INTRODUCTION

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E2F plays an important role in the regulation of genes required for cell cycle progression and entry into the S phase (reviewed in references 2, 12, and 19). The mammalian E2F protein family contains at least six members (E2F-1 to -6) that can associate with the two members of the DP protein family (DP-1 and DP-2), resulting in multiple heterodimers capable of binding E2F sequences. Control of E2F activity during the cell cycle is complex: expression of E2F-1 to -3 is low in quiescent cells and increases during growth stimulation, while E2F-4 and E2F-5 accumulate in quiescent cells or during differentiation (2, 12, 19). Various E2F-DP heterodimers also preferentially bind different members of the pRb tumor suppressor protein family: E2F-1 to -3 tend to associate with pRB, while E2F-4 and -5 are most often found in complexes with p107 and p130 (12). E2F-pRb family member complexes are dynamic during the cell cycle, with association and dissociation regulated by cyclin-dependent and other protein kinases. Moreover, members of the E2F and pRb families have been reported to be controlled by nuclear localization, phosphorylation, protein stability, and transcriptional regulation by E2F itself (reviewed in references 2, 12, and 19).

Of interest is the functional relationship between specific E2F-DP-pRb family member complexes and the regulation of specific E2F-dependent genes. In some instances, E2F appears more important for repression of gene expression during early G₁ than for activation after the G₁ restriction point (reviewed in references 2, 12, and 19). In contrast, E2F appears to play a role in both repression and activation of the major dhfr gene promoter (14). The dhfr major promoter is highly conserved in humans, mice, and hamsters; the core promoters lack obvious TATA and CAAT sequences but contain an 18-bp conserved sequence at the transcription start site that includes two overlapping and inverted E2F sites (reviewed in references 40 and 41). Upstream of the overlapping E2F sites are multiple binding sites for Sp1, a ubiquitous transcription factor found in association with E2F sites in several promoters that are regulated in late G₁ (reviewed in reference 2). Transcription of dhfr is low in quiescent cells and is activated after pRb protein family phosphorylation during growth stimulation (4, 15, 23, 42, 51, 52). In some settings the overlapping dhfr E2F sites alone are sufficient for induction of transcription after the G₁ restriction point (42). Other studies suggest that the reiterated Sp1 sites, which are required for selection of transcriptional start sites and which regulate basal levels of expression (reviewed in references 41 and 42), contribute to activation of dhfr during growth stimulation (3, 23, 36). Mutational analysis of the E2F-1 activation domain indicates that E2F-dependent activation of dhfr transcription is correlated with the binding of CREB binding protein (CBP), a transcriptional coactivator with histone acetylase activity (14). Proper control of dhfr requires the E2F sites to be precisely located relative to the Sp1 sites (15), suggesting that stereospecific interactions between proteins tethered to DNA through the Sp1 and E2F sites regulate dhfr transcription.

Cooperation between Sp1 and E2F in regulation of dhfr gene expression also is supported by observations that members of these two protein families interact with one another. For example, Sp1 has been reported to interact directly with p107 (9), pRb (35), and E2F-1 to -3 (24, 26, 38), as well as pRb binding protein histone deacetylase 1 (HDAC1) (11). The role of pRb in the expression of dhfr, however, has proven difficult to assess. Cotransfection of pRb expression plasmids with dhfr reporter genes stimulated expression in some studies (25, 47, 48), perhaps by titration of a negative repressor (8), while in others pRb repressed dhfr promoter activity in a manner dependent on the overlapping E2F sites (10). In serum-stimulated mouse embryo fibroblasts lacking various combinations of pRb family members, dhfr was regulated normally in cells lacking pRb and was up-regulated slightly earlier in G₁ in cells lacking p130 and p107 (21), suggesting that p130 and p107 function in repression of dhfr in a manner that is not entirely redundant with that in which pRb functions.

Here we have examined the effects of trichostatin A (TSA), a nonreversible inhibitor of HDAC activity (46), on activation and repression of the dhfr major promoter under various growth conditions. Our results support a dynamic model in which Sp1-HDAC1-pRb and E2F-4-DP-1-p130 complexes cooperate to establish stable repression of dhfr gene expression in quiescent cells.

	MATERIALS AND METHODS

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Cell culture and cell synchrony. CHOC 400 and U2OS cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS). For arrest in G₀/G₁ by serum deprivation, 3 × 10⁵ cells were plated in 60-mm-diameter culture dishes. After 24 h cells were washed and incubated in DMEM with 0.2% FBS for 72 h. Cells were stimulated to reenter the cell cycle with DMEM containing 10% FBS. As indicated in the Figure legends, 100nM TSA or 400 µM mimosine [-N(3-hydroxy-4-pyridone)--aminoproprionic acid] or both were added in culture medium. Cell cycle progression was monitored by flow cytometry as described previously (52). Cell culture supplies were from Life Technologies; mimosine and TSA were purchased from Sigma.

Plasmids. The [E2F]×4 luciferase reporter, which contains four E2F sites with the sequence TTTCGCGC and the TATA box from the adenovirus E1b gene, was generated by subcloning the promoter from [E2F]×4 chloramphenical acetyltransferase (20) into reporter plasmid pGL3-Basic (Promega). [E2F]×4 luciferase was a gift from F. Dick and N. Dyson (MGH Cancer Center). The dihydrofolate reductase (DHFR)-GAL4 reporter (14) and the GAL4-CBP constructs were a gift gift from C. Fry and P. Farnham. GAL4-pRb and GAL4-p130 plasmids (32) were a gift from J. Nevins.

Transfection and reporter gene assays. CHOC 400 or U2OS cells were plated at 3 × 10⁵ cells per 60 mm-diameter culture dish in DMEM with 10% FBS. After 16 h, cells were fed fresh medium and transfected with a total of 7.2 µg of DNA containing 2.4 µg of reporter plasmid and 4.8 µg of salmon sperm carrier DNA by calcium phosphate coprecipitation as described previously (29, 30). Cells were washed three times with phosphate-buffered saline 20 h after transfection to remove the DNA precipitates. Except for experiments with DHFR-GAL4, cotransfection with pCMV-GFP was used to evaluate transfection efficiency, which routinely exceeded 35% in both CHOC 400 and U2OS cells. For experiments with DHFR-GAL4, the cells were cotransfected with pCVM--gal as the control and -galactosidase activity was measured as described previously (29). In synchronization experiments, transfected cells were washed after 20 h and arrested in early G₁ by serum deprivation by incubation in DMEM with 0.2% FBS for 60 to 72 h. Serum-starved transfected cells were stimulated to reenter the cell cycle by incubation in DMEM with 10% FBS.

Northern blot analysis. Total RNA was isolated with Trizol reagent as described by the manufacturer (Life Technologies). Total RNA (10 µg per sample) was resolved by electrophoresis on 1% agarose gels containing formaldehyde, transferred to Hybond-N membranes (Amersham), and hybridized with ³²P-labeled probes derived from DHFR and/or GAPDH (glyceraldehyde-3-phosphate dehydrogenase) cDNAs. After the washing, hybridization signals were first visualized by exposure to Kodak X-Omat film and then quantified with a Bio-Rad GS250 molecular imager.

Immunoblotting. Cells were scraped from plates into Eppendorf tubes, lysed in 2 × sodium dodecyl sulfate (SDS) sample buffer (4% SDS, 20% glycerol, 200 mM dithiothreitol, 120 mM Tris-HCl [pH 6.8], and 0.002% bromphenol blue), and heated to 95°C for 10 min, and 10 to 20 µg of protein sample was resolved on 6, 8, or 12% denaturing polyacrylamide gels, depending on the size of the protein to be detected. Total protein was determined using Bio-Rad DC protein assays as described by the manufacturer. After transfer to Immobilon membranes (Millipore), immunoblots were incubated sequentially with primary antibodies and horseradish peroxidase-coupled secondary antibodies as described previously (30). Signals were generated by chemiluminescence with ECL substrate (Amersham) and visualized by exposure to Kodak X-Omat film.

Immunoprecipitations (IPs). Nuclear extracts were prepared as described for electrophoretic gel mobility shift assays (44). Extract from approximately 3 × 10⁶ cells (100 µl containing 200 µg of protein) was diluted to 1 ml in whole-cell extraction buffer (25 mM HEPES [pH 7.4), 1 mM MgCl₂, 100 mM KCl, 0.1 mM EDTA, 10% glycerol, 1 mM NaF, 2 µg of phenylmethylsulfonyl fluoride per ml, 0.1 µg of aprotinin and leupeptin per ml, and 1 mM dithiothreitol and incubated with 3 µl of antibody for 2 h at 4°C with gentle shaking. After addition of 50 µl of protein A-agarose beads (Santa Cruz Biotechnology), the suspension was incubated another 2 h at 4°C. The beads were pelleted by centrifugation, washed three times with 1.5 ml of cold extraction buffer, and resuspended in 50 µl of 2 × SDS sample buffer. After the suspension was heated to 95°C for 10 min, 20-µl samples were resolved on denaturing SDS-polyacrylamide gels, transferred to membranes, and probed for the presence of specific proteins by immunoblotting as described above.

Antibodies. Polyclonal rabbit antibodies to E2F-4 (C-108), pRb (C-15), p107 (C-18), and p130 (C-18) and p300 were purchased from Santa Cruz Biotechnology. Polyclonal rabbit antibodies to Sp1 were purchased from Geneka and Santa Cruz Biotechnology (pep2). Rabbit polyclonal antibodies to HDACI were purchased from Upstate Biotechnology and Santa Cruz Biotechnology (H-51).

Electrophoretic mobility shift assays. Gel shift assays were performed with nuclear extracts prepared as described above and a ³²P-labeled double-stranded DNA oligonucleotide probe representing the overlapping dhfr E2F sites as described previously (51, 52).

	RESULTS

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Effects of TSA on expression of the endogenous CHOC 400 dhfr genes. The promoter for the cyclin-dependent kinase inhibitor p21, which contains five Sp1 binding sites upstream of a TATA box, is markedly induced by TSA and other inhibitors of HDACs (7, 34, 39, 43). The dhfr gene promoter is similar to that of p21 but contains overlapping and inverted E2F sites downstream of the Sp1 sites rather than a TATA box. In some studies, inhibitors of HDACs have been reported to induce expression of dhfr (14), while in others HDAC inhibitors had no effect (6, 39). These differences may be due to cell type, growth and treatment conditions, or indirect effects of TSA on other processes.

View larger version (32K): [in this window] [in a new window]   FIG. 1.   Cell cycle kinetics and dhfr gene expression in the presence of mimosine and TSA. CHOC 400 cells were arrested in G0/G1 by incubation in 0.2% FBS for 60 h (zero time). Cells were then induced to reenter the cell cycle by the addition of fresh medium with 10% FBS (NT), 10% FBS plus TSA (TSA), 10% FBS plus mimosine (MIM), or 10% FBS plus TSA and mimosine (MIM + TSA). (A) Cultures were harvested at 4-h intervals, and the percentages of cells in S phase were determined by flow cytometry. (B) Total RNA was prepared from replicate cultures and probed simultaneously for dhfr and gapdh mRNA by Northern blotting. (C) Northern blot hybridization signals from two replicate experiments were quantified with a phosphorimaging system and are expressed as average increases in expression relative to that at time zero.

Phosphorylation of pRb family members is delayed by TSA. Because E2F is regulated by its association with pRb, p130, and p107, we examined the effects of TSA on the expression and phosphorylation of pRb family members and on E2F DNA binding activity in serum stimulated cells. In serum-starved CHOC 400 cells, phosphorylation of pRb family members occurred at 12 to 16 h after addition of medium with 10% FBS (Fig. 2A to C). After phosphorylation of pRb family members, cyclin-dependent kinase inhibitor p21 decreased in abundance (Fig. 2D). Mimosine blocked the phosphorylation of all three pRb family members completely, either in the presence or absence of TSA, likely by inducing expression of p21 (Fig. 2D). Thus, phosphorylation of pRb family members appears to be an obligate step for induction of dhfr in serum-stimulated cells.

View larger version (62K): [in this window] [in a new window]   FIG. 2.   Effect of TSA and mimosine on phosphorylation of pRb family members. CHOC 400 cells were arrested in G0/G1 by incubation in low serum for 60 h and then were stimulated to reenter the cell cycle in medium with 10% FBS, with or without TSA and/or mimosine (MIM) as for Fig. 1. Cultures were harvested at 4-h intervals, total cellular protein was resolved by electrophoresis under denaturing conditions, and the abundances and/or phosphorylation states of pRB (A), p107 (B), p130 (C), and p21 (D) were examined by immunoblotting. The phosphorylation patterns of pRb family members suggests that the G1 restriction point occurs 12 to 16 h after the addition of 10% FBS.

View larger version (94K): [in this window] [in a new window]   FIG. 3.   TSA does not block the dissolution of E2F-pRb and E2F-p130 complexes after serum stimulation. CHOC 400 cells were arrested in G0/G1 and induced to reenter the cell cycle in the presence of 10% FBS (A), 10% FBS plus TSA (B), 10% FBS plus mimosine (C), or 10% FBS plus aphidicolin (D). Nuclear extracts were prepared at 4-h intervals and examined for E2F DNA binding activity by gel mobility shift assays using the overlapping E2F sites from the dhfr promoter as a probe. Brackets, E2F-pRb family member complexes; arrows, E2F not bound to pRb, p130, or p107 (free E2F).

Differential sensitivity of the dhfr Sp1 and E2F sites to TSA. The insensitivity of the dhfr promoter to TSA suggested that the overlapping E2F sites may mask the sensitivity of the Sp1 sites to inhibitors of HDACs. To investigate the role of E2F and Sp1 sites in the sensitivity of the dhfr promoter to TSA, we used a series of model promoters linked to luciferase reporter genes. Plasmids containing the WT promoter (DHFR-WT), a reconstructed WT promoter (DHFR 2 Site), the four upstream Sp1 sites only (DHFR-SP1), or four TTTCGCGC E2F sites only ([E2F]×4) were transfected into CHOC 400 cells, and luciferase activity was measured after 20 h (Fig. 4). Under standard growth conditions constructs containing the reiterated dhfr Sp1 sites were about 10-fold more active for luciferase expression than the [E2F]×4 reporter (compare Fig. 4A, C, and D to B), supporting work that indicates that the Sp1 sites are critical for initiation of transcription of dhfr. Exposure of the transfected cells to 100 nM TSA for 20 h after the removal of DNA resulted in a 10-fold induction of luciferase activity from reporter constructs containing the DHFR Sp1 sites but had no effect on the [E2F]×4 construct (Fig. 4B).

View larger version (34K): [in this window] [in a new window]   FIG. 4.   Stimulation of dhfr promoter activity by TSA requires Sp1 binding sites. CHOC 400 cells were transfected with the indicated luciferase reporter constructs with or without cotransfection of pCMV-E2F-1 and pCMV-DP-1. Twelve hours after the addition of DNA, cultures were washed and incubated for 20 h in medium with 10% FBS with or without TSA as indicated. Luciferase activity was measured in cell extracts and is expressed in relative light units (RLU) (103) as an average of duplicate transfection samples. Error bars, standard errors of the means.

View larger version (24K): [in this window] [in a new window]   FIG. 5.   Induction of dhfr reporter genes by TSA during serum stimulation requires Sp1 sites. CHOC 400 cells were transfected with the indicated reporter constructs. After 20 h the cells were washed free of DNA and incubated in medium containing 0.2% FBS for 60 h. The cells then were treated with medium containing 10% FBS (NT) or 10% FBS plus TSA (TSA). At the indicated times duplicate plates were harvested and extracts were assayed for luciferase activity. Relative light units (RLU) (103) for the averages of duplicate samples are presented.

Interactions of Sp1 with HDAC1 and pRb during the cell cycle. Here the isolated dhfr Sp1 sites were markedly responsive to TSA, yet the endogenous dhfr gene and the [E2F]×4 reporter were not, suggesting that E2F may mask the effect of TSA on gene regulation by Sp1. In addition, conditions that blocked or delayed phosphorylation of pRb family members impaired dhfr gene expression (Fig. 1 to 3), while in certain other studies cotransfection of expression vectors for pRb stimulated expression from dhfr reporter plasmids (8, 25, 35). In an attempt to resolve the role of pRb and other regulatory factors in dhfr gene expression, we examined the interactions between endogenous Sp1, E2F, pRb family members, and HDAC1 during the cell cycle by IP experiments.

View larger version (50K): [in this window] [in a new window]   FIG. 6.   HDAC1 binds Sp1 and pRb but not p130 in Chinese hamster cell extracts. IPs were performed with nuclear extracts prepared from log phase CHOC 400 cells (zero time) or CHOC 400 cells incubated in 0.2% FBS for 48 h. (A) Antibodies (Ab) to the indicated proteins were used in the primary IP reaction, and the immunoprecipitates were probed for Sp1. Nonspecific rabbit serum (NS) was used as the control. (B) Antibodies to p130, E2F-4, or E2F-5 were used for the primary IP, and the immunoprecipitates were then probed by immunoblotting for p130. (C) Nuclear extracts from cells incubated in 0.2% FBS for 48 h were subjected to IP with the indicated antibodies, and the immunoprecipitates were then probed for HDAC1.

Binding of pRb but not HDAC1 to Sp1 is regulated during the cell cycle. The interactions of endogenous Sp1 with pRb proteins were examined during transition from G₀/G₁ to the S phase, when dhfr transcription is activated. Nuclear extracts were prepared from serum-starved cells or cells stimulated with serum for various periods of time, Sp1 was immunoprecipitated, and the proteins recovered in the Sp1 IPs were examined by immunoblotting. Immunoblotting of total-cell lysates and nuclear extract was used to assess the recovery, phosphorylation state, and cellular distribution of each protein during the IP experiments.

View larger version (73K): [in this window] [in a new window]   FIG. 7.   A cell cycle-regulated interaction of pRb with Sp1. Total cell lysate (total) and nuclear extract (NE) from serum-starved (zero time) cells or cells stimulated with 10% FBS for 4 to 24 h were probed for the presence of Sp1 (A), pRb (B), HDAC1 (C), and p130 (D) by immunoblotting (top two sections). Sp1 was immunoprecipitated from nuclear extract, and the IP reaction mixtures were probed for Sp1, pRb, HDAC1, and p130 (bottom section) by immunoblotting. In panels B and D the arrows indicate the hypophosphorylated forms of pRb and p130, respectively. Note that only hypophosphorylated pRb is found in nuclear extract or in association with Sp1. IgG, immunoglobulin G.

View larger version (61K): [in this window] [in a new window]   FIG. 8.   Effects of TSA on the interaction of pRb and HDAC1 with Sp1. (A) Nuclear extracts from serum-stimulated CHOC 400 cells treated with TSA were probed for Sp1 and pRb. (B) Sp1 was immunoprecipitated from nuclear extracts from serum-stimulated CHOC 400 cells treated with TSA, and the immunoprecipitates were probed for Sp1, pRb, and HDAC1. Note that TSA delays the loss of pRb from nuclear extract after serum stimulation (compare panel B to Fig. 6B). (C) Nuclear extracts from CHOC 400 cells stimulated with 10% FBS (FBS) or 10% FBS and TSA (TSA) were probed for p300 by immunoblotting. (D) TSA inhibits phosphorylation of pRb in serum-stimulated U2OS cells. Serum-starved U2OS cells were incubated with medium containing 10% FBS or medium with 10% FBS and TSA. Nuclear extracts were prepared and probed for pRb by immunoblotting. (E) Inhibition of pRb phosphorylation by TSA and delayed dissociation of pRb from Sp1 in U2OS cells. Sp1 was immunoprecipitated from nuclear extracts prepared from cells stimulated with serum or serum plus TSA as for panel D, and the immunoprecipitates were probed for Sp1 and pRb. In cells treated with TSA, hypophosphorylated pRb was recovered in association with Sp1 throughout the course of the experiment. IgG, immunoglobulin G.

Establishment of repression is inhibited by TSA. We then examined the effect of TSA on repression of dhfr gene expression during serum deprivation. CHOC 400 cells were plated in medium containing 0.2% FBS with or without TSA, RNA was isolated at 12-h intervals, and dhfr mRNA levels were measured by Northern blotting. GAPDH mRNA was used as a control. In 0.2% FBS dhfr levels dropped slowly over 48 to 72 h, whereas in medium with TSA dhfr levels rose about twofold during the first 24 h of serum deprivation and then dropped to control levels immediately thereafter (Fig. 9A and B). In low-serum conditions TSA transiently delayed the dephosphorylation of pRb but did not affect the accumulation of p130 or p21 by 24 h or thereafter (Fig. 9C).

View larger version (33K): [in this window] [in a new window]   FIG. 9.   TSA transiently stimulates endogenous dhfr promoter activity during serum withdrawal. Subconfluent CHOC 400 cells were incubated in fresh medium with 0.2% FBS (NT) or 0.2% FBS plus TSA (TSA) as indicated. At 12-h intervals cultures were harvested and total cellular RNA was examined for dhfr and gapdh mRNA levels by Northern blotting. (A) Autoradiographic signals of Northern blots for dhfr and gapdh mRNA as a function time in medium with 0.2% FBS with or without TSA. (B) The hybridization signals in panel A were quantified by phosphorimaging. dhfr mRNA levels first were normalized to gapdh mRNA levels and then plotted as a function of the percentage increase in signal relative to the levels of dhfr mRNA at zero time. (C) Total cell lysates from cells treated with or without TSA during serum deprivation were probed for pRb, p130, and p21 by immunoblotting.

View larger version (31K): [in this window] [in a new window]   FIG. 10.   E2F sites may contribute to transient stimulation of dhfr promoter activity by TSA during serum withdrawal. The indicated luciferase reporter plasmids were transfected into CHOC 400 cells. After being washed, the cultures were incubated in fresh medium containing 0.2% FBS (A) or 0.2% FBS plus TSA (B). Cultures were harvested at 12-h intervals, and duplicate samples were assayed for luciferase activity. The level of luciferase activity is plotted as a function of time relative to the 72-h value.

View larger version (18K): [in this window] [in a new window]   FIG. 11.   p130 renders the dhfr promoter insensitive to TSA. CHOC 400 cells were transfected with 0.5 µg of DHFR-GAL4, a luciferase reporter plasmid in which the E2F sites were replaced with a GAL4 DNA binding site, per plate and pCMV--gal (1 µg) as the control, with or without expression plasmids for GAL4-CBP (20 µg) (A) or GAL4-pRB (2 µg) and GAL4-p130 (2 µg) (B). After removal of DNA, cells were incubated for 24 h in fresh medium containing 10% FBS or 10% FBS with 100 nM TSA as indicated. Luciferase activity was determined in cell extracts, was normalized to expression of -galactosidase, and is expressed in relative light units (RLU) as averages of duplicate samples. Error bars, standard errors of the means. GAL4-CBP, GAL4-pRb, and GAL4-p130 influenced expression of pCMV--gal less than 5% under all conditions tested.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

While dhfr is considered a typical E2F-dependent gene (i.e., it is repressed in quiescent cells and activated late in G₁ after the phosphorylation of pRb family members), it is one of the few E2F-dependent genes in which E2F appears to play a prominent role in both repression and activation of transcription (14, 40, 41). For example, in serum starvation and stimulation experiments with mouse embryo fibroblasts lacking p130 and p107, the dhfr gene was not derepressed in G₀-G₁ as were cdc2, b-myb, and several others but rather was the only gene examined that showed premature induction after serum stimulation (21). The behavior of dhfr in these and other experiments likely results from the unique arrangement of Sp1 and E2F sites in the dhfr promoter. However, assigning specific roles for Sp1 and E2F in regulation of dhfr gene expression has generated considerable debate.

For instance, several studies have suggested different roles for Sp1 and E2F in activation of dhfr after growth stimulation. Experiments with the mouse promoter suggest that the reiterated Sp1 sites are not sufficient for activation after serum stimulation (42), while the experiments here support other studies that indicate that Sp1 sites alone respond to serum stimulation (23, 36). These and other experiments have inspired discussions about the relative contributions of E2F to repression and/or activation of the gene under various growth conditions. Here the DHFR-Sp1 reporter gene containing the four hamster dhfr Sp1 sites was 10-fold more active under standard growth conditions than the [E2F]×4 reporter, showed 2- to 4-fold increases in activity after serum stimulation, and displayed 10-fold increases after serum stimulation with TSA. In contrast, the [E2F]×4 reporter gene, although responsive to E2F, had weak basal activity, showed less than a twofold increase in activity after serum stimulation, and was completely unresponsive to TSA.

Importantly, no reporter construct employed here, including those with the WT promoter, precisely recapitulated the behavior of the endogenous dhfr gene. For example, elevated levels of E2F-1 and DP-1 expressed from tetracycline-responsive promoters in stable cell lines accentuate activation of dhfr gene expression, but only after the G₁ restriction point (28) (data not shown). When cotransfected with various reporter plasmids bearing either viral or cellular promoters, including those used here, E2F-1 reduced expression in a dose-dependent manner by transcriptional squelching (29). While under the same conditions the [E2F]×4 plasmid responds to E2F, it contains four consensus E2F binding sites upstream of a TATA box, a situation not yet encountered in nature.

Despite these limitations, transient transfection assays indicate that TSA is able to stimulate gene expression through the reiterated dhfr Sp1 sites, as has been observed for the Sp1 sites associated with the p21/WAF1/Cip1 promoter (7, 34, 43). Others have shown that the effect of HDAC inhibitors on Sp1 does not involve changes in Sp1 DNA binding activity (34, 43). Direct interactions between specific domains of Sp1 and HDAC1 (11) and Sp1 and E2F-1 to -3 (24, 26, 38) have been detected in vitro and in transfected cells. The interactions of Sp1 with HDAC1 and E2F are mutually exclusive, suggesting that expression of E2F-1 in late G₁ might displace HDAC1 from Sp1 and thereby relieve repression of gene expression (11).

Here HDAC1 was readily recovered in immunoprecipitates of Sp1, and this interaction persisted in serum-starved cells (Fig. 7), supporting the notion that Sp1 plays a role in repression of dhfr through HDAC1. However, our results suggest that activity of HDAC1 during the cell cycle may be regulated in a manner other than displacement from Sp1 by E2F-1. Our IP results indicate that HDAC1 is associated with Sp1 during all of G₁ in CHO and U2OS cells and that the association of pRb with Sp1 is regulated by pRb phosphorylation. The interaction of hypophosphorylated pRb with Sp1 may be mediated by HDAC1 because Sp1 and pRb do not interact directly in vitro (27, 49), and phosphorylation of pRb by cyclin D-cdk4 has been shown to disrupt pRb-HDAC1 complexes (18). Sp1 is also phosphorylated in mid-G₁ in C-terminal regions (3) that may be involved in binding HDAC1 (11). While we have not tested for Sp1 phosphorylation, no change in the binding of HDAC1 to Sp1 was observed in extracts from serum-stimulated cells. Thus it also appears that phosphorylation of Sp1 does not disrupt the Sp1-HDAC1 interaction during G₁. While it remains to be determined how (or if) the binding of pRb to the Sp1-HDAC1 complex affects HDAC activity, it is clear that TSA does not disrupt Sp1-HDAC1 interactions. Thus, we suggest that phosphorylation of pRb during the transition through G₁ releases pRb from the Sp1-HDAC1 complex and that this event contributes to relief of repression of the dhfr gene.

Our work also supports a role for E2F in activation of dhfr transcription. After phosphorylation of pRb and p130 at the G₁ restriction point, the genomic footprint of the E2F sites becomes occupied (51) and binding of E2F-1 to -3 to the promoter is detected by chromatin IP (ChIP) assays (50). Activation by E2F may occur by recruitment of CBP or p300 to the E2F transactivation domain (14), leading to modification of chromatin in the vicinity of the promoter by the HDAC activity of CBP, p300, or other E2F coactivators. ChIP experiments show that E2F-dependent transcriptional activity for several E2F-dependent genes is accompanied by histone acetylation (45), supporting a role for chromatin modification in activation of dhfr. Here TSA inhibited the accumulation of p300 in nuclear extract (Fig. 8C), providing a possible explanation for failure to fully activate the dhfr gene in serum-stimulated cells treated with TSA (Fig. 1). If recruitment of CBP or p300 is compromised by TSA, the levels of expression of dhfr in TSA at the G₁/S boundary (about 50% that in control cells) may represent that fraction of expression due solely to relief of repression.

Our experiments suggest a dynamic role for the E2F sites in the establishment of stable repression of dhfr during withdrawal from the cell cycle (Fig. 12). During the first 24 h of serum deprivation, we propose that dephosphorylated pRb accumulates and binds both Sp1-HDAC1 complexes and complexes of E2F-1 to -3 with DP, thereby dampening the transactivation capacity of both types of complexes. Note that during the early phases of cell cycle withdrawal, prior to expression of p130, both endogenous dhfr genes and dhfr reporter constructs containing functional E2F sites were susceptible to transient activation by TSA (Fig. 10 and 11), indicating that HDAC activity is important for repression during this time. In support of this interpretation, others have shown that ectopic expression of pRb from a tetracycline-regulated promoter for 24 h down-regulates dhfr transcription in cycling U2OS cells and that this inhibition is relieved by TSA (27).

View larger version (37K): [in this window] [in a new window]   FIG. 12.   A dynamic model for cooperative repression of the dhfr promoter by Sp1 and E2F. In cycling cells, Sp1 complexes and complexes of and E2F-1 to -3 with DP drive transcription of the dhfr gene. During the first 24 h of serum withdrawal, dephosphorylation of pRb results in the binding of pRb to Sp1-HDAC1 complexes and complexes of E2F-1 to -3 with DP, thereby dampening transcription. During the initial phases of serum deprivation, the dhfr promoter is sensitive to TSA, suggesting that HDAC activity associated with pRb is involved in repression at this time. After 24 h a TSA-insensitive state that correlates with the expression of p130 and E2F-4 is achieved, suggesting that E2F-4-DP-p130 complexes cooperate with Sp1-HDAC1-pRb complexes to establish stable repression of the promoter. Upon growth stimulation, repression is relieved by phosphorylation of pRb and p130 at the G1 restriction point. Activation occurs thereafter by recruitment of complexes of E2F-1 to -3 with DP and associated coactivators to the E2F sites. HDAC1 activity may be regulated by association with pRb or other mechanisms. The model posits that Sp1 and E2F cooperate in a dynamic fashion during the establishment of repression, relief of repression during G1, and activation of transcription after the G1 restriction point. Based on genomic footprinting experiments, the model suggests that a basal transcription complex (BTC) is present at the E2F sites throughout the cell cycle. The components of the BTC are not known.

After 24 h, increases in expression of p130 and E2F-4 lead to occupation of the dhfr sites by an E2F-4-DP-1-p130 complex, an interaction that has been documented by both gel mobility shift and ChIP experiments (46, 50). Recruitment of E2F-4-p130 complexes at E2F sites without displacement of hypophosphorylated pRb from Sp1 may explain why no E2F-dependent promoters other than dhfr and TK were recovered by pRB ChIP assays in serum-starved cells even though pRb was bound to chromatin (45, 50). In contrast to recruitment of pRb, recruitment of p130 to the dhfr promoter results in TSA-insensitive repression, perhaps by the binding of the E1A C-terminal binding protein (CtBP) corepressor complex to p130 (32). Thus, rather than a model of repression that invokes either HDAC-dependent or HDAC-independent activities, we suggest that interactions of pRb with Sp1 and p130 with E2F upon withdrawal from the cell cycle lead to inactivation of both transcription factors by complementary mechanisms. Because these mechanisms of repression are not redundant, they ensure that the promoter is stably repressed over long periods of time in quiescent cells.

One major issue remains unresolved for regulation of dhfr. Genomic footprinting shows that there is a large protein complex spanning 30 bp that is bound to the overlapping E2F and transcription initiation start sites during the entire cell cycle (51). Although the constituents of the basal complex at the E2F sites are not known, ChIP experiments indicate that E2F-4 and E2F-5 are bound to the dhfr promoter during the entire cell cycle (50), presenting the interesting possibility that E2F-4 (or E2F-5) is a constituent of the basal transcription complex for the dhfr gene. The next important step in understanding the regulation of dhfr will be to identify the components of the basal complex and to determine if these components include E2F and how this complex differs from those at TATA boxes during the cell cycle.

We note that our IP results differ from those of others who have reported interactions between HDAC1 and p130. After treatment of HaCaT cells with transforming growth factor  (TGF-), an interaction between endogenous HDAC1 and dephosphorylated p130 was observed (22), an interaction that we did not detect. Yet other studies have inferred an interaction between HDAC1 and p130 (13, 27, 53). Several factors may contribute to differences between our results and those from others. Use of different conditions for extract preparation or different antibodies in the IP experiments may account for some differences. However, we expect that the most important consideration in comparing results between systems is the likelihood that the interplay between regulatory proteins, which depends on numerous factors, including relative abundances of individual proteins, subcellular localization, and posttranslational modifications such as acetylation and phosphorylation, differs in various cell types and under different growth conditions. For example, treatment of cells with TGF- may result in distinct changes in the phosphorylation state of p130 that result in recruitment of HDAC1.

Finally, in transient transfection experiments, expression of pRb has been shown to superactivate several genes, including c-fos, c-myc, and dhfr, through Sp1 binding sites (25, 35, 47). Paradoxically, superactivation of Sp1 transcriptional activity by pRb required regions of pRb involved in growth suppression (48). One mechanism for superactivation involves titration of a negative regulator of Sp1 DNA binding activity by pRb (8). In addition to the observations that pRb stimulates Sp1-mediated transcription in transfection experiments, it has been reported that pRb does not repress the transcriptional activity of Sp1 tethered to an artificial promoter through a GAL4 DNA binding domain (27, 49). Yet in other studies pRb repressed dhfr reporter gene activity in a manner dependent on E2F sites (10). Here we have documented a cell cycle-regulated interaction between pRb and Sp1. This complex was present in early G₁ when dhfr transcription was low, and conditions that inhibited pRb phosphorylation also inhibited dhfr expression and dissolution of the Sp1-pRb complex. Together these data support a model for repression of dhfr that involves cooperation between pRb-HDAC1-Sp1 and p130-E2F-4-DP-1 complexes. We expect that the highly conserved architecture of the dhfr promoter reflects requirements for spacing, alignment, and orientation of proteins that are important for the assembly and maintenance of a stereospecific transcription complex. At other E2F-dependent promoters, different arrangements of regulatory elements may permit E2F and pRb family members to participate in other mechanisms of repression and activation.