p21WAF1/CIP1基因上游主要转录调控序列区及其相关功能

p21蛋白作为周期依赖性蛋白激酶抑制因子,调节细胞周期的进程,参与细胞生长、增殖、分化、衰老等多种活动.p21/WAF1/CIP1基因上游启动子中含有多个转录调控序列,包括p53,Sp1,Ap2,VDR及RAR,STAT,C/EBPα,β,E2A,MyoD和E2F等转录因子的顺式结合元件.在细胞的生长、分化,凋亡,衰老及疾病的发生发展中,这些转录因子通过与p21上游相应调控区相互作用,调节p21基因的表达.     

结肠癌细胞HCT116中p53的R213突变对靶基因p21表达的影响

目的确定在结肠癌细胞系中,p53的213位精氨酸突变后对其下游靶基因p21表达的影响。方法在p53缺失的结肠癌细胞系HCT116~(-/-)中转染p53的R213位突变的质粒;荧光素酶双报告基因系统检测p53靶基因启动子活性;EMSA检测p53与其靶基因p21的结合能力;real-time PCR和Western blot检测p53下游靶基因p21的表达。结果 p53蛋白213位精氨酸突变为赖氨酸后,明显降低了p53靶基因的启动子活性(P0.05);与靶基因p21启动子的结合能力明显降低(P0.05);并抑制了p21基因的表达(P0.05)。而213位突变为谷氨酰胺后,迁移率比突变前明显变快。结论在人的结肠癌细胞中,p53蛋白R213位的不同突变对下游靶基因p21有不同的影响,提示存在着不同的调控机制。     

转录因子Sp1和Sp3对食管癌细胞ezrin基因的表达调控作用

目的:明确转录因子Sp1和Sp3对食管癌细胞ezrin基因的表达调控作用.方法:将转录因子表达载体CMV-Sp1和CMV-Sp3分别转染食管癌EC109细胞,采用定量RT-PCR和Western blot技术检测过表达转录因子Sp1和Sp3对ezrin mRNA和蛋白表达的影响;将ezrin基因启动子驱动的报告基因表达载体与内参照质粒pRL-TK和转录因子表达载体共转染EC109细胞,采用双荧光素酶报告基因分析系统检测转录因子Sp1和Sp3对ezrin基因启动子的激活作用以及这种激活作用是否依赖于ezrin基因的Sp1结合位点-75/-69.结果:过表达转录因子Sp1和Sp3显著提高EC109细胞ezrin mRNA和蛋白表达水平以及启动子活性.Sp1和Sp3增强ezrin基因启动子活性的作用位点不同,只有Sp1通过-75/-69位点调控ezrin基因启动子活性.结论:转录因子Sp1和Sp3可调控EC109细胞ezrin基因的表达.     

Sp3对β-catenin基因转录调控作用初探

 目的:观察转录因子Sp3对β-catenin启动子转录活性的影响,探讨Sp3与Wnt/β-catenin信号通路的关系。方法:采用脂质体法将Sp1和(或)Sp3转录因子表达质粒单独/共同与β-catenin启动子（-410/-1 bp）报告基因质粒瞬时转染HEK293T细胞;通过双萤光素酶报告基因实验检测报告基因转录活性的变化。结果:（1）Sp1表达质粒在0.4 μg剂量时能提高启动子的活性2.4倍,Sp3表达质粒在0.4 μg剂量时能显著提高启动子的活性5.3倍。（2）0.4 μg总量的Sp1与Sp3表达质粒共转染293T细胞,随着Sp3/Sp1比例的递增,β-catenin启动子转录活性无明显变化。（3）共同转染Sp1 0.3 μg与Sp3 0.1 μg时,启动子活性提高3.5倍,比单独转染的转录活性强。结论: Sp3能促进β-catenin基因启动子（-410/-1 bp）的转录,Sp1的转录激活作用则较弱;在转录过程中Sp1和Sp3可能存在协同作用;Sp3可能在转录水平调控β-catenin的表达从而影响Wnt/β-catenin信号通路。     

DMD启动子在基因治疗中作用的研究进展

假肥大型进行性肌营养不良(DMD)是一类X连锁隐性退行性肌肉萎缩病。目前,关于DMD基因外显子缺失,内含子断裂点以及所编码dystrophin蛋白的结构已经明确,这对DMD的临床诊断有很大价值;但对于基因治疗来说,明确DMD基因启动子的顺式元件以及反式因子的结构及相互作用关系是十分关键的,这些元件的相互作用对于dystrophin蛋白的转录水平及表达起关键调节作用;研究表明,人工合成的DMD肌启动子活性比生物体内DMD基因启动子活性高,找出启动子关键元件构建特异性高效启动子,将其应用于基因治疗,这是一条新的途径。     

DMD启动子在基因治疗中作用的研究进展

假肥大型进行性肌营养不良(DMD)是一类X连锁隐性退行性肌肉萎缩病.目前,关于DMD基因外显子缺失,内含子断裂点以及所编码dystmphin蛋白的结构已经明确,这对DMD的临床诊断有很大价值;但对于基凶治疗来说,明确DMD基因启动子的顺式元件以及反式因子的结构及相互作用关系是十分重要的,这些元件的相互作用对于dystrophin蛋白的转录水平及表达起关键的调节作用;研究表明,人工合成的DMD肌启动子活性比生物体内DMD基因启动子活性高,找出启动子关键元件构建特异性高效启动子,将其应用于基因治疗,这是一条新的途径.     

Ⅰ型胶原基因转录调控的研究进展

调控Ⅰ型胶原(COL1)基因转录的顺式作用元件位于启动子和第一个内含子中。顺式作用元件及其结合的转录因子有生物种类特异性。一些转录因子以不同的亲和力与其部分潜在结合位点结合,它们间的相互作用稳定了DNA的环状结构,并引发转录。转化生长因子-β(TGF-β)、胰岛素样生长因子-Ⅰ(IGF-Ⅰ)、干扰素-γ(IFN-γ)、肿瘤坏死因子-α(TNF-α)等细胞因子在转录水平参与COL1基因的调控,在COL1基因启动子上有它们的反应元件。     

口腔鳞癌中survivin、cyclinD1和p53基因的表达及其相关性

目的:探讨survivin、cyclinD1和p53基因蛋白在口腔鳞癌中的表达及其相互关系.方法:应用免疫组化SP法检测95例口腔鳞癌组织和50例正常口腔黏膜组织中survivin、cyclinD1和p53蛋白的表达.结果:survivin、cyclinD1和p53在口腔鳞癌与正常口腔黏膜组织之间的差异均有统计学意义(P<0.05);在口腔鳞癌中,Survivin和CyclinD1蛋白的过表达均与临床分期及淋巴结转移有关(P<0.05或P<0.01),p53过表达与组织分化程度及临床分期有关(P<0.05或P<0.01);survivin的过表达与cyelin D1及p53蛋白表达均呈正相关(r=0.628,0.829),cyclin D1蛋白的过表达与p53蛋白表达也呈正相关.结论:survivin、cyclinD1和p53基因在口腔鳞癌的发生、发展中起着不同程度的作?三者之间可能具有协同作用.     

The zinc finger DNA-binding domain of K-RBP plays an important role in regulating Kaposi's sarcoma-associated herpesvirus RTA-mediated gene expression

K-RBP is a KRAB-containing zinc finger protein with multiple zinc finger motifs and represses Kaposi's sarcoma-associated herpesvirus (KSHV) transactivator RTA-mediated transactivation of several viral lytic gene promoters, including the ORF57 promoter. Whether K-RBP binds DNA through its zinc fingers and the role of zinc finger domain in repressing gene expression are unclear. Here we report that K-RBP binds DNA through its zinc finger domain and the target DNA sequences contain high GC content. Furthermore, K-RBP binds to KSHV ORF57 promoter, which contains a GC-rich motif. K-RBP suppresses the basal ORF57 promoter activity as well as RTA-mediated activation. The zinc finger domain of K-RBP is sufficient for the suppression of ORF57 promoter activation mediated by the viral transactivator RTA. Finally, we show that K-RBP inhibits RTA binding to ORF57 promoter. These findings suggest that the DNA-binding activity of K-RBP plays an important role in repressing viral promoter activity.     

Kaposi's sarcoma-associated herpesvirus RTA activates the processivity factor ORF59 through interaction with RBP-Jkappa and a cis-acting RTA responsive element

Kaposi’s sarcoma-associated herpesvirus (KSHV/HHV8) displays two life modes, latency and lytic reactivation in the infected host cells which are equally important for virus mediated pathogenesis. During latency only a small number of genes are expressed. Under specific conditions, KSHV can undergo lytic replication with the production of viral progeny. One immediate-early gene RTA, encoded by open reading frame 50 of KSHV, has been shown to play a critical role in switching the viral latency to lytic reactivation. Over-expression of RTA from a heterologous promoter is sufficient for driving KSHV lytic replication which results in production of viral progeny. In the present study, we show that RTA can activate the expression of the ORF59 which encodes the processivity factor essential for DNA replication during lytic reactivation. We also show that RTA regulates ORF59 promoter through interaction with RBP-Jκ as well as a cis-acting RTA responsive element within the promoter. In the context of KSHV infected cells, the upregulation of ORF59 is a direct response to RTA expression. Taken together, our findings provide new evidence to explain the mechanism by which RTA can regulate its downstream gene ORF59, further increasing our understanding of the biology of KSHV lytic replication.     

Reactivation of Kaposi's sarcoma-associated herpesvirus from latency requires MEK/ERK, JNK and p38 multiple mitogen-activated protein kinase pathways

Lytic replication of Kaposi's sarcoma-associated herpesvirus (KSHV) promotes the progression of Kaposi's sarcoma (KS), a dominant malignancy in patients with AIDS. While 12-O-tetradecanoyl-phorbol-13-acetate (TPA)-induced KSHV reactivation from latency is mediated by the protein kinase C delta and MEK/ERK mitogen-activated protein kinase (MAPK) pathways, we have recently shown that the MEK/ERK, JNK and p38 MAPK pathways modulate KSHV lytic replication during productive primary infection of human umbilical vein endothelial cells [Pan, H., Xie, J., Ye, F., Gao, S.J., 2006. Modulation of Kaposi's sarcoma-associated herpesvirus infection and replication by MEK/ERK, JNK, and p38 multiple mitogen-activated protein kinase pathways during primary infection. J. Virol. 80 (11), 5371-5382]. Here, we report that, besides the MEK/ERK pathway, the JNK and p38 MAPK pathways also mediate TPA-induced KSHV reactivation from latency. The MEK/ERK, JNK and p38 MAPK pathways were constitutively activated in latent KSHV-infected BCBL-1 cells. TPA treatment enhanced the levels of activated ERK and p38 but not those of activated JNK. Inhibitors of all three MAPK pathways reduced TPA-induced production of KSHV infectious virions in BCBL-1 cells in a dose-dependent fashion. The inhibitors blocked KSHV lytic replication at the early stage(s) of reactivation, and reduced the expression of viral lytic genes including RTA, a key immediate-early transactivator of viral lytic replication. Activation of MAPK pathways was necessary and sufficient for activating the promoter of RTA. Furthermore, we showed that the activation of RTA promoter by MAPK pathways was mediated by their downstream target AP-1. Together, these findings suggest that MAPK pathways might have general roles in regulating the life cycle of KSHV by mediating both viral infection and switch from viral latency to lytic replication.