Transcription Factor IID Recruitment and Sp1 Activation

DUAL FUNCTION OF TAF1 IN CYCLIN D1 TRANSCRIPTION*

Traci L. HiltonDagger and Edith H. Wang§

From the University of Washington, Department of Pharmacology, School of Medicine, Health Sciences Center, Box 357280, Seattle, Washington 98195-7280

Received for publication, January 14, 2003

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cyclin D1 is an oncogene that regulates progression through the G1 phase of the cell cycle. A temperature-sensitive missense mutation in the transcription factor TAF1/TAFII250 induces the mutant ts13 cells to arrest in late G1 by decreasing transcription of cell cycle regulators, including cyclin D1. Here we provide evidence that TAF1 serves two independent functions, one at the core promoter and one at the upstream activating Sp1 sites of the cyclin D1 gene. Using in vivo genomic footprinting, we have identified protein-DNA interactions within the cyclin D1 core promoter that are disrupted upon inactivation of TAF1 in ts13 cells. This 33-bp segment, which we termed the TAF1-dependent element 1 (TDE1), contains an initiation site that displays homology to the consensus motif and is sufficient to confer a requirement for TAF1 function. Electrophoretic mobility shift assays reveal that binding of ts13-TAF1-containing TFIID complexes to the cyclin D1 TDE1 occurs at 25 °C but not at 37 °C in vitro and involves the initiator element. Temperature-dependent DNA binding activity is also observed for TAF1-TAF2 heterodimers assembled with the ts13 mutant but not the wild-type TAF1 protein. These data suggest that a function of TAF is required for the interaction of TFIID with the cyclin D1 initiator. Our finding that recruitment of TFIID, by insertion of a TBP binding site upstream of the TDE1, restores basal but not activated transcription supports the model that TAF1 carries out two independent functions at the cyclin D1 promoter.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Understanding the control of cell proliferation at the molecular level is a central issue in cancer biology. Normal cells progress through a well-defined cell cycle consisting of four distinct stages, G1, S, G2, and M. Progression from one stage to the next is regulated in part by cyclins that bind and activate cyclin dependent kinases, an event necessary to drive cells through the proliferation cycle (1). Cyclin protein levels oscillate throughout the cell cycle and are thought to be controlled, in part, at the level of transcription. In support of this model, the deregulated expression of cyclins A and D1 has been found in several human tumors (2, 3). The overexpression of cyclin D1 is found in many breast cancers (~50%), mantle cell lymphomas (~100%), and squamous cell carcinomas (~64%) and is often associated with a poor prognosis (4-6). Therefore, a better understanding of the regulatory pathways governing cyclin D1 promoter activity may lead to the discovery of new therapeutic targets for the development of anti-cancer drugs.

RNA polymerase II-dependent gene transcription requires the assembly of more than 20 proteins at the promoter region to form a functional preinitiation complex. The general transcription factor IID (TFIID)1 is a large multisubunit complex consisting of the TATA-binding protein (TBP) and a set of TBP-associated factors (TAFs) (7). The recruitment of TFIID to the promoter is the first step in the assembly of the preinitiation complex. At promoters that contain a TBP binding site, the mode of recruitment is at least partly via TBP binding to the TATA element. DNase I protection studies have shown that TFIID, in contrast to TBP, protects an extended region that includes sequences ~30 bp downstream of the TATA box (8). These data suggest that one or more of the TAF subunits most likely contact DNA (9). However, the precise function of the TAF components of TFIID in the transcription process still remains to be elucidated.

Many promoters, including cyclin D1, lack a consensus TATA element. Under these circumstances the TAFs may be more actively involved in the recruitment of TFIID to promoter sequences. Several TAFs have been shown to interact directly with promoter DNA (9-11). UV cross-linking experiments with purified TFIID reveal that TAF1 and TAF2 are in close contact with the core promoter region (9). In a random site selection screen, TAF1-TAF2 heterodimers, but not the individual TAFs, were found to preferentially bind to DNA sequences that fit the initiator consensus motif (11). These data suggest that TAF1 and TAF2 could play an important role in TFIID binding under conditions suboptimal for TBP binding.

The ts13 cell line, derived from BHK-21 (baby hamster kidney) cells, is a temperature-sensitive mutant that arrests in late G1 at the nonpermissive temperature (39.5 °C) due to a missense mutation in the TAF1 protein (12-14). The single base pair change identified in ts13 cells results in a glycine to an aspartic acid substitution at amino acid 690 of the hamster TAF1 protein (15). Expression of wild-type human TAF1 is sufficient to complement the ts13 cell cycle arrest phenotype, suggesting that the point mutation is responsible for the proliferation defect (16).

Despite having a mutation in a component of the general transcriptional machinery, ts13 cells do not display a global defect in gene expression (16-19). Instead, transcription from a subset of genes, including the cell cycle regulators cyclins A, D1, and E, is dramatically reduced at 39.5 °C. Transfection of wild-type human TAF1 into ts13 cells not only complements the G1 arrest but also overcomes the cyclin A and D1 transcriptional defect (20). Conditional mutations in yeast TAF1 (yTAF1) also induce cells to arrest in G1 and to exhibit defects in gene transcription (21). DNA microarray analysis of yTAF1 mutant strains under permissive and restrictive conditions revealed that ~16% of yeast genes display a requirement for TAF1 function (22). Studies carried out with ts13 mRNA hybridized to a more limited mouse DNA microarray generated similar results (23). These data provide additional evidence that inactivation of the TAF1 subunit of TFIID leads to gene-specific defects in mRNA synthesis. Our current working hypothesis is that the inability of ts13 cells to progress through the G1/S phase checkpoint can be attributed to a disruption in TAF1 function, which compromises the transcription of a select group of cell cycle control genes. The availability of the ts13 cell line provides a unique model system to identify and characterize the specific activity of TAF1 required for cell cycle gene transcription and ultimately cell proliferation.

Cyclin D1 is a G1/S phase cell cycle regulatory protein that was isolated from murine bone marrow macrophages as an early responsive gene after stimulation with colony stimulating factor 1. Among the cyclin proteins, expression of the D-type cyclins is one of the earliest events to occur during the G1 phase of the cell cycle leading to cell division. Cyclin D1 has been shown to be transcriptionally up-regulated by numerous growth factors, including nerve growth factor in PC12 cells, estrogen and gestergen in endometrial cells, and epidermal growth factor in prostate cancer cells (24-26). The 5'-regulatory region of the cyclin D1 gene has been isolated from a number of species, including rat, mouse, and human (27-29). It consists of a TATA-less promoter with a cAMP-responsive element (CRE) and multiple Sp1 binding sites, through which several growth factors exert their proliferative effect (30).

For our studies, we have elected to focus on the cyclin D1 promoter, because decreases in cyclin D1 mRNA levels occur relatively quickly after ts13 cells have been shifted to 39.5 °C (19). The rapid response suggests that transcription of cyclin D1 is directly affected by the ts13 mutation in TAF1. Studies in yeast with conditional alleles of yTAF1 mapped the TAF1-dependent phenotype to the core promoter region of CLN2 (31). The analysis of chimeric promoter constructs between the TAF1-dependent cyclin A and TAF1 independent c-fos genes has revealed that the core promoter elements of cyclin A are also sufficient for temperature-sensitive expression in ts13 cells (20). These results indicate that TAF1 has a unique function at the core promoter of a subset of genes. The goal of our studies is to elucidate the function of TAF1 required for efficient promoter activity.

We report here that in vivo protein-DNA interactions within a 33-bp region of the cyclin D1 core promoter, the TDE1 (for TAF1-dependent element 1), are altered under conditions where TAF1 activity is compromised. We demonstrate that TFIID binds to the TDE1 of cyclin D1 in gel mobility shift assays. The DNA binding ability of TFIID as well as TAF1-TAF2 heterodimers is compromised at 37 °C in vitro by the ts13 mutation in TAF1. These results have led us to conclude that the lack of cyclin D1 transcription can be attributed at least in part to the inefficient recruitment of TFIID to the core promoter. The introduction of a properly positioned consensus TATA element in the minimal cyclin D1 core promoter is able to fully restore basal transcriptional activity at 39.5 °C in ts13 cells. However, full recovery of activated transcription was not observed when similar experiments were carried out with a longer cyclin D1 promoter fragment containing the upstream activating Sp1 sites. These findings suggest a dual function for TAF1 in cyclin D1 transcription, one of which is required for efficient TFIID binding to the core promoter to initiate the transcription cycle.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Tissue Culture Cell Lines-- ts13 and ts13R3 cells were grown in Dulbecco's modified Eagle's media (Invitrogen) supplemented with 10% fetal bovine serum (JRH Biosciences), 2 mM L-glutamine, and penicillin/streptomycin in a humidified incubator containing 10% CO2 at 33.5 °C and 39.5 °C, respectively. To inactivate TAF1 and induce cell cycle arrest, ts13 cells were shifted to 39.5 °C. ts13 and ts13R3 cells that constitutively express HA-epitope-tagged ts13 and wild-type alleles of human TAF1, respectively, were established as follows. Cells were transfected with a cytomegalovirus expression vector for the HA-tagged mutant or wild-type TAF1 protein along with a neomycin expression plasmid at a ratio of 10 to 1. After 24 h, cells were plated into media containing 1 mg/ml G418. G418-resistant colonies were isolated and clonally expanded, and the expression of the HA-tagged TAF1 was determined by Western blotting with the anti-HA mAb 12CA5. HA-positive cell lines were passaged in 200 µg/ml G418 to maintain expression of the HA-tagged TAF1. Sf9 insect cells were propagated in Hanks' TNM-FH insect media (JRH Biosciences) containing 10% FBS, L-glutamine, and penicillin/streptomycin. Cultures were grown in spinner flasks at 27 °C in the absence of CO2.

Cloning of Hamster Cyclin D1 Promoter Fragment-- An upstream fragment of the hamster cyclin D1 promoter was PCR amplified from ts13 genomic DNA using the following primer pair: 5'-AGGAAATAATGGCCACCATCTTG-3' and 5'-CTTTCAATTTCATCAACAGTG-3', designed from alignment of rat, mouse, and human cyclin D1 promoter sequences (GenBankTM accession numbers AF148946, AF212040, and Z29078, respectively). A hamster-specific primer 5'-TCTTGAGCTGTTGCTGGGATTTT-3', based on the above PCR product, and a primer (5"-GTCAGGGTACGCGCGGC-3') from alignment of downstream rat, mouse, and human cyclin D1 gene sequences available in GenBankTM (accession numbers AB042564, AF212040, and Z29078, respectively) were used to PCR-amplify an 895-bp fragment representing the hamster cyclin D1 promoter from ts13 genomic DNA. The PCR fragment was subcloned into pCRII TOPO vector using the TOPO TA cloning kit (Invitrogen), and the DNA sequence was determined. The sequence of the hamster promoter fragment has been deposited into GenBankTM under accession number AF539477.

Mapping of Hamster Cyclin D1 Transcription Start Sites by Primer Extension-- The 32P-labeled oligonucleotide (5'-TGCCTCGCGCTCTACTGCC-3'), complementary to the hamster cyclin D1 sequence, was used in reverse transcription reactions with 5 µg of total RNA from either asynchronously growing ts13 cells or ts13 cells synchronized in G1 ~ 8 h after release from serum starvation. Reaction products were separated on denaturing 6% polyacrylamide gels and visualized by autoradiography.

Reporter Plasmid Constructs-- Promoter fragments to be examined in transient transfection assays were subcloned into pGL2-basic (Promega), a reporter plasmid that contains the luciferase coding region. Mouse c-fos reporter contains -356 to +58 of the mouse gene as previously described (16). The 895-bp PCR fragment representing the hamster cyclin D1 promoter was restriction digested with NotI to remove the translational start codon, blunt-ended with Klenow, and subcloned into the SmaI site of pGL2-basic to produce -714/+109 cycD1-luc. The following cyclin D1 promoter fragments were also introduced into the SmaI site of pGL2-basic. The BstXI to NotI fragment from the cyclin D1 promoter was used to construct -439/+109 cycD1-luc. -55/-22 cycD1-luc was generated by annealing 5'-AGGGGAGTTTTGTTGAAGTTGCAAAGTCCTGC-3' and 5'-GCAGGACTTTGCAACTTCAACAAAACTCCCCT-3' and subcloning the double-stranded oligonucleotide into pGL2-basic. The remaining cyclin D1 deletion constructs were generated by PCR. The fragment for -183/+109 cycD1-luc was amplified using 5'-CGCTTTGGGCTTGTCCCCCT-3' and 5'-GTCAGGGTACGCGCGGC-3' and digested with NotI, and the ends were blunted with Klenow. The fragment for -183/+21 cycD1-luc was amplified using the primers 5'-CGCTTTGGGCTTGTCCCCCT-3' and 5'-ATCTGCCGCTCTCTGCTACGCG-3'. Primers 5'-TTCTCTGCCGGGCTTTGATC-3' and 5'-ATCTGCCGCTCTCTGCTACGCG-3' were used to synthesize the promoter fragment for -107/+21 cycD1-luc. -183/+21 wtTATA cycD1-luc and -183/+21 mtTATA cycD1-luc were constructed as follows. A PstI site was introduced into -183/+21 cycD1-luc upstream of the TDE1 by using the QuikChange site-directed mutagenesis kit (Stratagene) and primers 5'-GTCACACGGACTGCAGGGGAGTT-3' and 5'-AACTCCCCTGCAGTCCGTGTGAC-3'. The wild-type TDE1 was dropped out as a PstI fragment and replaced with a double-stranded oligonucleotide derived from one of the following two primer sets: (for wild-type TATA) 5'-GTATAAAGGGGAGTTTTGTTGAAGTTGCAAAGTCCTGCA-3' and 5'-GCAGGACTTTGCAACTTCAACAAAACTCCCCTTTATACTGCA-3'; (for mutant TATA) 5'-GGAGAAAGGGGAGTTTTGTTGAAGTTGCAAAGTCCTGCA-3'; and 5'-GCAGGACTTTGCAACTTCAACAAAACTCCCCTTTCTCCTGCA-3'. -107/+21 wtTATA cycD1-luc and -107/+21 mtTATA cycD1-luc were generated using -183/+21 wtTATA cycD1-luc and -183/+21 mtTATA cycD1-luc, respectively, and primers 5'-TTCTCTGCCGGGCTTTGATC-3' and 5'-ATCTGCCGCTCTCTGCTACGCG-3' in PCR reactions. The resulting PCR products were subcloned into the SmaI site of pGL2-basic.

Transient Transfection Assays-- Before the introduction of DNA, ts13 cells, seeded into 24-well plates, were maintained at 33.5 °C or shifted to 39.5 °C for 2 h. Cells were then switched to Dulbecco's modified Eagle's medium containing no FBS and no antibiotics. 0.1 µg of SV40-beta -galactosidase expression plasmid, and 0.25 µg of the cyclin D1 or c-fos reporter constructs were transfected into cells using the LipofectAMINE and Plus reagents as described by the manufacturer (Invitrogen) with the following modification: the amount of Plus reagent used was reduced by 50%. After 3 h, FBS to 10% final concentration and antibiotics were added to the cell culture media. Whole cell lysates were prepared using Report Lysis buffer (Promega) between 16 and 18 h post-serum addition. The amount of luciferase activity was determined using the Promega luciferase assay system and beta -galactosidase activity was measured as described (32). beta -Galactosidase activity was used to correct for differences in transfection efficiency.

In Vivo Genomic Footprinting of the Hamster Cyclin D1 Promoter-- ts13 and ts13R3 cells were arrested in G0 by serum starvation for 36 h at 33.5 °C, then released at either 33.5 °C or 39.5 °C by the addition of serum. Approximately 8 h post-serum addition, cells were treated with 0.01% DMS for 2 min. Genomic DNA was isolated and methylated at guanine/adenine nucleotides as described with the following minor modifications (33). For the preparation of genomic DNA, the ethyl ether extraction was eliminated from the protocol, and the isopropanol precipitation was replaced with an ethanol precipitation. Sites of guanine and adenine methylation were chemically cleaved and sites of cleavage in the hamster cyclin D1 promoter were mapped using the technique of ligation-mediated PCR (34). Briefly, 5 µg of genomic DNA served as template in a first-strand synthesis reaction with the following primers: upper strand, 5'-CTGCCTCGCCGTCTACTGCC-3'; lower strand, 5'-TCCCGAGCCCCCTCCCCCT-3'. Linkers were then ligated onto the blunt-ended products. Next, a linker and nested gene-specific primer (upper strand, 5'-GTCTCCGAGCGCGCGAATCT-3'; lower strand, 5'-GCGCCCGCCCAGTCCTTCC-3') were used to amplify the cleavage products. The resulting PCR products were end-labeled with the following 32P-labeled primers (upper strand, 5'-ATCTGCCGCTCTCTGCTACGCG-3'; lower strand, 5'-CCCCTCCCTTTCTCTGCCTGGCT-3'). Reaction products were separated on denaturing 6% polyacrylamide gels and visualized by autoradiography.

Preparation of Cell Lysates-- ts13 and ts13R3 nuclear extracts were prepared as previously described (16) from synchronized cells that had been serum-starved and released into the cell cycle for 8-12 h by serum addition. The preparation of HeLa nuclear extracts and fractionation of the extracts on phosphocellulose to obtain the TFIID-depleted extract and a partially purified TFIID fraction were carried out following the protocol of Dignam et al. (35).

Electrophoretic Gel Mobility Shift Assays-- ts13 or ts13R3 nuclear extracts to be used in binding reactions were equilibrated at 25 °C or 37 °C. Twenty micrograms of each extract was then preincubated at the appropriate temperature for 20 min in 20 mM HEPES, 50 mM NaCl, 10% glycerol, 2% polyethylene glycol 6000, 5 mM ammonium sulfate, 5 mM MgCl2, 3 mM beta -mercaptoethanol in the presence of 1 µg of dG-dC, and 300 µg/ml bovine serum albumin. 32P-Labeled double-stranded oligonucleotides encompassing the cyclin D1 TDE1 (5'-AGGGGAGTTTTGTTGAAGTTGCAAAGTCCTGCA-3') or c-fos core promoter (5'-TTCTATAAAGGCACCGGCTGAGGCGCCTACTACTCCAACCGCGACTGCAG-3') were added, and the reaction continued at 25 °C or 37 °C for an additional 20 min. Reaction mixtures containing the c-fos probe were separated on high ionic strength 1.2% Mg2+-agarose gels as described previously (36), and those containing the cyclin D1 TDE1 were run on 1.2% agarose/0.5× TBE gels (37). Agarose gels were run at 140 v for ~90 min. Mobility-shifted complexes were detected by autoradiography after the gels were dried onto DE81 filter paper. For the competition assays, 20 µg of extract was preincubated with the indicated molar excess of competitor for 20 min at 25 °C. 32P-Labeled double-stranded oligonucleotide was added, and the reaction continued at 25 °C for an additional 20 min. The sequences of the competitor DNAs are as follows: c-fos INR (5'-GGCACCGGCTGAGGCGCCTACTACTCCAACCGCGACTGCAG-3'), c-fos core (5'-TTCTATAAAGGCACCGGCTGAGGCGCCTACTACTCCAACCGCGACTGCAG-3'), NS (5'-AGCTAGGCCGCGAGCGCTTTCATTGGTCCATT), and TDE1 mtINR (5'-AGGGGAGTTTTGTTGAAGTTGCAAAGGGCTGCA-3'). Binding reactions with recombinant HA-TAF1, FLAG-TAF2, and HA-TAF1/FLAG-TAF2 heterodimers were performed essentially as described above and separated on 1.5% agarose/0.5× TBE gels.

Preparation of TAF1-TAF2 Heterodimers-- Recombinant baculovirus vectors used to express HA-tagged TAF1 and FLAG-tagged TAF2 have been previously described (9, 38). For protein production, Sf9 cells were seeded at ~7.5 × 106 cells/10-cm dish and infected with recombinant baculovirus at a multiplicity of infection of 1-2. Approximately 48 h post-infection, whole cell lysates were prepared by sonication in HEMG buffer (25 mM HEPES, pH 7.6, 12.5 mM MgCl2, 0.1 mM EDTA, 10% glycerol, 0.1% Nonidet P-40) containing 0.4 M KCl. HA-TAF1 and FLAG-TAF2 monomers were immunoaffinity purified from Sf9 whole cell extracts using the anti-HA (mAb 12C5) and anti-FLAG (M2 antibody) antibodies, respectively. TAF1-TAF2 heterodimers were assembled in vitro using the anti-HA and anti-FLAG antibodies as previously described (39). For gel mobility shift assays, all proteins were eluted under native conditions with peptides corresponding to the appropriate antibody epitope (HA: YPYDVPDYA; FLAG: DYKDDDDK) in HEMG buffer containing 0.1 M KCl.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cloning and Characterization of Hamster Cyclin D1 Promoter Fragment-- To investigate the function of TAF1 in the transcription of cyclin D1 in ts13 cells, we cloned a fragment of the hamster cyclin D1 promoter using a PCR-based approach. Analysis of the endogenous promoter will avoid limitations that may be inherent to the study of a human promoter in hamster cells. A short stretch of the hamster cyclin D1 promoter was PCR-amplified from ts13 genomic DNA using primers designed to regions of high sequence identity between the human, mouse, and rat promoters. To eliminate errors generated during the amplification reaction, several independent PCR products were subcloned and subjected to DNA sequencing. The sequence information available in GenBankTM and obtained from the PCR products were used to design hamster-specific primers that amplified a single 895-bp fragment from ts13 genomic DNA (data not shown). Comparison of this putative hamster cyclin D1 promoter fragment with the previously described human, rat, and mouse genes (27-29) revealed a remarkable 65.5%, 83.3%, and 83.7% sequence identity, respectively, in the noncoding region, with even greater sequence identity between -183 and +1, the region of transcriptional initiation (data not shown). The lower percentage of sequence identity found for the human promoter is primarily due to the insertion of short stretches of sequence unique to the human gene. Consistent with the cyclin D1 sequences isolated from other species, the hamster promoter lacks a consensus TBP binding site or TATA element. It also contains numerous binding sites for transcription factors, including the E-box proteins, Oct-1, Sp1, NF-kappa B, and CREB, most of which are conserved in other mammals, although their exact placements vary slightly (Fig. 1).


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Fig. 1.   Nucleotide sequence of the 5' regulatory region of the hamster cyclin D1 gene. The 895-bp nucleotide sequence of the putative hamster cyclin D1 promoter is provided. The sequence lacks a canonical TATA box element or TBP binding site. The positions of potential transcription factor binding sites, including sites for the E-box protein, Oct-1, CREB/ATF, NF-kappa B and Sp1, are indicated. The hamster promoter has more putative Sp1 binding sites than the mouse, rat and human promoters. The major sites of transcription initiation, as determined by primer extension (see Fig. 2), are indicated by the asterisks. The ATG start codon for translation is shown in large, bold type. The hamster cyclin D1 sequence has been deposited in GenBankTM under accession no. AF539477.

We also mapped the sites of transcription initiation by primer extension. Similar to the rat, mouse, and human promoters, hamster cyclin D1 utilizes multiple transcription initiation sites in ts13 cells (Fig. 2). As a reference point, we defined the most downstream transcriptional start site as +1, which is 124 nucleotides upstream of the translational start codon. This numbering system places all identified transcriptional initiation sites between +1 and -32. The two clusters at positions -32 to -29 and -13 to -12 appear to be the preferred sites of transcriptional initiation. We found that the same set of initiation sites are used at the nonpermissive temperature in ts13R3 cells, a ts13 subclone that expresses wild-type human TAF1 and no longer cell cycle arrests at 39.5 °C (data not shown). This finding suggests that the mechanism of cyclin D1 transcription is not altered by the increase in temperature and rules out any temperature-dependent shift to less efficient start sites of transcription.


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Fig. 2.   Hamster cyclin D1 promoter utilizes multiple sites for transcription initiation. Reverse transcriptase-mediated extension using a hamster cyclin D1 specific antisense primer (5'-TGCCTCGCGCTCTACTGCC-3') annealed to mRNA isolated from asynchronously grown (cycling) or G1 synchronized (G1) ts13 cells was carried out to identify sites of transcription initiation. Primer extension products were separated by denaturing 6% polyacrylamide gel electrophoresis and detected by autoradiography. Transcription initiation sites were found to cluster into five groups and to span a 30-bp region, with the two groups at -31 to -29 and -13 to -12 the preferred sites of initiation. The initiation site assigned the position of +1 is indicated.

Despite nearly 100% sequence identity within the initiation region, the start sites of transcription differs between species, although they tend to cluster within the same region of the promoter. Only one of the major clusters of transcriptional start sites in hamster cyclin D1 (between -32 and -29) displays any homology to the consensus initiator motif of YYAN(T/A)YY, suggesting that other factors are influencing the species-specific differences in initiation site selection. According to the consensus motif, position -30 would be the predicted site of transcriptional initiation. The two nucleotides proceeding the -30 position are also adenines and are used as initiation sites. Isolation of the hamster cyclin D1 promoter provides us with the tools to further analyze the role of TAF1 at the endogenous hamster promoter, by specifically investigating in vivo protein-DNA interactions under permissive and restrictive conditions for TAF1 function.

Hamster Cyclin D1 Transcription Requires TAF1 Function at the Core Promoter-- We have previously reported that transcription from the human cyclin D1 promoter is severely compromised when transiently transfected into ts13 cells at the nonpermissive temperature (20). To determine if the hamster promoter also demonstrates a requirement for TAF1, we cloned the 895-bp hamster cyclin D1 promoter fragment upstream of the luciferase gene to generate the reporter construct -714/+109 cycD1-luc (Fig. 3A). When transiently transfected into ts13 cells, -714/+109 cycD1-luc displayed high levels of transcriptional activity at 33.5 °C (Fig. 3B, left panel). As expected, transcription decreased ~25-fold when the cells were shifted to 39.5 °C. The dramatic reduction in promoter activity is not due to heat shock effects, because in ts13R3 cells, which express wild-type human TAF1, no decrease in promoter activity was observed at 39.5 °C (Fig. 3B, right panel). Using the identical experimental approach, we confirmed our previous finding that transcription from the TAF1-independent c-fos promoter is not altered at 39.5 °C in both ts13 and ts13R3 cells (data not shown). These results indicate that it is the inactivation of TAF1 and not the increase in temperature that is responsible for the defect in cyclin D1 transcription in ts13 cells.


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Fig. 3.   Mapping of the TAF1-dependent cis-element in the hamster cyclin D1 promoter. A, a schematic diagram of hamster cyclin D1 promoter constructs used in transient transfection assays is provided. Deletion constructs were engineered by either restriction enzyme digestion or PCR amplification. The location of putative transcription factor binding sites and the initiation sites of transcription and translation are shown. B, the indicated hamster cyclin D1 promoter constructs driving luciferase gene expression were transiently transfected into ts13 or ts13R3 cells incubated at either 33.5 °C or 39.5 °C, and luciferase activity was measured after 16-18 h. The level of activity detected with -714/+109 cycD1-luc at 33.5 °C was given a value of 100 and used as the reference point for normalizing the activity of all other reporter constructs tested. The data represent the average of eight independent experiments, each carried out in duplicate. Error bars indicate ±S.E.

A series of hamster cyclin D1 promoter deletions was constructed to determine what region is responsible for TAF1-dependent transcriptional activity (Fig. 3A). The successive removal of transcription factor binding sites between -714 and -183 led to progressively greater increases in transcription levels, a characteristic unique to the hamster promoter, but did not significantly alter TAF1 dependence (Fig. 3B, left panel). It wasn't until the Sp1 binding sites were deleted that a significant decrease (>80%) in promoter activity was observed at 33.5 °C (Fig. 3B, left panel). This finding is consistent with previously reported studies on the human and rat promoters demonstrating that Sp1 sites are critical for maximal activity. Despite the 4- to 5-fold decrease in promoter activity, the remaining core promoter fragment (-107/+21 cycD1) retained temperature-sensitive transcriptional activity and therefore a requirement for TAF1 function. Because all deletion constructs displayed similar levels of activity at 33.5 °C and 39.5 °C in ts13R3 cells (Fig. 3B, right panel), our data demonstrate that the core promoter of cyclin D1 is sufficient to confer a requirement for a function of TAF1.

Unexpectedly, deletion of sequences between +21 and +109 led to ~50% reduction in promoter activity (Fig. 3B). This region of the cyclin D1 gene contains no known transcription factor binding sites or apparent transcription initiation sites, whose loss could account for the reduction in transcription levels. The contribution of this segment of the cyclin D1 gene to promoter activity remains to be determined.

In Vivo Genomic Footprinting of the Cyclin D1 Core Promoter Region-- We have demonstrated by deletion analysis that transcription from the core promoter of cyclin D1 requires a function of TAF1. In yeast, TAFs are dispensable for the transcription of many genes (40, 41). However, yTAF1 is required at the core promoter for transcription of a subset of genes (31, 42). Therefore, we investigated if the decrease in cyclin D1 transcription at 39.5 °C is due to a defect in transcription factor binding to the core region. To address this issue, we utilized the technique of in vivo genomic footprinting to map protein-DNA interactions at the cyclin D1 core promoter in ts13 cells (Fig. 4). The advantage of this approach is that it allows one to map protein-DNA interactions that occur on the endogenous promoter assembled in native chromatin with nucleotide resolution.


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Fig. 4.   In vivo genomic footprint analysis of the hamster cyclin D1 core promoter. A, the in vivo DNA methylation pattern between nucleotides -79 and +3 of the hamster cyclin D1 promoter is shown. ts13 and ts13 R3 cells were subjected to DMS treatment at the indicated temperature. The modified genomic DNA was chemically cleaved, and the position of methylated nucleotide residues was mapped by ligation-mediated PCR using nested cyclin D1 gene-specific primers. Both the upper and lower strands were examined. The cleavage pattern obtained from genomic DNA methylated in vitro in the absence of cellular protein is shown in lanes 1 and 10. The solid lines between the ts13 and ts13R3 samples indicate the position of nucleotide residues (between nucleotides -55 and -22) that demonstrated increased methylation at 39.5 °C in ts13 but not in ts13R3 cells, in three independent experiments. Dashed lines indicate sites of increased methylation observed in two out of three experiments. The arrows indicate the position of transcriptional start sites. B, alignment of nucleotides -79 to +3 of the hamster cyclin D1 promoter with the rat, mouse, and human promoters shows nearly 100% sequence identity within this region. The large asterisks indicate nucleotides that repeatedly displayed increased methylation in ts13 but not in ts13R3 cells at 39.5 °C. The smaller asterisks indicate the location of changes observed in two out of three experiments. Indicated by the solid line underneath the nucleotide sequences is a 33-bp sequence that we defined as the TAF1-dependent element 1 (TDE1), a region that repeatedly displayed changes in methylation protection. Putative binding sites for cAMP-responsive element binding protein (CREB) and nuclear factor kappa B (NF-kappa B) are shown above the sequence. The sites of transcription initiation are indicated by the arrows.

Cyclin D1 transcription occurs during the G1 phase of the cell cycle. To maximize differences in transcription levels and thereby DNA binding activity, we analyzed synchronized populations of ts13 and ts13R3 cells at 33.5 °C and 39.5 °C. Approximately 8 h after release from G0 by serum addition, a time of maximal cyclin D1 transcription, cells were treated with 0.01% DMS to methylate accessible base residues, and the in vivo modified genomic DNA was isolated by standard procedures. The position of methylated guanines and adenines was determined by chemical cleavage followed by ligation-mediated PCR as described (34). The pattern of DNA methylation in the absence of protein was also determined in vitro (Fig. 4, lanes 1 and 10). Both the upper and lower strands were analyzed, because DMS methylates only adenine and guanine residues present in each DNA strand. The ts13R3 cell line, which expresses wild-type TAF1 at the higher temperature, was used in parallel to identify changes that are simply due to the increase in temperature (Fig. 4, lanes 4, 5, 8, and 9). Only differences detected between the DNA methylation patterns at the two temperatures in ts13 but not in ts13R3 cells can be attributed to changes in protein-DNA contacts dependent on TAF1 activity. We focused on the cyclin D1 core promoter between positions -107 and +21, because this region is sufficient to confer temperature-sensitive transcriptional activity in ts13 cells. Strong protection of a guanine residue at position -69, located within the CREB binding site, was detected at 33.5 °C and 39.5 °C, suggesting that the CREB/ATF site is occupied at the two temperatures. We found that the most predominant and reproducible changes localize to a 33-bp segment encompassing one cluster of transcription initiation sites for cyclin D1, which we termed the TAF1-dependent element 1 or TDE1 (Fig. 4). The changes detected on both strands of the TDE1 include enhancement of cleavage sites as well as the appearance of new cleavage products at 39.5 °C. Specifically, guanine residues between -55 and -49, a guanine at -29, and adenine residues at positions -31, -30, and -22 become more accessible at 39.5 °C, conditions at which TAF1 becomes partially inactive (Fig. 4, compare lanes 2 and 3 and lanes 6 and 7). Such changes were not detected when parallel experiments were carried out in ts13R3 cells (Fig. 4, compare lanes 4 and 5, and lanes 8 and 9). As an additional control, we analyzed the core promoter of the c-fos gene, which was strongly protected against DMS modification, and detected no significant TAF1-dependent changes in the pattern of DNA methylation (data not shown). Taken together, these findings suggest that, upon inactivation of TAF1 by the ts13 mutation at 39.5 °C, the efficient binding of one or more transcription factors to TDE1 in the cyclin D1 core promoter is compromised, thereby leading to a decrease in gene transcription.

Interestingly, the modification of cytosine residues was also observed but only within the TDE1. Unpaired cytosines can be methylated by DMS. Therefore, the modification of cytosines suggests the presence of structured DNA, with these regions being single-stranded in nature.

TDE1 Is Sufficient to Confer a Requirement for TAF1 in Cyclin D1 Transcription-- We have defined protein-DNA contacts within a segment of the cyclin D1 promoter or TDE1 that are altered at 39.5 °C in ts13 cells. Is this region of cyclin D1 sufficient to confer a requirement for TAF1 function in gene transcription? To address this question, the TDE1 of cyclin D1 was subcloned upstream of a luciferase reporter gene and the resulting reporter construct (-55/-22 cycD1-luc) was transiently transfected into ts13 and ts13R3 cells (Fig. 5). Expectedly, the level of transcription mediated by -55/-22 cycD1-luc was 20% of the "full-length" hamster promoter fragment (-714/+109 cycD1) at 33.5 °C. More importantly, the minimal construct still required TAF1 activity, displaying almost 20 times more activity at 33.5 °C than at 39.5 °C, which is similar to that observed for the full-length cyclin D1 promoter.


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Fig. 5.   TDE1 in the hamster cyclin D1 promoter is sufficient to confer TAF1-dependent transcriptional activity. The indicated cyclin D1-luc reporter constructs were transiently transfected into ts13 or ts13R3 cells at the indicated temperature. The amount of luciferase activity at 33.5 °C or 39.5 °C was measured after 16-18 h. The relative activity of each promoter was determined by normalizing to the activity of -714/+109 cycD1-luc in ts13 cells at 33.5 °C, which was given a value of 100. The data presented are the average of eight independent experiments, each carried out in duplicate. Error bars indicate ±S.E.

The TDE1 contains a major cluster of transcription initiation sites and a putative NF-kappa B binding site, both of which are conserved in the human, mouse, and rat cyclin D1 promoters. The consensus NF-kappa B site shows a large increase in DNA accessibility in vivo. However, we have been unable to demonstrate any involvement of NF-kappa B or its binding site using a number of different experimental approaches, including binding site mutation and inhibition of NF-kappa B activity. These data suggest that the transcription initiation site is the important feature of the TAF1-dependent element.

Temperature-sensitive DNA Binding Activity on the TDE1 of Cyclin D1-- Our in vivo genomic footprinting results suggest that protein binding to sequences within the core promoter of cyclin D1 is altered at 39.5 °C in ts13 cells. To identify the proteins responsible for the change in DNA methylation protection, nuclear extracts containing the mutant or wild-type allele of TAF1 were prepared from permissively grown ts13 or ts13R3 cells, respectively, and incubated with a radiolabeled DNA fragment containing the cyclin D1 TDE1. Binding reactions were carried out at either 25 °C or 37 °C, as these temperatures have been previously shown to be permissive and restrictive for in vitro transcription in ts13 nuclear extracts, respectively (16). One predominant retarded protein-DNA complex was detected with both extracts when the reactions were incubated at 25 °C (Fig. 6B, left panel). The formation of the shifted complex was disrupted when the reaction temperature was increased to 37 °C but only in extracts containing the mutant (ts13) TAF1 protein. The ts13 mutation in TAF1 decreases the expression of multiple genes. Therefore, the lack of DNA binding activity could be due to the absence of proteins responsible for complex formation. We have ruled out this possibility, because TDE1 binding activity was also observed at 25 °C but not at 37 °C in nuclear extracts prepared from ts13 cells grown at the nonpermissive temperature (Fig. 6B, right panel). These data suggest that TAF1 activity is required for efficient protein binding at the cyclin D1 core promoter.


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Fig. 6.   TFIID binds to the initiator element in the TDE1 of the hamster cyclin D1 promoter. A, nucleotide sequence of one strand of double-stranded oligonucleotides used in gel mobility shift assays is provided. The TATA element in the c-fos core is indicated in bold italics. The initiator elements in each sequence are boxed with the consensus motif given below. The initiator adenine is indicated in bold. B, electrophoretic gel mobility shift assays were performed using a radiolabeled double-stranded oligonucleotide that contains the 33-bp TDE1 from the hamster cyclin D1 promoter, as defined in Fig. 4. Twenty micrograms of nuclear extracts prepared from either permissively (left panel) or nonpermissively (right panel) grown ts13 or ts13R3 cells were equilibrated to 25 °C or 37 °C then incubated with the radiolabeled cyclin D1 TDE1 at the same temperature. Binding reactions were separated by agarose gel electrophoresis as described under "Experimental Procedures," and mobility shifted complexes were detected by autoradiography. The formation of shifted complexes on the cyclin D1 TDE1 was disrupted in ts13 nuclear extract at 37 °C but not in ts13R3 extracts. This decrease in binding activity was observed with both permissively and nonpermissively derived nuclear extracts. C, mobility shift assays with permissively derived extracts were carried out as described in B with a doubled-stranded radioactive probe containing the TATA element and initiator from the hamster c-fos promoter. No difference in binding activity was observed on the c-fos probe at the two temperatures. D, for competition assays, ts13 nuclear extracts were preincubated with different amounts (given in molar excess) of the indicated nonradioactive competitor DNA at 25 °C before the addition of the 32P-labeled cyclin D1 TDE1. Reaction mixtures were subjected to agarose gel electrophoresis followed by autoradiography. Left panel, the competitor DNAs included the cyclin D1 TDE1 (TDE1), the initiator of c-fos (c-fos INR), the TATA box and initiator of c-fos (c-fos core), and a nonspecific DNA fragment (NS). Right panel, competition assays were also carried out with the cyclin D1 TDE1 mutant containing base pair changes in the putative initiator (mt INR) as unlabeled competitor DNA. The position of shifted complexes is indicated by the arrows.

Transcription from only a subset of genes has been shown to be defective at 39.5 °C in ts13 cells. If the lack of promoter DNA binding is responsible for the decrease in transcription, we expect that protein binding to the c-fos core promoter, which displays no reduction in transcriptional activity at 39.5 °C, will be unaffected by temperature. On a DNA fragment containing the TATA box and initiator of the c-fos promoter, comparable amounts of complex formation was detected at 25 °C and 37 °C in ts13 and ts13R3 nuclear extracts (Fig. 6C). Therefore, the defect in promoter binding is specific to the cyclin D1 promoter and correlates with promoter activity as measured by transient transfection into ts13 cells.

Next, we assessed the sequence specificity of DNA binding by testing the ability of different DNA molecules to function as effective competitors. We found that increasing concentrations of the TDE1 as the nonradioactive competitor efficiently blocked retarded complex formation, whereas an unrelated double-stranded oligonucleotide had no detectable effect (Fig. 6D, left panel). Although the c-fos and cyclin D1 promoters have very little sequence similarity, we found that the c-fos TATA box and initiator (c-fos core) and the initiator alone (c-fos INR) competed for DNA binding activity at the cyclin D1 TDE1 (Fig. 6D, left panel). In fact, the c-fos core promoter fragment was more effective than the TDE1 in the competition assays. These data led us to speculate that the TATA box is responsible for the dramatic decrease in complex formation. Because TBP is known to bind the TATA box, we extended our results to suggest that TFIID is binding to the cyclin D1 TDE1 in gel mobility shift assays.

The cyclin D1 promoter contains no TBP binding sites. Components of TFIID have been reported to interact with DNA sequences that fit the initiator consensus motif. The hamster cyclin D1 promoter contains one region within the TDE1 that displays sequence homology to the initiator consensus. To test the importance of this putative initiator sequence in cyclin D1 promoter binding, we examined the ability of the cyclin D1 TDE1 fragment containing mutations in the initiator to function as a competitor in mobility shift assays. The bases mutated were chosen based on the consensus Inr motif YYAN(T/A)YY, with the last five bases conserved in the cyclin D1 TDE1. We elected to mutate the (T/A)Y to GG, because previous studies have reported that similar mutations disrupt initiator function (43). The resulting cyclin D1 initiator mutant was found to be an ineffective competitor suggesting that the protein-DNA interactions detected by gel mobility shift occur through the "initiator" motif in the cyclin D1 TDE1 (Fig. 6D, right panel).

TFIID Binds to TAF1-dependent Element of Cyclin D1-- Our finding that a promoter fragment containing a TATA box functions as a strong competitor for TDE1 binding led us to investigate if the TAF1-dependent DNA binding activity in ts13 cell extracts could be attributed to TFIID. TAFs originally identified in TFIID have been subsequently found in many other complexes. They may even exist as free subunits. Therefore, we wanted to convincingly demonstrate that the mobility-shifted complexes dependent on TAF1 activity were indeed TFIID. First, the ability of HeLa nuclear extracts and partially purified human TFIID to bind to the TDE1 of cyclin D1 was examined (Fig. 7A, lanes 2 and 3). Both protein preparations resulted in mobility shifts that migrated at similar positions on 1.2% agarose/0.5× TBE gels. Complex formation was dramatically reduced upon removal of TFIID by either phosphocellulose chromatography or immunodepletion using a monoclonal antibody against the TAF4 (previously TAFII135) subunit of TFIID (Fig. 7A, lanes 4 and 5). The addition of antibodies against human TBP, TAF1, and TAF4 was also found to prevent complex formation (data not shown). These findings place TFIID and thereby TAF1 at the cyclin D1 TDE1, suggesting that recruitment of TFIID and assembly of the transcription machinery is compromised by the ts13 mutation at 39.5 °C.


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Fig. 7.   Binding of TFIID to the cyclin D1 TDE1 requires TAF1 activity. A, binding assays were performed with the indicated source of human TFIID and the cyclin D1 TDE1 as probe. Lane 1, bovine serum albumin; lane 2, 20 µg of HeLa nuclear extract; lane 3, 100 ng of partially purified human TFIID fraction; lane 4, 20 µg of 0.4 M unbound HeLa nuclear fraction from a phosphocellulose column; lane 5, 20 µg of HeLa nuclear extract immunodepleted with an anti-human TAF4 mAb 3A6. Binding reactions were subjected to 1.2% agarose/0.5× TBE gel electrophoresis and autoradiography. B, twenty micrograms of nuclear extracts prepared from ts13 cells were preincubated with no antibody (lane 1) the anti-HA mAb 12CA5 (lane 2), anti-human TAF1 mAb 6B3 (lane 3), or anti-TBP polyclonal serum (lane 4) for 2 h on ice. Binding reactions were initiated by the addition of the 32P-labeled cyclin D1 TDE1. Reaction mixtures were incubated at 25 °C before agarose gel electrophoresis followed by autoradiography to visualize complex formation. The addition of the anti-TBP supershifted a portion of the protein-DNA complexes in the ts13 extracts. C: Left panel, 20 µg of ts13 or ts13R3 nuclear extract was preincubated in the absence (-) or presence (+) of the anti-HA monoclonal antibody for 2 h on ice. Binding reactions were initiated by the addition of the 32P-labeled cyclin D1 TDE1 and analyzed as described in B. Right panel, additional antibody supershift experiments were performed in which the binding reactions contained extracts from ts13 or ts13R3 cells that constitutively express an HA-epitope tagged ts13 mutant or wild-type human TAF1 protein, respectively. The addition of the anti-HA antibodies supershifted a portion of the protein-DNA complexes in the HA-tagged but not the un-tagged cell lysates.

Having demonstrated that TFIID in human extracts interacts with the cyclin D1 TDE1, we then tested for the components of TFIID in the mobility shifted complexes detected in ts13 nuclear extracts. The addition of a TBP polyclonal antiserum supershifted the complexes formed in ts13 extracts (Fig. 7B). However, the existing anti-TAF antibodies do not recognize the hamster subunits of TFIID. We found that addition of the anti-human TAF1 mAb 6B3 to gel mobility shift assays had no effect on the retarded complexes formed (Fig. 7B). To overcome this hurdle, we generated a ts13 cell line that stably expresses an HA-epitope tagged version of the ts13 allele of human TAF1. A cell line that expresses an HA-tagged wild-type human TAF1 was also established in parallel. Both subunits have been shown to incorporate into the endogenous TFIID complex assembled in the cell (13). Thus, the presence of the HA-epitope provides a means of tracking the TAF1 protein in electrophoretic mobility shift assays.

Nuclear extracts prepared from the HA-tagged cell lines produced a mobility shift at 25 °C that was indistinguishable from the shift detected in the untagged cell extracts (Fig. 7C). Addition of the anti-HA mAb 12A5 retarded the migration of a portion of the HA tag-shifted complexes. No such supershift was observed when the antibody was added to the untagged ts13 and ts13R3 cell extracts. The HA-tagged ts13 and ts13R3 cells continue to synthesize the endogenous untagged version of the largest TFIID subunit. Therefore, TFIID complexes containing the endogenously expressed TAF1 most likely account for the significant amount of retarded complexes that are unaffected by the presence of the anti-HA antibody. Consistent with this explanation, the amount of supershifted complex appears to be proportional to the expression levels of the HA-tagged proteins in the two cell lines, with higher levels detected in the HA-ts13 than the HA-ts13R3 cells. These data strongly suggest that TAF1 and TBP are components of the retarded protein-DNA complexes that bind to the initiator of the cyclin D1 core promoter and most likely contain TFIID.

Binding of TAF1-TAF2 Dimers to the Cyclin D1 Initiator Region-- A number of TAFs within the TFIID complex, in addition to TAF1, have been shown to contact DNA, thereby providing multiple mechanisms for recruiting TFIID to a promoter. To rule out any contribution of these TAFs to the defect in DNA binding, we examined recombinant TAF1-TAF2 heterodimers, which preferentially bind DNA sequences homologous to an initiator motif in gel mobility shift assays (11). Heterodimers of TAF1 and TAF2 were assembled with either the wild-type or mutant TAF1 protein (Fig. 8A), and their ability to bind to the TDE1 was tested at 25 °C and 37 °C in vitro (Fig. 8B). Although TAF1 and TAF2 alone displayed no DNA binding activity, heterodimers containing wild-type TAF1 exhibited significant DNA binding independent of the reaction temperature. By contrast, no detectable binding activity was detected with the ts13 mutant TAF1 containing heterodimers at 37 °C. We confirmed by coimmunoprecipitation analysis that both the wild-type and mutant heterodimers remain intact after incubation at 37 °C (data not shown). Because cyclin D1 lacks a TBP binding site, our data suggest that TAF1 plays a critical role in the efficient recruitment of TFIID through the cyclin D1 initiator.


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Fig. 8.   TAF1-TAF2 dimer binding to the cyclin D1 TDE1 requires TAF1 activity. A, assembly of recombinant TAF1-TAF2 dimers. FLAG-tagged TAF2 and HA-tagged TAF1 (wild-type and ts13 mutant alleles) were expressed in Sf9 cells using the recombinant baculovirus system and assembled into dimers as described under "Experimental Procedures." An aliquot of the individual immunopurified TAFs and the assembled wild-type and mutant TAF1-containing heterodimers were analyzed by SDS-PAGE and visualized by silver staining. The position of each TAF subunit is indicated. B, DNA binding activity of TAF1-TAF2 dimers. The purified wild-type and ts13 mutant TAF1-containing heterodimers and the individual TAFs were examined in electrophoretic gel mobility shift assays using a radiolabeled cyclin D1 TDE1 as probe. Reaction mixtures were incubated at 25 °C or 37 °C, followed by agarose gel electrophoresis and autoradiography. No DNA binding activity was detected at 37 °C with the mutant heterodimer while the wild-type form bound DNA.

Recruitment of TFIID to the Cyclin D1 Promoter Restores Basal Transcription in ts13 Cells-- Our transcription and DNA binding studies collectively suggest that failure to recruit TFIID to the cyclin D1 initiator is responsible for the transcription defect of cyclin D1 detected in ts13 cells. Therefore we tested if providing an alternative recruitment mechanism would restore cyclin D1 transcription at 39.5 °C in ts13 cells. The strategy we took was to insert a consensus TATA element upstream of the transcription initiation site at position -29 (Fig. 9A). For these studies we elected to use -183/+21 cycD1, the smallest fragment of the cyclin D1 promoter that still retained high levels of transcription and displayed a strong requirement for TAF1 activity. We found that insertion of the TATA sequence into -183/+21cycD1-luc only partially restored transcription at 39.5 °C (Fig. 9B). Replacement of the TATA element with a mutant TBP binding site had no effect on transcription levels or TAF1 dependence. Possible interpretations of these data are that 1) TFIID is not being effectively recruited to the promoter or that 2) recruitment of TFIID is not sufficient to replace the activity of TAF1 disrupted by the ts13 mutation.


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Fig. 9.   TFIID recruitment to the cyclin D1 promoter overcomes TAF1-dependent transcription in ts13 cells. A, a schematic diagram of cyclin D1 reporter constructs used in transient transfection assays is provided. The location at which a WT or mutant TATA element was inserted into each construct is shown. B, transcriptional activity in ts13 cells. The indicated cyclin D1 reporter construct, containing either no (-), a consensus (WT), or a mutant (mt) TATA element were transiently transfected into ts13 and ts13R3 cells. The amount of luciferase activity at 33.5 °C or 39.5 °C was determined after 16-18 h and normalized to the level of activity detected with -183/+21 cycD1-luc lacking a TATA element in ts13 cells at 33.5 °C. The data are the average of three independent experiments, each carried out in triplicate. Error bars represent ±S.E. C, the experiments described in B were also carried out with the -107/+21 cycD1-luc reporter construct. The data presented are the average of two independent experiments, each carried out in duplicate. ±S.E. is shown by the error bars. Insertion of a properly positioned TATA element restored basal transcription to the -107/+21 cycD1-luc construct. By contrast, activated transcription from -183/+21 cycD1-luc was not recovered.

We have gathered preliminary evidence suggesting that transcription mediated by the Sp1 sites, which are retained in -183/+21cycD1-luc, also requires TAF1 activity (data not shown). Thus, we repeated the "artificial recruitment" experiments using a cyclin D1 reporter construct (-107/+21 cycD1-luc) that lacks the Sp1 sites (Fig. 9A). Under these conditions, we found that introduction of the TBP binding site restored transcription at 39.5 °C to levels nearly comparable to those observed at 33.5 °C (Fig. 9C). As expected, the mutant TATA element had no effect on transcriptional activity. The absolute amount of activity restored by TFIID recruitment to -183/+21 at 39.5 °C was approximately equal to the level of transcription detected with the -107/+21 cycD1-luc construct at 33.5 °C. These data suggest that basal transcription from the cyclin D1 promoter requires TAF1 activity for efficient recruitment of TFIID to the core promoter through an initiator motif. The observation that artificial recruitment of TFIID was insufficient to overcome the requirement for TAF1 activity at a promoter containing the Sp1 sites and core region suggests that TAF1 serves two independent functions, one in basal and one in activated transcription, both of which are essential for maximal cyclin D1 expression.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The ts13 conditional mutation in TAF1 disrupts transcription from a subset of protein encoding genes resulting in a G1 cell cycle arrest. It still remains unclear, at the molecular level, how the mutation in TAF1 leads to the ts13 transcriptional defect. Chromatin immunoprecipitation studies in yeast have demonstrated that the C terminus of the yTAF1 protein is required for the association of TFIID with promoters (44). Here we provide evidence in mammalian cells that the recruitment of TFIID to the initiator requires a function of TAF1. In addition, we present data suggesting that TAF1 carries out an additional function required for activated transcription mediated by the upstream Sp1 sites. By in vivo genomic footprinting, we have defined a 33-bp DNA element in the cyclin D1 core promoter (TDE1 or TAF1-dependent element 1) that becomes more accessible upon inactivation of a function of TAF1. The TDE1 displays weak homology to a consensus initiator motif. It is also sufficient to support RNA pol II-mediated transcription in a TAF1-dependent manner suggesting this element contains a functional initiator. Gel mobility shift assays suggest that TFIID binds to the cyclin D1 TDE1 and that this binding event involves contacts with the initiator and requires an activity of TAF1. These data are the first pieces of evidence suggesting a molecular mechanism for TAF1 action at the core promoter. Based on our results, we propose that a lack of TFIID recruitment to the initiator is responsible for the decrease in cyclin D1 transcription in ts13 cells. This conclusion is further supported by our finding that the insertion of a consensus TBP binding site is able to fully restore basal but not activated cyclin D1 transcription at 39.5 °C in ts13 cells.

Interestingly, insertion of a consensus TATA element only partially restores transcription to a larger cyclin D1 promoter fragment (-183/+21 TATA cycD1-luc), whereas transcription from the basal promoter (-107/+21 cycD1-luc) can be fully recovered at the nonpermissive temperature. The only difference between the -183/+21cycD1-luc and -107/+21 cycD1-luc constructs is the presence of Sp1 sites in the larger of the two promoters. These Sp1 sites are critical for activated transcription of cyclin D1 in response to several growth factors. Similar results have been reported for the yeast RPS5 promoter, where transcription mediated by upstream activation sequences also requires TAF1 function (45). We have obtained in vivo footprinting data indicating that the interaction of proteins with the Sp1 sites in the hamster cyclin D1 promoter is also compromised at 39.5 °C in ts13 cells (data not shown). Therefore, it appears that TAF1 has a second independent function that is essential for recruitment of Sp1 or a related transcription factor and subsequent activation of cyclin D1 transcription.

The transcriptional control regions of eukaryotic promoters can be divided into two distinct domains: 1) a core promoter consisting of the transcriptional start site and flanking sequences that interact with the general transcriptional machinery and 2) upstream activation sequences (UAS) that are specific binding sites for transcriptional activators. Studies in yeast have demonstrated that UAS selectively recruits TAFs and thus TFIID to TAF-dependent promoters (46, 47). However, the requirement for TAF1 activity appears to be determined by core promoter elements. The UAS from a TAF-independent promoter is unable to recruit TAFs and mediate efficient transcription when inserted upstream of a TAF-dependent core promoter. Therefore, efficient transcription requires a compatible UAS-core promoter combination. It is the specific composition of the upstream and core promoter sequences that will govern whether a promoter depends on an activity of TAF1 for efficient transcription.

Whether or not transcription from a promoter will be compromised appears to depend on the specific allele of TAF1 being characterized. For example, ts2 mutation, isolated by Shen and Green (31), impaired RPS5 and CLN2 transcription. In contrast, the CLN2 promoter functioned normally in yeast harboring the T657K and N568Delta conditional mutations described by Tsukihashi et al. (42). The sets of genes affected by the different TAF1 mutations overlap but are not identical. Compared with the independently isolated yeast TAF1 mutants, the mammalian temperature-sensitive ts13 allele more closely phenocopies the T657 and N568Delta mutants by compromising core promoter function at select genes. More importantly, the introduction of a canonical TBP binding site into a TATA-less promoter was sufficient to overcome the requirement for TAF1 in cells harboring the T657, N568Delta , or ts13 mutation but not in cells containing the ts2 allele. One caveat of all these experiments is that the TATA sequence was inserted into different promoter constructs for the mutant alleles analyzed.

Another parallel between the ts13, T657, and N568Delta alleles is that all the mutations alter a single amino acid within the central histone acetyltransferase domain of the protein. The proteins produced are stably expressed at the restrictive temperature, and do not appear to strongly affect the structure of TFIID. The structural consequence of the glycine to aspartic acid missense mutation of ts13 cells has not been demonstrated. Heterodimers of TAF1 and TAF2 bind to initiator-like sequences and are thought to be important for the recruitment of TFIID to select promoters (11). At 39.5 °C, the ts13-TAF1 protein may undergo significant conformational changes resulting in the inability of TFIID to efficiently associate with the initiator of cyclin D1. In yeast, removal of the C terminus of TAF1 compromises the recruitment of TFIID to promoter DNA (44). Therefore, a more biophysical analysis of the consequences of different mutations in TAF1 will be required to determine which portion of the protein is structurally compromised.

An emerging model is that distinct core promoter elements form an array of binding sites for TFIID. The TATA box is bound by TBP (48, 49), the Inr by TAF1-TAF2 (9, 11), and the downstream promoter element, or DPE, by TAF6-TAF9 (TAFII60-TAFII40) (10). Many of these core promoter elements have relatively flexible sequence requirements. Therefore, the use of multiple correctly positioned elements increases the specificity of TFIID binding. In the case of TATA-less promoters, such as cyclin D1, RPS5, and TUB2, we predict that the contribution of the Inr for efficient TFIID binding is significantly increased compared with TATA-containing promoters. This assumption has led us to propose the following model for the function of TAF1 in RNA polymerase II transcription (see Fig. 10). The TAF1-independent c-fos promoter contains a consensus TATA element such that the primary mechanism for TFIID recruitment is mediated by TBP binding to the TATA box. Upon shifting ts13 cells to 39.5 °C, the ability of TAF1 to interact with the initiator region is disrupted. However, the binding of TBP to the TATA element remains intact and is sufficient to recruit TFIID and initiate transcription. In the cyclin D1 promoter, no high affinity binding site for TBP is present. Recruitment of TFIID depends on the interaction of TAF1 with the initiator. Therefore, at the nonpermissive temperature, the ts13 mutation hinders the primary mechanism by which TFIID can efficiently interact with the core promoter region of cyclin D1 to initiate assembly of the preinitiation complex. This model is based upon the assumption that promoters affected by the ts13 mutation in TAF1 will lack a strong TBP binding site. Although this criterion does not hold for all TAF1-dependent promoters, it does appear that many of the genes affected by the conditional mutations described in yeast and mammalian cells, do not possess a consensus TATA element. The requirement for TAF1 also appears to extend to upstream activation elements. In the case of cyclin D1, TAF1 may serve a dual function, one essential for efficient recruitment of TFIID to the initiation site of transcription and the second to mediate DNA binding to Sp1 sites and subsequent transcriptional activation.


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Fig. 10.   A model for TAF1 function in the recruitment of TFIID. Recruitment of TFIID to the TATA-containing c-fos promoter is via TBP binding to the consensus TATA element. At 39.5 °C in ts13 cells, the disruption of TAF1 initiator binding has no effect on TFIID binding and the initiation of c-fos transcription. By contrast, the cyclin D1 promoter lacks a TBP binding site such that recruitment of TFIID requires the interaction of TAF1-TAF2 with the initiator region. At 39.5 °C, TAF1 initiator binding is abrogated. The lack of an alternative mechanism for efficient TFIID binding compromises the transcriptional activity of the cyclin D1 promoter.


    ACKNOWLEDGEMENTS

We are indebted to K. Lewis for providing excellent technical and laboratory support. The generation of the HA-tagged ts13 cell line by K. S. White and assistance by M. Gelbert with the cloning of the hamster cyclin D1 promoter are also greatly appreciated. We thank R. Tjian for kindly providing the anti-HA ascites fluid and S. Hahn and R. Tjian for critical reading of the manuscript. We especially thank members of the Wang laboratory, including R. M. Squillace and E. L. Dunphy, for valuable discussions and support of this research.

    FOOTNOTES

* This work was supported in part by research project Grant RPG-98-201-CCG from the American Cancer Society.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.

Dagger Supported by National Research Service Award GM7750 from NIGMS, the National Institutes of Health.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AF539477.

§ To whom correspondence should be addressed: Dept. of Pharmacology, School of Medicine, Health Sciences Center, Box 357280, Seattle, WA 98195-7280. Tel.: 206-616-5376; Fax: 206-685-3822; E-mail: ehwang@u.washington.edu.

Published, JBC Papers in Press, February 4, 2003, DOI 10.1074/jbc.M300412200

    ABBREVIATIONS

The abbreviations used are: TFIID, transcription factor IID; TBP, TATA-binding protein; TAF, TBP-associated factors; yTAF, yeast TAF1; CRE, cAMP-responsive element; TDE1, TAF1-dependent element 1; HA, hemagglutinin; mAb, monoclonal antibody; FBS, fetal bovine serum; Inr, initiator; UAS, upstream activation sequence; DMS, dimethyl sulfate.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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