Tài liệu Báo cáo khoa học: FOXM1c transactivates the human c-mycpromoter directly via the two TATA boxes P1 and P2 docx

FOXM1c transactivates the human c-myc promoter directly via the two TATA boxes P1 and P2 Inken Wierstra 1 and Ju ¨ rgen Alves 2 1 Institute of Molecular Biology, Medical School Hannover, Germany 2 Institute of Biophysical Chemistry, Medical School Hannover, Germany c-Myc, a key regulator of proliferation, differentiation and apoptosis, plays a central role in cell growth control and can induce quiescent cells to enter into S-phase [1–7]. Because c-Myc potently stimulates pro- liferation and inhibits differentiation it possesses a high transformation potential that is supplemented by its cell growth and angiogenesis-promoting, cell-adhesion- reducing, immortality and genomic-instability-causing activities. c-myc expression correlates strictly with cell proliferation. c-Myc regulates target genes either by activation via E-boxes or by repression via initiator (Inr)-dependent and Inr-independent mechanisms. c-Myc acts as part of the Myc ⁄ Max ⁄ Mad network in which Max is the heterodimerization partner for c-Myc and Mad proteins, the c-Myc antagonists, which repress target genes via E-boxes. The forkhead ⁄ winged helix transcription factor FOXM1, expression of which correlates strictly with proliferation, stimulates proliferation by promoting S- and M-phase entry and regulates genes that control G 1 ⁄ S and G 2 ⁄ M transition [8–27]. The activity of FOXM1 as a conventional transcription factor is increased by proliferation signals and reduced by anti- proliferative signals. Furthermore, FOXM1 is assumed to be implicated in tumorigenesis [18,23–26,28]. We have previously shown that as a conventional Keywords c-myc; core promoter; FOXM1; TATA box; TATA-binding protein Correspondence I. Wierstra, Wißmannstr. 17, D-30173 Hannover, Germany Fax: +49 511 883 536 Tel. +49 511 883 536 E-mail: iwiwiwi@web.de (Received 29 June 2006, revised 9 August 2006, accepted 15 August 2006) doi:10.1111/j.1742-4658.2006.05468.x FOXM1c transactivates the c-myc promoter via the P1 and P2 TATA boxes using a new mechanism. Whereas the P1 TATA box TATAATGC requires its sequence context to be FOXM1c responsive, the P2 TATA box TATA- AAAG alone is sufficient to confer FOXM1c responsiveness to any minimal promoter. FOXM1c transactivates by binding to the TATA box as well as directly to TATA-binding protein, transcription factor IIB and transcrip- tion factor IIA. This new transactivation mechanism is clearly distinguished from the function of FOXM1c as a conventional transcription factor. The central domain of FOXM1c functions as an essential domain for activation via the TATA box, but as an inhibitory domain (retinoblastoma protein- independent transrepression domain and retinoblastoma protein-recruiting negative regulatory domain) for transactivation via conventional FOXM1c- binding sites. Each promoter with the P2 TATA box TATAAAAG is postulated to be transactivated by FOXM1c. This was demonstrated for the promoters of c-fos, hsp70 and histone H2B ⁄ a. A database search revealed almost 300 probable FOXM1c target genes, many of which function in proliferation and tumorigenesis. Accordingly, dominant-negative FOXM1c proteins reduced cell growth approximately threefold, demonstrating a pro- liferation-stimulating function for wild-type FOXM1c. Abbreviations BRE, TFIIB recognition element; ChIP, chromatin immunoprecipitation; DBD, DNA-binding domain; DPE, downstream promoter element; EDA, essential domain for activation; EMSA, electrophoretic mobility shift assay; FKH, forkhead domain; GST, glutathione S-transferase; GTF, general transcription factor; Inr, initiator; NE, neutrophile elastase; NLS, nuclear localization signal; NRD, negative regulatory domain; OHT, 4-hydroxy-tamoxifen; PIC, preinitiation complex; RB, retinoblastoma protein; SV40, simian virus 40; TAD, transactivation domain; TAF, TBP-associated factor; TBP, TATA-binding protein; TFIIB, transcription factor IIB; TK, thymidine kinase; TPA, 12-O-tetradecanoylphorbol-13- acetate; TRD, transrepression domain. FEBS Journal 273 (2006) 4645–4667 ª 2006 The Authors Journal compilation ª 2006 FEBS 4645 transcription factor the splice variant FOXM1c (MPP2) binds to FOXM1-specific DNA sequences via its fork- head domain and transactivates via its strong acidic transactivation domain (TAD) [29–31]. This strong TAD can be kept almost inactive by two different inhibitory domains. The N-terminus functions as a specific negative regulatory domain (NRD), named NRD-N, which completely inhibits the TAD by directly binding to it. The central domain functions as a retino- blastoma protein (RB)-independent transrepression domain (TRD) [29–31] and as RB-recruiting NRD-C [31]. Core promoters and basal transcription complexes were initially thought to be interchangeable at will, but are now viewed as active participants in gene regula- tion. Their diversity makes essential contributions to the specificity and variability in combinatorial gene regulation [32–34]. Core promoter elements are the TATA box, the initiator (Inr), the downstream promo- ter element (DPE), motif ten element (MTE) and the transcription factor IIB (TFIIB) recognition element (BRE). None of these elements is obligatory and sev- eral different combinations are operational. Enhancers can target certain core promoter elements so that their activating effect is limited to genes with these elements [32–35]. Basal transcription complexes are not uniform because of TATA-binding protein (TBP)-related fac- tors and alternative TBP-associated factors (TAF II s) [36,37]. It is believed that the basal transcription com- plex can adopt different conformations on different core promoters and that different core promoters can determine different rate-limiting steps in preinitiation complex (PIC) assembly and transcription initiation, as well as different reinitiation rates [32–34,38–48]. TBP plays a central role in the recognition of TATA box promoters. The C-terminal ⁄ core region of TBP has a saddle-like structure: its concave underside binds to DNA; the convex upper surface binds to a large variety of TAF II s, general trancription factors (GTFs), transcription factors, coactivators and general cofac- tors [38,49,50]. TBP binds to the minor groove of the TATA box, thereby bending the DNA 80° towards the major groove, unwinding the DNA by 120° and kink- ing the TATA box at both ends by intercalation of two phenylalanine residues. TFIIA interacts with the N-terminal TBP stirrup, which is orientated towards the 3¢-end of the TATA box, and with TBP helices H1 and H2. TFIIB interacts with the C-terminal TBP stir- rup, which is orientated towards the 5¢-end of the TATA box, and with TBP helix H1¢ [38,39,51]. The PIC can be assembled in a stepwise fashion in reconstituted in vitro systems [38,39]. In vivo, PIC assembly may vary among core promoters between two extremes: (a) the stepwise assembly of individual GTFs, and (b) recruitment of the complete holo- enzyme in one step [45]. However, PIC assembly will always require at least two separate steps, namely TFIID ⁄ TFIIA binding and TFIIB ⁄ Pol II binding [46]. Here, we describe a new transactivation mechanism by which FOXM1c transactivates the c- myc promoter via its P1 and P2 TATA boxes. It does so by binding to the TATA box and directly to TBP, TFIIB and TFIIA. The P1 TATA box TATAATGC requires its sequence context to be FOXM1c responsive. In con- trast, the P2 TATA box TATAAAAG alone is sufficient to confer FOXM1c responsiveness on any minimal promoter so that each promoter with this TATA box is postulated to be transactivated by FOXM1c as seen for c-fos, hsp70 and histone H2B ⁄ a. In addition to these new FOXM1c target genes, a database search revealed nearly 300 genes with such a TATA box sequence, many of which also play a role in proliferation and tumorigenesis. Accordingly, dom- inant-negative FOXM1c proteins reduce cell growth by approximately threefold demonstrating a proliferation- stimulating function for wild-type FOXM1c. Results FOXM1c transactivates the c-myc promoter, namely the minimal P1 and P2 promoters Human c-myc promoter was transactivated by wild- type FOXM1c and significantly more so by the mutant FOXM1c(189–762) (Fig. 1A), which lacks the negat- ive-regulatory N-terminus (see below). Therefore, FOXM1c(189–762) was used in this study. In contrast to c-myc, FOXM1c(189–762) did not transactivate the promoters of human c-jun, waf1(p21), ink4a(p16), murine neutrophile elastase (NE) or the simian virus (SV)40 early promoter (Fig. 1B; data not shown). To map the FOXM1c-responsive element, several c-myc–promoter constructs were analyzed (Fig. 1D). FOXM1c(189–762) strongly transactivated the P1 and P2 promoters, but not the P0 promoter. Because all potential FOXM1c-binding sites (C ⁄ T-AAA-C ⁄ T) of the c-myc promoter are positioned in the non- FOXM1c-responsive segment )2486 ⁄ )259 (Fig. 1D; data not shown), common elements of the P1 and P2 promoters were analyzed for FOXM1c responsive- ness. The P1 and P2 promoters both possess a TATA box and a GC-box-type Sp1-binding site. The Sp1-binding sites )44 (known; position )44 relative to the P1 transcription start site) and )66 (potential; position )66 relative to the P2 transcription start site), as well as overlapping binding sites for FOXM1c activates c-myc via its two TATA boxes I. Wierstra and J. Alves 4646 FEBS Journal 273 (2006) 4645–4667 ª 2006 The Authors Journal compilation ª 2006 FEBS other transcription factors were not FOXM1c respon- sive (Fig. 1D). Minimal promoters include only the TATA box and the transcription start (+1). These minimal c-myc P1 and P2 promoters were both strongly transactivated by FOXM1c(189–762) (Fig. 1C, D). By contrast, the minimal promoters of human D P1 P2 -44 -262 P1 +49 P2 P2 mintk -66 -66 GCTT GGCGGGAAA GCGGGAAA E2F gGGAA ETS-Core TTGGCGGGAAA STAT3 GGAAA NFATc1-Consensus GGCTT Smad GGAAAG METS-Consensus cGT 3x -95 +49 P2 -224 P1 -136 +49-2486 P1 P0 -259 mintk P0 -2486 pTATA-P1-luc p(-44)mintkluc pmyc(-224/-136)luc - + + TA b y FOXM1c (189-762) pTATA-P2-luc p(-66)mintkluc pmyc(-95/+49)luc pmycluc pmyc(-262/+49)luc TA b y FOXM1c (189-762) + - - + + + pmyc(-2486/-259) mintkluc C pTATA-WAF-luc pTATA-jun-luc pTATA-P2-luc pTATA-P1-luc pmintkluc y t ivitcaesareficulev i taler 0 10 20 30 40 0213456 μg pFOXM1c(189-762) B ytivitcaesareficulevitaler 0 2,5 5 7,5 10 pwaf1 (p21)luc pmyc luc pjun luc C FOXM1c(189-762) C )267-981(c1MXOF c1MXOF ytivitcaesareficulevi taler pmycluc 0 5 10 15 20 25 30 A mintk -44 3x ATCT CCGCCCACC Fig. 1. FOXM1c transactivates the minimal P1 and P2 promoters of c-myc. (A, B) RK13 cells were transiently transfected with expression plasmids for the FOXM1c proteins or as control (c) with the empty vector and with the indicated reporter constructs. The relative luciferase activity of each reporter construct in the control (c) was set as 1. (C) RK13 cells were transiently transfected with the indicated amounts of pFOXM1c(189–762) and with the indicated reporter constructs. The relative luciferase activity of each reporter construct in the absence of pFOXM1c(189–762) was set as 1. (D) c-myc sequences are shown as black lines, TATA boxes as black boxes, transcription start sites (+1) as arrows, Sp1-binding sites are shown as dark gray boxes and sequences of the thymidine kinase (TK) promoter of herpes simplex virus (HSV) as a light gray box. Numbers give the nucleotides of c-myc relative to the transcription start (+1) of P2. p()44)mintkluc and p()66)mintkluc contain three adjacent copies of the indicated nucleotide sequences. Sp1-binding sites are marked bold and underlined. Bind- ing sites for other transcription factors are indicated below. It is indicated whether the reporter constructs are transactivated by FOXM1c(189–762) (¼ +) or not (¼ –). TA, transactivation; P0, P1, P2, c-myc promoters; mintk, minimal TK promoter of HSV. I. Wierstra and J. Alves FOXM1c activates c-myc via its two TATA boxes FEBS Journal 273 (2006) 4645–4667 ª 2006 The Authors Journal compilation ª 2006 FEBS 4647 c-jun, waf1(p21) or herpes simplex virus (HSV) thymi- dine kinase (TK) were not transactivated by FOXM1c(189–762) (Fig. 1C). The P1 and P2 TATA boxes are the FOXM1c-responsive elements The existence of FOXM1c-responsive and -nonrespon- sive minimal promoters offered the possibility of con- structing hybrid minimal promoters (Fig. 2C) to map the responsive element exactly. Hybrids exchanging the TATA box half and the transcription start (+1) half between c-myc P1 or c-myc P2 and c-jun promoters showed that the TATA box halves of the P1 and P2 promoters both transfer FOXM1c responsiveness (Fig. 2A). Hybrids exchanging only the TATA boxes between P1 or P2 and the c-jun or waf1 ⁄ (p21) promot- ers, and vice versa, showed that the c-myc P1 and P2 TATA boxes are themselves the FOXM1c-responsive elements (Fig. 2B,C). Both are necessary for FOXM1c responsiveness because replacing them with the TATA box of a non-FOXM1c-responsive promoter abolished transactivation by FOXM1c(189–762) (Fig. 2B,C). The P2–TATA box is sufficient as the FOXM1c-responsive element because insertion of it into a nonresponsive minimal promoter resulted in very strong transactiva- tion by FOXM1c(189–762) (Fig. 2B). The P1 TATA box requires its sequence context to function as the FOXM1c-responsive element because insertion of it into the minimal promoters of c-jun and waf1(p21) did not result in transactivation by FOXM1c(189–762) (Fig. 2C). Figure 2D shows the sequence differences between the TATA boxes used. To our knowledge, transactivation of a promoter by a transcription factor via its TATA box has not been described previously and thus represents a new mechanism. FOXM1c domains required for transactivation of the c-myc promoter FOXM1c transactivates by two different mechanisms: (a) the reporter construct p(MBS) 3 -mintk-luc via its FOXM1c-binding sites as a conventional transcription factor [29–31]; and (b) the P1 and P2 promoters of c-myc via their TATA boxes by a new mechanism. Several FOXM1c mutants (Fig. 3F) the expression levels of which have been compared previously [30] were analyzed for transactivation of c-myc promoter constructs (Fig. 1D). Two mutants lacking either part of the TAD (amino acids 721–762) or part of the forkhead domain (amino acids 235–332), and thereby the complete recognition helix 3 (amino acids 277– 290) [53], repressed or did not transactivate the P1 and P2 promoters (Fig. 3A,B). Therefore, both the intact DNA-binding domain (DBD) and the intact TAD are essential for transactivation of the P1 and P2 promoters (Fig. 3E,F). Wild-type FOXM1c trans- activated the P1 and P2 promoters considerably less than FOXM1c(189–762) (Fig. 3A). The N-terminus (amino acids 1–232) in trans repressed transactivation of the P1 and P2 promoters by FOXM1c(189–762) (Fig. 3D), which can be explained by the direct interac- tion of the N-terminus (amino acids 1–194) with the TAD (amino acids 721–762) [30]. Therefore, the N-ter- minus as NRD represses transactivation of the P1 and P2 promoters by directly binding to the TAD. In sum- mary, the forkhead domain (i.e. the DBD) TAD and N-terminus, have the same functions for transactiva- tion of the c-myc promoter via its TATA boxes and for transactivation as a conventional transcription factor (Fig. 3E,F) [30]. FOXM1c(189–348; 573–762)NLS did not transacti- vate the P1 and P2 promoters (Fig. 3C). In contrast, FOXM1c(189–425; 568–762) transactivated the P1 and P2 promoters as strongly as FOXM1c(189–762) if the lower expression level of the former [30] was taken into account (Fig. 3A). Thus, these two mutants with deletions in the central domain (amino acids 349–572) showed that amino acids 349–425 are essential for transactivation of the P1 and P2 promot- ers. Therefore, amino acids 349–425 are referred to as the essential domain for activation (EDA). The cen- tral domain has opposing functions for transactiva- tion of the c-myc promoter via its TATA boxes, where it functions as the EDA, and for transactiva- tion as a conventional transcription factor, where it Fig. 2. The FOXM1c-responsive elements are the P1 and P2 TATA boxes. (A ,B) RK13 cells were transiently transfected with the indicated amounts of pFOXM1c(189–762) and with the indicated reporter constructs. The relative luciferase activity of each reporter construct in the absence of pFOXM1c(189–762) was set as 1. (C) TATA boxes and transcription start sites (+1) are bold and underlined. Symbols below the nucleotide sequences explain the composition of hybrid promoters. It is indicated whether the reporter constructs are transactivated by FOXM1c(189–762) (¼ +) or not (¼ –). TA, transactivation. (D) Differences of TATA boxes of non-FOXM1c-responsive (¼ –) promoters to the FOXM1c-responsive (¼ +) TATA boxes c-myc-P1 and c-myc-P2. Nucleotides that deviate from the c-myc TATA box are bold. Nucleotides that are identical to the c-myc TATA box are replaced by a dash. For c-jun and TK both possible TATA box positions are shown. c-myc-P0 and ink4a(p16) are TATA-less (¼ –) non-FOXM1c responsive promoters. TA by FOXM1c, transactivation by FOXM1c(189–762); NE, murine neutrophile elastase; TK, thymidine kinase of HSV; SV40early, early promoter of simian virus (SV)40. FOXM1c activates c-myc via its two TATA boxes I. Wierstra and J. Alves 4648 FEBS Journal 273 (2006) 4645–4667 ª 2006 The Authors Journal compilation ª 2006 FEBS I. Wierstra and J. Alves FOXM1c activates c-myc via its two TATA boxes FEBS Journal 273 (2006) 4645–4667 ª 2006 The Authors Journal compilation ª 2006 FEBS 4649 FOXM1c activates c-myc via its two TATA boxes I. Wierstra and J. Alves 4650 FEBS Journal 273 (2006) 4645–4667 ª 2006 The Authors Journal compilation ª 2006 FEBS functions as an inhibitory domain [29–31] (Fig. 3E,F). Consequently, FOXM1c(189–348; 573–762)NLS can be used to discriminate between these mechanisms: (a) if it transactivates considerably more strongly than FOXM1c(189–762), FOXM1c functions as a conventional transcription factor; and (b) if it does not transactivate, FOXM1c functions via the TATA box. FOXM1c transactivates other genes involved in cell proliferation that possess the c-myc P2 TATA box TATAAAAG The c-myc P2 TATA box is sufficient to transfer very strong transactivation by FOXM1c(189–762) to a non- responsive minimal promoter (Fig. 2). Consequently, it was postulated that each promoter with this TATA Fig. 4. FOXM1c transactivates other proliferation-associated genes with the c-myc P2 TATA box TATAAAAG. (A, B) RK13 cells were transiently transfected with expression plasmids for the FOXM1c proteins or as control (c) with the empty vector and with the indicated reporter con- structs. The relative luciferase activity of each reporter construct in the control (c) was set as 1. phsp70luc contains the hsp70 promoter sequence from )2400 to +150. phsp70-TATA-luc contains the hsp70 promoter sequence from )32 to +150, i.e. a ‘minimal’ hsp70 promoter. (C) Summary of the flanking nucleotides of the TATA box TATAAAAG (bold and underlined) in the six promoters that are activated (¼ +) by FOXM1c. The transcription start site (+1) is bold and underlined. Symbols below the sequences explain the composition of hybrid promoters. Fig. 3. FOXM1c domains required for c-myc promoter transactivation. (A–C) RK13 cells were transiently transfected with expression plas- mids for the indicated FOXM1c proteins or as control (c) with the empty vector and with the indicated reporter constructs. The relative lucif- erase activity of each reporter construct in the control (c) was set as 1. (D) RK13 cells were transiently transfected with the expression plasmid for FOXM1c(189–762) or as control (c) with the empty vector and with the indicated reporter constructs. The indicated amounts of pFOXM1c(1–232) were cotransfected. (E) Functions of FOXM1c domains for transactivation of the c-myc promoter via the P1 and P2 TATA boxes and for transactivation of p(MBS) 3 -mintk-luc as a conventional transcription factor [29–31] and whether their functions in these two dif- ferent transactivation mechanisms are equivalent or opposite. TA, transactivation; IA, interaction; P1, P2, P1- or P2-promoter of c-myc. (E, F) TAD, transactivation domain; DBD, DNA-binding domain; TRD, transrepression domain; EDA, essential domain for activation; NRD, negative regulatory domain. (F) FOXM1c(189–348; 573–762)NLS possesses the nuclear localization signal (NLS) of SV40 large T between amino acids 348 and 573. FKH, forkhead domain. p(MBS) 3 -mintk-luc is transactivated very strongly (+ + + + +), strongly (+ + +) or weakly (+) or repressed (–) and the c-myc-promoter is transactivated very strongly (+ + + + +), strongly (+ + +) or repressed (–) or neither transactivated nor repressed (). Note that the indicated transactivation for FOXM1(189–425; 568–762) is corrected by expression (see text). I. Wierstra and J. Alves FOXM1c activates c-myc via its two TATA boxes FEBS Journal 273 (2006) 4645–4667 ª 2006 The Authors Journal compilation ª 2006 FEBS 4651 box is transactivated by FOXM1c. Therefore, the pro- moters of human c-fos, hsp70 and histone H2B ⁄ a which all possess the c-myc P2 TATA box TATAAAAG (Fig. 4C) were tested. As postulated, these three pro- moters were transactivated by FOXM1c(189–762), but not transactivated or considerably less so (Fig. 4A,B) by FOXM1c(189–348; 573–762)NLS. This also held true for a ‘minimal’ hsp70 promoter (Fig. 4B) showing that FOXM1c transactivates the hsp70 promoter via its TATA box. The parental vectors used to construct the reporter plasmids were not FOXM1c responsive (Figs 1B,D and 4A,B; data not shown). This transacti- vation of the c-fos, hsp70 and histone H2B ⁄ a promoters confirmed that each promoter with the c-myc P2 TATA box is transactivated by FOXM1c. Comparison of the six promoters used showed that, in the sequences flank- ing the c-myc P2 TATA box, almost every nucleotide was found at almost every position (Fig. 4C). Thus the c-myc P2 TATA box TATAAAAG alone is sufficient as the FOXM1c-responsive element. A database search for promoters with this TATA box gave a list of almost 300 potential FOXM1c target genes (Fig. S1). FOXM1c binds directly to components of the basal transcription complex To characterize this new mechanism by which FOXM1c transactivates the c-myc P1 and P2 promoters we analyzed whether FOXM1c binds to their TATA boxes (Fig. 8) and whether it interacts with components of the basal transcription complex (Figs 5 and 6). In pull-down experiments (Fig. 5, Fig. S2), FOXM1c bound to TBP, TFIIB, TFIIAa ⁄ b, TFIIAc and TAF II 250 (TAF1) [52], but not to TFIIEa. These inter- actions are direct for TBP, TFIIB and TFIIAa ⁄ b because they could be verified using in vitro-translated proteins (Fig. 5). The respective interaction domains of FOXM1c were each mapped to its central domain (see below; Fig. 5, Fig. S2). Therefore, the interactions of TAF II 250 and ⁄ or TFIIAc with FOXM1c may be indi- rect via TBP or TFIIAa ⁄ b, respectively. The inter- actions of FOXM1c with TBP, TFIIAa ⁄ b, TFIIAc and TAF II 250 are also found in vivo because these proteins could be coimmunoprecipitated with FOXM1c (Fig. 6). TBP bound strongly to FOXM1c ( 28% of the input TBP was pulled down) (Fig. 5B). Deletion mutants of TBP showed that FOXM1c binds predom- inantly to the C-terminal half of the conserved TBP saddle (Fig. 5B,C), which is orientated towards the 5¢-end of the TATA box [38,49,50]. More detailed mapping (Fig. 5, Fig. S2) showed that TBP and TFIIB both bound to amino acids 380–425 of FOXM1c, i.e. to the EDA (amino acids 349–425) (Fig. 3F), but not to amino acids 1–379 or 574–762. TAF II 250 interacted with amino acids 380–477 of FOXM1c, but not with amino acids 1–379. TFIIAa ⁄ b and TFIIAc both probably interacted with amino acids 359–477 of FOXM1c. In summary, FOXM1c binds directly, via its essen- tially required EDA (amino acids 349–425) (Fig. 3F), to the components TBP, TFIIAa ⁄ b and TFIIB of the basal transcription complex, which are positioned at or near the TATA box, respectively. FOXM1c(189–762) and FOXM1c(189–425; 568–762), which bound to TBP and TFIIB, transactivated the c-myc P1 and P2 promoters, whereas FOXM1c(189–348; 573 762)NLS, which did not bind to TBP or TFIIB, failed to transac- tivate both promoters (Figs 3A,C,F, 5A, Fig. S2A,F,G; data not shown). Consequently, these interactions should be important for the new mechanism by which FOXM1c transactivates via the c-myc P1 and P2 TATA boxes. Binding of TBP and FOXM1c to the P1 and P2 TATA boxes Because TBP binds to all TATA boxes the question arose: what is the difference between the FOXM1c- responsive TATA boxes of c-myc P1 and c-myc P2 versus the non-FOXM1c-responsive TATA boxes of c-jun, waf1(p21) and HSV TK? The TBP ⁄ TFIIA com- plex bound to the c-myc P2 TATA box (P2) with the same very high affinity as to the identical TATA box of the adenovirus 2 major late promoter (AdML) (Fig. 7A), which is bound very strongly by TBP [50]. Its binding affinity for the c-myc P1 TATA box (P1) was lower, although still high (Fig. 7A). Its binding affinity for the FOXM1c-responsive TATA boxes of c-myc P1 and c-myc P2 was higher than for the non- responsive TATA boxes of c-jun (jun), waf1(p21) (WAF) and HSV TK (mintk) (Fig. 7B,C). GST–FOXM1c(233–334), which comprised the forkhead domain (amino acids 235–332), and GST– FOXM1c(195–596) bound to the c-myc P1 and c-myc P2 TATA boxes (Fig. 8C,D). These protein–DNA complexes were supershifted with an antibody [a-GST, a-FOXM1c(1B1)] that recognized the two GST– FOXM1c fusion proteins, but not with a control anti- body [a-FOXM1c(7E4)] (Fig. 8C,D; data not shown). These protein–DNA complexes were competed by an excess of unlabeled c-myc P1 TATA box or c-myc P2 TATA box, respectively, but not by an excess of unlabeled control oligonucleotides (Fig. 8A,B,D). Thus FOXM1c binds in a sequence-specific manner and with high affinity to the c-myc P1 TATA box and the c-myc P2 TATA box, and the forkhead domain FOXM1c activates c-myc via its two TATA boxes I. Wierstra and J. Alves 4652 FEBS Journal 273 (2006) 4645–4667 ª 2006 The Authors Journal compilation ª 2006 FEBS Fig. 5. Direct binding of FOXM1c to TBP, TFIIA and TFIIB. (A, B) Pull-down assays were performed in the presence of ethidium bromide [87] with purified GST or the indicated GST–fusion proteins and the indicated in vitro-translated proteins. Bound in vitro-translated proteins were detected following SDS ⁄ PAGE by autoradiography. The input control represents 1 ⁄ 10 of the volume used in the pull-down assays. (B) Amount (%) of the input bound to GST–FOXM1c(1–477). wt, wild-type. (C) (Upper) RASMOL drawing of the cocrystal structure of the C-ter- minal ⁄ core region of human TBP complexed with the TATA element of the adenovirus major late promoter [49]. TBP segments are colored as indicated in the table. DNA is shown in gray. (Lower) Quantification of the pull-down assay in (B). Contribution (%) made by the TBP seg- ments to total GST–FOXM1c(1–477) binding and which elements of the TBP saddle they included. H, a helix; S, b strand. I. Wierstra and J. Alves FOXM1c activates c-myc via its two TATA boxes FEBS Journal 273 (2006) 4645–4667 ª 2006 The Authors Journal compilation ª 2006 FEBS 4653 (amino acids 235–332) is sufficient for this DNA bind- ing. The order of binding affinities for the different TATA boxes was similar for GST–FOXM1c(195–596) as for the TBP ⁄ TFIIA complex (Fig. 7C; data not shown). For comparison, the best conventional FOXM1c-binding site HFH-11 [30] was bound by GST–FOXM1c(195–596) with lower affinity than the c-myc P1 and P2 TATA boxes (Fig. 8B). To examine in vivo binding of FOXM1c to the endog- enous c-myc promoter chromatin immunoprecipitation (ChIP) assays were performed. Figure 8E shows that the c-myc P1 ⁄ P2 TATA box region was enriched mark- edly more with a FOXM1c-specific antibody than with a control antibody (a-b-Gal), indicating that in vivo FOXM1c binds to the c-myc promoter. As a negative control, the NE promoter (TATA box region) was less immunoprecipitated with the FOXM1c-specific anti- body than with the control antibody (Fig. 8E), indicat- ing that in vivo this promoter is not bound by FOXM1c. Dominant-negative FOXM1c reduces cell growth c-Myc, a key factor for cell-growth control, potently stimulates cell proliferation, promotes apoptosis and represses differentiation and entry into quiescence. c-Fos also stimulates proliferation, HSP70 and histone H2B are required for its execution. Consequently, transactivation of the four respective genes by FOXM1c should increase proliferation. By contrast, repression of these genes by dominant-negative FOXM1c should reduce proliferation. FOXM1c(189–743)–Engr and FOXM1c(189–566)–Engr were constructed by replacing the TAD (amino acids 721–762) or its C-terminal half with the repressor domain of Drosophila Engrailed (Figs 9A and S3C). These two dominant-negative forms of FOXM1c repressed p(MBS) 3 -mintk-luc, the c-myc P1 promoter and the c-myc P2 promoter (Fig. S3A,B; data not shown). Thus they functioned as repressors for all FOXM1c target genes regardless whe- ther activation is via TATA box binding or binding to the conventional target sequences. In colony-formation assays, both FOXM1c(189– 743)–Engr and FOXM1c(189–566)–Engr reduced the HA-TBP FOXM1c (189-762) FOXM1c (189-762) WB: α-HA WB: α-FOXM1c HA-TBP FOXM1c (189-762) ++ HA-TBP ++ WB: α-HA IP: α-FOXM1c WB: α-FOXM1c IP: α-HA A HA-TFIIAγWB: α-HA FOXM1c (189-762) FOXM1c (189-762) WB: α-FOXM1c HA-TFIIAγ ++ FOXM1c (189-762) ++ IP: α-HA WB: α-FOXM1c D myc-TFIIAαβWB: α-myc myc-TFIIAαβ FOXM1c (189-762) WB: α-FOXM1c FOXM1c (189-762) ++ myc-TFIIAαβ ++ IP: α-FOXM1c WB: α-myc C HA- TAF II 250 WB: α-HA Co-IP input C ++ HA-TAF II 250 ++++ IP: α-FOXM1c ++ IP: α-C + B Fig. 6. In vivo binding of FOXM1c to TBP, TAF II 250 and TFIIA. Co- immunoprecipitations (Co-IP) were performed with total cell lysates of COS-7 cells transiently transfected with expression plasmids for the indicated proteins. The antibodies used in the coimmunoprecipi- tations (IP) and the primary antibodies used in the (following) west- ern blots (WB) are indicated. The input control represents 1 ⁄ 30 of the volume used in the coimmunoprecipitations. a-FOXM1c, a-FOXM1c(C-20). (B) The control antibody a-C was a-cytochrome c. FOXM1c activates c-myc via its two TATA boxes I. Wierstra and J. Alves 4654 FEBS Journal 273 (2006) 4645–4667 ª 2006 The Authors Journal compilation ª 2006 FEBS [...]... pXP-2 the annealed product of the oligonucleotides indicated in Table S1 pTATA -P2- luc, pTATA-jun-luc, pTATA-WAFluc, pP1-jun-luc, pP2-jun-luc, pjun -P1- luc, pjun -P2- luc, pP1(junTATA)luc, pP2(junTATA)luc, pP1(WAFTATA) luc, pP2(WAFTATA)luc, pjun(P 1TATA) luc, pjun(P 2TATA) luc, pWAF(P 1TATA) luc and pWAF(P 2TATA) luc were created by ligating into XhoI ⁄ HindIII-opened pXP-1 [84] the annealed product of the oligonucleotides... Wierstra and J Alves FOXM1c activates c-myc via its two TATA boxes P1 P1 B P2 mintk WAF P1 jun AdML P1 TBP+TFIIA CMD α-HA TBP+TFIIA α-TBP α-HA α-TBP c SV40 A T TBP+ TFIIA T TBP TBP+ TFIIA TBP F P2 F AdML P2 TBP+TFIIA TBP+ TFIIA CMD T TBP mintk WAF P2 jun T TBP+ TFIIA F AdML P1 SV40 P2 SV40 α-HA TBP+TFIIA α-TBP α-HA α-TBP c TBP F C DNA binding affinity c-myc -P2 c-myc -P1 waf1 (p21 ) TATAAAAG TATAATGC TATATCAG... [57,58] The c-myc P2 TATA box is bound by the TBP ⁄ TFIIA complex with the highest affinity (Fig 7) How could FOXM1c cooperate with TBP at such a good TATA box? Because the A-tract is very rigid [59] FOXM1c activates c-myc via its two TATA boxes and bent towards the minor groove [60] it is more difficult for TBP to bend the c-myc P2 TATA box TATAAAAG towards the major groove than it is to bend other more... TATA boxes [58] However, once TBP is bound to this TATA box the resulting complex is more stable than at other TATA boxes and accordingly this TATA box leads to higher reinitiation rates [41,42,50,57,61] so that, in vivo, TATAAAAG is the optimal TATA box Upon DNA binding, the forkhead domain of HNF-3c bends the DNA towards the major groove [53] so that FOXM1c is expected to also bend the c-myc P2 TATA. .. E7 enhances the transactivation by FOXM1c, i.e the relative luciferase activity of each reporter construct in the control (K) was always set to 1 contributes to transformation by HPV16 because c-Myc induces S-phase entry and inhibits differentiation [1–7] Discussion FOXM1c transactivates the human c-myc promoter via both its P1 TATA box TATAATGC and its P2 TATA box TATAAAAG (Figs 1,2) Thus FOXM1c can... groove of the A-tract in the c-myc P2 TATA box TATAAAAG make this TATA box a good target for FOXM1c, which may help TBP to bind this TATA box by pre-bending it towards the major groove Effects that depend specifically on the TATA box TATAAAAG have also been described for other transcriptional regulators and other genes demonstrating the special role of this TATA box in gene regulation [35,43,63–68] The existence... cell lines [23] it is unlikely that the strong negative effect on cell growth of the two dominant-negative forms of FOXM1c is based on an increased rate of apoptosis FEBS Journal 273 (2006) 4645–4667 ª 2006 The Authors Journal compilation ª 2006 FEBS 4655 FOXM1c activates c-myc via its two TATA boxes I Wierstra and J Alves Fig 8 FOXM1c binds to the P1 and P2 TATA boxes (A–D) EMSAs were performed with... specific for the c-myc P1 ⁄ P2 TATA boxes region Primers specific for the NE promoter (TATA box region) were used as control 4656 FEBS Journal 273 (2006) 4645–4667 ª 2006 The Authors Journal compilation ª 2006 FEBS I Wierstra and J Alves FOXM1c activates c-myc via its two TATA boxes A average construct OHT control - 100 1 762 FOXM1c( 189-762) - 94 1,06 762 FOXM1c( 189-587; 744-762) - 94 1,06 FOXM1c( 189-743)-Engr... different TATA boxes For c-jun and TK both possible TATA box positions are shown The TATA box definitions of Patikoglou et al [50] and Bucher [90] and the general TATA box consensus sequence are indicated (D) In the oligonucleotides TATA boxes (bold and underlined), E -boxes (CMD) and binding sites for Sp1 (SV40, )66, WAF), FOXM1c (HFH-11), E2F, STAT3, ETS, NFATc1, Smad and METS ()66) (underlined) are... 1,2) Thus FOXM1c can transactivate via two different mechanisms: (a) as a conventional transcription factor by binding to a conventional FOXM1c- binding site [29– 31]; and (b) using a new mechanism by binding to the TATA boxes of the c-myc P1 and P2 promoters The c-myc P2 TATA box alone is sufficient as the FOXM1c- responsive element, so that its insertion into a non -FOXM1c- responsive minimal promoter . only the TATA boxes between P1 or P2 and the c-jun or waf1 ⁄ (p21 ) promot- ers, and vice versa, showed that the c-myc P1 and P2 TATA boxes are themselves the. transactivates the c- myc promoter via its P1 and P2 TATA boxes. It does so by binding to the TATA box and directly to TBP, TFIIB and TFIIA. The P1 TATA box TATAATGC