【作者】 陈永军；

【导师】 吴宁华； 沈珝琲； 张业；

【作者基本信息】 中国协和医科大学 ， 分子生物学及生物化学， 2006， 博士

【摘要】 在染色质水平研究基因的表达调控是目前的热点之一，研究较多的两类染色质重塑复合物是ATP依赖的复合物和核小体共价修饰复合物，对通过形成非组蛋白染色质结构，改变核小体的正常排列类型的第三类蛋白质复合物却了解很少。果蝇GAF作为一种BTB／POZ结构域蛋白，它参与形成的GBP复合物是第三类染色质重塑复合物的典型代表。GBP复合物通过靶基因的GAGA元件介导形成非组蛋白染色质结构，导致染色质重塑，调控靶基因的表达。为观察人类细胞是否存在果蝇GBP类复合物，本实验室曾利用含有GAGA元件核心序列的报告基因质粒，通过酵母单杂交系统，鉴定了(?)种GAGA元件结合蛋白相关蛋白GRP。本文主要通过共转染及竞争性RT-PCR方法，检测果蝇ftz基因、人hsp90α和人hsp90β基因的启动子活性，主要在染色质水平研究了GRP对这些在启动子区含有GAGA元件的靶基因的表达活性的调控机制。另外，我们也通过流式细胞分析，初步观察了GRP对人Jurkat细胞周期分布和凋亡的影响。将启动子区含有GAGA元件的典型的GAF的靶基因ftz基因启动子(-617～＋124)构建到能模拟染色质结构的pREP4mCAT episome载体质粒上，转染人Jurkat细胞，通过竞争性RT-PCR方法检测其启动子活性，发现含有4个野生型GAGA元件的ftz基因启动子驱动的CAT报告基因可在人Jurkat细胞中有较好的表达，其相对启动子活性单位约为1.5。而4个GAGA元件突变的ftz基因启动子驱动的CAT报告基因表达明显减弱，相对启动子活性单位仅为0.5左右。提示人Jurkat细胞内可能存在与果蝇GAF类似的因子。在同样的实验条件下，GRP能显著增强GAF对野生型果蝇ftz基因启动子驱动的CAT报告基因的表达的促进作用，GAGA元件突变后该作用消失。提示人Jurkat细胞中可能有类似于果蝇GBP一样的复合物存在，GRP也是一种BTB／POZ结构域蛋白，很可能参与人的果蝇GBP复合物类似物的功能。许多研究表明，HSP90是热激蛋白家族(HSP)的重要成员，它在细胞中呈高组成性表达，热休克和其它应激条件可以进一步诱导其表达。HSP90作为分子伴侣广泛参与细胞的信号转导、激素应答及细胞周期调控等多种过程，对细胞在生理、病理及应激条件下的生存发挥了重要作用。因此研究HSP90蛋白基因表达调控机制具有重要的生物学意义。利用计算机软件对人hsp90α启动子(-1756～＋37)和人hsp90β启动子((?)943～＋(?)531)进行转录因子结合位点搜索，发现它们分别含有数个GAGA元件，因此人hsp90α和人hsp90β是否是GRP和／或GAF的靶基因是我们感兴趣的问题。用Rc／CMV-GAF和pREP4m CAT-hsp90α(-1756～＋37)或pREP4mCAT-hsp90β(-943～＋1531)共转染人Jurkat细胞，测定CAT报告基因的活性。结果发现37℃时，转染2、10和50μgRc／CMV-GAF时，hsp90α相对启动子活性分别为0.8、1.1和1.6，表明GAF能增强hsp90α报告基因的表达。经42℃热休克1小时，增强作用更加明显，hsp90α相对启动子活性分别为1.8、2.4和4.6。在与上述相同条件处理下，hsp90β与hsp90α相对启动子活性的变化趋势类似。提示人hsp90α和hsp90β基因可能是GAF类因子的侯选靶基因，可能通过它们调控序列中的GAGA元件发挥调控作用。转染正义GRP表达质粒，明显促进人hsp90启动子的热诱导。当转染50μg正义GRP质粒为时，hsp90α启动子活性热诱导倍数是6.4，而hsp90α对照组的热诱导倍数仅为2.0；转染反义GRP表达质粒，则抑制hsp90报告基因的热诱导表达，当转染50μg反义GRP质粒时，42℃热休克1小时，与对照组相比，hsp90α相对启动子活性抑制46％。在与上述相同条件处理下，hsp90β与hsp90α相对启动子活性的变化趋势类似。共转染Rc／CMV-GAF和pCDNA6-GRP，在37℃和42℃条件下，hsp90β相对启动子活性分别为6.5和11.2。仅转染Rc／CMV-GAF时，在37℃和12℃条件下，hsp90β相对启动子活性分别为1.9和4.0。在与上述相同条件处理下，hsp90α与hsp90β相对启动子活性的变化趋势类似。这些结果与GAF和GRP对果蝇ftz基因启动子表达调控的结果类似，提示GAF和GRP可能使用类似的机制，调控人hsp90基因和对果蝇ftz基因在人Jurkat细胞中的表达。丁酸钠是常见的组蛋白去乙酰化酶抑制剂。为研究乙酰化是否与GRP增强人hsp90基因表达的作用有关，用GRP正义或反义真核表达质粒与hsp90报告基因质粒共转染Jurkat细胞，培养36小时后用终浓度为2mM丁酸钠处理细胞12小时。37℃时，转染正义GRP细胞中，hsp90α相对启动子活性为0.8；转染空载体的对照组，hsp90α相对启动子活性为0.7。丁酸钠处理细胞，hsp90α相对启动子活性为1.5，表明丁酸钠对hsp90α-CAT报告基因表达有促进作用。若转染正义GRP后，经丁酸钠处理，hsp90α相对启动子活性为2.3，显示正义GRP和丁酸钠可共同增强hsp90α-CAT的表达，此时将细胞在42℃热休克1小时，增强作用更加明显，hsp90α相对启动子活性为5.2。在与上述相同条件处理下，hsp90β-CAT报告基因表达产生类似的变化。结果表明，丁酸钠处理增强GRP对hsp90基因启动子的热诱导活性的促进作用，提示组蛋白乙酰化参与GRP对hsp90基因启动子的热诱导调控。，p300是一种重要的组蛋白乙酰基转移酶。为了进一步研究组蛋白乙酰化在GRP对hsp90基因表达调控中的作用，设计了多种p300与GRP转染实验组合。仅转染GRP，37℃和42°条件下，hsp90β相对启动子活性为0.8和2.8。共转染GRP和p300，37℃和42°条件下，hsp90β相对启动子活性为1.5和6.7。共转染GRP和缺失HAT活性结构域的p300△h时，37℃和42°条件下，hsp90β相对启动子活性为0.8和2.9；仅转染GRP，37℃和42°条件下，hsp90α相对启动子活性分别为0.8和2.6。共转染GRP和p300，37℃和42°条件下，hsp90α相对启动子活性分别为0.8和2.8。共转染GRP和p300△h时，hsp90α相对启动子活性分别为0.7和2.7。说明无论热休克与否，p300或p300△h不影响GRP对hsp90α基因启动子活性的增强作用。表明在37℃时，野生型Pp300与GRP在某种程度上，共同促进hsp90β启动子驱动的报告基因表达，在42℃时，这种协同作用更加明显。缺失HAT结构域的突变型p300△h则无此作用。提示p300可能通过HAT结构域参与GRP对hsp90β基因启动子活性的的调控。初步通过流式细胞分析观察GRP对Jurkat细胞周期分布的影响。发现转染GRP48h后，G1期细胞所占比例由对照组的52.5％上升为68.7％，S期细胞所占比例由44.4％下降为25.5％，提示GRP可能阻滞Jurkat细胞G1／S过渡。综上所述，我们初步认为：(1) Jurkat细胞内可能存在类似果蝇GAF或GBP复合物：(2) GRP和GAF可能在染色质调控水平上参与人hsp90基因和果蝇ftz基因的表达：(3)组蛋白的乙酰化可能参与GRP对hsp90基因启动子的热诱导调控；(4) p300可通过HAT结构域参与GRP对hsp90β基因启动子活性的调控：(5) GRP可以影响Jurkat细胞G1向S期的过渡。更多还原

【Abstract】 The study of the expression and the control of gene on the chromatin level is one of hot spot at present. The two classes of complexes involved in chromatin remodelinghave been investigated more extensively. They are ATP-dependent complexes and chromatin-modifying complexes. Little is known about a third class of protein complexes that disrupt the normal nucleosome pattern by forming nonhistone chromatin structures. Drosophila GAF contains BTB/POZ domain. GBP, of that GAF is a component, is representative of this kind of nonhistone complexes. The complexes forming nonhistone chromatin structures regulate the expression of target gene containing GAGA element by chromatin remodeling.Fo observe if this kind of nonhistone complexes exists in human Jurkat cell, , we identified a GAGA element binding protein related protein, GRP using yeast one-hybrid system. By a competitive RT-PCR, this paper investigated mainly the effect of GRP on the promoter activity of its GAGA element-containing target gene ftz、hsp90αand hsp90βon the chromatin level.The promoter of Drosophila ftz gene is constructed into pREP4mCAf episomal vector capable of simulating chromatin structures. It is found that the CAT reporter gene driven by wild-type ftz promoter can express better than the CAT reporter gene driven by mutant-type ftz promoter in human Jurkat cell. The relative promoter activity of wild-type and mutant-type ftz gene is 1.5 (wild-type) and 0.5 (mutant-type) respectively ,suggesting that the factor similar to Drosophila GAF exists in human Jurkat cell.GRP enhances the promotion of GAF to the expression of CAT reporter gene driven by wild-type ftz gene promoter, but GRP does not about mutant-type ftz gene promoter. The results indicate that the complexes similar to GBP exist in human Jurkat cell.HsP90 is an important member of HSP protein family. HSP90 usually constitutively expressed in most mammalian cell types and can be further induced by heat shock and other stresses. HSP90 proteins are molecular chaperones that associated with cellar signal transduction, steroid hormone response, and cell cycle regulation, they are critical to the survival of cell in the normal and pathological conditions. So it is very meaningful for us to study the regulatory mechanisms of human hsp90 genes.We found a few of GAGA element in the hsp90αpromoter region ( -1756～+37 ) and hsp90βpromoter region ( -943～+1531 ) by searching them for the sites of transcriptional factor. Rc/CMV-GAF was transfected into human Jurkat cell with pREP4mCAT-hsp90α( -1756～+37 ) or pREP4mCAT- hsp90β( -943～+1531 ). When Rc/CMV-GAF is transfected at 37℃, GAF promotes the expression of CAT reporter gene driven by hsp90αpromoter in a dose-dependent manner. The effect is marked after heat shock for an hour at 42℃. Under the above experimental condition the change of CAT reporter gene driven by hsp90βpromoter is similar to that of hsp90α. Rc/CMV-GAF and pCDNA6-GRP was transfected into human Jurkat cell with pREP4mCAT-hsp90αor pREP4mCAT-hsp90β. GRP and GAF cooperatively promote the expression of CAT reporter gene driven by hsp90αor hsp90βpromoter. The mechanism regulating hsp90αor hsp90βpromoter by GRP and GAF may be similar to that regulating ftz gene promoter by GRP and GAF.Sodium butyrate is a common histone deacetylase inhibitor. The sense- or antisense- expressional plasmids of GRP were cotransfected into human Jurkat cell with pREP4mCAT-hsp90αor pREP4mCAT-hsp90β. 36 hours later the cells were treated with 2mM sodium butyrate for 12 hours. The results show that the 2mM sodium butyrate markedly enhances the promotion of GRP to the expression of CAT reporter gene driven by hsp90αor hsp90βpromoter. We assume that the promotion of GRP to hsp90αor hsp90βpromoter may involve histone deacetylation by Sodium butyrate. p300 is an irnportant histone acetyltransferase, p300 or p300△h and GRP were transfected into Jurkat cells, p300 and GRP synergistically promote the expression of CAT reporter gene driven by hsp90βpromoter, p300 and GRP do not about hsp90α, p300△h (histone acetyltransferase domain is deleted) can not affect the promotion of GRP to the expression of CAT reporter gene driven by hsp90αor hsp90βpromoter. These results suggest that p300 may take part in the regulation of GRP to hsp90αor hsp90βgene by histone acetyltransferase domain of it.GRP can regulate the distribtion of cell cycle. When the Jurkat cells were transfected with GRP, the percentage of G1-phase cells increases and the percentage of S-phase cells decreases, suggesting that GRP may blocks the G1-to-S transition. The overexpression of GRP can lead to cell apoptosis.Taken together, we get the following conclusion: The factor similar to Drosophila GAF or complexes similar to GBP may exist in human Jurkat cell. The mechanism regulating hsp90αor hsp90βpromoter by GRP and GAF may be similar to that regulating ftz gene promoter by GRP and GAF. The promotion of GRP to hsp90αor hsp90βpromoter may involve histone deacetylation by Sodium butyrate. P300 may take part in the regulation of GRP to hsp90αor hsp90βgene by histone acetyltransferase domain of it. GRP can regulate the distribtion of cell cycle. The overexpression of GRP can lead to cell apoptosis.更多还原