Transcriptional Coactivator PC4 Stimulates Promoter Escape and Facilitates Transcriptional Synergy by GAL4-VP16

Abstract

Positive cofactor 4 (PC4) is a coactivator that strongly augments transcription by various activators, presumably by facilitating the assembly of the preinitiation complex (PIC). However, our previous observation of stimulation of promoter escape in GAL4-VP16-dependent transcription in the presence of PC4 suggested a possible role for PC4 in this step. Here, we performed quantitative analyses of the stimulatory effects of PC4 on initiation, promoter escape, and elongation in GAL4-VP16-dependent transcription and found that PC4 possesses the ability to stimulate promoter escape in response to GAL4-VP16 in addition to its previously demonstrated effect on PIC assembly. This stimulatory effect of PC4 on promoter escape required TFIIA and the TATA box binding protein-associated factor subunits of TFIID. Furthermore, PC4 displayed physical interactions with both TFIIH and GAL4-VP16 through its coactivator domain, and these interactions were regulated distinctly by PC4 phosphorylation. Finally, GAL4-VP16 and PC4 stimulated both initiation and promoter escape to similar extents on the promoters with three and five GAL4 sites; however, they stimulated promoter escape preferentially on the promoter with a single GAL4 site. These results provide insight into the mechanism by which PC4 permits multiply bound GAL4-VP16 to attain synergy to achieve robust transcriptional activation.Transcription of mRNA-coding genes involves RNA polymerase II and six general transcription factors (TFIIA, TFIIB, TFIID, TFIIE, TFIIF, and TFIIH) which comprise the basal transcription machinery that recognizes the core promoter elements and elicits the basal level of transcription (50). Activated transcription requires the binding of activators to the regulatory DNA sequences typically present upstream of the core promoter and their interactions with the general transcription machinery (32,49). Despite the well-documented direct interactions of activators with the general transcription factors and RNA polymerase II, activated transcription requires yet another group of transcription factors, termed mediators or coactivators, that confer on the general transcription machinery a markedly enhanced responsiveness to activators (2,18,20,36,41).A wide array of coactivators may be grouped into two broad categories according to the requirement of chromatin for their action in biochemical assays. The coactivators which function on the templates without chromatin include the TATA box binding protein-associated factors (TAFs) present in TFIID (58), positive cofactors (PCs) (PC1, PC2, PC3, and PC4) derived from the upstream factor stimulatory activity (USA) cofactor fraction (20), and metazoan multiprotein complexes that are structurally related to the yeast mediator (40) (TRAP/ SMCC, ARC, DRIP, NAT, murine mediator, human mediator, CRSP, and PC2) (36, 41). The coactivators which require chromatin templates for their functions include CBP/p300, PCAF and its related GCN5 proteins, and p160 family proteins that display histone acetyltransferase activities (4,...

Heterogeneous Nuclear Ribonucleoprotein R Enhances Transcription from the Naturally Configured c-fos Promoter in Vitro

“…We added PC4 to in vitro transcription reactions, in which various combinations of the c-fos activators were examined. Consistent with our earlier study (19), PC4 enhanced GAL4-VP16-dependent transcription from 2.5-to 38-fold, giving rise to additional increase (over 10-fold) in activation (Fig. 3C).…”

“…2D) (19). The same transcription system, however, failed to activate transcription when one of the c-fos gene activators (SRF, Elk-1, CREB, and ATF1) was added individually (Fig.…”

“…PC4 exerts its coactivator activity by mediating interactions between activators and GTFs such as TFIIA (27), TFIIB (29), and TFIIH (19). Given the ability of PC4 to enhance the transcriptional activities of SRF, Elk-1, CREB, and ATF1 (Fig.…”

“…First, to reveal the intrinsic activation potential of these cellular activators, we employed an in vitro transcription system reconstituted only with RNAP II and six GTFs (TFIIA, TFIIB, TFIID, TFIIE, TFIIF, and TFIIH). The reconstituted in vitro transcription system was devoid of any known coactivators but capable of supporting activated transcription by GAL4-VP16 (19). A c-fos template, pfMC2AT, was constructed by replacing the upstream GAL4-binding sites and the TATA box of pG5HMC2AT with the c-fos promoter region ( Fig.…”

“…We employed a defined in vitro transcription system using purified GTFs (19), together with SRF, Elk-1, CREB, and ATF1 to analyze transcriptional activation from the c-fos promoter that retains cis-elements in its original configuration. Unlike the GAL4-VP16-based model promoter, activation of the c-fos promoter by SRF, Elk-1, CREB, and ATF1 was much more dependent on coactivators such as PC4 and Mediator.…”