title: High-frequency epigenetic repression and silencing of retroviruses can be antagonized by histone deacetylase inhibitors and transcriptional activators, but uniform reactivation in cell clones is restricted by additional mechanisms.
abstract: Integrated retroviral DNA is subject to epigenetic gene silencing, but the viral and host cell properties that influence initiation, maintenance, and reactivation are not fully understood. Here we describe rapid and high-frequency epigenetic repression and silencing of integrated avian sarcoma virus (ASV)-based vector DNAs in human HeLa cells. Initial studies utilized a vector carrying the strong human cytomegalovirus (hCMV) immediate-early (IE) promoter to drive expression of a green fluorescent protein (GFP) reporter gene, and cells were sorted into two populations based on GFP expression [GFP(+) and GFP(-)]. Two potent epigenetic effects were observed: (i) a very broad distribution of GFP intensities among cells in the GFP(+) population as well as individual GFP(+) clones and (ii) high-frequency GFP reporter gene silencing in GFP(-) cells. We previously showed that histone deacetylases (HDACs) can associate with ASV DNA soon after infection and may act to repress viral transcription at the level of chromatin. Consistent with this finding, we report here that treatment with the histone deacetylase inhibitor trichostatin A (TSA) induces GFP activation in GFP(-) cells and can also increase GFP expression in GFP(+) cells. In the case of the GFP(-) populations, we found that after removal of TSA, GFP silencing was reestablished in a subset of cells. We used that finding to enrich for stable GFP(-) cell populations in which viral GFP reporter expression could be reactivated by TSA; furthermore, we found that the ability to isolate such populations was independent of the promoter driving the GFP gene. In such enriched cultures, hCMV IE-driven, but not the viral long terminal repeat-driven, silent GFP reporter expression could be reactivated by the transcriptional activator prostratin. Microscopy-based studies using synchronized cells revealed variegated reactivation in cell clones, indicating that secondary epigenetic effects can restrict reactivation from silencing. Furthermore we found that entry into S phase was not required for reactivation. We conclude that HDACs can act rapidly to initiate and maintain promoter-independent retroviral epigenetic repression and silencing but that reactivation can be restricted by additional mechanisms.
authors.affiliation: Institute for Cancer Research, Fox Chase Cancer Center, 333 Cottman Avenue, Philadelphia, PA 19111, USA. Richard.Katz@fccc.edu
authors.forename: Richard A
authors.initials: RA
authors.lastname: Katz
authors.forename: Emily
authors.initials: E
authors.lastname: Jack-Scott
authors.forename: Anna
authors.initials: A
authors.lastname: Narezkina
authors.forename: Ivan
authors.initials: I
authors.lastname: Palagin
authors.forename: Pamela
authors.initials: P
authors.lastname: Boimel
authors.forename: Joseph
authors.initials: J
authors.lastname: Kulkosky
authors.forename: Emmanuelle
authors.initials: E
authors.lastname: Nicolas
authors.forename: James G
authors.initials: JG
authors.lastname: Greger
authors.forename: Anna Marie
authors.initials: AM
authors.lastname: Skalka
mesh_terms: D002999:Clone Cells / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D044127:Epigenesis, Genetic; D020868:Gene Silencing; D017930:Genes, Reporter; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006367:HeLa Cells; D056572:Histone Deacetylase Inhibitors; D006801:Humans; D006877:Hydroxamic Acids / Q000494:pharmacology; D010703:Phorbol Esters / Q000494:pharmacology; D012190:Retroviridae / Q000235:genetics / Q000378:metabolism*; D015534:Trans-Activators / Q000378:metabolism*
chemical_list: D004791:Enzyme Inhibitors; D056572:Histone Deacetylase Inhibitors; D006877:Hydroxamic Acids; D010703:Phorbol Esters; D015534:Trans-Activators; D049452:Green Fluorescent Proteins; C012589:trichostatin A; C070999:prostratin