| Summary: | 博士 === 國立陽明大學 === 微生物及免疫學研究所 === 85 === B型肝炎病毒的複製及基因表現具有組織特異性，這種特異性主要是受到多重轉錄調控序列及具有組織特異性之轉錄蛋白的控制。已知B型肝炎病毒的基因群中包含了四個起動子及兩個具有組織特異性的加強子。其中第二加強子(核甘酸序列1636至1741)，簡稱ENII位於核心抗原起動子的上游，它可以加強表面抗原起動子(SPI)，X蛋白起動子及異源性起動子(如SV40早期起動子)的轉錄活性，同時其加強能力與距離及方向性無關，因此其作用方式為加強子模式。但是同樣這段序列只能以正向且位於上游方式增強核心抗原起動子的轉錄，所以作用方式為上游調控序列(core promoter upstream regulatory sequence，簡稱為CURS)模式。
為了更進一步研究核心抗原起動子的轉錄調控，本論文以刪減序列的方法分析ENII/CURS的上游序列，發現ENII/CURS的上游有負調控子，其中核甘酸1613至1636可以抑制ENII/CURS的轉錄加強能力，因此將這段序列稱為負調控子(NRE;negative regulatory element)。以連續突變掃讀的方式，定出核甘酸1616至1621為NRE的必要序列。NRE單獨存在時對起動子的抑制力不強。並且突變ENII使其失去加強子功能時，NRE會失去抑制力，因此NRE須在具有加強子功能的ENII存在下才有抑制力。此亦可解釋為何只在具有ENII/CURS加強活性的細胞株(如HepG2及HuH-7)才有明顯的NRE抑制作用。同時其抑制力對ENII/CURS的距離有關。根據這些特性我們推測NRE的作用機制可能屬於quenching mechanism。
以band-shift方法分析時發現，HepG2細胞核蛋白可結合在NRE序列上；進一步以heparin-Sepharose column及各種濃度NaCI分離結合親和力不同的DNA結合蛋白，發現0.4M核蛋白只能結合在NRE原型(wild type)序列上，而不能結合在NRE突變序列MT1上，因此HepG2 0.4M fraction含有具NRE結合專一性的核蛋自，稱為專一性NREBP。進一步分析不同細胞株的NREBP，發現專一性NREBP廣泛地分布於高度分化的肝癌細胞株(如HepG2及HuH-7)，低度分化的肝癌細胞株(如HA22T/VGH)，及非肝細胞株(如HeLa)中，因此推測它可能不具有組織及分化特異性。
以NRE序列為探針，自HepG2表現cDNA庫中選殖出四種不同的DNA結合蛋白基因，表現這四組cDNA的GST融合蛋白，測試其DNA結合專一性，其中NRE結合專一性最高的一組，稱為NREBP18o。NREBP180具有下列特性:(一)對NRE序列原型的結合力較高，對NRE突變序列MT1突變型的結合力較低；(二)以binding site selection assay發現其DNA結合序列為[GA(G/T)AN(C/G)(A/G)CCN]，HBV NRE序列(1613-1636)完全符合此序列；(三)以其多源抗體在HepG20.4M fraction核蛋白中偵測到180kDa的蛋白。因此稱此蛋白為NREBP180；(四)將NREBP180 cDNA轉染入HuH-7及HepG2細胞來表現NREBP180時，可以加強NRE對CURS-BCP的抑制力；(五)以northernblot analysis偵測，發現其mRMA廣泛地表現於各種組織及器官中，其主要mRNA的長度為9至9.5kb。NREBP180蛋白可結合於NRE上且可以加強NRE的抑制力，但其是否就是在細胞中作用於NRE序列上抑制ENII/CURS作用的蛋白則仍待更多的研究證明之。
The replication and gene expression of hepatitis B virus (HBV) exhibit a liver specificity. This liver specificity is mostly regulated at the transcriptional level by the liver-specific regulatory elements and their binding transcriptional factors. HBV genome consists of four promoters and two liver-specific enhancers. One of these two enhancers, enhancer II (nt 1636 to 1741; ENII), is located to the upstream of basal core promoter. ENII can enhance the transcriptional activity of large surface promoter (SPI) and X promoter of HBV and a heterologous promoter SV40 early promoter in a position-and orientation-independent manner. In contrast, the same sequence activates the basal core promoter in a position-and orientation-dependent manner, that is, functions as upstream regulatory element (core promoter upstream regulatory sequence; CURS).
Using deletion mutants to analyze the sequence upstream to the CURS/ENII, a negative regulatory element (located at nt 1613 to 1636; NRE) which represses the ENII/CURS activity was identified. A linker-scanning analysis reveal that the sequence from nt 1616 to 1621 essential for the repressive activity of the NRE. NRE itself has only a marginal inhibitory effect. Mutation of the enhancer II sequence which destroies the enhancer activity can abolish the repressive activity of the NRE, demonstrating that the repressive function of the NRE dependents on the presence of a functional enhancer II. These results are consistent with the observation in which the repressive effect of NRE occurs only in the highly differentiated hepatoma cell line. Furthermore, a proximity distance to enhancer II is required for the repressive function of NRE. These results suggest that NRE represses the enhancer activity of ENII via a quenching mechanism.
HepG2 crude nuclear extract was fractionated by the heparin Sepharose column and eluted with increasing concentrations of NaC1. Band-shift analysis in the presence of specific and nonspecific competitors was performed to examine the interaction of the cellular factor(s) with the NRE sequence. Using the NRE sequence as a probe, a specific DNA-protein complex was detected, by 0.4 M fraction and designated as NRE binding protein (NREBP). NREBP is present in highly differentiated hepatoma cell line (such as HepG2 and HuH-7), poorly-differentiated hepatoma cell line (such as HA22T/VGH) and non-liver cell line (HeLa). These results indicate that NREBP is probably an ubiquitous protein.
To clone the NREBP, a concatemer of NRE was used as a probe to screen the HepG2 expression cDNA library. 12 cDNA clones which belong to four different kinds of genes were identified. The DNA binding specificity of GST fusion protein of these four genes was examined. One clone which displays the highest binding specificity toward NRE designated as NREBP180. NREBP180 exhibits the following characteristics: (1) It binds to the NRE wild type better than the NRE mutant; (2) The binding site selection ssay determines the consensus sequence as GA(G/T)AN(C/G)(A/G)CCN, the NRE sequence of HBV fits it very well; (3) A 180 kDa protein was detected in the HepG2 0.4M fraction by polycloned antibody against NREBP180; (4) Overexpression of the NREBP180 in HuH-7 and HepG2 cell lines can further increase the repressive effect of NRE; (5) Northern blot analysis of RNAs from different human tissues indicated the major transcript of NREBP180 gene (about 9~9.5 kb) is ubiquitously expressed in human tissues. These results together strongly suggest that NREBP180 is a candidate for specific NREBP.
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