This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Brd2 is a double bromodomain-containing nuclear-localized transcription factor kinase, related to the basal transcription factor TAFII250, that participates in an incompletely described multiprotein transcriptional complex involved in the cell cycle. Its double bromodomain, a motif often found in various regulators of transcription such as co-activators, histone acetylases (HATs), histone deacetylases (HDACs), and other chromatin remodeling machines, consists of a 110 amino acid motif that binds to acetylated amino functional groups of lysine residues in nucleosomal histone proteins. Brd2 complexes recruit E2Fs and histone H4-directed histone acetyltransferase to the cyclin A promoter, contributing to cell cycle control in normal B cells. B cell-restricted constitutive expression of Brd2 in transgenic mice transcriptionally activates cyclin A, leading to B cell leukemia and lymphoma. We hypothesize that Brd2 provides a scaffold for transcription or chromatin remodeling machinery and that the time-ordered identity of Brd2-associated proteins will help to reveal the components and functions of this machinery. Therefore, we have undertaken a mass spectrometric (MS) proteomic analysis of Brd2-containing protein complexes purified from B cells through immunoaffinity chromatography. Mouse splenic B cells were isolated by MACS-based magnetic bead separation with anti-CD43 negative selection. Cytoplasmic and nuclear extracts were pooled and subjected to Brd2 immunoaffinity column chromatography, consisting of Protein A agarose that was covalently linked to anti-Brd2 antibodies to minimize antibody/complex co-elution. Extracts were passed over the column, washed extensively with ice-cold buffer and subjected to a pH drop to elute the complex. After pH neutralization, complexes were dried down, or subjected to intact protein MALDI-TOF MS or in-solution tryptic digestion followed by peptide MALDI-TOF MS. Dried samples were reconstituted and subjected to further separation by SDS-PAGE. Discrete protein bands visualized by Coomassie staining were excised and digested with trypsin in-gel. Eluted peptides were analyzed by MALDI-TOF MS and by LC-MS/MS. Using these techniques, we have purified Brd2-containing transcription factor complexes from B cells and have subjected them to MS analyses. Through peptide mass fingerprinting and LC-MS/MS peptide sequencing, we have identified several known and many novel components of these complexes, including basal transcription factors, chromatin and chromatin-remodeling proteins (Swi/Snf), nuclear kinases and other nucleic acid-associated proteins. The Brd2-associated set of Swi/Snf components we have found may define a specific Brd2-dependent chromatin- remodeling complex that regulates transcription. Immunoblots have been used to confirm the MS-based assignments of several of the transcription co-activators and co-repressors which contribute to transcriptional control of cyclin A. Ongoing studies of these complexes throughout the cell cycle will reveal their dynamic changes and provide great insight into their functionality. Comparative analyses of the Brd2 complexes in B cell lymphoma are likely to be informative of mechanisms of proliferation and lymphomagenesis. This multiprotein complex we have described is likely to contribute to cell cycle control and play a role in proliferation and cancer. The results will have broad significance for our understanding of adaptive immunity and B cell malignancy.
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