Analysis of BCL6-interacting proteins by tandem mass spectrometry

B-cell lymphoma 6 (BCL6) is a 95-kDa nuclear phosphoprotein and member of the Pox virus zinc finger/bric-a-brac, tramtrack, broad complex (POZ/BTB) family of transcription factors. BCL6 is a transcriptional repressor required for germinal center formation, and the gene encoding it is frequently altered in diffuse large B-cell and follicular lymphomas. The dysregulation of BCL6 has therefore been implicated in lymphomagenesis. A limited number of proteins is known to interact with BCL6 and modulate its activity or participate in its role in transcriptional regulation. Identification of additional BCL6-binding proteins could reveal potential signaling targets and previously undescribed functional roles for BCL6. We used a functional proteomic approach to determine the identity of proteins that interact with BCL6. Proteins were isolated by co-immunoprecipitation with an anti-BCL6 antibody and identified using MS/MS. We identified 61 proteins in the BCL6 immunocomplex from the following Gene Ontology categories: transcription regulator activity (n = 18), binding activity (n = 11), signal transducer activity (n = 10), catalytic activity (n = 8), structural molecule activity (n = 3), enzyme regulator activity (n = 3), transporter activity (n = 2), motor activity (n = 2), chaperone activity (n = 1), and unknown function (n = 3). Importantly we identified BCL6 and several previously reported BCL6-interacting proteins in the BCL6 immunocomplex. The remaining proteins have not been shown previously to be associated with BCL6. MS/MS results were validated on four proteins using immunoprecipitation and Western blotting. Two of these protein interactions were further confirmed by reciprocal immunoprecipitation. This study demonstrates the utility of antibody immunoprecipitation and subsequent peptide identification by MS/MS for the elucidation of BCL6-binding proteins. Many of the novel proteins identified in this study suggest additional functional roles for BCL6 beyond transcriptional repression.