Long-distance transcriptional enhancement by the histone acetyltransferase PCAF

Abstract

Enhancers are defined by their ability to stimulate gene activity from remote sites and their requirement for promoter-proximal upstream activators to activate transcription. Here we demonstrate that recruitment of the p300/CBP-associated factor PCAF to a reporter gene is sufficient to stimulate promoter activity. The PCAF-mediated stimulation of transcription from either a distant or promoter-proximal position depends on the presence of an upstream activator (Sp1). These data suggest that acetyltransferase activity may be a primary component of enhancer function, and that recruitment of polymerase and enhancement of transcription are separable. Transcriptional activation by PCAF requires both its acetyltransferase activity and an additional activity within its N terminus. We also show that the simian virus 40 enhancer and PCAF itself are sufficient to counteract Mad-mediated repression. These results are compatible with recent models in which gene activity is regulated by the competition between deacetylase-mediated repression and enhancer-mediated recruitment of acetyltransferases.

Figure 1

SV40 enhancer-activated transcription is not stimulated in the presence of the deacetylation inhibitor TSA. Nuclease S1 analysis of RNA from mouse B78 cells transfected with the reporter constructs SP1*-TATA-MG and SP1-TATA-MG-SVE in the presence and absence of the deacetylase inhibitor TSA. Cells were transfected with 12.5 μg of SP1*-TATA-MG and different amounts of SP1-TATA-MG-SVE (lanes 1 and 2, 12.5 μg; lanes 3 and 4, 2.5 μg; lanes 5 and 6, 0.5 μg). Half of the transfected cells was treated with TSA (100 ng/μl) for 16 hr, and RNA from treated and nontreated cells was subjected to nuclease S1 analysis using an end-labeled probe derived from the SP1*-TATA-MG template (see Materials and Methods). The transcribed regions of SP1-TATA-MG-SVE (SVE) and SP1*-TATA-MG (MG*) differ through a 30-nt sequence; therefore, transcripts can be distinguished by using the same probe.

Figure 2

Direct recruitment of PCAF enhances Sp1-promoter activity similar to the SV40 enhancer. (A) Stimulation of the SP1-TATA-MG-Gal reporter construct by the acetyltransferase PCAF fused to the Gal4-DNA binding domain was compared with the transcriptional enhancement by the known transactivators Gal4-Sp1 and Gal4-p300. The reporter construct SP1-TATA-MG-Gal was transiently transfected with empty vector DNA, Gal4-Sp1, Gal4-p300, or Gal4-PCAF and with the CMV-hGH gene as an internal control for efficiency of transfection. Steady-state RNA levels of the MG fusion genes and the human growth hormone (hGH) gene were determined with the S1-nuclease assay as described in Materials and Methods. (B) The activity of the reporter gene TATA-MG-Gal, lacking upstream-Sp1 binding sites, in the presence of Gal4-VP16, Gal4-p300, and Gal4-PCAF, is shown. For comparison, the stimulation of transcription by Gal4-PCAF in the presence of Sp1 upstream activating sequences is shown in the left two lanes. (C) Gal4-PCAF does not activate transcription by itself. The steady-state levels of the transfected Gal-TATA-MG construct in the presence of Gal4-PCAF were compared with the activity of the reporter gene after cotransfection of empty vector DNA, Gal4-Sp1, Gal4-p300, and Gal4-VP16. The samples derived from cotransfections with different Gal4-fusion proteins were mapped with the same end-labeled probe in the nuclease-S1 assay; however, the Gal4-VP16 derived sample was run on a separate gel. (D) Stimulation by Gal4-PCAF bound at a proximal position, adjacent to the Sp1 upstream activator sites. The reporter gene Gal-SP1-TATA-MG, which contains three copies of Gal4 recognition sites just upstream of multiple Sp1-sites, was cotransfected with Gal4-Sp1 or the Gal4-PCAF expression plasmid.

Figure 3

The HAT domain of PCAF is required, but not sufficient, to stimulate promoter activity. (A) Deletions within the N or C terminus of PCAF abrogate the ability of PCAF to enhance the Sp1-driven reporter gene. Two deletions within Gal4-PCAF were tested: ΔHAT(574–608), a Gal4-PCAF mutant containing a deletion within the HAT domain of the C-terminal portion of PCAF; and ΔN(65–112), a Gal4-PCAF mutant containing a deletion of amino acids 65–112 within the N-terminal portion of PCAF. RNA from transfected B78 cells was tested with S1-nuclease protection assays. (B) HAT activity in vitro. Wild-type and mutant PCAF proteins were tested for acetyltransferase activity in vitro.

Figure 4

Gal4-PCAF-mediated stimulation of transcription is resistant to suppression by the adenoviral protein E1a. B78 cells were transfected with the reporter construct SP1-TATA-MG-Gal and expression vectors for Gal4-p300 and Gal4-PCAF. The transfection mixtures contained the CMV-promoter driven human growth hormone (hGH) gene as an internal control. The E1a gene was expressed from the Rous sarcoma virus (RSV) promoter in pRc/RSV (Invitrogen). (Upper) S1-nuclease assay using end-labeled probes homologous to the hGH gene and the MG reporter gene. (Lower) Quantitative PhosphorImager analyses of the S1-nuclease assay reveals a 4-fold decrease in Gal4-p300 mediated activation in the presence of E1a. In contrast, Gal4-PCAF stimulated transcription remains unaffected or increases slightly.

Figure 5

The SV40 enhancer or the recruitment of PCAF counteracts HDAC-mediated repression. (A) The reporter constructs Gal-TATA-MG (Left, −SVE) and Gal-TATA-MG-SVE (Right, +SVE), which differ through SV40 enhancer sequences (SVE) downstream of the transcribed MG gene, were transfected into mouse B78 cells along with the CMV-hGH gene as an internal control and with Gal4-VP16, Gal4-VP16 mad, or Gal4-VP16 madpro as transactivators. Gal4-VP16 mad contains the Sin3 interaction domain (SID) of the Mad protein, whereas Gal4-VP16 madpro is mutated within SID, and therefore does not interact with Sin3 (30). RNA from transfected cells was analyzed with S1-nuclease assays. Transactivation by Gal4-VP16 mad is significantly less (12-fold) than transactivation by Gal4-VP16 and Gal4-VP16 madpro. (B) Gal4-PCAF counteracts Mad-mediated repression of VP16 transactivation. The LEX-TATA-MG-Gal reporter construct was cotransfected into B78 cells with expression vectors encoding Lex-VP16 madpro (left lane), LexVP16 mad (middle and right lanes), and Gal4-PCAF (right lane). The CMV-hGH plasmid was included as an internal control for transfection efficiency. RNA was analyzed in S1-nuclease assays.