BRD2-MULTIPROTEIN COMPLEXES IN MAMMALIAN CELL CYCLE TRANSCRIPTIONAL CONTROL

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。Brd2是一种含有双溴结构域的核定位转录因子激酶，与基础转录因子TAFII250相关，参与一个未完全描述的多蛋白转录复合物，参与细胞周期。它的双溴结构域是一种常在各种转录调节因子中发现的基序，如共激活剂、组蛋白乙酰化酶（HATs）、组蛋白去乙酰化酶（hdac）和其他染色质重塑机器，它由110个氨基酸基序组成，与乙酰化？核小体组蛋白中赖氨酸残基的氨基官能团。Brd2复合物招募e2f和组蛋白H4？将组蛋白乙酰转移酶定向到周期蛋白A启动子，有助于正常B细胞的细胞周期控制。转基因小鼠B细胞限制性Brd2组成性表达转录激活细胞周期蛋白A，导致B细胞白血病和淋巴瘤。我们假设Brd2提供了一个“支架”brd2相关蛋白的时序特性将有助于揭示这一机制的组成和功能。因此，我们通过免疫亲和层析对从B细胞中纯化的含brd2的蛋白复合物进行了质谱（MS）蛋白质组学分析。采用抗cd43阴性选择磁珠分离技术分离小鼠脾B细胞。将细胞质和细胞核提取物混合并进行Brd2免疫亲和柱层析，由与抗Brd2抗体共价连接的蛋白A琼脂糖组成，以减少抗体/复合物的共洗脱。提取液通过色谱柱，用冰冷的缓冲液广泛洗涤，并使pH值下降以洗脱复合物。pH中和后，将配合物干燥，或进行完整蛋白MALDI-TOF MS或溶液中胰蛋白酶消化，然后进行多肽MALDI-TOF MS。取考马斯氏染色显示的离散蛋白带，用胰蛋白酶凝胶消化。洗脱肽采用MALDI-TOF质谱和LC-MS/MS分析。利用这些技术，我们从B细胞中纯化了含有brd2的转录因子复合物，并对其进行了质谱分析。通过肽质量指纹图谱和LC-MS/MS肽测序，我们已经鉴定了这些复合物的几个已知的和许多新的组分，包括基础转录因子、染色质和染色质重塑蛋白（Swi/Snf）、核激酶和其他核酸相关蛋白。我们发现的与brd2相关的Swi/Snf组分可能定义了一种特定的brd2依赖性染色质重塑复合体，该复合体调节转录。对这些复合物在整个细胞周期内的持续研究将揭示它们的动态变化，并对它们的功能提供深入的了解。Brd2复合物在B细胞淋巴瘤中的比较分析可能为增殖和淋巴瘤发生的机制提供信息。该结果将对我们对适应性免疫和B细胞恶性肿瘤的认识具有广泛的意义。

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. 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. The results will have broad significance for our understanding of adaptive immunity and B cell malignancy.