【作者】 沈为群；

【导师】 施蕴渝； 吴季辉；

【作者基本信息】 中国科学技术大学 ， 生物化学与分子生物学， 2006， 博士

【摘要】 本论文工作的重点是人染色质重构复合物SWI／SNF的核心亚基Brg1的Bromodomain结构域的克隆、表达、纯化和溶液结构测定以及它与乙酰化组蛋白尾巴的相互作用研究。论文分为以下两个部分：第一部分对染色质重构复合物（第一章）、组蛋白密码（第二章）和Brg1蛋白质的生物学功能（第三章）进行全面的综述。染色质重构复合物是真核生物基因转录的共调控因子，它通过改变染色质结构而使相应的基因“裸露”出来，使其它转录因子或转录起始复合物能够在正确的位置装配，从而激活基因表达。真核生物的染色质重构复合物至少可分为4类：SWI／SNF类，ISWI类，Mi-2类和DOMINO类，它们对染色质的重构都依赖于ATP的水解。染色质重构复合物还在DNA复制、DNA损伤修复和基因的转录抑制中起作用。组蛋白密码是指一条或几条组蛋白尾巴的顺序修饰（乙酰化、甲基化、磷酸化、SUMO化、泛素化等）及其组合。这些密码可以被含有特定结构域如Bromodomain和chromodomain的蛋白质解读。这些蛋白质又招募其效应蛋白质，如通用转录因子、RNA聚合酶等，从而启动下游生物学反应如染色质凝集、DNA修复或转录激活／抑制。不同的修饰方式及其组合产生不同的局部结构和不同的生物学功能。组蛋白密码是表观遗传的重要内容之一。作为SWI／SNF复合物的核心组分，Brg1在基因调控，细胞因子应答，肿瘤发生，发育和分化过程中起重要作用。5-6％的酵母基因受Brg1调控，哺乳动物中至少有100多个基因受其影响。最显著的是Brg1可促进CDK抑制蛋白p21和p15的表达，同时通过与pRB的相互作用抑制E2F目标蛋白质（包括cyclin E，cyclin A和CDC2）的表达，从而导致细胞周期中止。因此，Brg1功能的丧失可能导致肿瘤发生。10％的肺癌同时伴有BRG1的缺失。乳腺癌、胰腺癌和前列腺癌细胞系中也有较高比例的BRG1基因突变。Brg1还在发育和分化中起重要作用。许多发育分化相关的调控因子都通过SWI／SNF发挥其作用。红细胞、骨髓细胞、脂肪细胞、成骨细胞、肌肉细胞和神经细胞发育的调控基因的激活都需要SWI／SNF的参与。如生肌决定因子（MyoD）的转录激活作用需要Brg1的参与；神经元分化因子NeuroD和Ngnr1的促神经生成作用依赖于Brg1。第二部分详细介绍了Brg1 Bromodomain的溶液结构及其与乙酰化组蛋白的相互作用研究。Bromodomain主要存在于染色质调控蛋白质中，专一性结合乙酰化赖氨酸，在组蛋白密码的解读中起重要作用。它有约110个氨基酸残基，是左手四螺旋束的拓扑结构。我们通过基因重组表达了¹⁵N-标记和¹⁵N／¹³C双标记的Brg1 Bromodomain蛋白质。用Ni离子亲和层析和分子筛两步纯化获得了较稳定的NMR实验样品。通过异核三维核磁共振的方法，测定了Brg1 Bromodomain的溶液结构。解析出的Brg1Bromodomain溶液结构同其它Bromodomain蛋白质相似，都具有Bromodomain特征的左手四螺旋结构，在螺旋束的一端有一个疏水的乙酰化赖氨酸结合口袋；不同的是Brg1 Bromodomain的第一个螺旋αZ比其它Bromodomain短约4个氨基酸。用CE方法，我们发现在已知结构中，它与scGCN5的结构相似性最高（RMSD≈2.1A）。用化学位移干扰实验，我们研究了Brg1 Bromodomain对不同位点乙酰化的组蛋白N-端多肽结合专一性并计算了解离常数（K_D）。NMR滴定结果显示乙酰化的组蛋白多肽对Brg1 Bromodomain的化学位移都有不同程度的干扰，但所需的多肽浓度都较高。这表明Brg1 Bromodomain对乙酰化组蛋白有比较弱的相互作用。其中，H3-AcK14的亲和力最大（K_D≈1.2mM），H4-AcK8（K_D≈4.0mM）和H4-AcK12（K_D≈3.6mM）次之，H4-AcK16和H2B AcK5最弱。非乙酰化的H4 N-端多肽不能结合Brg1 Bromodomain。以scGcn5与组蛋白H3-AcK16多肽复合物的晶体结构（PDB ID：1E6I．pdb）为模板，用分子动力学模拟（MD）的方法，我们构建了Brg1 Bromodomain与组蛋白N-端多肽H3-AcK14的复合物模型。分析发现残基F1485、L1488、L1494、F1539、N1540与H3-AcK14有相互作用；T1538与H3-AcK14的Arg17之间存在氢键。为验证化学位移干扰实验和复合物模型所揭示的相互作用，我们进行了Brg1Bromodomain和H3-AcK14多肽的突变研究。我们获得了三个Brg1 Bromodomain突变体（V1484／A、F1539／A、N1540／A）和一个H3-AcK14突变体（H3-R17／A），并进行了一系列化学位移干扰实验。突变分析证实，F1539和N1540在H3-AcK14结合中起重要作用，而V1484不影响结合；T1538与H3-AcK14的Arg17之间的氢键是选择性结合H3-AcK14的关键相互作用。更多还原

【Abstract】 This dissertation reports on the solution structure and the acetylated histone binding property of the bromodomain from human Brg1, the core catalytic subunit of the chromatin remodeling complex SWI/SNF.The first part of the dissertation is a substantive review of chromatin remodeling complex （chapter 1）, histone code （chapter 2） and the biological function of Brg1 （chapter 3）. Chromatin remodeling complexes are coregulators in eukaryotic gene expression. They remodel nucleosomes to expose the promoter of particular genes, enabling the assembly of transcription activators or transcription initiation complexes. Chromatin remodeling complexes can be classified into four groups: SWI/SNF, ISWI, Mi-2 and DOMINO. They are all ATP dependent chromatin modifiers functioning in DNA replication, DNA repair and transcription activation/inhibition.The histone code hypothesis refers to the sequential modification of histone tails including acetylation, methylation, phosphorylation, sumolyzation and ubiqitination. Defined patterns of modifications, possibly acting in combination, can be recognized by specific factors such as bromodomain proteins and chromodomain proteins, which in turn, induce the recruitment of particular effectors , thereby translating the histone-modification pattern into a particular chromatin state to bring about downstream biological reactions including chromatin condensation, DNA repair or transcription activation/inhibition. Histone code is an important content of epigenetic.As the core catalytic subunit of SWI/SNF complex, Brg1 play a critical role in gene regulation, cell cycle control, tumorigenesis, cell proliferation and differentiation. About 5-6% of yeast genes are regulated by Brg1; at least more than 100 genes are affected by Brg1 in mammals. Brg1 can inhibit the expression of E2F target genes including Cyclin E, Cyclin A and CDC2 by up-regulating the expression of p21 and p15, and by interaction with pRB, leading to cell cycle arrest. Therefore, loss of Brg1 function may promote tumor development. 10% of lung cancers are accompanied with loss of Brg1; high ratio of BRG1 mutations are also found in cell lines from breast cancer, pancreas caner and prostate cancer. Many development regulators function by interacting with SWI/SNF complex. SWI/SNF complex involves in the activation of regulatory genes for the development of heamatopoitic cell, myeloid cell, adipose cell, neural cell and myocyte. For example, MyoD recruits Brg1 to the myogenin promoter; the proneural activities of Ngnr1 and NeuroD are Brg1 dependent.The second part of the dissertation details the solution structure of Brg1 Bromodomain and its interaction with acetylated histone peptides. Bromodomain proteins largely associate with chromatin regulation. They recognize histones acetylated at specific lysine residues in deciphering the histone code. It has about 110 amino acids and adopts a left-handed four-helix bundle topology.We expressed ¹⁵N and/or ¹⁵N/¹³C labled Brg1 Bromodomain and obtained purified samples by Ni⁺ affinity chromatography and size-exclusion chromatography. Using 3D NMR methodology, we resolved the solution structure of Brg1 Bromodomain, which conserves the left-handed four-helix bundle topology; on top of which resides the hydrophobic acetyl-lysine binding pocket. The distinct feature of Brg1 Bromodomain is that itsαZ helix is about 4 residues shorter than published bromodomain structures. Wefound it shares the highest structural similarity with scGCN5 （RMSD≈2.1 A）.Using NMR perturbation studies, we demonstrate that Brg1 Bromodomain binds acetyl-lysine in the context of histone tails, with no comparable affinity for unacetylated peptides. The bindings are generally weak. The estimated dissociation constants （K_D） for acetylated histone peptides H4-AcK8 and H4-AcK12 are 4.0 mM and 3.6 mM respectively. The dominant substrate was H3-AcK14 （K_D≈1.2mM）.Using Molecular Dynamics （MD） simulation with the crystal structure of scGCN5-H4-AcK16 （PDB ID 1E6I） complex as a template, we built a model of the Brg1 Bromodomain in complex with H3-AcK14. The model reveals that residues F1485, L1488, L1494, F1539 and N1540 are interacting with H3-AcK14 peptide; and the backbone carbonyl of T1538 forms a hydrogen bond with the side-chain NH₂ of Arg 17 of H3-AcK14.To verify the interactions indicated by NMR titration and the complex model, we performed mutagenesis research both in Brg1 Bromodomain and H3-AcK14. We obtained three Brg1 Bromodomain mutants （V1484/A, F1539/A, N1540/A） and one H3-AcK14 mutant （R17/A）, with which NMR titration experiments were carried out. The mutagenesis analysis confirms that F1539 and N1540 are essential for H3-AcK14 binding, while V1484 is irrelevant, and that the hydrogen bond between T1538 and Arg 17 of H3-AcK14 is critical for H3-AcK14 selectivity.更多还原