人乳头瘤病毒16型长控制区上YY1结合位点的破坏可增强病毒诱导细胞永生化能力

细胞转录调节因子 Ｙ Ｙ１ 可抑制人乳头瘤病毒１６ 型（ Ｈ Ｐ Ｖ １６） 癌基因启动子 Ｐ９７ 的活性, Ｙ Ｙ１ 位点的突变和缺失不仅可诱导 Ｐ９７ 活性增强而且可在全基因组内增强 Ｅ６ 癌基因转录,同时使病毒对啮齿类动物纤维细胞的转化能力增强。为了观测人乳头瘤病毒１６ 型长控制区（ Ｈ Ｐ Ｖ１６ Ｌ Ｃ Ｒ） 序列上 Ｙ Ｙ１ 蛋白特异性结合位点破坏在完整基因组范围内对人原代包皮角源细胞永生化能力的影响,将 Ｈ Ｐ Ｖ １６ Ｙ Ｙ１ 位点突变株和野毒株转染至人原代包皮角源细胞。筛选结果表明,突变株可诱导形成永生化细胞,永生化能力明显高于野毒株。对４ 株永生化细胞系 Ｄ Ｎ Ａ检测发现,均含有呈整合状态的 Ｈ Ｐ Ｖ １６ Ｄ Ｎ Ａ,其中３ 株的 Ｅ１／ Ｅ２ 区域有缺失。 Ｒ Ｎ Ａ 检测显示,４株细胞内均有 Ｅ６／ Ｅ７ ｍ Ｒ Ｎ Ａ 的转录。这表明, Ｈ Ｐ Ｖ １６ Ｌ Ｃ Ｒ 上 Ｙ Ｙ１ 蛋白特异性结合位点的破坏,可在完整基因组范围内增强病毒使人原代包皮角源细胞永生化的能力。     

YY1抑制效应的破坏可促进人乳头瘤病毒16型癌基因的转录

人乳头瘤病毒１６型（ＨＰＶ１６）癌基因的表达受病毒早期启动子Ｐ９７的控制。位于ＬＣＲ上ＹＹ１蛋白结合位点的破坏可明显提高Ｐ９７的活性。为了观测ＹＹ１位点破坏在全基因组范围内对病毒ｅ６／ｅ７基因转录的影响,将构建的带有ＬＣＲ特异性突变的重组ＨＰＶ１６全基因组ＤＮＡ和ＨＰＶ１６野毒株ＤＮＡ转染至培养细胞,同时组建ＨＰＶ１６Ｅ６反向序列ＲＮＡ体外转录质粒。ＲＮａｓｅ保护试验证实,突变ＨＰＶ１６ＤＮＡ在短     

构建来自宫颈癌并带有突变及缺失LCR的HPV16重组体

为研究ＨＰＶ１６转录调节蛋白ＹＹ１结合位点改变对病毒致癌性的影响,以ＨＰＶ１６野毒株质粒ｐ１２０３为基础,经多次克隆,将来自宫颈癌组织并带有缺损突变的ＬＣＲ重组到地ＨＰＶ１６基因组中。核酸序列分析证实,质粒ｐＤＶ３９０在第２个ＹＹ１位点上有一Ｇ→Ａ点穷变,质粒ｐＤＶ３２６和ｐＤＶ４０１分别带有１１５ｂｐ和１４３ｂｐ的缺失突变,从而涉及２和４个ＹＹ１结合位点,重组质粒其它核苷酸序列与ＨＰＶ１６标准序列一致。这些重组质粒的组建为进一步研究ＹＹ１蛋白对ＨＰＶ１６致癌基因表达的调控及ＨＰＶ１６致癌作用的影响打下基础。     

YY1 activates Msx2 gene independent of bone morphogenetic protein signaling

Msx2 is a homeobox gene expressed in multiple embryonic tissues which functions as a key mediator of numerous developmental processes. YY1 is a bi-functional zinc finger protein that serves as a repressor or activator to a variety of promoters. The role of YY1 during embryogenesis remains unknown. In this study, we report that Msx2 is regulated by YY1 through protein–DNA interactions. During embryogenesis, the expression pattern of YY1 was observed to overlap in part with that of Msx2. Most notably, during first branchial arch and limb development, both YY1 and Msx2 were highly expressed, and their patterns were complementary. To test the hypothesis that YY1 regulates Msx2 gene expression, P19 embryonal cells were used in a number of expression and binding assays. We discovered that, in these cells, YY1 activated endogenous Msx2 gene expression as well as Msx2 promoter–luciferase fusion gene activity. These biological activities were dependent on both the DNA binding and activation domains of YY1. In addition, YY1 bound specifically to three YY1 binding sites on the proximal promoter of Msx2 that accounted for this transactivation. Mutations introduced to these sites reduced the level of YY1 transactivation. As bone morphogenetic protein type 4 (BMP4) regulates Msx2 expression in embryonic tissues and in P19 cells, we further tested whether YY1 is the mediator of this BMP4 activity. BMP4 did not induce the expression of YY1 in early mouse mandibular explants, nor in P19 cells, suggesting that YY1 is not a required mediator of the BMP4 pathway in these tissues at this developmental stage. Taken together, these findings suggest that YY1 functions as an activator for the Msx2 gene, and that this regulation, which is independent of the BMP4 pathway, may be required during early mouse craniofacial and limb morphogenesis.     

The DNA binding domain of a papillomavirus E2 protein programs a chimeric nuclease to cleave integrated human papillomavirus DNA in HeLa cervical carcinoma cells

Viral DNA binding proteins that direct nucleases or other protein domains to viral DNA in lytically or latently infected cells may provide a novel approach to modulate viral gene expression or replication. Cervical carcinogenesis is initiated by high-risk human papillomavirus (HPV) infection, and viral DNA persists in the cancer cells. To test whether a DNA binding domain of a papillomavirus protein can direct a nuclease domain to cleave HPV DNA in cervical cancer cells, we fused the DNA binding domain of the bovine papillomavirus type 1 (BPV1) E2 protein to the catalytic domain of the FokI restriction endonuclease, generating a BPV1 E2-FokI chimeric nuclease (BEF). BEF introduced DNA double-strand breaks on both sides of an E2 binding site in vitro, whereas DNA binding or catalytic mutants of BEF did not. After expression of BEF in HeLa cervical carcinoma cells, we detected cleavage at E2 binding sites in the integrated HPV18 DNA in these cells and also at an E2 binding site in cellular DNA. BEF-expressing cells underwent senescence, which required the DNA binding activity of BEF, but not its nuclease activity. These results demonstrate that DNA binding domains of viral proteins can target effector molecules to cognate binding sites in virally infected cells.