转录因子E2F1调控KIR3DL1基因表达的分子机制  

Molecular regulation of KIR3DL1 gene expression by transcription factor E2F1

作　　者：高晓宁[1] 于力[1] 

机构地区：[1]解放军总医院血液科,北京100853

出　　处：《中华血液学杂志》2008年第12期806-810,共5页Chinese Journal of Hematology

基　　金：基金项目：国家自然科学基金（30670898、3057108）;国家973计划（2005CB522408） 志谢：感谢解放军总医院分子生物室韩为东博士、广西医科大学第一附属医院胃肠腺体外科肖强教授和李雷博士对本工作的支持

摘　　要：目的研究转录因子E2F1对KIR3DL1基因启动子转录活性的影响，明确其调控KIR3DL1基因表达的分子机制。方法采用PCR方法从K562细胞基因组DNA中扩增突变型KIR3DL1基因启动子序列，将产物连接到pGL3-Basic荧光素酶载体，构建报告重组子；采用染色质免疫共沉淀方法检测E2F1与KIR3DL1启动子在细胞内的结合；采用阳离子脂质体SuperFect包裹突变型和野生型KIR3DL1启动子-荧光素酶报告重组子，然后转染K562细胞，染色质免疫共沉淀方法检测E2F1与重组子在细胞内的结合，双荧光素酶检测试剂盒测定荧光素酶活性；将E2F1真核表达载体与野生型KIR3DL1启动子-荧光素酶报告重组子共转染K562细胞，双荧光素酶检测试剂盒测定荧光素酶活性。结果成功构建突变型KIR3DL1启动子-荧光素酶报告重组子，测序证实，在K562细胞KIR3DL1启动子区1个潜在的转录因子E2F1结合位点有1个自然发生的点突变（TTTGGCGC→TrCGGCGC）；E2F1完全不能与K562细胞中突变型KIR3DL1启动子结合，但能与NK-92MI细胞中没有突变的野生型KIR3DL1启动子结合；突变型KIR3DL1启动子-荧光素酶报告重组子保留了部分与E2F1的结合能力，但其相对荧光素酶活性较野生型降低了50％；共转染E2F1真核表达载体使野生型KIR3DL1启动子-荧光素酶报告重组子的相对荧光素酶活性增加为对照组的2倍以上。结论转录因子E2F1参与调控KIR3DL1基因转录激活，E2F1结合位点上CpG二核苷酸的数量和甲基化模式可能影响转录因子E2F1与靶序列的结合。Objective To study the role of transcription factor E2F1 in the transcription of KIR3DL1 promoter, and to identify its molecular mechanism of regulation of KIR3 DL1 gene expression. Methods The mutant promoter fragment of KIR3DL1 gene was amplified from genomie DNA of K562 cells by PCR. The PCR product was cloned into pGL3-basic reporter vector to construct KIR3DL1 promoter-luciferase reporter plasmid（PLRP）. The binding of E2F1 to KIR3DL1 promoter was detected by chromatin immunoprecipitation （CHIP） assays. The KIR3DL1 PLRP construction was transfected into K562 cells using cationic liposome Snperfect. The binding of E2F1 to the construction was detected by CHIP assays and reporter activity was quantitated by the dnal-luciferase reporter assay system. The mammalian expression vector containing E2F1 cDNA was co-transfected into K562 cells with wild-type KIR3DL1 PLR construction and reporter activity was qnantirated . Results The mutant KIR3DL1 PLR recombinant was constructed successfully and a naturally point mutation （TTTGGCGC→TTCGGCGC） within a putative E2F1 binding site in the KIR3DL1 promoter region was authenticated by DNA sequencing. E2F1 absolutely could not bind to the mutant KIR3DL1 promoter in K562 cells, but could bind to the wild-type one in NK-92MI cells. The binding of E2F1 to the mutant KIR3DL1 PLR construction was partially reserved, however, its relative luciferase activity was decreased by 50% than that of wild-type. On the other hand, when eo-transfected with E2F1 mammalian expression vector, the relative luciferase activity of wild-type construction was increased, over 2-fold higher than that of control group. Conclusion E2F1 participates in the regulation of the transcriptional activation of KIR3DL1 gene. The number of CpG dinucleotide and methylation pattern within the E2F1 binding site probably influence the binding of E2F1 to target sequence.

关 键 词：基因 KIR3DL1 转录因子 E2F1 基因表达 

分 类 号：R686[医药卫生—骨科学]