【作者】 刘华英；

【导师】 李桂源；

【作者基本信息】 中南大学 ， 病理学与病理生理学， 2007， 博士

【摘要】 溴区结构(Bromodomain)是近年来发现的广泛存在于多种生物中的一种高度保守结构域，可特异性地与组蛋白末端乙酰化的赖氨酸位点结合。Bromodomain蛋白通过与乙酰化组蛋白结合，改变染色质构象，协调多个转录复合物与染色质模板的有序结合而参与信号依赖性基因转录调控。BRD7基因是利用cDNA代表性差异分析法筛选出的Bromodomain基因。它在鼻咽癌细胞和组织中表达下调。前期研究工作表明：BRD7是一个细胞周期特异性转录相关因子，一方面通过调节Ras／MEK／ERK和Rb／E2F信号通路中的关键分子而参与细胞周期调控，另一方面通过与乙酰化组蛋白H3结合，参与核内组蛋白乙酰化信号传递。过表达BRD7基因可抑制鼻咽癌细胞增殖和细胞周期进程，并部分地逆转鼻咽癌细胞的恶性表型。为了揭示BRD7基因在鼻咽癌细胞和组织中表达下调的分子机制，更深入地阐明BRD7基因的生物学功能，本课题展开了BRD7基因的转录调控研究。【BRD7基因启动子的克隆、鉴定及精细定位】生物信息学分析为基因转录调控研究提供了一个很好的切入点。在线程序PromoterInspector分析结果表明BRD7调控区-375／+416的区间为其候选启动子区，而在线程序PromoterScan的预测结果表明-391／-141的区间为BRD7的候选启动子区。以正常人外周血细胞基因组DNA为摸板，利用PCR技术，获得了BRD7调控区长片段-711／+496，并证实该区域具有与病毒SV40启动子同等强度的启动活性。进一步以该长片段调控区为模板，利用缺失突变体报告质粒构建技术和荧光素酶活性分析系统，定位了BRD7基因的近距离启动子区-404／+46，并发现BRD7启动子区-293／-168的125bp区域是其发挥启动子活性的必需序列。为了进一步获得BRD7基因的最小启动子序列，分别在其近距离启动子片段-404／+46的5’、3’或内部缺失部分序列，构建一系列缺失突变体报告载体，并将它们分别转染不同细胞系中，发现BRD7基因-266／-212的55bp区间为其最小启动子区域，而且这一最小启动子选择性地在c-Myc缺失的细胞株中发挥启动活性。【BRD7启动子区顺式作用元件和反式作用因子的鉴定和功能研究】真核基因的转录调控是一个非常复杂的生化反应过程，数目惊人的蛋白因子参与了基因的转录调控。在线软件MatInspector分析结果表明BRD7启动子区是一个不含TATA盒，也不含CAAT盒的GC富集区。它含有多个GC盒，多种转录因子结合位点，如：KLF，Sp1，E2F，MYC-MAX和AP2等结合位点。凝胶迁移率分析(EMSA)证实了BRD7基因启动子区的Sp1／MYC-MAX(-223／-198)、E2F-6(-243／-229)以及E2F(-183／-169)结合位点。超变动(Supershift Assay)和免疫染色质共沉淀(ChIP)实验证实了转录因子c-Myc、E2F6和Sp1对这些位点的特异性结合。利用转基因技术，发现过表达c-Myc能大幅度地下调BRD7启动子活性并明显抑制BRD7基因的内源性mRNA表达水平；封闭内源性c-Myc的表达使BRD7启动子活性在鼻咽癌5-8F细胞中提高了10倍以上，同时也明显上调了BRD7基因内源性mRNA的表达水平。采用MicroRNA芯片分析发现，封闭内源性c-Myc的表达在鼻咽癌细胞中上调了14个miRNA分子的表达，其中hsa-miR-224上调了66.49倍，是上调幅度最大的miRNA基因，同时还下调7个miRNA分子的表达，hsa-miR-200c和hsa-miR-141的表达下调了10倍以上。在线程序预测这些差异表达miRNA分子的靶基因，发现它们大多涉及细胞周期和凋亡等信号分子通路。过表达转录因子Sp1以浓度依赖性方式在鼻咽癌细胞中上调BRD7启动子活性，但并不足以使BRD7启动子活性上调至全长启动子-404／+46的活性；转录因子Sp1结合位点的特异性竞争剂光辉酶素A(Mithramycin A)可大幅度地抑制BRD7启动子活性，并明显下调BRD7基因内源性mRNA表达水平。但转录因子E2F6对BRD7基因最小启动子片段没有活性调节作用。为了进一步确定BRD7启动子区-223／-198的MYC-MAX／Sp1重叠结合位点和-243／-229的E2F6结合位点的重要性，分别构建了这两个结合位点的点突变和缺失突变报告基因载体。荧光素酶分析系统结果表明：突变MYC-MAX／Sp1(-223／-198)重叠结合位点中的三个关键碱基(C／A，G／T，G／A)使BRD7最小启动子活性在鼻咽癌5-8F细胞中增加了4个数量级；但缺失MYC-MAX／Sp1结合位点并不影响BRD7最小启动子的活性。而突变E2F6结合位点的4个关键碱基不影响BRD7最小启动子活性，但缺失E2F6结合位点的15个关键碱基却大幅度地增加了BRD7最小启动子活性。另外，免疫共沉淀和免疫共定位实验都确证了鼻咽癌5-8F细胞中蛋白因子c-My与Sp1的直接交互作用，提示蛋白因子c-Myc结合于Sp1从而竞争或覆盖了Sp1与其相应位点结合的基序可能是c-Myc抑制BRD7启动子活性和内源性表达的另一调节因素。【DNA甲基化抑制BRD7基因的表达，去甲基化恢复其表达】c-Myc和Sp1对BRD7启动子活性调节至关重要，但在鼻咽癌细胞中抑制c-Myc的表达或过表达Sp1蛋白因子都不足以使BRD7最小启动子活性上调至全长启动子-404／+46的活性或完全恢复BRD7基因的mRNA表达水平，提示在BRD7基因转录调控机制中还存在未知的调节因素如：CpG岛或Sp1结合位点甲基化等。利用欧洲分子生物学开放软件包(European Molecular Biology Open Software Suite，EMBOSS)和美国softberry软件公司推出的CpGFinder程序在线扫描BRD7基因转录起始位点上游2000bp的gDNA序列，分别发现其5’上游-418／-56或-374／-4的区间为—CpG岛。这两个软件的预测结果大部分重叠，且与BRD7近距离启动子-404／+46重叠。利用BRD7基因启动子区特异性甲基化和非甲基化引物进行甲基化特异性聚合酶链反应(MSP-PCR)，发现BRD7启动子在所有鼻咽癌细胞系中都呈部分甲基化状态，且其甲基化程度与BRD7基因mRNA的表达水平呈负相关。MSP-PCR测序结果表明BRD7基因启动子区共有10个甲基化CpG位点，它们分别涉及位于-353／-337的Sp1结合位点、-330／-317的Sp1结合位点、-260／-246的MYC-MAX结合位点、-223／-198的Sp1结合位点以及-243／-229的E2F结合位点，另有两个位于翻译起始位点ATG的附近。利用SssI甲基转移酶能将腺苷甲硫氨酸(H．SAM)中的甲基转移至基因组DNA CpG位点的非甲基化胞嘧啶“C”上，使原本未甲基化的“C”发生甲基化的特点，分别将野生型双链寡核苷酸探针Wt E2F-6(-243／-229)和WtSp1／MYC-MAX(-223／-198)经SssI甲基转移酶处理后，进行EMSA分析，发现这些甲基化的寡核苷酸探针不能与核蛋白结合。同样，将BRD7启动子报告载体pGL3-404／+46、pGL3-404／+46／GFP经SssI甲基转移酶处理后，发现它们在COS7和BHK-21细胞以外的所有癌细胞株中丧失了全部的启动子活性。3.75μM的甲基化酶抑制剂5-脱氧杂氮胞昔(ADC)就足以在鼻咽癌5-8F细胞中完全逆转BRD7启动子甲基化状态，并最大幅度地上调BRD7基因mRNA和蛋白质表达水平，其上调幅度分别为76.7％和63.3％。流式细胞仪分析结果表明，3.75μM的5-脱氧杂氮胞苷具有阻滞鼻咽癌细胞周期G2／M、S期进程和诱导鼻咽癌细胞凋亡的作用。MicroRNA芯片分析结果发现，3.75μM的5-脱氧杂氮胞苷在鼻咽癌5-8F细胞中上调了10个miRNA分子的表达，其中hsa-miR-122a上调了63.2倍，是上调幅度最大的miRNA分子，同时还下调7个miRNA分子的表达，其中hsa-miR-203下调了7.79倍。利用在线程序预测其靶基因，发现这些靶基因主要涉及细胞周期和凋亡等信号分子通路。更为重要的是：通过检测36例鼻咽癌病人组织和16例正常人外周血细胞中BRD7基因的甲基化状态，发现BRD7基因启动子区的甲基化频率在鼻咽癌病人的鼻咽组织中为100％，而在正常人群外周血细胞中为50％，且在正常人群中呈微弱甲基化状态。总之，通过本课题研究，我们获得了BRD7启动子及其精细位置，较系统地阐述了BRD7启动子的特征及其顺式作用元件和反式作用因子的组成和功能，较详细地探索了DNA甲基化对BRD7启动子活性、mRNA和蛋白质表达水平的影响及分子机制，并获得了BRD7基因启动子甲基化可能成为界定鼻咽癌病人和正常人群的生物分子标志物之一的重要信息。这些研究结果将有助于全面地了解BRD7基因的生物学功能，明确BRD7基因在鼻咽癌癌变中的作用机制，并为其潜在的临床应用提供科学的理论和实验依据。更多还原

【Abstract】 Bromodomain is an evolutionally conserved domain that wasrecently identified in many living organisms. Bromodomain containingprotein has specific affinity to combine with acetylated lysines onN-terminal tails of histones. It has been demonstrated that bromodomainwas characteristics of proteins that regulated signal-dependenttranscriptional regulation through the mechanism that bromodomainproteins may modulate chromatin remodelling and facilitate the accessionof transcription factors to chromatin. BRD7 is a bromodomain gene thathas been recently cloned by cDNA RDA （cDNA RepresentationalDifference Analysis）. It is down-expressed in Nasopharygeal Carcinoma（NPC） biopsies and their derived cell lines. Previous studies showed thatBRD7 is a cell cycle related transcription factor. It participates in cellcycle regulation by regulating some of the cell cycle associated genesthrough Ras/MEK/ERK and Rb/E2F pathways. Moreover, it can transmithistoric acetylation signal and modulate chromatin remodeling throughbinding to acetylated lysines of histone H3. Over-expression of BRD7inhibits cell growth and cell cycle progression of NPC cells, and partlyreverses malignant phenotype of NPC cells. To uncover the molecularmechanisms underlying down-expression of BRD7 in NPC cells, in thisstudy, we investigated the transcriptional regulation of BRD7.【Cloning, characterization and fine mapping of BRD7 promoter】Bioinformatics approaches provide a breakthrough point foranalyzing of transcriptional regulation. A 792 bp region spanning frompositions -375 to +416 was identified as potential promoter region ofBRD7 gene by using PromoterInspector, whereas a 252 bp region located positions from -393 to -141 was identified as BRD7 promoter by usingPromoterScan program. With human genomic DNA prepared fromhuman blood cells as templates, a fragment spanning from positions -711to +496 of BRD7 gene was amplified by PCR. This fragment expressesas strong promoter activity as SV40 promoter. Further analysis withdeletion constructs demonstrated that the region spanning from positions-404 to +46 is the proximal promoter of BRD7 gene, and that thefragment from positions -293 to -168 was indispensable for the basalpromoter activity of BRD7.To define minimal promoter of BRD7, a series of 5’, 3’ or internaldeletion constructs were generated from pGL3-404/+46, andcotransfected with the SV40β-galactosidase vectors into cultured cells.Luciferase assay revealed that the shortest 55 bp region from positions-266 to -212 is the minimal promoter of BRD7 gene, and that thisminimal promoter selectively functions in c-Myc-null cell lines【Identification and characterization of the cis-acting elements andtrans-acting factors in BRD7 promoter】Eukaryotic gene transcription is a remarkably intricate biochemicalprocess. An astounding number of protein factors were found to beresponsible for transcriptional control. Searches for binding sites oftranscription factors in BRD7 promoter were performed by usingMatInspector Professional program. No canonical TATA or CAAT boxeswere found, while several GC boxes and putative transcription bindingsites for KLF, Sp1, IFRF-2, AP2, MYC-MAX, E2F and E2F6 were foundin BRD7 promoter. The results of EMSA confirmed the specificoverlapping site of Sp1/MYC-MAX at -223/-198, consensus E2F-6 site at-243/-229 and E2F binding site at -183/-169. Supershift and ChIP assay revealed the specific binding of transcription factor c-Myc, E2F6 and Sp1with their corresponding binding sites in BRD7 minimal promoter.To explore the regulation role of transcription factor Sp1, c-Myc andE2F on BRD7 promoter activity and mRNA expression, we generated theexpression constructs of c-Myc, E2F6 and Sp1, and carded out a series ofrelated experiments. It was found that over-expression of c-Mycsignificantly inhibits BRD7 promoter activity and obviously reducesendogenous mRNA expression of BRD7 gene, while knockdown ofendogenous c-Myc increases more than ten fold of BRD7 promoteractivity and mRNA expression of BRD7 in NPC 5-8F cells. Additionally,MicroRNA chip results revealed that knockdown of endogenous c-Mycup-regulated 14 miRNA expression including hsa-miR-224, whichexpression was increased more than 66.49 fold, and down-regulated 7miRNA expression including hsa-miR-200c and hsa-miR-141. Onlineprediction results revealed that most of these miRNA target genes areinvolved in cell cycle and cellular apoptosis.Over-expression of transcription factor Sp1 increases BRD7promoter activity in a dose-dependant manner in NPC HNE1 and 5-8Fcells, but not enough to the promoter activity of the full-length promoter-404/+46, while mithramycin A, the specific competitor of Sp1, inhibitsthe promoter activity and endogenous expression of BRD7. Butover-expression of E2F6 has no effects on BRD7 minimal promoter.To further confirm the importance of the overlapping site ofMYC-MAX/Sp1 at -223/-198 and E2F site at -243/-229, reporterconstructs containing site mutant or deletion mutant of these two bindingsites were generated. It was found that site mutating of three keynucleotides （C/A, G/T, G/A） of the overlapping binding site of MYC-MAX/Sp1 increases 10000 fold of BRD7 promoter activity in NPC5-8F cells, whereas deletion of this overlapping region didn’t change thepromoter activity of pGL3-266,-212 either in COS7 or 5-8F cells.Similarly, mutation of four key nucleotides in E2F6 binding site didn’taffect the promoter activity of BRD7 gene, but deletion of E2F6 bindingsite significantly increased BRD7 promoter activity in NPC 5-8F cells.Moreover, immunoprecipitation along with immuno-localizationassay revealed the direct interaction between c-Myc and Sp1 in 5-8F cells,suggesting that c-Myc binds to Sp1 to cover or compete the domain ofSp1 for binding to overlapping site of Sp1/MYC-MAX at -223/-198 maybe another mechanism underlying the negative regulation effect of c-Mycon BRD7 promoter.【Methylation of BRD7 promoter suppresses its expression, whiledemethylation of BRD7 promoter increases its expression】Knock-down of c-Myc expression or over-expression of Sp1 is notenough to increase BRD7 promoter activity to that of the full-lengthpromoter -404/+46 or completely reverse BRD7 expression in NPC 5-8Fcells, indicating that other mechanisms such as methylation status,especially methylation of GC boxes （Sp1 sites）, might be related totranscriptional regulation of BRD7 gene. A CpG island spanning frompositions -418 to -56 bp or from -374 to -4 bp was revealed by usingCpGplot and the CpGFinder program, respectively. The CpG islandspredicted by these two programs overlap with each other, and overlapwith BRD7 promoter. Methylation specific primers and unmethylationspecific primers were designed to detect the methylation status of BRD7promoter. It was found that BRD7 promoter was partly methylated in allanalyzed NPC cell lines, and that the methylation status of BRD7 promoter is reversely correlated with BRD7 mRNA expression.MSP-PCR sequencing results revealed ten methylated CpG sites in BRD7promoter region, among which one was included in Sp1 binding site at-353/-337, one in MYC-MAX binding site at -330/-317, one in Sp1binding site at -229/-198, one in E2F binding site at -243/-229, and theother two near the translation start site. As CpG methylase SssI cantransmit the -CH₃ of SAM to Cytosine of CpG site, we incubated probesof oligonucleotide wt E2F6 （-243/-229） and wt Sp1/MYC-MAX（-223/-198） with SssI. EMSA results showed that DNA methylationinhibited the formation of DNA-protein complex. Luciferase and directGFP fluorescence assays showed that methylation of pGL3-404/+46 andpGL3-404/+46/GFP with SssI inhibits BRD7 promoter activity inanalyzed cell lines except for COS7 and BHK-21. 3.75μM of5’-aza-2’-deoxycytine （ADC） was sufficient to reverse the methylatedstatus of BRD7 promoter, and increased the mRNA and proteinexpression of BRD7 up to 66.7% and 63.3%, respectively. Results offlow cytometry analysis showed that 3.75μM ADC can inhibit cell cycleprogression of G₂/M and S phase, and induce cellular apoptosis.Moreover, MicroRNA chip results revealed that 3.75μM ADCup-regulated 10 miRNA expression including hsa-miR-122a, whichexpression was increased more than 63.2 fold, and down-regulated 7miRNA expression including hsa-miR-200c and hsa-miR-203. Onlineprediction results showed that most of these miRNA target genes areinvolved in cell cycle and cellular apoptosis. More importantly, wedetected methylation status of BRD7 promoter in 36 cases of NPCpatients and 16 normal individuals, and found that the methylation rate ofBRD7 promoter in NPC patients is 100%, while 50% of weak promoter methylation was revealed in normal individuals.In summary, we have identified BRD7 promoter and finely mappedits positions, demonstrated the characteristics of BRD7 promoter,confirmed and investigated the functions of cis-acting elements andtrans-acting factors of BRD7 promoter, explored the effects of DNAmethylation on BRD7 promoter activity and mRNA expression, andfound that the methylation status of BRD7 promoter may be served asone of the biomarkers to distinguish NPC patients from normalindividuals. These results will help to better understand the biologicalfunctions of BRD7, elucidate the molecular mechanisms of BRD7involved in the carcinogenesis of NPC, provide evidences for thepromising clinical application of BRD7.更多还原