核受体作为转录因子的重要成员，在调节生物体的生长发育和保持体内稳态等诸多方面有十分重要的作用。核受体与其特异性配基结合后将发生结构的变化，结合于靶基因特异性序列之后将招募大量的基因转录激活因子及其复合体。核受体的转录激活包括解除抑制和激活两个方面。核受体的抑制作用是在无配基或配基拮抗剂存在时通过招募辅助抑制因子，如 N-CoR ( nuclear receptor corepressor ) 和SMRT ( silencing mediator for RAR and TR )来实现的，而配基的结合导致核受体的构像改变幷导致抑制因子的解离和协同激活因子的募集。 到目前为止，许多核受体的协同激活因子已被发现 ，其中包括CBP / p300 (cAMP responsive element binding protein-binding protein/protein 300)、p300/CBP相关蛋白因子 pCAF ( p300 / CBP-associated protein factor )、RIP140 ( receptor -interacting protein 140 )、 RIP-160 ( receptor-interacting protein 160)、 ERAP-140 (estrogen receptor-associated protein 140) 及SRC家族 (steroid receptor coactivators，SRCs)。协同激活因子通过蛋白质相互作用起到联系转录因子和转录起始复合体的作用，幷通过其染色质重塑的活性来促进基因的转录激活。 SRC家族 (又称类固醇激素受体协同激活因子) 是在结构和功能上都有许多相似性的功能蛋白 ，其中包括 SRC-1/NcoA1 ( steroid receptor-interacting protein 1/nuclear receptor coactivatior 1 )、GRIP1/ TIF2 /SRC-2 / NcoA2 ( glucocorticoid receptor - interacting protein1 / transcriptional Intermediary factor 2 / steroid receptor coactivator 2 / nuclear receptor coactivator-2 ) 和 pCIP/ACTR/AIB1/RAC3 / TRAM1/ SRC-3 / NcoA3 ( p300 / CBP interacting protein / activator for thyroid hormone and retinoid receptors / amplified in breast cancer 1 / receptor-associated coactivator 3 / thyroid hormone receptor activator molecule 1/ steroid receptor coactivator 3 / nuclear receptor coactivator 3) 三种成份。它们通过直接或间接与其它协同激活因子的相互作用来增强核受体介导的转录激活。尽管SRC家族在结构和功能上都有相似之处，但病理学证据和SRCs敲除小鼠的不同表型变化提示它们在动物的生理功能方面有不同作用。然而目前在分子水平仍没有明显的证据支持它们的特异性功能，因此，本研究从以下三个方面对SRC家族协同激活因子在基因调控中不同作用的分子机理进行了探讨。 在第一部分研究了在雌激素受体和雄激素受体相关肿瘤细胞中，SRC-1、GRIP1和 AIB1在核受体介导的基因调控中的特异性。本研究结果提示：(1) 在含有经典激素反应元件 (Classical hormone responsive element, HRE)的晚期基因，SRCs以AIB1:GRIP1 或AIB1:SRC-1异二聚体的形式结合于靶基因启动子；(2) 而在不含激素经典反应元件的早期基因， GRIP1 或 SRC-1以单体形式结合于靶基因启动子，而这种结合方式是通过间接与启动子结合的类固醇激素受体而实现的，并且GRIP1 或 SRC-1与早期基因启动子的结合依赖于它们在细胞中的表达丰度；(3) SRCs参与类固醇受体介导的早期反应基因和晚期反应基因的时序激活；(4) 在ER阳性的乳腺癌组织中，AIB1与c-myc的高表达有明显的正相关，阐明了AIB1高表达与雌激素受体阳性的乳腺癌发生的联系。 实验第二部分主要通过酵母双杂交实验来筛选与SRC家族成员相互作用的不同蛋白，预期从另一个侧面对SRC家族在基因表达中的特异性作用提供线索。我们分别构建缺失核受体相互作用结构域C末端片段的Gal4 DBD酵母表达融合蛋白质粒pGBKT7-SRC-1C (pGBKT7-SRC-1N), pGBKT7-Grip1C (pGBKT7-Grip1N) 和 pGBKT7-AIBC ( pGBKT7-AIB1N)，幷用Matchmaker Gal4 two-hybrid system 3对人乳腺癌cDNA文库进行筛选。经过不同的筛选程序后，共筛选出16个已知和未知功能的基因。其中LMP2 (large multifunctional protease 2) 被pGBKT7-SRC-1N 和pGBKT7-Grip1N 在不同实验中随机筛选出来，同时pGBKT7-SRC-1N杂出的两种基因STATA 5B和c-fos与文献报道的结果一致。这些结果说明酵母双杂交的结果能为SRC家族在基因调控中的不同作用提供一些信息。 实验第三部分研究了LMP2在SRC家族介导的基因调控中的作用。LMP2是分别通过pGBKT7-SRC-1N和pGBKT7-Grip1N SRC-1N 酵母表达质粒筛选出的基因。应用GST Pull-down、免疫共沉淀、染色质免疫沉淀 (ChIP)，RNA 干涉及荧光素酶测定等方法，我们发现LMP2能在体内外与SRC家族成员相互作用，并通过SRC家族参与激活雌激素受体介导的基因表达；同时，ChIP结果表明pS2基因转录激活后，LMP2结合于从启动子到终止子的pS2基因全长。这些结果表明， LMP2不仅能通过SRC家族参与雌激素受体介导的基因激活，同时还在基因转录起始、延长及终止的全过程起一定作用。 总之，本课题研究了SRC家族成员在核受体基因调控中不同作用的分子机理；筛选出了与SRC家族成员相互作用的不同蛋白；证明了LMP2参与SRC家族介导的基因转录激活,并在基因转录的全过程起作用。

Nuclear receptors are ligand-dependent transcription factors that regulate diverse biological activities including growth, development, and homeostasis by binding as homodimers or heterodimers to their cognate DNA elements. Transcriptional activation by nuclear receptors involves at least two separate processes: derepression and activation. Repression is effected in part by association of unliganded receptors with nuclear receptor corepressor (N-CoR) and/or silencing mediator for RAR and TR (SMRT). Ligand binding induces conformational changes in the receptors and triggers dissociation of the corepressors and recruitment of coactivators. To date, a number of nuclear receptor-associated coactivators have been identified, including CBP/p300(cAMP responsive element-binding protein-binding protein/protein 300), pCAF (p300/CBP-associated protein factor), RIP140(receptor-interacting protein 140), RIP-160 ( receptor -interacting protein 160), ERAP-140 (estrogen receptor-associated protein 140), and SRC family members. Coactivator proteins fulfil their functions through protein-protein interactions that bridge transcription factors and the basal transcription machinery and through their chromatin remodeling activities that facilitate the assembly of a transcription initiation complex. SRC family, also referred to as Steroid Receptor Coactivator , consists of three members including SRC-1/NCoA1 (steroid receptor - interacting protein 1 / nuclear receptor coactivatior 1 ) , GRIP1 / TIF2 / SRC-2 / NcoA2 ( glucocorticoid receptor -interacting protein1/transcriptional intermediary factor 2/steroid receptor coactivator 2/ nuclear receptor coactivator-2), and pCIP/ACTR/AIB1/RAC3/TRAM1/SRC-3/NCoA3 ( p300/CBP interacting protein/ activator for thyroid hormone and retinoid receptors / amplified in breast cancer 1 / receptor - associated coactivator 3 / thyroid hormone receptor activator molecule 1/steroid receptor coactivator 3/nuclear receptor coactivator 3), which share structural and functional domains and were shown to enhance nuclear receptor-mediated gene transcription through direct or indirect recruitments of other coactivators. In spite of their structural and functional similarities, pathological evidences and phenotypic manifestations in gene knockout mice for SRC-1, GRIP1 and AIB1 of the SRC family indicate their differential roles in mammal physiology, and there is no clear evidence, at the molecular level, to support a functional specificity for these proteins. Therefore, our researches were focused on three-related topics to investigate the differential gene regulation by the SRC family of coactivators. Part I mainly deals with the differences among the SRC family members in gene regulation. We demonstrated that two species of SRC coactivators, either as AIB1:GRIP1 or as AIB1:SRC-1, are recruited, possibly through heterodimerization, on the promoter of genes that contain a classical hormone responsive element (HRE). In contrast, on non-HRE-containing gene promoters on which steroid receptors bind indirectly, either GRIP1 or SRC-1 is recruited as a monomer, depending on the cellular abundance of the protein. Typically, non-HRE-containing genes are early genes activated by steroid receptors, whereas HRE-containing genes are activated later. Our results also showed that SRC proteins contribute to the temporal regulation of gene transcription. In addition, our experiments revealed a positive correlation between AIB1/c-myc overexpression in ER+ breast carcinoma samples, suggesting a possible mechanism for AIB1 in breast cancer carcinogenesis. Part II is about finding different proteins that are associated with each member of SRC family. In order to find the basis for the functional specificity of the SRC family members, three Gal4 DBD fusion plasmids containing pGBKT7-SRC-1N, pGBKT7-Grip1N, and pGBKT7-AIB1N were constructed by insertion of SRC family N-terminal fragments into the pGBKT7 vector and were used to screen the human mammary cDNA library (CLONTECH) using the Matchmaker Gal4 two-hybrid system 3. After several rounds of selection, sixteen known and unknown genes were identified, including the gene encoding for LMP2 (large multifunctional protease 2), which showed positive reaction with both pGBKT7-SRC-1N and pGBKT7-Grip1N plasmids. Among other positive clones, STAT5B and c-fos were identified to interact with pGBKT7-SRC-1N, consistent with data published by other investigators. The functional significance of the two-hybrid results will be a subject of future study. Part III is about the role of LMP2 in SRC family-mediated gene regulation. As already stated, LMP2 was identified with pGBKT7-SRC-1N and pGBKT7-Grip1N SRC-1N DBD fusion protein expression plasmids. We were interested in whether LMP2 was involved in gene regulation by the SRC family. Using GST pull-down, co-immunoprecipitation, chromatin immunoprecipitation (ChIP), siRNA transfection, and reporter assays, we confirmed that LMP2 interacted with SRC family members in vitro and in vivo and demonstrated that LMP2 stimulated estrogen receptor (ER)-mediated gene transcription through its interaction with SRC family. ChIP results showed that LMP2 existed on the entire pS2 gene after estrodial (E2) addition. These results suggested that LMP2 is involved in the whole process of gene transcription including initiation, elongation, and termination. In summary, we investigated the differential gene regulation by SRC family members at molecular level, screened for proteins that may differentially interact with members of SRC family by two-hybrid system, and demonstrated that LMP2 was involved in ER-mediated gene regulation. These results are of significance in better understanding of the roles of SRC proteins in animal physiology and pathophysiology.

中文摘要………………………………………………………………………..1
英文摘要………………………………………………………………………..4
前 言………………………………………………………………………..7
第一部分………………………………………………………………………..9
材料方法………………………………………………………………………..10
实验结果………………………………………………………………………..24
讨 论…………………………………………………………………….….38
第二部分………………………………………………………………………..46
材料方法………………………………………………………………………..46
实验结果………………………………………………………………………..53
讨 论…………………………………………………………………….….57
第三部分………………………………………………………………………..61
材料方法………………………………………………………………………..62
实验结果………………………………………………………………………..67
讨 论…………………………………………………………………….….75
全文总结………………………………………………………………………..82
文献综述………………………………………………………………………..83
附 录………………………………………………………………………..127
致 谢…………………………………………………………………….….133
简 历………………………………………………………………………..134