FIG 5.

KRAB-ZFPs repress EBV lytic genes through the transcriptional corepressor TRIM28. (A and B) BL cells were transfected with empty vector and scrambled siRNA, SZF1 (A) or ZNF557 (B) plasmid and scrambled siRNA, or SZF1 (A) or ZNF557 (B) plasmid and siTRIM28. Cells were exposed to NaB and harvested for determination of relative amounts of transcripts from the EBV latency-to-lytic-cycle switch gene BZLF1 and the early lytic gene BMRF1 by qRT-PCR. Data represent results from two independent experiments with 3 technical replicates each; error bars indicate standard errors of the means (*, p ≤ 0.05). Samples were also harvested for immunoblotting and reacted with indicated antibodies. Representative data from two independent experiments are shown. (C and D) ChIP was performed on BL cells containing doxycycline (dox)-inducible shTRIM28 cassette. Cells were left untreated (open bars) or treated with doxycycline (black bars) for 12 h. DNA precipitated by using anti-H3K9Ac and anti-H3K9Me3 antibodies was analyzed via qPCR using primers spanning predicted SZF1-binding sites in the promoters of lytic genes BZLF1 (C) and BMRF1 (D) and normalized to input. Immunoblot analysis of TRIM28 after doxycycline induction of shTRIM28 was performed to assess knockdown efficiency. Data represent the averages of results from three independent experiments; error bars indicate standard errors of the means (*, p ≤ 0.05).