Early growth response gene 1 (Egr-1) regulates HSV-1 ICP4 and ICP22 gene expression

Abstract

The molecular mechanisms mediating herpes simplex virus type 1 (HSV-1) gene silencing during latent infection are not clear. Five copies of early growth response gene 1 (Egr-1) binding elements were identified in the intron of HSV-1 ICP22 (infected cell protein No. 22) gene, leading to the hypothesis that Egr-1 binds to the viral genome and regulates the viral gene expression. Transient co-transfection assays indicated that Egr-1 negatively regulated the transcription of both full-length and intron-removed ICP22 promoters. The same assays also revealed that Egr-1 repressed ICP4 (infected cell protein No. 4) promoter activity in a dose-dependent manner but showed less inhibition when the intron was removed. Histone deacetylation was not involved in this regulation since histone deacetylase inhibitor trichostatin A did not exhibit any effect on Egr-1-mediated repression. Chromatin immunoprecipitation assays showed that Egr-1 reduced the binding of Sp1 to the promoters and that the co-repressor Nab2 (NGFI-A/EGR1-binding protein) was recruited to the proximity of ICP4 in the presence of Egr-1. These results suggested that the multifunctional transcription factor Egr-1 can repress HSV-1 immediate-early gene expression through the recruitment of co-repressor Nab2 and reduction of Sp1 occupancy, and thus may play a critical role in HSV-1 gene silencing during latency.

Figure 1

Schematic representation of promoter regions in plasmids. All plasmids are constructed based on the pREP-SEAP vector, and promoters were introduced into the multiple cloning sites. ICP4 and ICP22 promoters share the same regulatory sequence but direct transcription in opposite orientations. The Sp1 binding sites in the regulatory sequence are shown in black boxes.

Figure 2

Over-expression of Egr-1 in HEK293 cells. HEK293 cells were transfected with the Egr-1 expression vector pJDM948. Lane 1: Cells transfected with the control plasmid. Lane 2: Cells transfected with pJDM948. Immunoblot was performed using anti-Egr-1 antibody and anti-α-tubulin antibody as a control.

Figure 3

Regulatory effect of Egr-1 on HSV-1 promoters. HEK293 cells were co-transfected with pH4-2 and pJDM948. Lane 1: Cells tarnsfected with pH4-2 and the control plasmid (molar ratio, 1:1). Lane 2: Cells transfected with pH 4-2 and pJDM948 (molar ratio, 1:1). The cDNA of LAT, ICP4, ICP0, and actin were labeled. The quantitative analysis of RT-PCR signals was performed by Kodak Gel Logic 100 system.

Figure 4

Regulation of ICP22 and ICP4 promoter activity by Egr-1 in HEK293 cells and neuronal PC-12 cells. (A) Co-transfection of pICP4 or pICP4-ID with different amounts of Egr-1 expression vector was performed followed by SEAP assays to analyze the regulatory effect of Egr-1 on promoters. 1, pICP4 and control plasmid (1:1.5); 2, pICP4 and pJDM948 (1:0.5); 3, pICP4 and pJDM948 (1:1.5); 4, pICP4-ID and control plasmid (1:1.5); 5, pICP4-ID and pJDM948 (1:0.5); 6, pICP4-ID and pJDM948 (1:1.5). Note that the SEAP activities of control in lanes 1 and 4 were arbitrarily set to 100 and the SEAP activities of other lanes were normalized against the controls. The error bars represent SDs and the data were calculated and graphed using Microsoft Excel. (B) Co-transfection of pICP22 or pICP22-ID with different amounts of pJDM948 plasmids was performed followed by SEAP assays. 1, pICP22 and control plasmid (1:1.5); 2, pICP22 and pJDM948 (1:0.5); 3, pICP22 and pJDM948 (1:1.5); 4, pICP22-ID and control plasmid (1:1.5); 5, pICP22-ID and pJDM948 (1:0.5); 6, pICP22-ID and pJDM948 (1:1.5). The data were analyzed by the same strategy as described in (A). (C) Co-transfection of all promoters with the Egr-1 expression vector pJDM948 at the molar ratio of 1:1 was performed followed by SEAP assay to analyze the regulatory effect of Egr-1 in neuronal cells PC-12. The arrangement was labeled as shown in the figure. Note that the SEAP activity in lane 1 was arbitrarily set to 100 and the SEAP activity of other lanes was normalized against that of lane 1. The error bars represent SDs and the data were calculated and graphed using Microsoft Excel.

Figure 5

Regulation of ICP4 and ICP22 promoter activity by Egr-1 in the presence of TSA. (A) HEK293 cells were co-transfected with pICP22 or pICP22-ID and the control plasmid or pJDM948 in the presence (white bar) or absence (gray bar) of 100 nM TSA. 1, pICP22 co-transfected with control plasmid; 2, pICP22 co-transfected with pJDM948; 3, pICP22-ID co-transfected with control; 4, pICP22-ID co-transfected with pJDM948. The SEAP activity in lane 1 without TSA was arbitrarily set to 100 and the SEAP activity of others was normalized against this sample. The error bars represent SDs and the data were calculated and graphed using Microsoft Excel. (B) The effects of TSA on Egr-1 mediated regulation of ICP4 and ICP4-ID promoters were accessed using the strategy in (a) except that pICP4 and pICP4-ID were used instead.

Figure 6

Co-repressor Nab2 was recruited to the ICP4 promoter in the presence of Egr-1 and Egr-1 significantly reduced the occupancy of Sp1 to the promoter when the intron was removed. HEK293 cells were co-transfected with the pICP4 (or pICP4-ID) and pJDM948. Lane 1, pICP4 and control; lane 2, pICP4 and pJDM948; lane 3, pICP4-ID and control plasmid, lane 4, pICP4-ID and pJDM948. The antibodies used for immunoprecipitation were labeled. Input control indicated the PCR products from samples prior to immunoprecipitation. Anti-Ig control confirmed the specificity of antibodies used in the experiments. The quantitative analysis was performed by Kodak Gel Logic 100 system. White bar: no Egr-1; gray bar: plus Egr-1.

Figure 7

Proposed mechanism of Egr-1 action. Egr-1 has two binding locations, the intron or the Sp1 sites. Egr-1 recruits the co-repressor Nab2, exerts a negative effect on gene expression, and participates in gene silencing possibly via chromatin modeling. The competition of Egr-1 binding to the Sp1 sites may reduce the Sp1 occupancy to the promoter and also contributes to the inhibition of transcription.