人肝细胞核因子4基因Ⅱ型启动子的双向转录调控的特性

Characterization of bidirectional transcription of human hepatocyte nuclear factor 4 alpha P2 promoter

目的探讨人肝细胞核因子4α基因Ⅱ型启动子(hHNF4α-P2)双向调控基因表达的作用。方法克隆hHNF4α-P2–23~–1291(1268 bp)序列;构建由该段正、反序列驱动的荧光蛋白真核表达载体;将其通过细胞转染和显微注射导入细胞或斑马鱼体内,考察其表达的时空特性。通过DNA缺失实验和序列预测分析寻找hHNF4α-P2的正、反向核心启动序列。结果 hHNF4α-P2的正向和反向DNA序列(–23~–1291)均可驱动其下游报告基因在人正常肝细胞L02及肝癌细胞Huh7中表达;在斑马鱼体内正向启动子驱动的红色荧光蛋白基因可在耳石、嗅泡和神经丘等感觉器官中高表达,在肝区可见较弱的红色荧光。反向启动子驱动的绿色荧光蛋白基因可在血细胞、神经细胞、体节及脊索等组织表达。该启动子正链存在一段核心序列,反链存在两段核心序列。结论首次发现hHNF4α-P2的–23~–1291序列具有双向调控基因在不同组织表达的功能。

Objective To explore the bidirectional transcriptional function of human hepatocyte nuclear factor 4 alpha（hHNF4α） P2 promoter. Methods The P2 promoter partial sequence of h HNF4α from –23 to –1291, totally 1268 bp was cloned. Bidirectional transcription vectors were constructed in eukaryotic expression vectors via linking the fluorescent protein genes, EGFP and DsRed, to both the upstream or/and downstream of the P2 DNA sequence, respectively. They were named pP2f/hnf4-EGFP（pP2f-EGFP） for the forward transcription, pP2r/hnf4-EGFP（pP2r-EGFP） for the reverse transcription and pP2bi/hnf4-EGFP/Ds Red（pP2bi-EGFP/DsRed） for the bidirectional transcription. Then, pP2f-EGFP and pP2r-EGFP were separately transfected into L02 and Huh7 cells, and pP2bi-EGFP/Ds Red was microinjected into zebrafish embryos to profile the time-and tissue-specific expression patterns of P2 promoter at both transcriptional directions in zebrafish embryonic development. Core promoter regions were identified by a series of P2 sequence deletion experiments and combining with sequence prediction of promoter core regions by http：//www. fruitfly.org/online. Results Fluorescent reporters was expressed driven by either the forward or the reverse directional transcription of h HNF4α-P2 promoter in human normal hepatocyte cell line L02 and hepatoma cell line Huh7. Furthermore, transcriptional patterns in the forward and reverse directional transcription of the P2 promoter were examined in zebrafish embryos by the P2 bidirectional transcription construct pP2bi-EGFP/Ds Red. The results showed that EGFP, as the reverse-directional reporter, started to express at the stage of 16 hpf in embryonic organs derived from mesoderm and ectoderm, such as blood cells, neurons, epidermis, somites and notochord cells; meanwhile, Ds Red, as the forward-directional reporter, expressed intensively in otolith, olfactory bulb and hair cells of neuromasts in the lateral lines which developed from ectoderm, and also expressed moderately in liver bud which originated from endoderm. The expressions of both forward-and reverse-directional transcriptions sustained to at least 10 dpf. Only one core promoter region（–529-–587） was found in the P2 sequence of forward transcriptional direction, and its expression pattern was as same as that of pP2f-Ds Red construct. Also, two core promoter regions existed in the complementary strand sequence of P2 promoter（–23-–1291）, and one of them（–797-–976） showed the same expression pattern as pP2r-EGFP pattern, while the other one（–355-–529） lacked signal in neurons and showed relatively delayed expression compared to pP2r-EGFP pattern. Conclusion This is the first report on the bidirectional transcription of h HNF4α-P2 promoter（–23-–1291） in developmental stageand tissue-specific manner in zebrafish.