Transcription termination factor Pcf11 limits the processivity of Pol II on an HIV provirus to repress gene expression

Abstract

Many elongation factors in eukaryotes promote gene expression by increasing the processivity of RNA polymerase II (Pol II). However, the stability of RNA Pol II elongation complexes suggests that such complexes are not inherently prone to prematurely terminating transcription, particularly at physiological nucleotide concentrations. We show that the termination factor, Pcf11, causes premature termination on an HIV provirus. The transcription that occurs when Pcf11 is depleted from cells or an extract is no longer sensitive to 6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB), a compound that causes premature termination. Hence, Pcf11 can act as a negative elongation factor to repress RNA Pol II gene expression in eukaryotic cells.

Figure 1.

Depletion of Pcf11 increases HIV replication in latently infected U1 cells. (A) U1 cells were either untreated or treated with 10 ng/mL PMA, control siRNA, or Pcf11 siRNA. Relative levels of virus production were determined by measuring viral protein p24 in the culture medium using an ELISA assay. (B) ChIP analysis for the p65 subunit of NF-κB. Cells were treated as described by the matrix above the lanes. (C) RT–PCR analysis of Pcf11 and actin mRNA shows that Pcf11 mRNA is diminished in cells treated with Pcf11 siRNA. (D) Western blot analysis of Pcf11 and actin protein in crude nuclear preparations shows that Pcf11 protein is diminished in cells treated with Pcf11 siRNA. Pcf11 migrated slightly slower than a 175-kDa marker protein on 8% SDS-PAGE.

Figure 2.

Depletion of Pcf11 increases the density of Pol II downstream from the promoter region. Interactions of Pol II, TBP, and Pcf11 with the provirus were determined by ChIP. (A) Representative data for one ChIP experiment. Immunoprecipitated DNA, 10% input DNA, and 2% input DNA were subjected to PCR amplification for the regions corresponding to the promoter region in the 5′ LTR (+1 to +248) or a downstream region (+2415 to +2690). (B) Quantification of ChIP data from three independent experiments. The control samples recovered with nonspecific antibody are shown in Figure 1B.

Figure 3.

HIV induction by PMA results in recruitment of Pcf11 to the 3′ LTR. (A) Schematic showing the 5′ and 3′ LTRs, the location of PCR-amplified regions corresponding to the 5′ LTR and both the 5′ and 3′ LTRs, the transcription start site (arrow), potential polyadenylation sites. (B) Representative data for one ChIP experiment. Immunoprecipitated DNA and the indicated percentages of input DNA were subjected to PCR amplification of the 5′ LTR alone or both the 5′ and 3′ LTRs. The amount of input for amplification of both the 5′ and 3′ LTRs was half that of the input for the 5′ LTR. (C) Quantification of ChIP data from three independent experiments. The signals for the 3′ LTR were calculated by subtracting the intensity of the signals for the 5′ LTR alone from the signals for the sum of the 5′ and 3′ LTRs.

Figure 4.

Depletion of Pcf11 from cells or cell extracts renders transcription insensitive to DRB. (A) RT–PCR analysis of transcripts in U1 cells. Double-stranded RNA was transfected into U1 cells using an Amaxa Nucleofector II. Approximately 2 million cells (2 mL of cells treated with siRNA or not) were placed in a culture dish. Twelve hours post-transfection (or not), cells were incubated with or without 5 μM DRB. After 12 h, cells were stimulated with 2 ng/mL PMA or not for 12 h. Total RNA was isolated, and transcripts spanning +1 to +40 and +5396 to +5531 of HIV RNA and actin were detected by RT–PCR. (B) Western blot analysis of Pcf11-immunodepleted and mock-depleted HeLa nuclear extracts. Spt5 and NELF-F B are subunits of DSIF and NELF, respectively. (C) In vitro transcription of the 5′ LTR in Pcf11-immunodepleted (lanes 3–13) or mock-depleted (lanes 1,2) HeLa nuclear extracts. DRB at 50 μM was present in those samples marked with plus signs. dPcf11 and dmPcf11, which were isolated from Escherichia coli, encompass amino acids 1–283. The amounts of exogenously added Pcf11 correspond to 0.1 μg, 0.5 μg, and 1 μg.