title: Identification of cellular proteins that maintain retroviral epigenetic silencing: evidence for an antiviral response.
abstract: 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.
authors: Fox Chase Cancer Center, Institute for Cancer Research, 333 Cottman Avenue, Philadelphia, PA 19111, USA.
authors: Andrey
authors: A
authors: Poleshko
authors: Ivan
authors: I
authors: Palagin
authors: Rugang
authors: R
authors: Zhang
authors: Pamela
authors: P
authors: Boimel
authors: Carolyn
authors: C
authors: Castagna
authors: Peter D
authors: PD
authors: Adams
authors: Anna Marie
authors: AM
authors: Skalka
authors: Richard A
authors: RA
authors: Katz
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics / Q000502:physiology; D001483:Base Sequence; D000090266:Chromobox Protein Homolog 5; D002868:Chromosomal Proteins, Non-Histone / Q000502:physiology; D056970:Co-Repressor Proteins; D017931:DNA Primers; D044127:Epigenesis, Genetic / Q000502:physiology*; D020868:Gene Silencing / Q000502:physiology*; D006367:HeLa Cells; D056572:Histone Deacetylase Inhibitors; D006655:Histone Deacetylases / Q000235:genetics / Q000502:physiology; D006801:Humans; D018832:Molecular Chaperones; D009687:Nuclear Proteins / Q000235:genetics / Q000502:physiology; D011401:Promoter Regions, Genetic; D011506:Proteins / Q000502:physiology*; D034741:RNA, Small Interfering; D012190:Retroviridae / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D002868:Chromosomal Proteins, Non-Histone; D056970:Co-Repressor Proteins; C106985:DAXX protein, human; D017931:DNA Primers; D056572:Histone Deacetylase Inhibitors; D018832:Molecular Chaperones; D009687:Nuclear Proteins; D011506:Proteins; D034741:RNA, Small Interfering; D000090266:Chromobox Protein Homolog 5; D006655:Histone Deacetylases