Transcription activation by targeted recruitment of the RNA polymerase II CTD phosphatase FCP1

Abstract

Human FCP1 in association with RNAP II reconstitutes a highly specific CTD phosphatase activity and is required for recycling RNA polymerase II (RNAP II) in vitro. Here we demonstrate that targeted recruitment of FCP1 to promoter templates, through fusion to a DNA-binding domain, stimulates transcription. We demonstrate that a short region at the C-terminus of the FCP1 protein is required and sufficient for activation, indicating that neither the N-terminal phosphatase domain nor the BRCT domains are required for transcription activity of DNA-bound FCP1. In addition, we demonstrate that the C-terminus region of FCP1 suffices for efficient binding in vivo to the RAP74 subunit of TFIIF and is also required for the exclusive nuclear localization of the protein. These findings suggest a role for FCP1 as a positive regulator of RNAP II transcription.

Figure 1

(A) Artificial recruitment of FCP1 protein to promoter template effectively activates transcription. Transfections of 293 cells were carried out using 100 ng of the indicated reporters together with 500 ng of GAL4–FCP1 or GAL4–FIN13 expression vectors as indicated. Each histogram bar represents the mean of at least three independent transfections made in duplicate. (B) Synergy between hTBP and FCP1 recruited to promoter DNA. The G5–38HIV reporter (100 ng) was transfected into 293 together with the indicated effectors (500 ng). In both panels, data are presented as luciferase activity relative to the sample without effector after normalization.

Figure 2

Identification of FCP1 domains critical for activation. A schematic representation of FCP1 protein and the GAL4–FCP1 fusion constructs is presented on the left. G5–83HIV reporter (100 ng) was co-transfected into 293 cells with the indicated expression vectors (500 ng). Data are presented as fold activation relative to the sample without effector after normalization. Each histogram bar represents the mean of at least three independent transfections.

Figure 3

Interactions between FCP1 and RAP74. (A) FCP1-containing complexes from cells expressing FLAG–FCP1wt, FLAG–FCP1(1–833) or FLAG–FCP1(833–961) were immunoprecipitated (IP) with agarose-conjugated M2 anti-FLAG antibody and analyzed for the presence of RAP74 by western blotting. Lane 1, volume corresponding to 20% of the input was loaded on the gel; lane 3, 5-fold more cell extract was immunoprecipitated with the anti-FLAG antibody and loaded on the gel to obtain a similar intensity of signal. (B) Artificial recruitment of RAP74 protein to promoter template does not activate transcription. G5–83HIV reporter (100 ng) was co-transfected with GAL4–RAP74 or GAL4–FCP1 expression vectors (500 ng) as indicated. Data are presented as fold activation relative to the sample without effector after normalization. Each histogram bar represents the mean of at least three independent transfections. (C) Western blot of untransfected and GAL4–RAP74 293 transfected cells with RAP74 antibody displays the relative level of expression of GAL4–RAP74 compared to the endogenous RAP74 protein.

Figure 4

Nuclear localization of FCP1. Cos cells were transfected with the GFP fusion expression vectors encoding the indicated region of FCP1. (Top) Fluorescence microscopy images of GFP-positive cells from each transfection along with nuclear DAPI staining. (Bottom) Schematic representation of the GFP fusion constructs with their respective subcellular localization indicated as (N) nuclear, (C) cytoplasmic or (N/C) nuclear and cytoplasmic.