BRD2-MULTIPROTEIN COMPLEXES IN MAMMALIAN CELL CYCLE TRANSCRIPTIONAL CONTROL

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 Brd2是一种双溴结构域核定位转录因子激酶，与基础转录因子TAFII250相关，参与细胞周期的未完全描述的多蛋白转录复合体。它的双溴结构域是一个经常存在于多种转录调控因子中的基序，如共激活因子、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，由110个氨基酸基序组成，与核小体组蛋白中赖氨酸残基的乙酰化氨基功能基团结合。Brd2复合体将E2F和组蛋白H4导向的组蛋白乙酰转移酶募集到细胞周期蛋白A启动子，有助于正常B细胞的细胞周期控制。B细胞限制性的Brd2在转基因小鼠中的结构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为转录或染色质重塑机制提供了一个支架，Brd2相关蛋白的时序同一性将有助于揭示这一机制的组成和功能。因此，我们对通过免疫亲和层析从B细胞中纯化的含Brd2的蛋白质复合体进行了MS蛋白质组学分析。用MACS磁珠分离法分离小鼠脾B细胞，抗CD43阴性选择。将细胞质和细胞核提取液混合在一起，进行Brd2免疫亲和柱层析，其中蛋白A琼脂糖共价连接到抗Brd2抗体上，以最大限度地减少抗体/复合体的共洗脱。萃取物通过色谱柱，用冰冷缓冲液广泛洗涤，然后进行pH下降以洗脱络合物。在pH中和后，将复合物干燥，或用完整蛋白MALDI-TOF MS或溶液中胰酶消化，然后再用多肽MALDI-TOF MS重组干燥样品，并用SDS-PAGE进一步分离。取Coomassie染色显示的离散蛋白条带，用胰酶凝胶内消化。用MALDI-TOF MS和LC-MS/MS对洗脱肽进行了分析，利用这些技术，我们从B细胞中纯化了含有Brd2的转录因子复合体，并对它们进行了MS分析。通过多肽质量指纹图谱和LC-MS/MS多肽测序，我们已经鉴定了这些复合体的几个已知成分和许多新的成分，包括基础转录因子、染色质和染色质重塑蛋白(Swi/Snf)、核蛋白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依赖的染色质重塑复合体，调节转录。免疫印迹已经被用来确认几个转录辅助激活因子和辅助抑制因子的MS分配，这些转录辅助激活因子和辅助抑制因子有助于细胞周期蛋白A的转录控制。对这些复合体在整个细胞周期中的持续研究将揭示它们的动态变化，并为了解它们的功能提供更好的见解。对B细胞淋巴瘤中Brd2复合体的比较分析可能有助于了解其增殖和淋巴肿大的机制。我们已经描述的这种多蛋白复合体可能有助于细胞周期控制，并在增殖和癌症中发挥作用。这一结果将对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 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.