BRD2-MULTIPROTEIN COMPLEXES IN MAMMALIAN CELL CYCLE TRANSCRIPTIONAL CONTROL

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目及 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 Brd 2是一种双溴结构域的核定位的转录因子激酶，与基础转录因子TAFII 250相关，参与细胞周期中涉及的不完全描述的多蛋白转录复合物。它的双溴结构域是一种经常在各种转录调节因子如共激活因子、组蛋白乙酰化酶（HAT）、组蛋白脱乙酰酶（HDAC）和其他染色质重塑机器中发现的基序，由110个氨基酸的基序组成，其结合核小体组蛋白中赖氨酸残基的乙酰化氨基官能团。Brd 2复合物募集E2 Fs和组蛋白H4-定向的组蛋白乙酰转移酶到细胞周期蛋白A启动子，有助于正常B细胞的细胞周期控制。转基因小鼠中Brd 2的B细胞限制性组成型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Brd 2为转录或染色质重塑机制提供了一个支架，并且Brd 2相关蛋白的时间顺序身份将有助于揭示该机制的组成和功能。因此，我们对通过免疫亲和层析从B细胞纯化的含Brd 2的蛋白质复合物进行了基于MSW的蛋白质组学分析。小鼠脾B细胞通过基于MACS的磁珠分离与抗CD 43阴性选择分离。合并细胞质和细胞核提取物，并进行Brd 2免疫亲和柱层析，该柱层析由与抗Brd 2抗体共价连接的蛋白A琼脂糖组成，以使抗体/复合物共洗脱最小化。将提取物通过柱，用冰冷的缓冲液充分洗涤，并使pH下降以使复合物稳定。pH中和后，将复合物干燥，或进行完整蛋白质MALDI-TOF MS或溶液中胰蛋白酶消化，然后进行肽MALDI-TOF MS。将干燥的样品重构并通过SDS-PAGE进行进一步分离。切下通过考马斯染色可视化的离散蛋白质条带，并用胰蛋白酶在凝胶中消化。通过MALDI-TOF MS和LC-MS/MS分析洗脱的肽。使用这些技术，我们从B细胞纯化含Brd 2的转录因子复合物，并对其进行MS分析。通过肽质量指纹图谱和LC-MS/MS肽测序，我们确定了这些复合物的几个已知的和许多新的组件，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Brd 2相关的Swi/Snf组件集可能定义了一个特定的Brd 2依赖的染色质重塑复合物，调节转录。免疫印迹已被用于确认基于MS的分配的几个转录共激活因子和共抑制因子，有助于细胞周期蛋白A的转录控制。对这些复合物在整个细胞周期中的持续研究将揭示它们的动态变化，并提供对其功能的深入了解。在B细胞淋巴瘤的Brd 2复合物的比较分析可能是信息的增殖和淋巴瘤发生的机制。我们描述的这种多蛋白复合物可能有助于细胞周期控制，并在增殖和癌症中发挥作用。这些结果对于我们理解获得性免疫和B细胞恶性肿瘤具有广泛的意义。

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 an MSW-based 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 purified Brd2-containing transcription factor complexes from B cells and subjected them to MS analyses. Through peptide mass fingerprinting and LC-MS/MS peptide sequencing, we 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.