Stepwise recruitment of components of the preinitiation complex by upstream activators in vivo

Abstract

Recently, it was found that if either the TATA binding protein or RNA polymerase II holoenzyme is artificially tethered to a promoter, transcription is activated. This finding provided presumptive evidence that upstream activating proteins function by recruiting components of the preinitiation complex (PIC) to the promoter. To date, however, there have been no studies demonstrating that upstream factors actually recruit components of the PIC to the promoter in vivo. Therefore, we have studied the mechanism of action of two disparate transactivating domains. We present a series of in vivo functional assays that demonstrate that each of these proteins targets different components of the PIC for recruitment. We show that, by targeting different components of the PIC for recruitment, these activating domains can cooperate with each other to activate transcription synergistically and that, even within one protein, two different activating subdomains can activate transcription synergistically by cooperating to recruit different components of the PIC. Finally, considering our work together with previous studies, we propose that certain transcription factors both recruit components of the PIC and facilitate steps in transcriptional activation that occur subsequent to recruitment.

FIG. 1

Activators target different components of the PIC for recruitment. (a) Overexpression of TBP inhibits the activity of promoter-bound TBP. When TBP is overexpressed with a reporter that contains Gal4 binding sites and a TATA box, transcription is activated. However, activation of the same promoter by Gal4-TBP is inhibited by overexpression of TBP. Gal4-TBP activity is unaffected by overexpression of LexA-VP16Δ456, even though LexA-VP16Δ456 squelches Gal4-VP16Δ456. (b) Cells were transfected in exactly the same manner as for panel a, and primer extensions for CAT and GAPDH mRNA were performed in parallel for each sample. The extension products were of the expected sizes. Numbers at left show sizes of DNA standards. (c) Overexpression of TBP does not affect the expression of Gal4-TBP protein, and expression of LexA-VP16Δ456 does not affect the expression of Gal4-VP16Δ456 protein when assessed by immunoblotting. (d) One possible model for the mechanism of inhibition of Gal4-TBP activity by TBP overexpression (see text for details). (e) Overexpression of TBP inhibits the activity of Gal4-Sp1 but potentiates the activity of Gal4-VP16Δ456. (f) Cells were transfected in exactly the same manner as for panel e, and primer extensions for CAT and GAPDH mRNA were performed in parallel for each sample. The extension products were of the expected sizes. The contrast of the image was increased to facilitate the visualization of the CAT mRNA in the cells transfected with the Gal4-Sp1 expression vector and the cells cotransfected with the Gal4-Sp1 and TBP expression vector. Numbers at left show sizes of DNA standards. (g) Overexpression of TBP does not affect the expression of the Gal4-Sp1 or Gal4-VP16Δ456 proteins when assessed by immunoblotting. (h) One possible model for the mechanisms of transcriptional inhibition and potentiation by TBP overexpression (see text for details).

FIG. 1

Activators target different components of the PIC for recruitment. (a) Overexpression of TBP inhibits the activity of promoter-bound TBP. When TBP is overexpressed with a reporter that contains Gal4 binding sites and a TATA box, transcription is activated. However, activation of the same promoter by Gal4-TBP is inhibited by overexpression of TBP. Gal4-TBP activity is unaffected by overexpression of LexA-VP16Δ456, even though LexA-VP16Δ456 squelches Gal4-VP16Δ456. (b) Cells were transfected in exactly the same manner as for panel a, and primer extensions for CAT and GAPDH mRNA were performed in parallel for each sample. The extension products were of the expected sizes. Numbers at left show sizes of DNA standards. (c) Overexpression of TBP does not affect the expression of Gal4-TBP protein, and expression of LexA-VP16Δ456 does not affect the expression of Gal4-VP16Δ456 protein when assessed by immunoblotting. (d) One possible model for the mechanism of inhibition of Gal4-TBP activity by TBP overexpression (see text for details). (e) Overexpression of TBP inhibits the activity of Gal4-Sp1 but potentiates the activity of Gal4-VP16Δ456. (f) Cells were transfected in exactly the same manner as for panel e, and primer extensions for CAT and GAPDH mRNA were performed in parallel for each sample. The extension products were of the expected sizes. The contrast of the image was increased to facilitate the visualization of the CAT mRNA in the cells transfected with the Gal4-Sp1 expression vector and the cells cotransfected with the Gal4-Sp1 and TBP expression vector. Numbers at left show sizes of DNA standards. (g) Overexpression of TBP does not affect the expression of the Gal4-Sp1 or Gal4-VP16Δ456 proteins when assessed by immunoblotting. (h) One possible model for the mechanisms of transcriptional inhibition and potentiation by TBP overexpression (see text for details).

FIG. 1

Activators target different components of the PIC for recruitment. (a) Overexpression of TBP inhibits the activity of promoter-bound TBP. When TBP is overexpressed with a reporter that contains Gal4 binding sites and a TATA box, transcription is activated. However, activation of the same promoter by Gal4-TBP is inhibited by overexpression of TBP. Gal4-TBP activity is unaffected by overexpression of LexA-VP16Δ456, even though LexA-VP16Δ456 squelches Gal4-VP16Δ456. (b) Cells were transfected in exactly the same manner as for panel a, and primer extensions for CAT and GAPDH mRNA were performed in parallel for each sample. The extension products were of the expected sizes. Numbers at left show sizes of DNA standards. (c) Overexpression of TBP does not affect the expression of Gal4-TBP protein, and expression of LexA-VP16Δ456 does not affect the expression of Gal4-VP16Δ456 protein when assessed by immunoblotting. (d) One possible model for the mechanism of inhibition of Gal4-TBP activity by TBP overexpression (see text for details). (e) Overexpression of TBP inhibits the activity of Gal4-Sp1 but potentiates the activity of Gal4-VP16Δ456. (f) Cells were transfected in exactly the same manner as for panel e, and primer extensions for CAT and GAPDH mRNA were performed in parallel for each sample. The extension products were of the expected sizes. The contrast of the image was increased to facilitate the visualization of the CAT mRNA in the cells transfected with the Gal4-Sp1 expression vector and the cells cotransfected with the Gal4-Sp1 and TBP expression vector. Numbers at left show sizes of DNA standards. (g) Overexpression of TBP does not affect the expression of the Gal4-Sp1 or Gal4-VP16Δ456 proteins when assessed by immunoblotting. (h) One possible model for the mechanisms of transcriptional inhibition and potentiation by TBP overexpression (see text for details).

FIG. 2

Sp1 recruits TBP, and VP16Δ456 targets a step that is downstream of TBP recruitment to activate transcription. (a) Gal4-Sp1 is inert if TBP is tethered to the promoter by LexA, but Gal4-VP16Δ456 cooperates with LexA-TBP to activate transcription synergistically. (b) Synergistic activation induced by the combination of Gal4-VP16Δ456 and LexA-TBP is dependent upon the VP16Δ456 and TBP moieties in these chimeric proteins. This is evidenced by the fact that the Gal4 and LexA DNA-binding domains are inactive in this assay (left panel) even though the Gal4 and LexA DNA-binding domains are readily expressed in transfection assays when assessed by immunoblotting (right panels). NS, nonspecific bond. (c) Gal4-Sp1 and Gal4-TBP are less dependent upon the TATA box than Gal4-VP16Δ456 for their transactivation function. “fold decrease” represents the ratio of the activity of each activator in the presence to that in the absence of the TATA box. The cell lysates for the assays depicted in the left panel were diluted 10-fold to facilitate the comparison between the two reporter constructs.

FIG. 3

Overexpressed TBP must be incorporated into the PIC to cooperate with VP16Δ456. (a) TBP_RS binds to both a canonical TATA box and a mutant TATA box (TATA_RS); TBP binds only a canonical TATA box. (b) Overexpression of either TBP_RS or TBP increases the activity of a basal promoter that contains a canonical TATA box (left panel); only TBP_RS increases the activity of the basal promoter when the TATA box is replaced by TATA_RS (right panel). Both TBP_RS and TBP cooperate with VP16Δ456 with equal effectiveness to activate a promoter that contains a canonical TATA box (left panel); TBP_RS cooperates with VP16Δ456 much more effectively than does TBP to activate a promoter that contains TATA_RS (right panel).

FIG. 4

VP16Δ456 recruits a component(s) that is assembled into the PIC downstream of TBP to activate transcription. (a) When Gal4-VP16Δ456 was tethered to the promoter, it potentiated the activity of promoter-bound LexA-TBP in a synergistic manner (left panel). Gal4-VP16Δ456 does not cooperate with LexA-TBP unless it is tethered to the promoter, even when high concentrations of the Gal4-VP16Δ456 expression vector are transfected (right panel). (b) Even when Gal4-VP16Δ456 is not tethered to the promoter, it can still interact with its transactivation target(s), as indicated by the fact that it squelches the activity of LexA-VP16Δ456 (left panel) but not that of LexA-TBP (a). Overexpression of Gal4-VP16Δ456 does not affect cellular levels of LexA-VP16Δ456 as assessed by immunoblotting (right panel). (c) A model for the interactions suggested by the findings that Gal4-VP16Δ456 squelches the activity of promoter-bound LexA-VP16Δ456 but not promoter-bound LexA-TBP.

FIG. 5

Transcriptional synergy. (a) Sp1 cooperates with VP16Δ456 but not with itself to activate transcription synergistically. Relative promoter activity is indicated above each assay. The cell lysate from one assay was diluted as indicated to facilitate quantification of activity. (b) Activating subdomains within one protein can cooperate to activate transcription synergistically. A single chimeric protein, encoded by Gal4-(Sp1-VP16Δ456), containing the activation domains of Sp1 and VP16Δ456 fused to the Gal4 DNA-binding domain is a strong transcriptional activator.