métodos , Histona Acetiltransferases/antagonistas & inibidores , Histona Acetiltransferases/metabolismo , Acetilcoenzima A/metabolismo , Animais , Linhagem Celular , Humanos , Cinética
Mol Cell Biol ; 22(20): 7337-50, 2002 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-12242308


E47 and Pip are proteins crucial for proper B-cell development. E47 and Pip cooperatively bind to adjacent sites in the immunoglobulin kappa chain 3' enhancer and generate a potent transcriptional synergy. We generated protein-DNA computer models to visualize E47 and Pip bound to DNA. These models predict precise interactions between the two proteins. We tested predictions deduced from these models by mutagenesis studies and found evidence for novel direct interactions between the E47 helix-loop-helix domain (Arg 357 or Asp 358) and the Pip N terminus (Leu 24). We also found that precise spatial alignment of the binding sites was necessary for transcriptional synergy and cooperative DNA binding. A Pip dominant negative mutant that cannot synergize with E47 inhibited enhancer activity in plasmacytoma cells and could not activate transcription in pre-B cells. Using electrophoretic mobility shift assays, we found that Pip can bind to the heavy-chain intron enhancer region. In addition, we found that in fibroblasts Pip greatly increased E47 induction of germ line I micro transcripts associated with somatic rearrangement and isotype class switching. However, a Pip dominant negative mutant inhibited germ line I micro transcripts. The importance of these results for late B-cell functions is discussed.

Proteínas de Ligação a DNA/metabolismo , Elementos Facilitadores Genéticos , Sequências Hélice-Alça-Hélice , Cadeias mu de Imunoglobulina/genética , Fatores de Transcrição/metabolismo , Células 3T3 , Sequência de Aminoácidos , Animais , Linfócitos B/citologia , Linfócitos B/fisiologia , Linhagem da Célula , DNA/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/fisiologia , Humanos , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias kappa de Imunoglobulina/genética , Fatores Reguladores de Interferon , Íntrons , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Insercional , Fatores de Transcrição TCF , Proteína 1 Semelhante ao Fator 7 de Transcrição , Fatores de Transcrição/química , Fatores de Transcrição/genética , Transcrição Genética