RESUMO
The E-protein transcription factors play essential roles in lymphopoiesis, with E12 and E47 (hereafter called E2A) being particularly important in B cell specification and maturation. The E2A gene is also involved in a chromosomal translocation that results in the leukemogenic oncoprotein E2A-PBX1. The two activation domains of E2A, AD1 and AD2, display redundant, independent, and cooperative functions in a cell-dependent manner. AD1 of E2A functions by binding the transcriptional co-activator CBP/p300; this interaction is required in oncogenesis and occurs between the conserved Ï-x-x-Ï-Ï motif in AD1 and the KIX domain of CBP/p300. However, co-activator recruitment by AD2 has not been characterized. Here, we demonstrate that the first of two conserved Ï-x-x-Ï-Ï motifs within AD2 of E2A interacts at the same binding site on KIX as AD1. Mutagenesis uncovered a correspondence between the KIX-binding affinity of AD2 and transcriptional activation. Although AD2 is dispensable for oncogenesis, experimentally increasing the affinity of AD2 for KIX uncovered a latent potential to mediate immortalization of primary hematopoietic progenitors by E2A-PBX1. Our findings suggest that redundancy between the two E2A activation domains with respect to transcriptional activation and oncogenic function is mediated by binding to the same surface of the KIX domain of CBP/p300.
Assuntos
Fator 3 de Transcrição/química , Ativação Transcricional , Fatores de Transcrição de p300-CBP/química , Sítios de Ligação , Células da Medula Óssea/metabolismo , Modelos Moleculares , Domínios e Motivos de Interação entre Proteínas , Estrutura Terciária de Proteína , Fator 3 de Transcrição/metabolismo , Fatores de Transcrição de p300-CBP/metabolismoRESUMO
E-proteins are critical transcription factors in B-cell lymphopoiesis. E2A, 1 of 3 E-protein-encoding genes, is implicated in the induction of acute lymphoblastic leukemia through its involvement in the chromosomal translocation 1;19 and consequent expression of the E2A-PBX1 oncoprotein. An interaction involving a region within the N-terminal transcriptional activation domain of E2A-PBX1, termed the PCET motif, which has previously been implicated in E-protein silencing, and the KIX domain of the transcriptional coactivator CBP/p300, critical for leukemogenesis. However, the structural details of this interaction remain unknown. Here we report the structure of a 1:1 complex between PCET motif peptide and the KIX domain. Residues throughout the helical PCET motif that contact the KIX domain are important for both binding KIX and bone marrow immortalization by E2A-PBX1. These results provide molecular insights into E-protein-driven differentiation of B-cells and the mechanism of E-protein silencing, and reveal the PCET/KIX interaction as a therapeutic target for E2A-PBX1-induced leukemia.
Assuntos
Proteínas de Homeodomínio/química , Leucemia/genética , Proteínas de Fusão Oncogênica/química , Fatores de Transcrição de p300-CBP/química , Motivos de Aminoácidos , Sequência de Aminoácidos , Fatores de Transcrição Hélice-Alça-Hélice Básicos/química , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Transformação Celular Neoplásica/genética , Sequência Conservada , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Leucemia/metabolismo , Modelos Moleculares , Simulação de Acoplamento Molecular , Dados de Sequência Molecular , Mutação , Proteínas de Fusão Oncogênica/genética , Proteínas de Fusão Oncogênica/metabolismo , Ligação Proteica/genética , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Fatores de Transcrição de p300-CBP/metabolismoRESUMO
E-proteins are basic helix-loop-helix transcription factors that function in cell type specification. The gene E2A encodes two E-proteins, E12 and E47, which are required in B-lymphopoiesis. E2A proteins can interact directly with the transcriptional co-activators and lysine acetyltranferases (KATs) CBP, p300 and PCAF to induce target gene transcription. Prior investigations have shown that the E2A-encoded isoform E2-5 is acetylated by CBP, p300 or PCAF in vitro or in vivo. However, E2-5 lacks the important N-terminal activation domain AD1. Furthermore, the acetylated residues in E-proteins have not been mapped, and the functional consequences of acetylation are largely unknown. Here, we use mutagenesis to show that a lysine residue at position 34 within AD1 of E12/E47 is acetylated by CBP/p300 and PCAF. Lys34 lies adjacent to a conserved helical LXXLL motif that interacts directly with the KIX domain of CBP/p300. We show that acetylation at Lys34 increases the affinity of AD1 for the KIX domain and enhances AD1-driven transcriptional induction. Our results illustrate for the first time that AD1 can both recruit, and be acetylated by, KATs and that KAT recruitment may promote transcriptional induction in part through acetylation of AD1 itself.