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J. Biol. Chem., Vol. 277, Issue 40, 37191-37200, October 4, 2002

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CCAAT Binding Factor (CBF) Binding Mediates Cell Cycle Activation of Topoisomerase II

CONVENTIONAL CBF ACTIVATION DOMAINS ARE NOT REQUIRED^*

Qianghua Hu, Chitralekha Bhattacharya, and Sankar N. Maity^§

From the Department of Molecular Genetics, The University of Texas, M. D. Anderson Cancer Center, Houston, Texas 77030

Received for publication, June 17, 2002, and in revised form, July 29, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

To understand the role of the CCAAT binding factor (CBF) in transcription during the cell cycle, we studied the mouse topoisomerase II (topo II) promoter, which is activated during the late S and G₂/M phases of the cell cycle and contains multiple CBF binding sites. Mutational analysis of the promoter shows that CBF binding to an inverted orientation of the CCAAT motif in the topo II promoter, but not to a direct orientation, is required for transcription activation during the cell cycle. In contrast, analysis of the promoter in an in vitro reconstituted transcription system shows that CBF activates transcription of the topo II promoter irrespective of the orientation of the CBF binding sites. This analysis demonstrates that only one of the three transcription start sites of the topo II promoter is stimulated by CBF, indicating that transcription activation by CBF is dependent on basal promoter structure. Interestingly, mutations of the start site that abolish CBF-dependent transcription activation in vitro do not inhibit activation of the promoter during the cell cycle. Consistent with this observation, expression of a truncated CBF-B subunit lacking a transcription activation domain, which inhibits activity of a collagen promoter, does not affect activity of the topo II promoter in fibroblast cells. In contrast, expression of an allele-specific CBF-B mutant that binds high affinity to a mutant CBF binding site containing a CCAAC motif revives transcription activation of an inactive mutant topo II promoter containing CCAAC during the cell cycle. Altogether, this study indicates that CBF binding, but not conventional CBF activation domains, are required for activation of the topo II promoter during the cell cycle. Considering these results together with results of another recent study, we hypothesize that binding of CBF that disrupts the nucleosomal structure in the topo II promoter is a major function of CBF by which it regulates the cell cycle-dependent transcription of the topo II promoter and possibly many other cell cycle-regulated promoters containing CBF binding sites.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

DNA topoisomerase II (topo II)¹ is a ubiquitous nuclear enzyme that is involved in various cellular functions, such as replication, chromatin condensation, and segregation of newly synthesized chromatin pairs. Mammalian cells contain two topo II isoforms, 170-kDa topo II and 180-kDa topo II. Although the two isoforms are structurally very similar, they differ with respect to several biochemical and pharmacological properties, including sensitivity to topo II-targeting drugs and regulation of synthesis during cell cycle. Whereas the level of topo II is constant throughout the cell cycle, the expression of topo II varies during the cell cycle. The topo II level is very low at G₀/G₁ phase, begins to increase in late S phase, and is high at G₂/M phase. Moreover, the expression of the topo II gene is almost undetectable in quiescent or differentiated cells but is present in proliferating cells of all normal tissues of mice and humans as well as in various human tumors. Thus, it is believed that the topo II isoform plays a major role during chromosome segregation in proliferating cells (1, 2).

To understand transcriptional regulation of the topo II gene during cell proliferation, the promoter of the topo II gene was isolated from various mammalian species. The topo II promoter does not contain a consensus TATA motif. The most conserved feature of this promoter is that it contains multiple CCAAT motifs, which are located mostly in inverted orientation. The activity of the promoter is regulated by various external stimuli, such as heat shock, growth arrest, and stages of the cell cycle, and also by the p⁵³ tumor suppressor protein. Studies have shown that the CCAAT motifs play a critical role in the transcription regulation of this promoter by these various agents (3-6).

In a recent study (7), we showed that the mammalian heterotrimeric CCAAT binding factor (CBF) regulates expression of the cellular topo II gene as well as transcription activity of the topo II promoter. We also showed that inactivation of CBF in mouse fibroblast cells by expression of a dominant-negative CBF subunit results in retardation of cell growth. Analysis of the mRNA profile showed that the inactivation of CBF in fibroblasts decreases the expression of only a small number of cellular genes, including topo II and E2F1, which are regulated during cell growth. The results implied that CBF might be primarily involved in transcription of growth-regulated genes in cultured mammalian cells. Because the promoters of various growth-regulated genes, such as topo II, cyclin B1, CDC25C, E2F1, and thymidine kinase, are activated at different stages of the cell cycle and contain multiple CBF binding sites (8-12), we hypothesized that these promoters are highly dependent on CBF activity in vivo. One paradox of this hypothesis is that the DNA binding activity of CBF is unchanged during the cell cycle; thus, it remains unclear how constitutively expressed CBF is involved in regulating transcription of various promoters in specific cell-cycle stages.

We recently analyzed the transcription activity of the topo II promoter using an in vitro reconstituted nucleosomal assembled DNA template (13). The study showed that binding of CBF to the nucleosomal topo II promoter disrupts the regular nucleosomal structure over the CBF binding sites as well as over the downstream promoter region containing the transcription start site. Interestingly, binding of CBF to the nucleosomal topo II promoter strongly activated transcription. The study also revealed that although the topo II promoter contains three major transcription start sites, CBF activates transcription primarily through one of them. The CBF-mediated activation requires activation domains of CBF, which, however, do not play any role in the CBF-mediated nucleosomal disruption. When a mutant nucleosomal promoter containing mutations in all CBF binding sites was used in the in vitro transcription reaction, no transcription from any of the three start sites was observed. This observation suggested that binding of CBF to the topo II promoter disrupts the nucleosomal structure, which allows transcription from all three start sites, whereas the activation domains of CBF stimulate transcription through only one of the start sites. Altogether, the results of the study indicate that CBF controls topo II promoter activity by two mechanisms, nucleosomal disruption and direct transcription activation. However, the mechanism by which CBF controls activation of the topo II promoter during the cell cycle remains to be determined.

In the present study, we show that mutations in all CBF binding sites located in inverted but not in direct orientation that are present in the topo II promoter completely abolish transcription activation of the promoter during cell-cycle progression. Analysis of the promoter in an in vitro reconstituted transcription system shows that CBF activates transcription of topo II regardless of the orientation of the CBF binding site and controls transcription of only one of the three start sites present in the promoter. Mutations of the transcription start site completely abolish transcription activation by recombinant CBF in vitro but do not affect activation of the promoter during the cell cycle. Two CBF-B mutants, one containing a truncated CBF-B lacking transcription activation domain and the other containing an allele-specific CBF-B mutant, were expressed in mouse fibroblast cells to determine the role of CBF in the cell cycle-dependent transcription activation. This study reveals that the cell cycle-dependent activation of the topo II promoter is exclusively dependent on CBF binding but not on CBF-dependent transcription activation mediated by the glutamine-rich domains of CBF.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Plasmids-- To construct the 7CCAAT reporter plasmid, the sequence between 250 and +110 of the mouse topo II gene promoter (6) was amplified by PCR and cloned between the SacI and XhoI sites of the pGL3-basic vector. The reporter plasmid constructs 4CCAAT, 3CCAAT, and 2CCAAT with deletions at the 5' end of the topo promoter were generated by PCR using the 7CCAAT template. The constructs W12, W34, W1, W2, W3, W4, and M1-4 harboring point mutations (CCAAT to CCAAA) in the CBF binding sites were obtained by PCR using the 4CCAAT template as described previously (13). The locations of mutations in the promoter is indicated in Fig. 2B. The construct W3mt carrying point mutation (CCAAT to CCAAC) was generated by PCR using the W3 template. The constructs 170/+29, 170/+16, 170/+4, and 170/18 with deletions at the 3' end of the topo II promoter were generated by PCR using the 4CCAAT template. The constructs M15/13, M11/9, M8/6, M5/3, M2/+1, and M+2/+4 carrying nucleotide substitutions in the start site II region were obtained by PCR using 170 + 4 as a template. Two plasmid constructs, pTRE-FLAGBdbd and pTRE-FLAGBmut307, which carry a deleted mutant and an amino acid-substituted mutant of the CBF-B subunit, were generated by PCR using the pTRE-FLAGB template as reported before (7). The reporter construct FC1 containing four CBF binding sites of mouse 2 (1) collagen promoter was described previously (14).

Cell Culture and Transfection-- Mouse fibroblast NIH3T3 cells were transfected with plasmids using the LipofectAMINE reagent (Invitrogen), and expression of the luciferase gene was measured as described previously (7). For the experiments illustrated in Figs. 8 and 9, a mouse fibroblast cell line, 3T3TtA, expressing tetracycline-responsive transcription activator (TtA), which was generated in a previous study (7), was used for DNA transfection. To make a stably integrated 7CCAAT reporter plasmid into NIH3T3 cells, both 7CCAAT and ptk-Hyg plasmids (9:1 ratio) were first transfected, and then cells were selected in the presence of 200 µg/ml hygromycin. Each of the topo II promoter-luciferase plasmid constructs were analyzed by a minimum of three independent DNA transfection experiments, and an average reporter activity with standard deviations was calculated from results of all the experiments, which are shown in Tables I and II and Figs. 8 and 9.

Cell Cycle Analysis-- For cell-cycle analysis, fibroblast cells were starved by incubation in media containing a low concentration of serum (0.5%) for 48 h and then stimulated for growth in media containing 10% serum. To determine the cell cycle stage, cells were harvested at different times after serum stimulation and then analyzed by flow cytometry using propidium iodide staining as described previously (7). To analyze the activity of the reporter constructs during the cell cycle, fibroblast cells were first transfected with plasmids, and then 24 h after transfection, cells were starved with 0.5% serum for 48 h and subsequently activated with 10% serum.

Recombinant CBF Proteins-- Full-length and truncated recombinant CBF subunits were generated as fusion proteins with glutathione S-transferase and purified as described previously (15). The mutant CBF-B subunit (Bmut) was expressed as a fusion with His₆ tag and was purified as described before (16). For the experiment in Fig. 9C, the wild-type and the mutant CBF-B (Bwt and Bmt307) were expressed in the 3T3TtA fibroblast cell line after transient transfection of either pTRE-FLAGB or pTRE-FLAGBmut307 plasmid construct. Each of the expressed CBF-B polypeptides was purified from fibroblast cellular extracts using anti-FLAG-agarose affinity resin (Sigma). Each CBF-B was copurified with the cellular CBF-A and CBF-C subunits present in the fibroblast cells.

In Vitro Transcription-- The in vitro transcription reactions were performed using nuclear extracts prepared from HeLa cells. Preparation of nuclear extracts, condition of the in vitro transcription reactions, and subsequent analysis of RNAs using the primer extension method were done as described previously (17).

DNA Binding-- The binding of CBF with the topo II promoter was analyzed using both electrophoretic mobility shift assay and DNase I footprinting methods as previously described (18). For DNase I footprinting analysis shown in Fig. 3, a topo II promoter fragment was isolated from either the 4CCAAT or the M1-4 construct by SacI and XhoI digestion, labeled at the XhoI end using Klenow, and then used in the DNA binding reactions. For the experiment in Fig. 4, 35-bp double-stranded oligonucleotides w1, w2, w3, and w4 corresponding to the CCAAT motifs located at 26, 46, 66, and 117 (respectively) in the topo II promoter were synthesized, labeled using Klenow, and then used in the DNA binding reactions. In the w1, w2, w3, and w4 oligonucleotides, the CCAAT motif is located in the middle and is flanked by 15-bp topo II promoter sequences on both sides. Nuclear extracts used for DNA binding were prepared according to a method described by Schreiber et al. (19).

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Topo II Promoter but Not Collagen Promoter Containing Multiple CCAAT Motifs Is Activated During the Late S and G₂/M Phases of the Cell Cycle-- Previously, analysis of the mouse topo II promoter showed that it contains seven CCAAT motifs that are binding sites of transcription factor CBF/NF-Y (6, 13). To understand the role of the CCAAT motifs in transcription during the cell cycle, we compared the activity of the topo II promoter with that of a collagen promoter, which contains four CBF binding sites, during cell cycle progression in mouse fibroblasts. In previous studies, we showed that the collagen promoter is strongly activated by CBF in an in vitro reconstituted transcription system (14, 17). The transcription activity of the topo II promoter in fibroblast cells was determined after either the promoter was stably integrated in chromosome or the promoter construct was transiently transfected in fibroblast cells. The cells were first synchronized in a quiescent state (G₀/G₁) by serum starvation, and cell-cycle progression was initiated by the addition of serum. The state of synchronization and cell-cycle progression was analyzed by flow cytometry methods using both propidium iodide and bromodeoxyuridine labeling. The results showed that after serum starvation, about 85% of fibroblast cells enter into G₀/G₁ phase and that the induction of S phase starts at 12 h and reaches the maximum level at 16 h after serum stimulation, similar to what was seen in our earlier studies (7). The activity of stably integrated topo II promoter, which is very low in quiescent cells, increased 5-fold at 18 and 24 h after serum addition (Fig. 1A). Similarly, the activity of transiently transfected topo II promoter was increased about 6-fold at 18 and 24 h after serum addition. This result shows that the activity of the topo II promoter is induced at late S phase to G₂/M phase. This observation is in good agreement with earlier studies of both human and mouse topo II promoters (1, 6). In contrast, the transcription activity of the collagen promoter was not induced after serum stimulation (Fig. 1B). Instead, the collagen promoter activity was lower at late S and G₂/M phase compared with the activity of quiescent phase cells before serum stimulation. This indicates that although both the topo II and the collagen promoters contain multiple CCAAT motifs, the cell cycle-dependent stimulation of promoter activity is specific to the topo II promoter.

View larger version (30K): [in this window] [in a new window]   Fig. 1.   Activity of 7CCAAT, a topo II promoter (A), and FC1, a 2(1) collagen promoter (B) constructs during cell cycle progression. The activity of each of the promoter constructs was measured after transfection in mouse fibroblast cells followed by starvation in a 0.5% serum concentration and then stimulation in a 10% serum concentration. Activity of the 7CCAAT promoter construct was measured using either a transient transfection method (white bar) or after isolation of cell clones containing a stably integrated promoter construct (black bar). The activity of the FC1 promoter construct was measured only after transient transfection. The activity of each promoter construct at each time point was measured at least three times, and the average relative value with S.D. (error bar) is shown. Because of low values at 0 h in A and at 18 and 24 h in B, the standard deviations are below the range of the y axis scale.

Role of Multiple CCAAT Motifs in Transcription Activation of Topo II Promoter during Cell Cycle Progression-- To determine the role of multiple CCAAT motifs in the topo II promoter activity during cell-cycle progression, we generated several deletion promoter constructs containing decreasing numbers of CCAAT motifs (Fig. 2A). The activity of the deleted constructs was analyzed during the cell cycle after transient transfection. This shows that the promoter containing four CCAAT motifs (4CCAAT) is activated very similar to the promoter containing seven CCAAT motifs during the cell cycle (Table I). The promoter containing three CCAAT motifs (3CCAAT) is also activated during the cell cycle but to a lesser extent than the promoter containing either four or seven CCAAT motifs. In contrast, the promoter containing two CCAAT motifs (2CCAAT) is activated very little. This shows that the four proximal CCAAT motifs in the topo II promoter are required for a high level of promoter activation during the cell cycle.

View larger version (38K): [in this window] [in a new window]   Fig. 2.   Schematic diagram of luciferase reporter constructs of the topo II promoter. The promoter constructs containing deletions in different parts of the 5' region are shown in A. The position of various CCAAT motifs in the promoter are shown with respect to start site II, which is shown in B. Mutant constructs containing a single nucleotide substitution in the CCAAT motif, which were introduced in the 4CCAAT promoter, are shown in B. The CCAAT motifs that are changed either from ATTGG to TTTGG or CCAAT to CCAAA are indicated by ×'s within the box.

View this table: [in this window] [in a new window]   Table I Activity of a collagen promoter and various deletion and nucleotide substitution mutants of the topo II promoter during the cell cycle Each of the promoter constructs was analyzed after transient transfection in mouse fibroblast cells followed by starvation with a 0.5% serum concentration and subsequent stimulation with a 10% serum.

To determine the role of individual CCAAT motifs in the promoter activity, single nucleotide substitution mutation was introduced in each of the four CCAAT motifs in the 4CCAAT topo II promoter construct (Fig. 2B). The interaction of recombinant CBF with both wild-type 4CCAAT promoter and a mutant M1-4 promoter containing mutations in all four CCAAT motifs was analyzed by the DNase I footprinting method. This shows that CBF interacts with all four CCAAT motif regions in the 4CCAAT promoter (Fig. 3, lanes 1 and 2). In contrast, no interaction of CBF with the M1-4 promoter was observed (lanes 3 and 4), indicating that single nucleotide substitution mutation in each of the four CCAAT motifs results in complete abolition of CBF binding. When a mutant promoter containing a single wild-type CCAAT motif at various locations (W1, W2, W3, or W4) was analyzed, CBF only interacted with the respective wild type but not with the mutant sites (data not shown). In this regard, it is important to mention that the CBF binding sites in the W1, W3, and W4 promoters are located in inverted orientation as ATTGG, whereas the site in W2 is located in direct orientation.

View larger version (43K): [in this window] [in a new window]   Fig. 3.   Footprinting analysis of the interaction of recombinant CBF (rCBF) with the wild-type (4CCAAT) and the mutant (M1-4) topo II promoters. Labeled promoter fragments from each construct were first incubated with (lanes 2 and 4) or without (lanes 1 and 3) 200 ng of purified recombinant CBF protein and then analyzed by the DNase I footprinting method. The position of the DNA fragments with respect to the transcription start site II and the position of the CCAAT motifs are indicated at the left side of the figure and by boxes located between lanes 2 and 3, respectively.

Analysis of promoter activity for each of the mutants shows that each of the three promoters, W1, W3, and W4, containing a single CBF binding site in the inverted orientation is activated during the cell cycle in a manner very similar to the 4CCAAT promoter (Table I). In contrast, the M1-4 promoter containing mutations in all four CBF sites was not activated during the cell cycle. Interestingly, the W2 promoter containing a single CBF site in the direct orientation was not activated during the cell cycle. This result indicates that at least one CBF binding site in the inverted orientation but not in the direct orientation is required for cell cycle-dependent activation of the topo II promoter.

It is possible that binding of CBF to the inverted CCAAT motif may have increased during the cell cycle and that this contributes to the activation of the topo II promoter. To test this possibility, we prepared nuclear extracts from fibroblast cells at 0, 12, 18, and 24 h after serum stimulation. Four double-stranded oligonucleotides, w1, w2, w3, and w4, corresponding to each of the CCAAT motifs located at the 26, 46, 66, and 117 positions, respectively, in the topo II promoter were made, labeled, and used in the DNA binding assay. This shows that the CBF binding activity in fibroblast nuclear extracts before serum stimulation (0 h) binds to each of the CCAAT motifs with indistinguishable affinity (Fig. 4), indicating that all four CCAAT motifs in the 4CCAAT promoter interact with CBF with similar affinity. Comparison of CBF binding activity at various time points after serum stimulation shows that a smaller increase of CBF binding to the w3 CCAAT motif is observed 18 h after serum stimulation. However, no significant alteration of CBF binding to the w1, w2, or w4 CCAAT motifs was observed at different time points of serum stimulation. This result indicates that activation of the topo II promoter during the cell cycle is not caused by an increase of CBF binding to the inverted CCAAT motifs in the promoter.

View larger version (93K): [in this window] [in a new window]   Fig. 4.   The activity of CBF during cell cycle progression of mouse fibroblast cells. Four oligonucleotides, w1, w2, w3, and w4, corresponding to the CCAAT motifs located at 26, 46, 66, and 117, respectively, in the topo II promoter, were labeled and used in the DNA binding reactions containing nuclear extracts prepared from fibroblast cells after 0, 12, 18, and 24 h of serum stimulation. The DNA binding was analyzed by electrophoretic mobility shift assay. Each of the oligonucleotides binds specifically to CBF present in the nuclear extracts, which was confirmed by a supershift assay using anti-CBF-B antibodies. The figure shows only CBF-DNA complexes for each of the oligonucleotides.

Analysis of Topo II Promoter Constructs in an in Vitro Reconstituted Transcription System-- It is possible that CBF-dependent transcription activation of the 4CCAAT promoter may be regulated during the cell cycle. Our previous study showed that although the 4CCAAT promoter contains three major start sites, recombinant CBF activates transcription mostly through one of the start sites of the nucleosomal promoter template (13). To understand CBF-dependent topo II promoter activity, we analyzed 7CCAAT and 4CCAAT promoters in an in vitro reconstituted transcription system. In a previous study, expression of a mutant CBF-B, Bmut, which inhibits DNA binding of cellular CBF, decreases expression of the topo II gene in fibroblast cells (7). The recombinant Bmut polypeptide was expressed and purified from bacteria (16). As expected, the addition of recombinant Bmut to HeLa cell nuclear extracts inhibited DNA binding of cellular CBF (Fig. 5A). Analysis of transcription reactions containing either the 7CCAAT promoter or the 4CCAAT promoter shows that each of the promoters is transcribed from three different start sites, which are designated I, II, and III (Fig. 5B, lanes 1 and 5). Interestingly, the addition of increasing amounts of recombinant Bmut in the transcription reactions results in almost complete inhibition of transcription from start site II but not from start sites I and III of both the promoters (Fig. 5B, lanes 2-4 and 6-8). Consistent with this observation, analysis of transcription from the mutant M1-4 promoter shows that almost no transcription from start site II is observed, whereas start sites I and III are transcribed similar to the wild-type 4CCAAT promoter. Altogether, these results indicate that transcription from start site II of the topo II promoter in vitro is dependent on CBF binding to the promoter.

View larger version (58K): [in this window] [in a new window]   Fig. 5.   Analysis of topo II promoter in an in vitro transcription reaction in the presence of a dominant negative CBF-B mutant. A, DNA binding activity of CBF in HeLa cell nuclear extracts in the presence of a dominant negative CBF-B mutant, the Bmut polypeptide. Labeled w1 oligonucleotide is used in the DNA binding reactions containing HeLa cell nuclear extracts with increasing amounts of the Bmut polypeptide. The reactions in lanes 1-6 contain 0, 400, 200, 40, 20, and 10 ng (respectively) of the Bmut polypeptide, which is added in each DNA binding reaction together with the nuclear extracts. B, activity of the topo II promoter templates in an in vitro transcription reaction. The 7CCAAT, 4CCAAT, and M1-4 topo II promoter templates are each transcribed in the HeLa cell nuclear extracts together with a control template containing 1(III) collagen promoter. The transcribed RNA is analyzed by the primer extension method. The transcripts from the three transcription start sites of the topo II promoter are indicated by I, II, and III. Increasing amounts of the Bmut polypeptide are added in the transcription reactions containing the 7CCAAT and 4CCAAT promoter templates. The amounts of Bmut polypeptide in lanes 2 and 7, lanes 4 and 8, and lanes 5 and 9 are 400, 200, and 80 ng, respectively.

Consistently, the addition of purified recombinant CBF in the in vitro transcription reaction results only in activation of start site II (Fig. 6A, lanes 1 and 2). We have also determined that transcription of the topo II promoter occurred in the presence of three truncated forms of CBF; one contains a truncated CBF-B that lacks amino-terminal glutamine-rich domain, the second is composed of truncated CBF-C, which lacks carboxyl-terminal glutamine-rich domains, and the third contains both truncated CBF-B and CBF-C. We previously showed that these three truncated forms of CBF interact with DNA similar to wild-type CBF (17). The addition of the first two forms of truncated CBF containing either truncated CBF-B or truncated CBF-C in the in vitro transcription reactions resulted in activation of transcription from the start site II (lanes 3 and 4). However, the level of transcription activation by these two truncated CBFs was much lower (3- and 2-fold lower, respectively) compared with the transcription activation by the wild-type CBF. In contrast, the addition of the third form of CBF containing both truncated CBF-B and CBF-C resulted in no activation from start site II (lane 5). These results indicate that the two glutamine-rich activation domains of CBF-B and CBF-C subunits mediate transcription activation of the topo II promoter through start site II in vitro. The four mutants of the topo II promoter, W1, W2, W3, and W4, each of which contains a single CBF binding site, are also analyzed in the in vitro transcription reaction. This shows that the addition of recombinant CBF in the transcription reaction results in activation of each of the promoters specifically from start site II, similar to the wild-type 4CCAAT promoter (Fig. 6B). Altogether, these results indicate that CBF activates the topo II promoter in vitro mostly through a specific start site and that the in vitro CBF-mediated activation is not dependent on orientation or location of the CBF binding site in the topo II promoter.

View larger version (48K): [in this window] [in a new window]   Fig. 6.   A, analysis of the 4CCAAT topo II promoter in the in vitro transcription reaction in the presence of full-length and truncated recombinant CBF subunits. The transcription reactions are performed in the presence of 551 ng of the full-length CBF-B subunits (lanes 2 and 4), 342 ng of CBF-Bd lacking amino-terminal glutamine-rich domain of CBF-B (lanes 3 and 5), 450 ng of full-length CBF-A/CBF-C subunits (lanes 2 and 3), and 350 ng of CBF-A/CBF-Cd lacking the carboxyl-terminal glutamine-rich domain of CBF-C. B, analysis of the W1, W2, W3, and W4 topo II promoter templates in the in vitro transcription reaction. Each of the promoter templates is transcribed in vitro in the absence (lanes 1, 3, 5, and 7) and in the presence of full-length CBF subunits (lanes 2, 4, 6, and 8). The recombinant CBF (rCBF) contains 551 ng of full-length CBF-B and 450 ng of full-length CBF-A/CBF-C. The transcription reactions in both A and B are analyzed according to the method described in Fig. 5B.

Mutational Analysis of the Transcription Start Site II Region-- Each of the three start sites of the topo II promoter likely contains a basal promoter element. Because none of the start site regions contain a consensus TATA motif, it is most likely that these basal promoter elements contain binding sites for initiator protein. It is not known whether all the start sites of the topo II promoter are utilized in the in vivo transcription or, more importantly, whether any of these start sites plays a role in the activation of the topo II promoter during the cell cycle. Indeed, our earlier observation showed only that start site II can be activated by CBF, suggesting that start site II is different from start sites I and III. To determine whether start site II plays a role in activation of the topo II promoter during the cell cycle, we have generated deletions and nucleotide substitution mutations in the 3' downstream region of the 4CCAAT promoter (Fig. 7, A and B). Each of the deletion mutants is analyzed by the in vitro transcription assay and also during the cell cycle in fibroblast cells. This shows that similar to the 4CCAAT promoter, each of the deleted promoters (170/+29, 170/+16, and 170/+4), which contains both start sites I and II, is activated by recombinant CBF in vitro (Fig. 7C, lanes 1-8). In contrast, a deleted promoter (170/18) containing start site I but not start site II is not activated by CBF in vitro (lanes 9 and 10). Similarly, analysis of promoter activity during the cell cycle showed that promoters 170/+29, 170/+16, and 170/+4, but not 170/18, are activated during the cell cycle (Table II; data not shown for the 170/+29 and 170/+16 promoters). Altogether, these results indicate that the nucleotide sequences in the start site II region of the topo II promoter are essential for both the CBF-dependent transcription activation in vitro and the activation of the promoter during the cell cycle in fibroblast cells.

View larger version (37K): [in this window] [in a new window]   Fig. 7.   The role of the start site II region of the topo II promoter in the CBF-dependent transcription activation in vitro. A and B, schematic diagram of the reporter constructs containing deletions in the 3' end region (A) and nucleotide substitution mutations in the start site II region (B) of the topo II promoter. C and D, each of the deletions and the substitution mutants are analyzed in the in vitro transcription reaction with or without the addition of recombinant full-length CBF (rCBF) as described in Fig. 6B. Arrows show the transcripts from each start site for each of the constructs.

View this table: [in this window] [in a new window]   Table II Activity of various mutants in the transcription start site II region of the topo II promoter during the cell cycle Relative promoter activity during the cell cycle for each construct was determined as described in Table I. The promoter activity in the unsynchronized fibroblast cells was determined 48 h after transient transfection. The fold increase of each promoter at 24 h compared with its activity at 0 h after serum stimulation is shown.

For further analysis, several mutants containing nucleotide substitutions in the start site II region were generated in the 170/+4 promoter (Fig. 7B). These mutants are also analyzed by the in vitro transcription reaction assay and also during the cell cycle in fibroblast cells. This shows that similarly to the 170/+4 promoter, the mutant promoters m15/13, m11/9, and m8/6, which are transcribed from both start sites I and II, are activated by CBF in vitro (Fig. 7D, lanes 1-8). In contrast, the mutant promoters m5/3, m2/+1, and m+2/+4, which are transcribed only from start site I, are not activated by CBF in vitro (lanes 9-14). Surprisingly, analysis of promoter activity during the cell cycle shows that the mutant promoters m5/3, m2/+1, and m+2/+4, but not m-11/9 or m8/6, are activated during the cell cycle (Table II). These results demonstrate that the nucleotide sequences between 5 to +4, which are important for CBF-mediated transcription activation in vitro, are dispensable for cell cycle-dependent transcription activation in fibroblast cells. Furthermore, this study identifies a promoter element between 11 and 6 that is required for the cell cycle-dependent activation but not for CBF-dependent activation in vitro. It is important to note that although the m5/3, m2/+1, and m+2/+4 promoters are not activated by CBF in vitro, mutations of the CBF binding sites in these promoters abolish the cell cycle-dependent activation in fibroblast cells (data not shown). We interpret the results as indicating that CBF binding, but not conventional CBF transcriptional activation domains, is required for the cell cycle-dependent activation of the topo II promoter in fibroblast cells.

Evaluation of CBF Binding and CBF-dependent Transcription Activation in the Topo II Promoter Activity in Fibroblast Cells-- It is possible that mutations of the CBF binding sites in the topo II promoter might inhibit binding of other transcription factors that could result in abrogation of cell cycle-dependent activation in fibroblasts. To determine whether CBF-mediated transcription activation plays a role in the topo II promoter activity in fibroblast cells, we have expressed a truncated form of CBF-B lacking a glutamine-rich activation domain under control of a tetracycline-inducible promoter (Fig. 8A). Nuclear extracts prepared from fibroblast cells expressing truncated CBF-B were used in a DNA binding experiment with the labeled w1 oligonucleotide. As a control, nuclear extracts were also prepared from cells transfected with either vector alone or vector expressing the full-length CBF-B subunit. The DNA binding of the control nuclear extracts resulted in generation of a specific CBF-DNA complex (Fig. 8B, first and third lanes). In contrast, DNA binding of nuclear extracts containing the truncated CBF-B forms an additional faster moving complex (second lane). The addition of anti-CBF-B antibodies in the DNA binding reaction containing truncated CBF-B resulted in concomitant inhibition of the faster mobility complex and formation of a supershift complex. We interpret this result as indicating that the faster mobility complex is formed due to the binding of truncated CBF-B together with cellular CBF-A/CBF-C. When truncated CBF-B is cotransfected with the 4CCAAT topo II promoter in fibroblast cells, no change of topo II promoter activity was observed (Fig. 8C). In contrast, cotransfection of the truncated CBF-B with the collagen promoter (FC1) resulted in an almost 4-fold inhibition of promoter activity. This result indicates that the transactivation region of CBF-B is required for activity of the collagen promoter but is dispensable for activity of the topo II promoter in fibroblast cells.

View larger version (25K): [in this window] [in a new window]   Fig. 8.   The effect of the expression of a truncated CBF-B lacking an activation domain in fibroblast cells. A, schematic of a truncated CBF-B (Bdbd) containing 240-336 amino acids (aa) of the CBF-B subunit, which was constructed as a fusion with FLAG epitope under control of the tetracycline-inducible promoter (TRE PminCMV). B, DNA binding of CBF present in nuclear extracts prepared from fibroblast cells transfected with either expression vectors without insert (lane 1) or with Bdbd (lane 2) or with full-length CBF-B (lane 3). The DNA binding was performed with labeled w3 oligonucleotide and then assayed using the electrophoretic mobility shift assay. The DNA binding reaction in lane 4 contains nuclear extracts from Bdbd fibroblast cells, which was first incubated with anti-CBF-B antibodies (Ab) and then assayed for DNA binding. C, activity of the 4CCAAT topo II and the FC1 collagen promoters in fibroblast cells after expression of Bdbd. The promoter activity of each construct was determined after transient transfection in fibroblast cells together with either the expression vector alone (pTRE-FLAG) or with the vector expressing Bdbd polypeptide (pTRE-FLAGBdbd). For each construct, the average relative value of three experiments with S.D. is shown.

To address whether CBF binding to the topo II promoter is required for transcription activation during the cell cycle, we have generated an allele-specific mutant of CBF-B, Bmt307 (Fig. 9A). Previous studies of HAP2, the yeast homologue of CBF-B, showed that a mutant HAP2 containing mutation of a lysine to leucine in the DNA binding domain interacts with a high affinity to a mutant CBF site with a CCAAC motif (20). The wild-type CBF interacts very weakly to DNA containing the CCAAC motif. The Bmt307 mutant was generated by a similar mutation from lysine to leucine at the corresponding position in the DNA binding domain of CBF-B. To examine DNA binding activity in vitro, Bmt307 was expressed in bacteria and purified as described earlier for the wild-type CBF polypeptide (17). As expected, purified Bmt307 together with the wild-type CBF-A and CBF-C interacts strongly with DNA containing CCAAC but not DNA containing the CCAAT motif (data not shown).

View larger version (27K): [in this window] [in a new window]   Fig. 9.   The activity of a mutant topo II promoter in the presence of an allele-specific CBF-B mutant. A, schematic of a mutant CBF-B, Bmt307, containing single amino acid substitutions from arginine to leucine at position 307, which was constructed as a fusion with the FLAG epitope under control of the tetracycline-inducible promoter. B, schematic diagram of a mutant topo II promoter, W3mt, in which the ATTGG (CCAAT) motif at 66 position was mutated to GTTGG (CCAAC). The CCAAT motifs at 26, 46, and 117 positions were mutated to CCAAA similar to the W3 topo II promoter. C, DNA binding of CBF containing Bmt307. The wild-type CBF-B (Bwt) and the mutant Bmt307 were first expressed after transfection in fibroblast cells, and then each of the polypeptides was purified from the fibroblast cell extracts using anti-FLAG antibody affinity resin. Each of the polypeptides was copurified with the cellular CBF-A/CBF-C complex and used in a DNA binding assay with a labeled promoter fragment of W3mt (lanes 2 and 3). The DNA binding reaction in lane 4 contained anti-CBF-B antibody, which was first incubated with the Bmt307 complex and then assayed for DNA binding. D, activity of the W3mt promoter in the presence of Bmt307 in fibroblast cells. The promoter activity was measured with or without expression of the mutant CBF-B, Bmt307, both in the unsynchronized cells and at different time points of serum stimulation of the synchronized cells. Analysis of promoter activity during serum starvation and stimulation was done as described in Table I. The activity of the W3 promoter is shown for comparison. The fold activation of the W3 promoter during the cell cycle is different from that shown in Table I and is possibly due to a different fibroblast cell line.

To test the transcription activity of Bmt307, the mutant was expressed under the control of the tetracycline-inducible promoter in fibroblast cells. We have also generated a mutant W3 promoter (W3mt) containing a single nucleotide substitution mutation of ATTGG (CCAAT) to GTTGG (CCAAC) (Fig. 9B). To examine DNA binding of Bmt307 to the W3mt promoter, we purified the Bmt307 polypeptide that was expressed as a fusion protein with FLAG epitope from fibroblast cell extracts using anti-FLAG immunoaffinity resin. This purification method allows copurification of cellular CBF-A and CBF-C together with the Bmt307 polypeptide. The addition of the purified Bmt307 complex in a DNA binding reaction containing the labeled W3mt promoter results in formation of a DNA-protein complex that can be supershifted with anti-CBF-B antibodies (Fig. 9C, lanes 3 and 4). When the DNA binding reaction is performed with a wild-type CBF-B complex that was expressed and purified similar to Bmt307, a DNA-protein complex is formed that has similar mobility but much weaker intensity than that with Bmt307 (lane 2). This result indicates that the Bmt307 complex interacts with W3mt with much higher affinity than does the wild-type CBF-B complex. The promoter activity of W3mt is about 3.7-fold lower than that of the W3 promoter in unsynchronized fibroblasts cells, and unlike the W3 promoter, no increase in promoter activity of W3mt is observed during the cell cycle (Fig. 9D). Interestingly, the expression of Bmt307 results in a 2.7-fold increase in the activity of the W3mt promoter in the unsynchronized fibroblasts and in a 2.3-fold induction of W3mt promoter during the cell cycle. We interpret this result as indicating that the expression of Bmt307 directs formation of a high affinity CBF-DNA complex in the W3mt promoter that results in the induction of W3mt promoter during the cell cycle. Altogether, these results strongly indicate that CBF binding to the topo II promoter is essential for activation of the promoter during the cell cycle in fibroblast cells.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Our results demonstrate that among the multiple CBF binding sites in the topo II promoter, a minimum of a single CBF site in the inverted orientation is required for transcription activation during the cell cycle. The fact that a collagen promoter containing multiple CBF binding sites in the inverted orientation is not activated during the cell cycle is strongly indicative that the role of CBF in transcription activation during the cell cycle is specific to the topo II promoter. Analysis of DNA binding shows that CBF interacts very similarly, irrespective of orientation of the CBF binding sites in the topo II promoter. We speculate that CBF-dependent transcription activation of the topo II promoter is regulated during the cell cycle, and such activation might be specific with respect to the orientation of the CBF binding site. To test this possibility in this study, we have analyzed in more detail the CBF-dependent activation of the topo II promoter in vitro. In this regard, our previous study showed that recombinant CBF activated transcription of a nucleosomal topo II promoter template in vitro (13). Our present study shows that inhibition of CBF binding to the topo II promoter either by mutations in the CBF sites or by a dominant negative CBF-B mutant results in repression of transcription of only one (start site II) of the three start sites present in the topo II promoter. This result is consistent with the observation that recombinant CBF activates transcription of the topo II promoter in vitro only through start site II but not through the other start sites. These results indicate that CBF-mediated transcription activation only regulates part of the topo II promoter activity. This study prompted further analysis of the CBF-dependent start site II. To our surprise, this shows that specific mutations of start site II that completely inhibit CBF-dependent activation in vitro do not affect cell cycle-dependent activation in fibroblast cells. It is important to note that although the mutant promoters with the start site II mutation are not activated by CBF in vitro, the CBF binding sites in these promoters are still required for the cell cycle-dependent activation in fibroblast cells.

To date, the mechanism of CBF-dependent transcription in vivo in a mammalian cell is not clearly understood. Two factors that have restricted our study of CBF in mammalian cells are 1) no mammalian cell line lacking CBF has been isolated, and 2) because of multiple subunits, reconstitution of recombinant CBF in a mammalian cell cannot be done easily. Our earlier studies demonstrated that the association between CBF-B and CBF-A/CBF-C heterodimer is reversible. Thus, when a dominant negative CBF-B mutant was expressed in fibroblast cells, it formed an inactive complex with the cellular CBF-A/CBF-C by displacing the cellular CBF-B subunit from the CBF protein (7). Consistent with the previous studies, our present results show that a truncated CBF-B lacking a glutamine-rich activation domain but containing a subunit interaction and DNA binding domain also interacts with cellular CBF-A/CBF-C, which binds to the CCAAT motif. This allowed us to perform an assay to determine whether the activation domain of CBF-B mediates the transcription activation of the CBF-dependent promoter in fibroblast cells. Interestingly, this experiment shows that the activation domain of CBF-B does not play any role in transcription activity of the topo II promoter but it is required for the transcription activity of the collagen promoter in fibroblast cells. This result is in contrast with the in vitro transcription analysis that showed that the activation domain of CBF-B mediated about 60% of the CBF-dependent transcription activation of the topo II promoter. Altogether, these results support a model that the transcription activity of the topo II promoter in fibroblasts cells is not dependent on the transcription activation function of CBF that is mediated by the two glutamine-rich domains of CBF subunits.

Expression of an allele-specific CBF-B also results in formation of a complex with cellular CBF-A/CBF-C that binds high affinity to a CCAAC motif DNA. Our results show that mutation of CCAAT to CCAAC in the topo II promoter decreases promoter activity and inhibits the cell cycle-dependent activation. Interestingly, expression of the allele-specific CBF-B mutant increases promoter activity of the mutant topo II promoter and restores activation of the mutant promoter during the cell cycle in fibroblast cells. Thus, this observation is in good agreement with a conclusion that CBF binding to the topo II promoter is essential for the cell cycle-dependent activation of the promoter in fibroblast cells.

It is not clear why CBF binding but not CBF-mediated transcription activation is utilized for the cell cycle-dependent activation of the topo II promoter. We speculate that because the activity of CBF is present throughout all stages of the cell cycle, the transcription activation function of CBF should be regulated to avoid constitutive activation of the topo II promoter during the cell cycle. Indeed, the data in Table II indicate that the basal promoter element at start site I of the topo II promoter that is not responsive to CBF-dependent activation in vitro is utilized in the cell cycle-dependent transcription. This indicates that the activation function of CBF is differentially regulated by the basal promoter elements present in the topo II promoter. Because none of the basal promoter elements of the topo II promoter contains the TATA motif, it is likely that the differential function of the basal elements is due to binding of different initiator proteins. It is also possible that the specific location and architecture of the basal element in the promoter may play a role in mediating CBF-dependent transcription activation. Recent studies of two adenovirus promoters showed that the basal promoter sequences surrounding the TATA and the initiator elements play an important role in the responsiveness of the promoter toward the upstream activator (21). This suggests that besides an assembly site for the general transcription factor, the basal promoter elements might also contribute a physiologic response that leads to temporal transcription activation. Our study also underscores that the basal promoter structure plays an important role in CBF-dependent transcription activation. The fact that the majority of basal promoter elements of topo II are not responsive to direct transcription activation by CBF suggests that this may be a mechanism by which the activity of the topo II promoter is dependent only on CBF binding but not on the activation function of CBF. It is not known whether CBF interacted in vivo to the topo II promoter during all stages of the cell cycle. However, the in vivo interaction of CBF to the cyclin B1 promoter, which is also activated during the late S and G₂/M phases of the cell cycle and whose activity is also dependent on the CBF binding sites in the promoter, was recently studied using the chromatin immunoprecipitation method (12). The result was that CBF interacts with this promoter during all stages of the cell cycle in vivo irrespective of activation of the promoter, implying that the activity of the cyclin B1 promoter during the cell cycle is regulated by the CBF binding but not by the activation function of CBF.

It is possible that binding of CBF to the topo II promoter results in formation of an active promoter structure which may allow binding of other transcription factors that leads to activation of the promoter during the cell cycle. Indeed, our recent study showed that binding of CBF to the nucleosomal topo II promoter disrupts the regular nucleosomal structure not only in the promoter region containing CBF binding sites but also in a large portion of the downstream promoter region that does not contain the CBF binding site (13). Interestingly, binding of a truncated CBF lacking the two glutamine-rich activation domains also similarly disrupted regular nucleosomal structure in the topo II promoter, indicating that the CBF-mediated nucleosomal disruption does not require the activation domains of CBF subunits. Altogether, the results of our previous studies strongly indicated that only CBF binding, not CBF-mediated transcription activation to the topo II promoter, is essential for disruption of the nucleosomal structure in the topo II promoter. Although a direct relationship between CBF-mediated nucleosomal disruption and transcription activation of the topo II promoter was not established, comparison of the in vitro transcription activity of the nucleosomal 4CCAAT and M1-4 promoters indicated that mutation of the CBF binding sites results in inhibition of transcription through all three start sites in the topo II promoter (13). This indicated that in a chromatin promoter template, binding of CBF to the topo II promoter is required to maintain transcription from all the start sites in the promoter irrespective of whether the start sites are activated by CBF. This implies that CBF-mediated nucleosomal disruption is a predominant mechanism by which CBF controls transcription of the topo II promoter.

Our present study demonstrates that interaction of CBF to the binding site located in inverted but not in direct orientation in the topo II promoter leads activation of the promoter during the cell cycle. The orientation-specific CBF function is possibly due to asymmetric CBF binding to the CCAAT motif. Our previous analysis of CBF-DNA interaction using the photocross-linking method showed that all the three CBF subunits interact at the 5' side of the CCAAT motif, whereas only CBF-B and CBF-C, but not CBF-A, interact at the 3' side (16). This finding indicates that the contact between CBF and the CCAAT motif is asymmetric. In this regard, CBF-mediated transcription activation in vitro, which is mediated by the two glutamine-rich segments of CBF subunits, is not dependent on the orientation of the CBF binding site, suggesting that the orientation-specific CBF function mediates by regions outside the activation domains of CBF subunits, possibly through the conserved segments of the CBF subunits involved to form the CBF-DNA complex. It is also possible that the binding of CBF in the inverted orientation may differentially disrupt the nucleosomal structure in the topo II promoter. Indeed, our analysis of CBF-mediated nucleosomal disruption showed that CBF binding results in strong disruption, more in the downstream than in the upstream region of the topo II promoter (13). In the present study, mutational analysis in the downstream promoter region showed that a GC box element is required for the cell cycle-dependent activation of the topo II promoter. This supports the hypothesis that the binding of CBF in the inverted orientation may allow possible cross-talk between CBF and the GC box binding factor, which may lead activation of the topo II promoter during the cell cycle.

In summary, our study revealed that CBF regulates transcription of the topo II promoter by two separate mechanisms. The first one requires only CBF binding without action of the CBF activation domains; the second utilizes CBF binding followed by transcription activation mediated by the glutamine-rich domains of CBF subunits. Our study demonstrated the first mechanism by which CBF regulates activation of the topo II promoter during the cell cycle and suggests that it is possibly a common mechanism by which CBF regulates transcription of other cell cycle-regulated genes.

	ACKNOWLEDGEMENTS

DNA sequencing of plasmid constructs was performed at The University of Texas M. D. Anderson Cancer Center Core Sequencing Facility. DNA sequencing and the flow cytometry facilities of the institution were supported by NCI, National Institutes of Health Grant CA16672. We acknowledge Françoise Coustry for providing allele-specific CBF-B mutant construct. We thank Vickie Williams for editorial assistance.

	FOOTNOTES

^* This work was supported by National Institutes of Health Grant R01 AR46264 (to S. N. M.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

These authors contributed equally to this study.

^§ To whom correspondence should be addressed: Dept. of Molecular Genetics, Box 11, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Blvd., Houston, TX 77030. Tel.: 713-792-8943; Fax: 713-794-4295; E-mail: smaity@mdanderson.org.

Published, JBC Papers in Press, July 30, 2002, DOI 10.1074/jbc.M205985200

	ABBREVIATIONS

The abbreviations used are: topo II, topoisomerase II; CBF, CCAAT binding factor.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES