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: 8170865
• 负责人: Gerald V Denis
• 金额: $ 0.93万
• 依托单位: BOSTON UNIVERSITY MEDICAL CAMPUS
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-06-01 至 2011-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8170865
• 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; Chronic Lymphocytic Leukemia; 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; Immunoblotting; Institution; Link; 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; adaptive immunity; base; chromatin remodeling; comparative; functional group; histone acetyltransferase; immunoaffinity chromatography; insight; leukemia/lymphoma; magnetic beads; novel; promoter; protein aminoacid sequence; protein complex; 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 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.

该副本是利用众多研究子项目之一 由NIH/NCRR资助的中心赠款提供的资源。子弹和 调查员（PI）可能已经从其他NIH来源获得了主要资金， 因此可以在其他清晰的条目中代表。列出的机构是 对于中心，这不一定是调查员的机构。 BRD2是一种与基础转录因子TAFII250相关的含双溴脱域的转录因子激酶，它参与了细胞周期中涉及的不完整描述的多蛋白转录复合物。它的双溴结构域是在各种转录剂中经常发现的基序，例如共激活剂，组蛋白乙酰基酶（帽子），组蛋白脱乙酰基酶（HDAC）和其他染色质重塑机，由110个氨基酸基图像组成，由110个氨基酸模式组成，与乙酰化氨基蛋白酶蛋白酶的蛋白酶蛋白酶蛋白酶蛋白蛋白酶构成组成。 BRD2复合物可募集E2F和组蛋白H4指导的组蛋白乙酰转移酶到Cyclin A A启动子，从而有助于正常B细胞​​的细胞周期控制。 B细胞限制的BRD2在转基因小鼠中的本构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械提供了支架，并且与BRD2相关蛋白的时间顺序的身份将有助于揭示该机械的组件和功能。因此，我们对通过免疫亲和力色谱从B细胞纯化的含BRD2的蛋白质复合物进行了基于MSW的蛋白质组学分析。通过基于MAC的磁珠分离，抗CD43阴性选择分离小鼠脾脏B细胞。汇集了细胞质和核提取物，并进行BRD2免疫亲和力色谱柱色谱法，由蛋白A琼脂糖组成，该琼脂糖与抗BRD2抗体共价相关，以最大程度地减少抗体/复杂共洗脱。提取物通过色谱柱，用冰冷的缓冲液广泛洗涤，并经过pH下降以洗脱络合物。 pH中和后，将复合物干燥，或者经过完整的蛋白质MALDI-TOF MS或溶液内胰蛋白酶消化，然后进行肽MALDI-TOF MS。干燥样品被重构，并通过SDS-PAGE进一步分离。切除通过Coomassie染色可视化的离散蛋白带，并用胰蛋白酶内凝胶消化。通过MALDI-TOF MS和LC-MS/MS分析洗脱的肽。 使用这些技术，我们纯化了B细胞中含有BRD2的转录因子复合物，并对它们进行了MS分析。通过肽质量指纹和LC-MS/MS肽测序，我们确定了这些复合物的几种已知和许多新成分，包括基础转录因子，染色质和染色质复发蛋白（SWI/SNF），核激酶和其他核酸酸搭配蛋白。 我们发现的与BRD2相关的SWI/SNF组件集可能定义了调节转录的特定BRD2依赖性染色质复制复合物。免疫印迹已被用来确认几个转录共激活因子和共抑制剂的基于MS的分配，这些分配有助于细胞周期蛋白A的转录控制A。在整个细胞周期中，对这些复合物的正在进行的研究将揭示其动态变化，并提供对其功能的极大洞察力。 B细胞淋巴瘤中BRD2复合物的比较分析可能会使增殖和淋巴细胞化的机理有用。我们所描述的这种多蛋白质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。结果对于我们对适应性免疫和B细胞恶性肿瘤的理解将具有广泛的意义。