BRD2-MULTIPROTEIN COMPLEXES IN MAMMALIAN CELL CYCLE TRANSCRIPTIONAL CONTROL

哺乳动物细胞周期转录控制中的 BRD2-多蛋白复合物

• 批准号: 7722965
• 负责人: Gerald V Denis
• 金额: $ 0.58万
• 依托单位: BOSTON UNIVERSITY MEDICAL CAMPUS
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-06-01 至 2009-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7722965
• 关键词: Amino Acid Motifs; Antibodies; B lymphoid malignancy; B-Cell Lymphomas; B-Lymphocytes; Binding; Bromodomain; Buffers; Cell Cycle; Cell Cycle Regulation; Chromatin; Chromatin Remodeling Factor; Column Chromatography; Complex; Computer Retrieval of Information on Scientific Projects Database; Cyclin A; Digestion; Drops; Fingerprint; Funding; Gel; General Transcription Factors; Genetic Transcription; Grant; Histone H4; Histones; Ice; Immunity; Immunoblotting; Institution; Link; Localized; Lymphomagenesis; Lysine; Malignant Neoplasms; Mammalian Cell; Multiprotein Complexes; Mus; Nuclear; Nuclear Extract; Nucleic Acids; Peptides; Phosphotransferases; Play; Proteins; Proteomics; Recruitment Activity; Research; Research Personnel; Resources; Role; Sampling; Sepharose; Solutions; Source; Staining method; Stains; TAF1 gene; Techniques; Time; Transcriptional Regulation; Transgenic Mice; Trypsin; United States National Institutes of Health; base; chromatin remodeling; comparative; functional group; histone acetyltransferase; immunoaffinity chromatography; insight; leukemia/lymphoma; magnetic beads; novel; promoter; protein aminoacid sequence; reconstitution; scaffold; transcription factor

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.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目及 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 Brd2 是一种含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，参与细胞周期中未完全描述的多蛋白转录复合物。它的双溴结构域是一种常见于各种转录调节因子的基序，例如共激活因子、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，由 110 个氨基酸基序组成，可与乙酰化氨基功能结合核小体组蛋白中的赖氨酸残基组。 Brd2 复合物将 E2F 和组蛋白 H4 定向的组蛋白乙酰转移酶募集至细胞周期蛋白 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 Brd2 的 B 细胞限制性组成型表达会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支架，并且 Brd2 相关蛋白的时间顺序身份将有助于揭示该机制的组件和功能。因此，我们对通过免疫亲和层析从 B 细胞中纯化的含 Brd2 的蛋白质复合物进行了质谱 (MS) 蛋白质组分析。通过基于 MACS 的磁珠分离和抗 CD43 阴性选择来分离小鼠脾 B 细胞。合并细胞质和核提取物并进行 Brd2 免疫亲和柱层析，该层析由与抗 Brd2 抗体共价连接的 Protein A 琼脂糖组成，以最大限度地减少抗体/复合物共洗脱。 提取物通过柱，用冰冷的缓冲液充分洗涤，并降低 pH 值以洗脱复合物。 pH 中和后，将复合物干燥，或进行完整蛋白质 MALDI-TOF MS 或溶液内胰蛋白酶消化，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。通过考马斯染色观察到的离散蛋白质条带被切除并用凝胶内胰蛋白酶消化。 通过 MALDI-TOF MS 和 LC-MS/MS 分析洗脱的肽。 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，并对它们进行了 MS 分析。通过肽质量指纹图谱和 LC-MS/MS 肽测序，我们鉴定了这些复合物的几种已知成分和许多新成分，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 -相关蛋白质。 我们发现的 Brd2 相关 Swi/Snf 组件集可能定义了调节转录的特定 Brd2 依赖性染色质重塑复合物。免疫印迹已用于确认几种有助于细胞周期蛋白 A 转录控制的转录共激活因子和共抑制因子的基于 MS 的分配。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深入的见解进入他们的功能。 B 细胞淋巴瘤中 Brd2 复合物的比较分析可能为增殖和淋巴瘤发生机制提供信息。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。该结果对于我们理解适应性免疫和 B 细胞恶性肿瘤具有广泛的意义。