Fig. 2

Trim28 is enriched in PFV 5′LTR region. a Illustration of the primer sets at the PFV LTR and IP promoter used in the ChIP assays. The numbers are relative to the transcription start site nucleotide+1. b Trim28 was enriched in the PFV 5′LTR regions during PFV infection. ChIP assay was performed on cells infected with PFV for 48 h using IgG, or anti-Trim28 Ab. ChIP-qPCR data were normalized by the percent input method (%input with IgG as control) with GAPDH promoter region as the negative control. The data presented are means the standard errors of the means of three independent experiments. c Trim28 is enriched in PFV 5′LTR U5 region in the absence of Tas. Trim28 was enriched in the PFV 5′LTR regions. ChIP assay was performed on PFV-5′LTR-transfected cells using IgG, or anti-Trim28 antibody. ChIP-qPCR data were normalized by the percent input method (%input with IgG as control) with GAPDH promoter region as the negative control. The data presented are means the standard errors of the means of three independent experiments. d ChIP assays of Trim28 in the presence (+) or absence (−) of Tas expression. Plasmid pGL3-basic-PFV-LTR containing the LTR promoter was transfected into cells with Tas expression plasmid or empty-vector controls. Cells were collected for ChIP assay at 24 h post-transfection. 8 h before harvest, the cells were treated with MG132 (5 μM). ChIP-qPCR data were normalized by the fold enrichment method (ChIP signals were divided by the IgG signals) with GAPDH promoter region as the negative control. e The effect of Trim28 in regulating the activity of Tas-dependent transactivation of the PFV LTR and truncated-LTR promoter activity. Schematic represent of the PFV LTR or truncated-LTR-driven expression of firefly luciferase. HEK293T cells seeded in 24-well plates were transfected with pCMV-Flag or pCMV-Flag-Trim28 (400 ng), pRL-TK (3 ng), pTK-Tas (50 ng) and truncated pGL3-PFV-LTR-luc (70 ng) firefly luciferase reporter. Luciferase activities were measured as described in the Materials and Methods. (paired t-test; * p < 0.05, ** p < 0.01, *** p < 0.001). f, g Trim28 promotes H3K9me3 recruiting to the PFV 5′LTR regions. ChIP assays of H3K9me3 with the overexpression or knockdown of the Trim28 expression in PFV infected HEK293T cells. ChIP-qPCR data were normalized by the fold enrichment method (ChIP signals were divided by the IgG signals). The data are presented as the means ± SD. The telomere (Tel) genomic region acted as a positive control, and GAPDH promoter region acted as a negative control when Trim28 was overexpressed during PFV infection