RESUMO
The class I helix-loop-helix (HLH) proteins, which include E2A, HEB, and E2-2, have been shown to be required for lineage-specific gene expression during T and B lymphocyte development. Additionally, the E2A proteins function to regulate V(D)J recombination, possibly by allowing access of variable region segments to the recombination machinery. The mechanisms by which E2A regulates transcription and recombination, however, are largely unknown. Here, we identify a novel motif, LDFS, present in the vertebrate class I HLH proteins as well as in a yeast HLH protein that is essential for transactivation. We provide both genetic and biochemical evidence that the highly conserved LDFS motif stimulates transcription by direct recruitment of the SAGA histone acetyltransferase complex.
Assuntos
Sequência Conservada , Sequências Hélice-Alça-Hélice/genética , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Ativação Transcricional/genética , Sequência de Aminoácidos , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Proteínas de Ligação a DNA/genética , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica , Células HeLa , Humanos , Dados de Sequência Molecular , Recombinação Genética , Alinhamento de Sequência , Fatores de Transcrição TCF , Transativadores/genética , Proteína 2 Semelhante ao Fator 7 de Transcrição , Fatores de Transcrição/genética , TransfecçãoRESUMO
A number of transcriptional coactivator proteins have been identified as histone acetyltransferase (HAT) proteins, providing a direct molecular basis for the coupling of histone acetylation and transcriptional activation. The yeast Spt-Ada-Gcn5-acetyltransferase (SAGA) complex requires the coactivator protein Gcn5 for HAT activity. Identification of protein subunits by mass spectrometry and immunoblotting revealed that the TATA binding protein-associated factors (TAF(II)s) TAF(II)90, -68/61, -60, -25/23, and -20/17 are integral components of this complex. In addition, TAF(II)68 was required for both SAGA-dependent nucleosomal HAT activity and transcriptional activation from chromatin templates in vitro. These results illustrate a role for certain TAF(II) proteins in the regulation of gene expression at the level of chromatin modification that is distinct from the TFIID complex and TAF(II)145.
Assuntos
Acetiltransferases/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas Fúngicas/metabolismo , Nucleossomos/metabolismo , Proteínas de Saccharomyces cerevisiae , Fatores Associados à Proteína de Ligação a TATA , Fatores de Transcrição/metabolismo , Transcrição Gênica , Histona Acetiltransferases , Histonas/metabolismo , Proteínas Quinases/metabolismo , Saccharomyces cerevisiae , Proteína de Ligação a TATA-Box , Fator de Transcrição TFIIDRESUMO
Prior to ligand activation, the unactivated aryl hydrocarbon receptor (AhR) exists in a heterotetrameric 9S core complex consisting of the AhR ligand-binding subunit, a dimer of hsp90, and an unknown subunit. Here we report the purification of an approximately 38-kDa protein (p38) from COS-1 cell cytosol that is a member of this complex by coprecipitation with a FLAG-tagged AhR. Internal amino acid sequence information was obtained, and p38 was identified as the hepatitis B virus X-associated protein 2 (XAP2). The simian ortholog of XAP2 was cloned from a COS-1 cDNA library; it codes for a 330-amino-acid protein containing regions of homology to the immunophilins FKBP12 and FKBP52. A tetratricopeptide repeat (TPR) domain in the carboxy-terminal region of XAP2 was similar to the third and fourth TPR domains of human FKBP52 and the Saccharomyces cerevisiae transcriptional modulator SSN6, respectively. Polyclonal antibodies raised against XAP2 recognized p38 in the unliganded AhR complex in COS-1 and Hepa 1c1c7 cells. It was ubiquitously expressed in murine tissues at the protein and mRNA levels. It was not required for the assembly of an AhR-hsp90 complex in vitro. Additionally, XAP2 did not directly associate with hsp90 upon in vitro translation, but was present in a 9S form when cotranslated in vitro with murine AhR. XAP2 enhanced the ability of endogenous murine and human AhR complexes to activate a dioxin-responsive element-luciferase reporter twofold, following transient expression of XAP2 in Hepa 1c1c7 and HeLa cells.
Assuntos
Antígenos da Hepatite B/química , Proteínas Quinases/química , Proteínas/química , Receptores de Hidrocarboneto Arílico/química , Ativação Transcricional , Sequência de Aminoácidos , Animais , Sequência de Bases , Células COS , Dimerização , Genes Reporter , Proteínas de Choque Térmico HSP90/metabolismo , Células HeLa , Antígenos da Hepatite B/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Luciferases/genética , Luciferases/metabolismo , Substâncias Macromoleculares , Dados de Sequência Molecular , Conformação Proteica , Proteínas Quinases/metabolismo , Proteínas/metabolismo , Receptores de Hidrocarboneto Arílico/metabolismoRESUMO
The SAGA histone acetyltransferase/transcriptional adaptor complex is composed of multiple transcriptional regulators including Ada, Spt, and TAFII proteins. Here we identify an additional novel subunit of the complex, Tra1, an ATM/PI-3-kinase-related homolog of the human TRRAP cofactor, which is essential for c-Myc and E2F-mediated oncogenic transformation. Mass spectrometry, immunoblotting, and immunoprecipitation experiments confirm the stable association of this protein within SAGA. In addition, the Tra1 protein is a component of at least two other histone acetyltransferase protein complexes. These results indicate a role for Tra1 in the regulation of transcriptional activation through the recruitment of HAT activity to an activator-bound promoter.
