The Chromatin Structure of the Long Control Region of Human Papillomavirus Type 16 Represses Viral Oncoprotein Expression
The Chromatin Structure of the Long Control Region of Human Papillomavirus Type 16 Represses Viral Oncoprotein Expression
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DOI：
10.1128/jvi.73.3.1918-1930.1999
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发表时间：
1999-03
影响因子：
5.4
通讯作者：
W. Stünkel;H. Bernard
中科院分区：
文献类型：
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作者：
W. Stünkel;H. Bernard
摘要人乳头瘤病毒16型（HPV-16）的长控制区（LCR）长约850 bp（约占病毒基因组的12%），调控病毒DNA的转录和复制。LCR的5′片段含有转录终止信号和核基质附着区，中央片段含有上皮细胞特异性增强子，3′片段含有复制起点和E6启动子。在这里，我们报告的HPV-16基因组的这一部分的染色质组织的观察。用甲基丙基-EDTA-Fe（II）处理CaSki细胞（一种具有500个染色体内拷贝的HPV-16的细胞系）的细胞核，揭示了位于LCR和E6和E7基因上的特定位置的核小体。其中一个核小体，我们称之为Ne，与病毒增强子的中心重叠，而第二个核小体Np 16与复制起点和E6启动子重叠。在克隆的HPV-16 LCR上体外组装染色质后，两个核小体位于完全相同的片段上。DNA酶I切割的染色质的引物延伸图谱显示Np 16位于E6启动子元件的中心，延伸到复制起点。Ne覆盖增强子的中心，但留下AP-1位点，增强子的最强顺式反应元件之一，未受保护。Np 16或Np 16和Ne的组合在HPV-16染色质的体外转录期间抑制E6启动子的活性。通过添加Sp1和AP-1转录因子缓解抑制。Sp1在体外改变了Np 16的结构，而在AP-1的结合过程中没有观察到变化。HPV-18具有与HPV-16相似的顺式反应元件排列，尽管其进化上与HPV-16不同，但在体外显示出核小体Np 18在E1结合位点和E6启动子元件上的特异性组装，但位于HPV-16同源序列上Np 16位置的约90 bp 5′处。HPV-16和HPV-18基因组的染色质组织表明核小体在病毒生命周期中的重要调节作用。
ABSTRACT The long control region (LCR) of human papillomavirus type 16 (HPV-16) has a size of 850 bp (about 12% of the viral genome) and regulates transcription and replication of the viral DNA. The 5′ segment of the LCR contains transcription termination signals and a nuclear matrix attachment region, the central segment contains an epithelial cell-specific enhancer, and the 3′ segment contains the replication origin and the E6 promoter. Here we report observations on the chromatin organization of this part of the HPV-16 genome. Treatment of the nuclei of CaSki cells, a cell line with 500 intrachromosomal copies of HPV-16, with methidiumpropyl-EDTA-Fe(II) reveals nucleosomes in specific positions on the LCR and the E6 and E7 genes. One of these nucleosomes, which we termed Ne, overlaps with the center of the viral enhancer, while a second nucleosome, Np16, overlaps with the replication origin and the E6 promoter. The two nucleosomes become positioned on exactly the same segments after in vitro assembly of chromatin on the cloned HPV-16 LCR. Primer extension mapping of DNase I-cleaved chromatin revealed Np16 to be positioned centrally over E6 promoter elements, extending into the replication origin. Ne covers the center of the enhancer but leaves an AP-1 site, one of the strongestcis-responsive elements of the enhancer, unprotected. Np16, or a combination of Np16 and Ne, represses the activity of the E6 promoter during in vitro transcription of HPV-16 chromatin. Repression is relieved by addition of Sp1 and AP-1 transcription factors. Sp1 alters the structure of Np16 in vitro, while no changes can be observed during the binding of AP-1. HPV-18, which has a similar arrangement ofcis-responsive elements despite its evolutionary divergence from HPV-16, shows specific assembly in vitro of a nucleosome, Np18, over the E1 binding site and E6 promoter elements but positioned about 90 bp 5′ of the position of Np16 on the homologous HPV-16 sequences. The chromatin organization of the HPV-16 and HPV-18 genomes suggests important regulatory roles of nucleosomes during the viral life cycle.