Identification of an insulator in AAVS1, a preferred region for integration of adeno-associated virus DNA
Identification of an insulator in AAVS1, a preferred region for integration of adeno-associated virus DNA
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DOI：
10.1128/jvi.77.16.9000-9007.2003
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发表时间：
2003-08-01
影响因子：
5.4
通讯作者：
Kanda, T
中科院分区：
文献类型：
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作者：
Ogata, T;Kozuka, T;Kanda, T
在潜伏性腺相关病毒(AAV)感染中，病毒基因组优先整合到人类染色体19Q臂上的一个特定区域，称为AAVS1，该区域具有开放的染色质构象，其存在的DNase I超敏位点(DHS-S1)表明。我们检查了在本研究中使用的2.6kBP AAVS1相关DNA片段的中间是否存在绝缘体，该绝缘体通过定向阻断增强子的作用和防止异染色质的扩散来定义基因表达的结构域。将该片段克隆到基于EB病毒(EBV)的真核细胞上，将其导入HEK293细胞。在细胞核中复制的质粒上的DHS-S1对DNase I酶切高度敏感，因此，EBV质粒系统被用于增强子阻断实验，其DNA为2.6-KBP，两个短DNA分别为1.6KBP和336BP，含有DHS-S1。当这三个DNA片段以正确的方向插入巨细胞病毒即刻早期增强子和最小启动子之间时，抑制了报告基因的表达。因此，增强子阻断活性位于包含DHS-S1整个区域(300bp)的336个碱基对的DNA中。在HEK293和HeLa细胞中引入外源异染色质阻遏基因，构建了外源基因表达盒，其两侧分别位于增强子阻断方向。用整合到细胞DNA中的盒检测的所有细胞克隆都继续表达转基因，这表明这对336bP的DNA明显阻止了异染色质的扩散。结果表明，在AAVS1的DHS-S1附近，17号和354号核苷酸之间存在绝缘子。在凝胶漂移试验中，336个碱基的DNA没有结合体外制备的CCCTC结合因子，该因子结合到鸡β-珠蛋白绝缘子上，这表明AAVS1绝缘子需要一个未知的结合蛋白。新发现的AAVS1绝缘体可能有助于维持开放的染色质构象，从而影响AAV的生命周期。
In latent adeno-associated virus (AAV) infection, the viral genome is integrated preferentially into the human chromosome 19 q arm at a specific region designated AAVS1, which has an open chromatin conformation as indicated by the presence of a DNase I-hypersensitive site (DHS-S1). We examined whether an insulator, which defines the domain of gene expression by directionally blocking the action of enhancers and by preventing the spread of heterochomatin, is present near the DHS-S1 in the middle of a 2.6-kbp AAVS1-related DNA fragment used in this study. The fragment, cloned into an Epstein-Barr virus (EBV)-based eukaryotic episomal plasmid, was introduced into HEK293 cells. The DHS-S1 on the plasmid replicating in the nuclei was hypersensitive to DNase I digestion, and thus, the EBV plasmid system was used in an enhancer-blocking assay with the 2.6-kbp DNA and two shortened DNAs, of 1.6 kbp and 336 bp, containing DHS-S1. The three DNA fragments, when inserted in the proper direction between the cytomegalovirus immediate-early enhancer and minimal promoter, repressed the expression of a reporter gene. Thus, the enhancer-blocking activity was located within the 336-bp DNA containing the entire region (300 bp) of DHS-S1. To investigate the prevention of repression caused by heterochromatin, a transgene-expressing cassette flanked by the two 336-bp DNAs placed in the enhancer-blocking direction was introduced into HEK293 and HeLa cells. All the cell clones examined with the cassette integrated into cell DNA continued to express the transgene, which indicates that the pair of 336-bp DNA apparently prevented the spread of heterochromatin. The results show that an insulator lies between nucleotides 17 and 354 near the DHS-S1 in AAVS1. In a gel shift test, the 336-bp DNA did not bind an in vitro-prepared CCCTC-binding factor that binds to the chicken beta-globin insulator, suggesting that the AAVS1 insulator requires an as yet unidentified binding protein. The newly identified AAVS1 insulator is likely to contribute to the maintenance of an open chromatin conformation that affects the life cycle of AAV.