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.

FIG. 1.

Knockdown of HDAC1 or Daxx results in reactivation of the silent GFP reporter. (A) HeLa TI-C cells (28) were transfected with the indicated siRNA SMARTpools (100 nM) (Dharmacon) and incubated for 96 h. GFP expression was analyzed by FACS. NT, not transfected. (B) Histograms of GFP intensities (x axis) from the experiment shown in panel A. Percentages of GFP-positive cells are indicated by the numerical values. Autoscaling was used to portray the distribution of GFP intensities.

FIG. 2.

Determination of specificity of siRNA knockdown. (A) qRT-PCR analysis of target mRNAs. For each target mRNA measurement, the values were normalized to a control which was treated with transfection reagent only (DharmaFECT 1 [DF1]). For HDAC1 and Daxx siRNA treatments, the levels of HDAC1, HDAC2, HDAC3, HDAC4, and Daxx mRNAs were measured. For HDAC2, HDAC3, and HDAC4 siRNAs, only the cognate target mRNA levels were measured (*). (B) Assessment of knockdown by Western blotting. TI-C cells were treated with 100 nM of siRNAs indicated above the panel and cells were processed for Western blotting after 72 h. GAPDH antibody was used to monitor recovery. Mock siRNA treatments were performed in duplicate. (C) Transfection of a plasmid encoding an siRNA-resistant form of HDAC1 mRNA. Silent mutations that destroy the HDAC1 siRNA 01 annealing site were introduced into an HDAC1 expression plasmid, as described in Materials and Methods. Mutant plasmids prepared in duplicate (R1, R2) or a wt control plasmid was introduced into TI-C cells along with the HDAC1 siRNA 01. GFP expression was monitored by FACS. As shown, HDAC 01 siRNA was capable of stimulating GFP reactivation after transfection of the wt HDAC1 plasmid. In contrast, the siRNA-resistant plasmids were able to repress GFP expression in the presence of the siRNA. (D) Localization of Daxx at the GFP promoter. ChIP analysis was carried out as described in Materials and Methods. Two primer sets were used, targeting the silent viral GFP promoter region or the active cellular β-actin gene. Experiments shown are representative, and the Daxx results are averages of triplicate immunoprecipitations. IgG, immunoglobulin G.

FIG. 3.

Knockdown of several candidate proteins or treatment with various control siRNAs fails to reactivate the silent GFP reporter. (A and B) HeLa TI-C cells were treated with the indicated siRNAs and the percentages of GFP-positive cells were determined by FACS at 96 h posttransfection. Single siRNAs were used for H3.3A, H3.3B, and HIRA. Two independent single siRNAs (designated a and b) were tested for HIRA. DF1, DharmaFECT 1 transfection reagent. (C) TI-C cells were treated with 100 nM siRNAs as indicated above the panels and cells were processed for Western blotting after 72 h. (D) Treatment and analysis with the indicated siRNAs was as for panel A. Negative control siRNAs RISC−, RISC+, and GAPDH were analyzed. (E) The HDAC1 siRNA SMARTpool was titrated to determine the lowest effective concentration versus the negative control siRNA RISC⁺. Analysis was as for panel A.

FIG. 4.

Analysis of TI-C silent cell clones. (A) Clones were treated with TSA or a dimethyl sulfoxide (DMSO) control, and GFP was monitored by FACS after 24 h. (B) Cell clones were transfected with the indicated siRNAs, and GFP reactivation was monitored by FACS after 96 h. Representative results are shown.

FIG. 5.

Analysis of the role of HP1 isoforms in silencing maintenance. (A) HeLa TI-C cells were transfected with the indicated HP1 isoform siRNA SMARTpools, and GFP reactivation was monitored by FACS analysis after 96 h. Abbreviations: NT, not transfected; DF1, DharmaFECT 1 transfection reagent. (B and C) Western blot analyses of siRNA knockdown of the HP1 family of proteins. Cells were transfected with the siRNAs indicated above the panels. The detection of HP1α required loading of 10-fold more protein.

FIG. 6.

Expression of a dnHP1 reactivates silent GFP. (A) Map of dnHP1. Chromo, chromodomain. (B) A retroviral vector encoding a dnHP1 (60) was used to infect HeLa TI-C cells. Cells were placed under selection with puromycin and were monitored for reactivation of GFP expression by FACS. The FACS profile obtained at 5 days postinfection shows GFP expression in cells selected with the dnHP1 expression vector (no fill) versus the empty vector (filled). The expression of dnHP1 produced a population of GFP-positive cells (24%), some of which were very bright and appear off scale in the graph. A representative experiment is shown.

FIG. 7.

Reactivation of silent GFP by viral proteins. (A and B) HeLa TI-C cells were transfected with expression plasmids encoding the indicated proteins and GFP reactivation was monitored after 48 h by FACS analysis.

FIG. 8.

Analysis of HeLa cells harboring silent GFP under the control of the ASV LTR. (A and B) TI-L cells were transfected with the indicated siRNA SMARTpools and GFP expression was measured after 96 h by FACS analysis. NT, not transfected; two independent single siRNAs tested for HIRA are designated HIRA a and b.