Poleshko Andrey, Palagin Ivan, Zhang Rugang, Boimel Pamela, Castagna Carolyn, Adams Peter D, Skalka Anna Marie, Katz Richard A
Fox Chase Cancer Center, Institute for Cancer Research, 333 Cottman Avenue, Philadelphia, PA 19111, USA.
J Virol. 2008 Mar;82(5):2313-23. doi: 10.1128/JVI.01882-07. Epub 2007 Dec 19.
Integrated retroviral DNA is subject to epigenetic gene silencing, resulting in loss of expression of viral genes as well as reporter or therapeutic genes transduced by retroviral vectors. Possible mediators of such silencing include the histone deacetylase (HDAC) family of cellular proteins. We previously isolated HeLa cell populations that harbored silent avian sarcoma virus-based green fluorescent protein (GFP) vectors that could be reactivated by treatment with HDAC inhibitors. Here, we developed a small interfering RNA (siRNA)-based approach to identify specific host factors that participate in the maintenance of silencing. Knockdown of HDAC1, the transcriptional repressor Daxx (a binding partner of HDAC1), or heterochromatin protein 1 gamma resulted in robust and specific GFP reporter gene reactivation. Analyses of cell clones and diverse GFP vector constructs revealed that the roles of HDAC1 and Daxx in retroviral silencing are largely independent of the integration site or the promoter controlling the silent GFP reporter gene. Previous findings from our laboratory and those of others have suggested that Daxx and HDAC proteins may act broadly as part of an antiviral response to repress viral gene transcription. Expression of presumptive viral "countermeasure" proteins that are known to inhibit Daxx or HDACs (pp71, IE2, and Gam1) resulted in the reactivation of GFP reporter gene expression. This study has identified individual host factors that maintain retroviral silencing and supports the proposal that these factors participate in an antiviral response. Furthermore, our results indicate that siRNAs can be used as specific reagents to interrupt the maintenance of epigenetic silencing.
整合的逆转录病毒DNA会发生表观遗传基因沉默，导致病毒基因以及逆转录病毒载体转导的报告基因或治疗基因表达缺失。这种沉默的可能介导因子包括细胞蛋白的组蛋白去乙酰化酶（HDAC）家族。我们之前分离出了携带沉默的基于禽肉瘤病毒的绿色荧光蛋白（GFP）载体的HeLa细胞群体，这些载体可通过HDAC抑制剂处理而重新激活。在此，我们开发了一种基于小干扰RNA（siRNA）的方法来鉴定参与维持沉默的特定宿主因子。敲低HDAC1、转录抑制因子Daxx（HDAC1的结合伴侣）或异染色质蛋白1γ会导致强大且特异性的GFP报告基因重新激活。对细胞克隆和多种GFP载体构建体的分析表明，HDAC1和Daxx在逆转录病毒沉默中的作用在很大程度上独立于整合位点或控制沉默GFP报告基因的启动子。我们实验室以及其他实验室之前发现，Daxx和HDAC蛋白可能广泛作为抗病毒反应的一部分来抑制病毒基因转录。已知能抑制Daxx或HDACs的假定病毒“对策”蛋白（pp71、IE2和Gam1）的表达导致了GFP报告基因表达的重新激活。本研究鉴定出了维持逆转录病毒沉默的单个宿主因子，并支持了这些因子参与抗病毒反应的提议。此外，我们的结果表明，siRNA可作为特异性试剂来中断表观遗传沉默的维持。
