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Molecular Biology and Genetics |
Department of Medicine, Division of Medical Oncology, University of Texas Health Science Center, San Antonio, Texas 78229-3900
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The promoter of the TßRII gene has been cloned and partially characterized (18) . The TßRII promoter lacks TATA box, and four major regulatory elements have been identified. They are two positive regulatory elements [PRE-1 and PRE-2 (located at -219 to -172 and +1 to +50, respectively)], a negative regulatory element [NRE-2 (located at -100 to -67)], and a core promoter (located at -47 to -1). Similar to other promoters that lack TATA boxes, Sp1 is required for transcription of TßRII. There are two consensus Sp1 binding sites, one in the core promoter (located at -25), and one upstream of the NRE-2 (located at -147; Ref. 18 ). Recently, two novel regulatory sites at position -102 and -59 were identified that bind Sp family proteins (19) . An AP-1/cAMP-responsive element-binding protein site is located in the PRE-1 of the TßRII promoter, and an ets binding site is located in the PRE-2 of the TßRII promoter (16) .
Transcriptional repression can be mediated by alterations in the activity or DNA binding properties of trans-activators or repressors or by epigenetic mechanisms that alter DNA structure. A suboptimal level of the transcription factor Sp1 due to methylation is responsible for the lack of TßRII expression in the late passage of MCF-7 cells (20) . Similarly, we recently showed that increasing Sp1 expression was sufficient to reverse transcriptional repression of TßRII in MIA PaCa-2 cells (21) . More recently, two separate studies indicate that HDAC causes loss of TßRII expression in lung and breast cancer cells (22 , 23) . HDACs, together with the opposing enzymes, HATs, control acetylation of histones (24 , 25) . Histone acetylation by HATs mediates transcription by facilitating the binding of transcription factors to DNA. Histone deacetylation by HDACs opposes this effect by restricting the access of transcription factors (25) . HDAC activity is reported to be increased in some tumors compared with normal tissue, and this increase in HDAC activity has been associated with transcriptional repression of tumor suppressor genes that cause growth inhibition and apoptosis (24 , 25) .
We demonstrate here that treatment of PDAC cells with a HDAC inhibitor, TSA, increases TßRII promoter activity and TßRII expression. The aim of the present study was to determine the mechanism of HDAC-mediated repression of TßRII gene expression. During the course of our study, reports on lung (22) and breast cancer cells (23) showed that an inverted CCAAT box was required for HDAC-mediated transcriptional repression of TßRII. We show here, for the first time, that a specific Sp1 site (Sp1C) adjacent to the inverted CCAAT box is required for HDAC-mediated silencing of TßRII. Furthermore, we found that HDAC1 is complexed with Sp1 in PDAC cells and that TSA treatment interferes with this association. These findings indicate a novel mechanism by which HDAC1 mediates transcriptional repression of TßRII.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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HDAC Activity Assay.
Cellular HDAC enzymatic activity was determined using a HDAC assay kit (Upstate Biotechnology, Lake Placid, NY) per the manufacturer’s instructions. Briefly, 10 µg of nuclear extracts from TSA-treated and untreated cells were incubated with [3H]acetate-labeled histone H4 peptide in HDAC assay buffer for 24 h. The released [3H]acetate was extracted with 600 µl of ethyl acetate and separated by centrifugation. Aliquots (200 µl) of ethyl acetate phase in duplicates were measured for radioactivity.
TGF-ß Cross-linking Assay.
Exponentially growing cells were treated with 100 ng/ml TSA or vehicle control for 24 h. Cell monolayers were then incubated with 200 pM 125I-TGF-ß1 (Amersham Pharmacia) at 4°C for 3 h followed by cross-linking with disuccinimidyl suberate for 15 min. The labeled cells were solubilized in 200 µl of 1% Triton X-100 with 1 mM PMSF. Equal amounts of cell lysates were separated by a 4–10% gradient SDS-PAGE under the reducing condition and exposed for autoradiography.
ChIP Assay.
ChIP assay was carried out as described by Boyd et al. (26)
, with some modifications. Briefly, 2 x 107 cells treated with and without 100 ng/ml TSA were cross-linked in 1% formaldehyde for 15 min at room temperature, and cells were collected in cold PBS. Cells were then lysed on ice in cell lysis buffer [5 mM PIPES (pH 8.0), 85 mM KCl. 0.5% NP40, 1 mM PMSF, and 10 µg/ml of both leupeptin and aprotinin] for 10 min. Nuclei were collected by centrifugation and resuspended in nuclear lysis buffer [50 mM Tris-HCl (pH 8.1), 1% SDS, 10 mM EDTA, and protease inhibitors as indicated above]. Samples were then sonicated on ice to break the chromatin DNA to an average length of about 500–600 bp and precleaned with protein A-agarose. The chromatin-protein complexes were immunoprecipitated with anti-acetylated histone H4 antibody (Upstate Biotechnology). The cross-links were reversed by heating at 65°C for 5 h. DNA was then extracted with phenol/chloroform/isoamyl alcohol and subjected to PCR. The primers used for TßRII promoter were as follows: forward primer, 5'-GTAAATACTTGGAGCGAGGAAC-3' (-182/-161); and reverse primer, 5'-ACTCACTCAACTTCAACTCAGC-3'(+54/+33).
Construction of the TßRII Promoter-luciferase Reporter Plasmids with Deletions and Site Mutations.
The 1.8-kb full-length TßRII promoter region from the TßRII promoter-CAT vector was subcloned into pGL3 basic luciferase vector. The serial deletion constructs of the TßRII promoter-luciferase reporter vectors were generated from the full-length construct by PCR using primers with an integrated XhoI site and HindIII site for the forward and reverse primers, respectively. The amplified TßRII promoter regions were then inserted into pGL3 basic vector. The site mutation constructs of the TßRII promoter were generated using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) based on the TßRII promoter-luciferase construct (-291/+50) that contains the major regulatory elements. The mutations were confirmed by DNA sequencing.
Immunoprecipitation and Western Blotting Analysis.
Nuclear extracts of the cells were prepared as described previously (21)
. Nuclear extracts (100 µg) from BxPC-3 and MIA PaCa-2 cells were incubated with 0.5 µg of the respective antibodies [Sp1 and c-Jun/Ap-1 from Santa Cruz Biotechnology (Santa Cruz, CA) and HDAC1 from Upstate Biotechnology] for 16 h at 4°C in 300 µl of radioimmunoprecipitation assay buffer [20 mM Tris-HCl (pH 8.0), 100 mM NaCl, 1 mM EDTA, 1% NP40, 1 mM PMSF, 1 µg of aprotinin, and 1 µg of leupeptin]. Protein A-agarose beads (25 µl) were used to precipitate the associated proteins. Samples were then separated by SDS-PAGE and detected by Western blot analysis using the indicated antibodies.
EMSA.
EMSA was performed as described previously (21)
. Briefly, 5 x 104 cpm of 32P-labeled probes (the sequences from -112 to -65 of the TßRII promoter) and 6 µg of nuclear extracts from MIA PaCa-2 cells were incubated at room temperature for 20 min in binding buffer [10 mM Tris-HCl (pH 7.6), 50 mM NaCl, 1 mM EDTA, 1 mM DTT, 5% glycerol, 1 µg/µl BSA, and 1.5 µg of poly(dI-dC)·(dI-dC)]. For competition analysis, a 100-fold molar excess of unlabeled oligonucleotides was used. For the supershift assay, 2 µg of Sp1 antibody (Santa Cruz Biotechnology) or, as a control, mouse IgG were added and incubated for another 30 min. Samples were resolved on a 5% nondenaturing polyacrylamide gel and exposed for autoradiography.
DAPA.
DAPA was performed as described by Walker et al. (27)
. Briefly, 1 µg of biotin end-labeled double-strand oligonucleotides corresponding to the sequence from -112 to -65 of the TßRII promoter was incubated with 100 µg of nuclear extracts from untreated or TSA-treated MIA PaCa-2 cells for 16 h in DAPA buffer [25 mM HEPES (pH 7.6), 60 mM KCl, 5 mM MgCl, 7.5% glycerol, 0.1 mM EDTA, 1 mM DTT, and 0.25% Triton X-100]. The DNA-protein complexes were then precipitated with 50 µl of Neutravidin-coated agarose beads (Pierce Chemical Co., Rockford, IL). Complexed proteins were resolved by 7.5% SDS-PAGE and detected by Western blot using antibodies to Sp1, HDAC1, and NF-YA.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. An increase in the cell surface expression of TßRII was also observed in TSA-treated cells as determined by a TGF-ß cross-linking assay (Fig. 1B)
. To determine whether TSA-induced expression of TßRII was due to transcriptional activation, cells were transiently transfected with the TßRII promoter-luciferase reporter constructs and treated with and without 100 ng/ml TSA for 24 h. As shown in Fig. 1C
, the TßRII promoter activity was dramatically increased after TSA treatment. No significant induction in basic vector and the insulin-like growth factor I receptor promoter activity was observed (data not shown), suggesting that transcriptional activation of TßRII by TSA is gene specific and that this activation leads to reconstitution of TßRII expression.
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. TSA treatment also caused accumulation of acetylated histone H4 in these cells, whereas HDAC1 protein levels were unchanged (Fig. 2B)
. Correspondingly, an increase in association of the TßRII promoter with acetylated histone H4 protein was determined by a ChIP assay (Fig. 2C)
. These results suggest that repression of TßRII in PDAC cells may be caused, at least in part, by HDAC-mediated condensation of chromatin.
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. To characterize the regions and cis elements that are required for TSA-mediated activation of the TßRII promoter, serial deletion mutants of the TßRII promoter-luciferase reporter constructs were transiently transfected into MIA PaCa-2 cells. Twenty-four h after transfection, cells were treated with either TSA or vehicle control for 24 h, and luciferase activities were measured. A strong induction of the TßRII promoter activity by TSA depends on the -137 to -47 bp region that contains the NRE-2 (-100 to -67) of the TßRII promoter. When the NRE-2 region was deleted, the TSA-mediated induction of the TßRII promoter activity was completely abolished (Fig. 3B)
. To further define the cis elements that regulate responsiveness of TSA, specific site mutations of TßRII promoter-luciferase constructs were generated by site-directed mutagenesis. Sequences of the primers used to generate these site mutations are shown in Table 1
. As shown in Fig. 3C
, mutations of either Sp1C (-102) or inverted CCAAT box (-83) attenuated the TSA-mediated activation of TßRII promoter. Mutation of other Sp1 sites (Sp1A, Sp1B, and Sp1D) or the AP-1 site caused only a minor reduction of TSA-induced promoter activity. These results suggest that the Sp1C site and the inverted CCAAT box play a key role for TSA-mediated activation of TßRII promoter.
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, three specific complexes (complexes A, B, and C) bound to this region as determined by the competition with unlabeled wild-type competitor (Lane 4). We found that both Sp1 and NF-Y bind to this region. Complex A contains Sp1 because it was partially competed from binding by the unlabeled consensus Sp1 oligonucleotides (Fig. 4A
, Lane 5). The identity of the Sp1 protein in this complex was further confirmed by a supershift assay using an antibody specific to Sp1 (Fig. 4A
, Lane 8). Although it may also be possible that other Sp family proteins are present in some of these complexes, we did not analyze for their presence in this study. TSA treatment appears to cause a minor reduction of complex A (Fig. 4A
, Lane 3). However, whether this minor reduction is due to a lack of binding of other Sp family proteins, such as Sp3, should be further verified. Complex B was diminished by competition with unlabeled consensus CCAAT oligonucleotides (Fig. 4A
, Lane 6); whereas oligonucleotides with mutations in the CCAAT sequences failed to compete with the binding of this complex (Fig. 4A
, Lane 7), suggesting that complex B contains CCAAT-binding protein. To determine whether NF-Y protein binds to this region, DAPA was performed using the same oligonucleotides that were end labeled with biotin. NF-YA binds to the -112 to -65 region of the TßRII promoter as detected by using a monoclonal antibody specific to NF-YA (Fig. 4B)
. However, no obvious altered binding was observed between untreated and TSA-treated cells.
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. Treatment of the cells with TSA diminished the association of HDAC1 with Sp1 (Fig. 5B)
. To confirm these results, we performed DAPA. The same oligonucleotides (-112 to -65) used in the EMSA were end-labeled with biotin and incubated with nuclear extracts from untreated and TSA-treated MIA PaCa-2 cells. As shown in Fig. 5C
, both Sp1 and HDAC1 were present in these complexes. TSA treatment reduced the amount of HDAC1 present in the complexes, suggesting that inhibition of HDAC activity affects the binding of HDAC1 to this region of TßRII promoter.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Therefore, it is possible that a separate TSA-responsive element may reside in this region.
The TßRII promoter lacks a TATA box and has several Sp1 binding sites. Two typical Sp1 sites located at -147 and -25 bp upstream of transcription start site are critical in regulating TßRII transcription (18
, 20)
. Another two sites, located at -102 and -59, have recently been reported to bind Sp family proteins and also to play an important role in TßRII transcription (19)
. We have shown previously that overexpression of Sp1 protein restored TßRII expression in MIA PaCa-2 cells (21)
. These results support the premise that Sp1 is a key transcriptional regulator of the TßRII promoter. In this regard, the BxPC-3 cell line is reported to have high levels of Sp1 expression and Sp1 activity (21
, 29)
. This is consistent with our observation that BxPC-3 cells show a greater level of TßRII promoter activity compared with MIA PaCa-2 cells (Fig. 1C)
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We found that both Sp1 and NF-Y bind to the -112/-65 region (which contains both Sp1C and NF-Y sites) of the TßRII promoter. The Sp1C site and the inverted CCAAT box are located close together (-102 and -83, respectively) and lie within a region originally described as a negative regulatory element termed NRE-2 (16) . Thus, the NRE-2 possesses a Sp1 and inverted CCAAT sites that are generally considered to positively regulate promoter activity (30 , 31) . However, HDAC activity appears to selectively block the ability of these sites to activate the TßRII promoter. Studies indicate that HDAC1 can repress transcription by binding to Sp1 (32) and that Sp1 sites are involved in HDAC inhibitor-induced promoter activity (27 , 28 , 32) . Sp1 and HDACs can directly interact in vivo and in vitro (32) . In our study, immunoprecipitation assays of PDAC cells also suggest that Sp1 and HDAC1 are present in the same complex. Thus, Sp1 may serve as a scaffold to recruit HDAC to the promoter and causes chromatin condensation leading to transcriptional repression. NF-Y has also been reported to recruit HDACs (23) . Sp1 and NF-Y have been reported to interact and lead to synergistic transcriptional activation of some genes (31 , 33) . It is therefore possible that these two factors may cooperatively regulate TSA-mediated TßRII transcription. Additional studies are required to determine whether HDAC1 recruited to the NRE-2 region is mediated independently or cooperatively through both of these transcription factors. Moreover, studies are necessary to assess whether a direct interaction of Sp1 with HDAC1 exists in these cells.
It is generally believed that the Sp family of transcription factors (Sp1, Sp2, Sp3, and Sp4) binds to the same consensus sequences. Sp3 is considered to be a transcriptional repressor in some instances (34) . It is possible that Sp3 may also be involved in repression of TßRII or recruitment of HDAC to the TßRII chromatin. Whether an interaction of other Sp family proteins and HDACs occurs in PDAC cells also needs to be determined.
In this study, we provide the first evidence that HDAC1 mediates repression of TßRII transcription by associating with Sp1 in PDAC cells. Inhibition of HDAC activity by TSA interferes with the association of HDAC1 and Sp1. Based on these results, we propose a model in which Sp1 and NF-Y cooperatively regulate TSA-mediated trans-activation of the TßRII (Fig. 6)
. In this model, Sp1 may serve as a scaffold to recruit HDAC1 to the promoter and cause chromatin condensation and gene silencing. Inhibition of HDAC activity by TSA disrupts the association of HDAC1 with Sp1. This favors Sp1 and NF-Y interaction by recruitment of factors that possess HAT activity, such as p300 and PCAF, to the promoter, thus activating TßRII transcription. Because other transcription factors also interact with Sp1, competition between transcriptional regulators and HDACs might be a more general way to regulate gene expression via reversible chromatin modification.
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FOOTNOTES |
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1 Supported by NIH Grant CA96122 and the Pancreatic Cancer Research Network (J. W. F.). 
2 To whom requests for reprints should be addressed, at Department of Medicine, Division of Medical Oncology, University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, TX 78229-3900. Phone: (210) 567-5298; Fax: (210) 567-6687; E-mail: freemanjw{at}uthscsa.edu
3 The abbreviations used are: TGF-ß, transforming growth factor ß; PDAC, pancreatic ductal adenocarcinoma; HDAC, histone deacetylase; HAT, histone acetyltransferase; TßRII, transforming growth factor ß type II receptor; TSA, trichostatin A; RT-PCR, reverse transcription-PCR; PMSF, phenylmethylsulfonyl fluoride; ChIP, chromatin immunoprecipitation; EMSA, electrophoretic mobility shift assay; DAPA, DNA affinity precipitation assay; CMV, cytomegalovirus; AP-1, activator protein 1; NF-Y, nuclear factor-Y.
Received 11/ 5/02. Accepted 3/12/03.
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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