Volume 5, Issue 2, Pages 331-341 (February 2000) The FBP Interacting Repressor Targets TFIIH to Inhibit Activated Transcription  Juhong Liu, Liusheng He, Irene Collins, Hui Ge, Daniel Libutti, Junfa Li, Jean-Marc Egly, David Levens  Molecular Cell  Volume 5, Issue 2, Pages 331-341 (February 2000) DOI: 10.1016/S1097-2765(00)80428-1

Figure 1 FIR Interacts with FBP's Central Domain In Vitro and In Vivo and Enhances FUSE Binding (A) FBP, FBPN, FBPΔCD, and FBPΔC (central domain or carboxy-terminal deletions, respectively) were tagged with hexa-histidine at their amino termini and expressed by transient transfection in HeLa cells. Tagged proteins were recovered with NiSO4 beads, and coprecipitated proteins were analyzed by anti-FIR immunoblot following SDS-PAGE. FBPCD directs FIR binding. (B) FUSE bottom strand ssDNA was used as an EMSA probe to test the effect of FIR on FBP DNA binding. Recombinant FBP (0.5 ng) was used for sequence-specific binding. FIR alone did not form a specific complex (lane 2); however, FIR augmented binding by FBP. A slower complex also formed in a FIR dose-dependent manner (lanes 3–6). Addition of either anti-FIR (lanes 7–10) or anti-FBP (lanes 11–14) antibody abolished the complex indicating the involvement of both FBP and FIR. Molecular Cell 2000 5, 331-341DOI: (10.1016/S1097-2765(00)80428-1)

Figure 2 FIR Is Expressed in All Tissues Examined (A) Amino acid sequence of FIR. (B) Northern blot analysis of FIR mRNA expression in different tissues. A human multiple tissue mRNA blot (Clontech) was hybridized with a full-length FIR cDNA probe. Molecular Cell 2000 5, 331-341DOI: (10.1016/S1097-2765(00)80428-1)

Figure 3 FIR Represses Activated Transcription in cis and in trans (A) FIR repression of FBPC-activated transcription in cis does not require FBPCD. Increasing amounts (25, 50, and 150 ng) of a plasmid expressing a lexAFIR chimera were cotransfected with the indicated amount of a second plasmid expressing either G4FBPC or G4VP16, and a reporter plasmid with one lexA site and one GAL4 UAS upstream of a TATA box driving a CAT gene. lexAFIR alone failed to activate expression (lane 9). Activation by G4FBPC was progressively inhibited by lexAFIR (lanes 5–8) whereas G4VP16 was not inhibited (lanes 1–4). (B) G4FIR blocks activation by G4PBFC and G4E1a, but not by G4VP16. pG4FIR (25 ng or 100 ng) was cotransfected with pG4:FPBC (25 ng), pG4:E1a (20 ng), or pG4:VP16 (5 ng) into HeLa cells. Plasmid expressing the GAL4 DNA-binding domain was included to compensate for mixed dimer formation or competition for UAS binding. Similar to the experiment in (A), G4FIR repressed activation by FBPC (lanes 1–3) as well as E1a (lanes 4–6) but failed to repress VP16 (lanes 7–9). (C) The amino terminus of FIR is necessary and sufficient for repression. To map the repression domain, 25 ng or 100 ng of plasmids expressing truncations from both amino and carboxyl termini of FIR as GAL4 chimeras were cotransfected with 25 ng of pG4:FBPC. CAT assays show that carboxy-terminal deletions to amino acid 121 had no effect on FIR's ability to repress transcription (lanes 2 and 3). Deletion of the first 55 amino acid residues incapacitated FIR as a repressor (lanes 6–9). (D) The amino terminus of FIR is capable of repression in trans. Either 1 or 4 μg of truncations of FIR was cotransfected with 25 ng of pG4:FBPC into HeLa cells. Deleting the carboxyl terminus yielded a more effective repressor than full-length FIR (lanes 4 and 5 versus 2 and 3). An amino-terminal deletion completely abolished trans-repression (lanes 6 and 7). Molecular Cell 2000 5, 331-341DOI: (10.1016/S1097-2765(00)80428-1)

Figure 4 Repression of c-myc Transcription by FIR Is FUSE Dependent (A) FIR represses c-myc promoter activity in a dose-dependent manner. CAT assays were done in duplicates. Overexpression of FIR by transfecting 4 μg of FIR expression plasmid reduced CAT activity up to 5.5-fold (lanes 1, 2 versus 3, 4, and 5, 6). (B) FIR expression plasmid (4 μg) was cotransfected with a reporter containing a CAT gene under the control of myc regulatory sequence with or without the FUSE element (myc-CAT and myc-ΔFUSE-CAT, respectively) into U2OS cells. Overexpression of FIR repressed transcription driven by the c-myc promoter (lanes 3 and 4). The repression is FUSE dependent; deletion of 68 base pairs of the FUSE element abolished the FIR effect (lanes 1 and 2). Molecular Cell 2000 5, 331-341DOI: (10.1016/S1097-2765(00)80428-1)

Figure 5 FIR Represses Activated, but Not Basal, Transcription In Vitro (A) Two supercoiled plasmids were used for in vitro transcription assays. pΔ56, which contained the minimal mouse c-fos promoter, was used to program basal transcription. pG45CAT, the same plasmid used in transfection assays, was the template for activator-dependent transcription. 25 ng of GAL4 DNA-binding domain, 20 ng of recombinant GAL4FBPC, 25 ng of GAL4FBPΔN, or 5 ng of GAL4AH was added to transcription reactions as indicated. The transcripts were analyzed by primer extension using an oligonucleotide primer complementary to the coding strand of the CAT gene. Recombinant FIR (50 or 150 ng) was also added to the transcription reaction. FIR similarly repressed transcription activated by either G4FBPC (lanes 3–5) or G4FBPΔN (lanes 6–9). G4AH is more resistant to FIR repression (lanes 9–11). Basal transcription from the c-fos promoter, however, is not affected by FIR. (B) To find out which complex of the transcription machinery interacts with FIR, immunoprecipitations were performed with affinity-purified anti-FIR antibody cross-linked to protein–A agarose beads. Anti-GST was used as the control antibody. Purified proteins were analyzed with immunoblots using a variety of antibodies against components of the basal machinery. Beside FBP and FIR, three subunits of TFIIH complex, p62, p89, and cyclin H, were consistently copurified with anti-FIR but not with anti-GST. Molecular Cell 2000 5, 331-341DOI: (10.1016/S1097-2765(00)80428-1)

Figure 6 FIR Blocks Transcription Activation through TFIIH and Suppresses p89/XPB 3′–5′ Helicase (A) Full-length FIR and FIR repression domain deletions were HA tagged and transfected into HeLa cells. The overexpressed proteins were purified with monoclonal anti-HA and subjected to Western blot analysis. HA-FIR and HA-FIRΔN55 were similarly expressed in HeLa cells (upper panel). Unlike HA-FIR, HA-FIRΔN, missing 55 amino-terminal residues, failed to copurify TFIIH. Neither HA-FIR nor HA-FIRΔN coimmunoprecipitated with TBP. (B) Excess of TFIIH overcomes transcription repression by FIR. Purified TFIIH was added to in vitro transcription assays together with FIR. Adding extra TFIIH to 50 μg of HeLa nuclear extract had no effect on either basal or activated transcription (lane 4 versus 3), indicating that TFIIH was not limiting in untreated nuclear extract. FIR (150 ng) repressed activated transcription to basal levels (lanes 5 and 6). Addition of purified TFIIH restored activated transcription but did not perturb transcription from the basal promoter (lanes 7 and 8). (C) FIR does not alter CTD phosphorylation. GST-CTD (5 ng) was mixed with TFIIH in the presence of 150 ng of GST-FIR or GST. FIR has no effect on TFIIH's ability to phosphorylate CTD. (D) The repression domain of FIR interacts directly with the p89 subunit of TFIIH. GST, GSTFIR113, or GSTFIRΔ183, respectively possessing or lacking the FIR repression domain, was incubated with baculovirus expressed and purified p89/XPB. p89/XPB was efficiently bound by the amino terminus of FIR (GSTFIR113); deleting the amino terminus (GSTFIRΔ183) abolished this interaction. (E) Left, the repression domain of FIR suppresses p89/XPB helicase activity. Purified p89/XPB was examined for 3′–5′ helicase activity in the presence of increasing amounts of GSTFIR113 (20–100 ng). GSTFIR113 was purified by HPLC to eliminate possible bacterial contaminants. FIR's repression domain suppressed, but did not abolish, the helicase activity of purified p89/XPB (lane 1 versus 2–6). Right, GSTFIR113 blocked p89/XPB helicase (lanes 3–5), but not p80/XPD (lanes 8–10). Lane 2, no protein additons; lanes 6 and 7, FIR only. Molecular Cell 2000 5, 331-341DOI: (10.1016/S1097-2765(00)80428-1)

Figure 7 Proposed Scheme for FIR-Modulated Transcription via TFIIH (A) Because FIR represses activated but not basal transcription, and suppresses but does not abolish p89/XPB 3′–5′ helicase activity, we suggest that there are activated and basal states of TFIIH that support fast or slow promoter escape. FIR would drive more TFIIH to the basal state whereas FIR-resistant activators, such as VP16, would drive more TFIIH to the activated state. (B) Activators bound to DNA and engaging TFIIH prior to promoter escape must form a loop and define a topological domain. As transcription occurs, the engaged activators must be dragged around the promoter-proximal template. Negative supercoils would be introduced, at least transiently, behind the translocating transcription machinery that includes TFIIH. This direct transmission of torsional stress into the loop through FIR and/or FBP is proposed to control single-strand/supercoil-dependent binding to FUSE (Michelotti et al. 1996b). Active FIR repression would lead to regulation of paused polymerases such as are found on the c-myc and other promoters (Krumm et al. 1995). Molecular Cell 2000 5, 331-341DOI: (10.1016/S1097-2765(00)80428-1)