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Departments of Biochemistry and Molecular Biology and Oncology, University of Calgary, Calgary, Alberta, T2N 4N1 Canada [H. K., P. B., D. V., X. F., Y. H., K. R.]; Molecular Neurobiology [Y. M.] and Department of Internal Medicine and Bioregulation [T. J.], Nagoya City University Graduate School of Medical Sciences, Nagoya, 467-8601 Japan; Department of Biochemistry Hokkaido University Graduate School of Medicine, Sapporo, 060-8638 Japan [H. N.]; Ontario Cancer Institute, Toronto, Ontario, M5G 2M9 Canada [H. V.]; and National Cancer Institute, NIH, Bethesda, Maryland 20892 [C. C. H.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-fetoprotein (AFP) promoter activity but stimulated the p21WAF1 promoter in parallel experiments in the same cell type, similar to the effects of p53. The p47ING1a isoform also repressed AFP promoter activity, but in contrast to other ING isoforms, it repressed the p21WAF1 promoter. p47ING3 up-regulated p21WAF1 promoter activity, but it did not have any effect on the AFP promoter. ING1b and ING2 also repressed the AFP promoter in Hep3B p53-null cell lines, and p53 coexpression enhanced this transcriptional repression. Suppression of AFP gene transcription by ING was strongly dependent on AT-motifs that bind to the hepatocyte nuclear factor 1 (HNF1) transcription factor. Indeed, electrophoretic mobility shift assays confirmed that HNF1 binds to AT-motifs, but we found, surprisingly, that the ING1 complexes binding to these AT-motifs were devoid of HNF1 protein. Both ING1 and p53 were able to suppress AFP transcription and cause p21 induction; hSIR2, a negative regulator of the p53 protein, showed the opposite effects on the AFP promoter and, like HDAC1, repressed p21 promoter activity. In addition, we found that p33ING1b physically interacts with hSIR2, reverses its ability to induce the AFP promoter, and induces acetylation of p53 residues at Lys373 and/or Lys382. These findings provide novel evidence that p33ING1b represses AFP transcription by at least two mechanisms, one of which includes p53. The first is by binding to the AT-motif and excluding HNF1 binding while possibly targeting HAT activity to promoter regions, and the second is by increasing the levels of active, acetylated p53 via binding and inhibiting the ability of hSIR2 to deacetylate p53 protein. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Based upon results from a mouse cDNA microarray assay using antisense ING1b that showed that the albumin and AFP genes were strongly up-regulated (15) and the fact that both genes are regulated by HNF1 binding to the AT-motifs in promoter regions of both genes (16) , it was possible that p33ING1b played an important role in AFP gene regulation through association with HNF1 and/or AT-motifs. Aberrant expression of AFP is characteristic of hepatocellular carcinoma cases and serves as a diagnostic tumor-specific marker (17) . The AFP gene is regulated primarily by the positive transcriptional regulatory factor HNF1 and a negative transcriptional regulatory factor, ATBF1, both of which competitively bind to AT-motifs (18 , 19) . p53 also negatively regulates mouse AFP gene expression through alteration of chromatin structure at the core promoter (20 , 21) .
The SIR2, which links chromatin silencing, metabolism, and cell aging, belongs to the class III family of HDACs (22 , 23) . Recently, the hSIR2SIRT1 protein, the human homologue of the Saccharomyces cerevisiae SIR2 protein, was shown to bind to and deacetylate the p53 protein with specificity for its COOH-terminal Lys382 (24 , 25) . This modification has been implicated in the inactivation of p53 as a transcription factor (26) . Thus, we speculated that ING and hSIR2 might interact with each other and play a role in regulating p53 function.
In this study, we show results from experiments designed to examine the mechanism by which ING family members contribute to regulating the AFP gene through direct association with a region of the AFP promoter and by modifying p53 function through a physical interaction with hSIR2.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Plasmid Construction.
ING1 cDNAs (13)
were subcloned into pCI vector (Promega). pCI-ING2 was constructed by modifying pcDNA3.1-ING2 (7)
. HDAC1 was a gift from Dr. David Bazett-Jones. We isolated ING3 cDNA from Hs68 human diploid fibroblasts by reverse transcription-PCR and ligated it into pCI expression vector (Promega), producing pCI-ING3. HNF1 (pSV2-LFB1S) was constructed by inserting full-length HNF1 DNA into pSV2-CAT vectors so that HNF1 cDNA is controlled by the SV40 promoter, SV40 enhancer, and also used the SV40 polyadenylation signal. ATBF1-A expression vectors (pHßME) were constructed as described previously (27)
. pcDNAhSIR2 and pcDNAhSIR2HY were constructed by inserting full-length hSIR2 and dominant-negative hSIR2HY DNA from pYESir2-puro (24)
into the pcDNA3.1 (+) expression vector (Invitrogen). The pGL3-basic luciferase reporter gene (Promega) was used as the base vector for inserting various human AFP-regulatory elements (28)
. The p21WAF1 promoter-reporter construct containing a p53-binding site inserted into the pGL2-basic luciferase reporter gene (Promega; pGl p21 H2320 vector) was a gift from Dr. Phyllis LuValle (29)
.
Transfection Experiments and Luciferase Assays.
Cells were grown in 12-well tissue culture plates (1.5 x 105cells/well; Becton Dickinson), and the reporter and effector plasmids were cotransfected with the control vector pRL-TK (Promega) using Lipofectamine 2000 (Invitrogen). The latter was used to control for transfection efficiency. Twenty-four or 48 h after transfection, the activities of luciferase were quantified in a luminometer (Junior LB 9509; Berthold Technology) using the Dual Luciferase Reporter Assay System (Promega). All experiments were performed in triplicate. TSA (Sigma) treatments were started 4 h after transfection and continued for another 20 h. Vector controls were always the same plasmid DNA as used in matched expression constructs.
EMSAs.
The 31-mer oligonucleotides corresponding to both strands of the AT-motif in the AFP enhancer region (5'-AGGGAGCCTGATTAATAATTACACTAAGTCA-3'; Ref. 30
) were synthesized and annealed. These double-stranded oligonucleotides were radiolabeled with [
-32P]ATP and T4 polynucleotide kinase (New England BioLabs) and 20,000 cpm of radiolabeled DNA probe, 2 µg of poly(dI-dC) (Pharmacia), 3 µg of BSA in addition to 5 µg of nuclear extracts from cultured Hep G2 cells were used in binding reactions. Reactions were incubated for 30 min at 37°C in binding buffer containing 10 mM Tris (pH 7. 5), 50 mM NaCl, 0.5 mM EDTA, 0.5 mM DTT, 1 mM MgCl2, and 4% glycerol. Competition assays contained a 5- or 50-fold excess of unlabeled double-stranded AT-motif oligonucleotide and an Sp-1-binding site sequence (5'-CTAACTCCGCCCATCT-3') was used as nonspecific competitor. For supershift experiments, four monoclonal antibodies against ING1 (CAb1, CAb2, CAb3, and CAb4; Ref. 31
) and control mouse IgG were concentrated 10-fold using Centricon YM-100 (Millipore) tubes. Two µl of the concentrated monoclonal antibody against ING1 (1.3 µg/µl) or 2 µl of antibody recognizing HNF1 (sc-8986 X; Santa Cruz Biotechnology) were incubated with nuclear extracts for 30 min at room temperature before addition of radiolabeled probe. The reactions were analyzed on 4% nondenaturing gels using 0. 5x Tris-borate-EDTA buffer (pH 8. 0).
Western Blotting and IP.
Cells grown in 6-cm diameter cell culture dishes (7.5 x 105 cells/dish; Corning, Inc.) were cotransfected with pCI-p53 (2.5 µg) and the indicated expression vectors (7.5 µg) using Lipofectamine 2000 (Invitrogen). Cell lysates were electrophoresed through 12.5% SDS polyacrylamide gels, transferred to nitrocellulose membranes, probed with rabbit anti-acetyl-p53 (Lys373 and Lys382) antibody (Upstate biotechnology), stripped, and were reprobed with anti-p53 (DO-1) monoclonal antibody.
IPs were carried out to assess the interaction between p33ING1b and hSIR2 in vivo. Harvested cells were sonicated in radioimmunoprecipitation assay buffer containing 10 mM Tris (pH 7.4), 150 mM NaCl, 10 mM KCl, 1 mM EDTA, 0.5% Tween 20, 0.5% NP40, and 1 µg/ml of the protease inhibitors leupeptin, aprotinin, and pepstatin plus 1 mM phenylmethylsulfonyl fluoride. Cells were centrifuged at 14,000 x g for 5 min to pellet debris at 4°C. Supernatants were incubated for 4 h at 4°C with protein G-Sepharose beads (Amersham Pharmacia Biotech) conjugated with a mixture of four ING1 monoclonal antibodies (31) or control mouse IgG. The Sepharose beads were washed with radioimmunoprecipitation assay buffer and then boiled in SDS sample buffer and electrophoresed through 10% SDS polyacrylamide gels. Gels were transferred to membranes that were probed with rabbit polyclonal antibodies to hSIR2 protein (24) . IP-Western blots of hSIR2 IPs followed by Western blots with ING1 monoclonal antibodies were also performed.
Statistical Analyses.
We performed ANOVA (one-way ANOVA) and Student-Newman-Keuls Multiple Comparisons Tests for analysis among more than three groups and unpaired t tests (two-tail P) for analysis between two groups using Instat Biostatistics, Graph Pad Software, Inc.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. HNF1, which is a positive regulator of AFP gene transcription via binding to the AT-motifs in the enhancer and promoter regions (32)
, strongly up-regulated AFP promoter activity. In contrast, ATBF1, which is a negative regulator of the AFP gene via binding to AT-motifs competitively with HNF1 (18)
, down-regulated AFP promoter activity (Fig. 1A)
. Because some ING family members have been reported to activate p53 as a transcription factor (7)
, we also tested the effects of ING proteins on the activity of a p21WAF1 promoter construct containing a p53-binding site. As expected, p33ING1b, p24ING1c, p33ING2, and p47ING3 modestly up-regulated p21WAF1 promoter activity (Fig. 1B)
but not with effects as great as seen for inhibition of the AFP promoter. Unlike the other family members, p47ING1a repressed both the AFP and p21WAF1 promoters, whereas p47ING3 did not repress the AFP promoter. The functional differences seen between the ING family members suggested that they regulated gene expression by at least two distinct mechanisms. The repressive effects of ING1b and ING2 on the AFP promoter were much weaker in the Hep3B p53-null cell line (Fig. 1C)
, and when p53 activity was restored by cotransfection of wt p53, the repressive effect was enhanced but not to the degree seen in the HepG2 (p53 wt) cells. These results are consistent with ING proteins affecting gene expression through both p53-dependent and -independent pathways.
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, several important regulatory sites in the human AFP gene have been identified including the Enhancer b (Eb) element, two Silencer (S) elements, and a Promoter (P) element (33)
. Both the Eb and P elements possess AT-motifs where HNF1 and ATBF1 competitively bind (16
, 18)
, and silencer binding protein binds to both of the silencer elements and represses transcription (33)
. To test the contributions of these elements to ING protein inhibitory activity on the AFP promoter, we first prepared various truncated AFP reporter plasmids (28)
and measured promoter activities in Hep G2 cells. As shown in Fig. 2B
, the AT-motifs in Eb are important for AFP promoter activity because activity is strongly suppressed in phAF[-2953/+45]-Luc reporter plasmid, which lacks the Eb domain. Increased promoter activity of phAF[-4995/-2953]-[-178/+45]-Luc, which lacks two silencer domains compared with phAF[-4995/+45]-Luc, confirms that these silencer elements exhibit strong repressive effects. The phAF[-178/+45]-Luc reporter also showed relatively high activity (
80% of phAF[-4995/+45]-Luc) because the P region also possesses two AT-motifs but no silencer. As shown in Fig. 2C
, p47ING1a, p33ING1b, p24ING1c, and p33ING2 repressed all of the truncated forms of the reporter plasmids. These results suggest that AT-motifs are important regions for repressive effects by p47ING1a, p33ING1b, p24ING1c, and p33ING2 because they repressed reporters with no silencer elements, whereas even the shortest reporter plasmid, phAF[-178/+45]-Luc, contained AT-motifs.
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. Excess amounts of AT-motif oligo DNA (specific competitor) diminished the bands (Fig. 3
, Lanes 2 and 3), but GC-rich Sp-1-binding site double-stranded oligo DNA (nonspecific competitor) did not compete (Fig. 3
, Lanes 4 and 5). Preincubation with nonspecific antibodies increased the intensity of a rapidly migrated nonspecific band (Fig. 3
, Lane 6). Preincubation with four kinds of ING1 monoclonal antibodies also increased the nonspecific band and diminished the bottom band (Fig. 3
, Lane 7), and anti-HNF1 antibody supershifted the top band (Fig, 3
, Lane 8; HNF1 supershift). This experiment suggested that ING1 and HNF1 were in distinct protein complexes that formed on the AT-motifs. To determine whether direct protein-protein interactions occurred between ING1 and HNF1, we performed IP-Western analysis between ING1 and HNF1, but we obtained no evidence for association (data not shown). We were also unable to detect binding of ING1b produced in an in vitro transcription-translation system to the AT-motif, suggesting that ING1 binds indirectly to the AT-motif, perhaps through binding another protein such as ATBF1 or p53 or that posttranslational modification of ING1b is required for binding (data not shown). Thus, ING1 can bind to the AT-motif independently of HNF1 and represses AFP promoter activity.
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. Previous reports have also indicated that the ING proteins may exert their effects on transcription through altering histone and/or p53 acetylation and have been found in protein complexes containing both HATs and HDAC1 (10, 11, 12, 13)
. To test if the effects of ING proteins on AFP expression were sensitive to acetylation levels, the effects of p33ING1b, p33ING2, and p53 were tested in the presence of the HDAC inhibitor TSA. As shown in Fig. 4
, TSA blocked the inhibitory effect of p53 and completely neutralized the inhibitory effect of ING proteins to the point that TSA, even in the presence of p33ING1b and p33ING2, stimulated the reporter construct. This suggests that the ING proteins and p53 inhibit AFP expression by a mechanism that is sensitive to acetylations levels because ING1b has been previously noted to regulate histone acetylation (13)
. Because the inhibitory effect of p53 is reduced by TSA, which should activate p53 as a transcription factor by promoting p53 acetylation, the loss of inhibitory activity is likely via a mechanism distinct from p53 acting as a gene-specific transcriptional activator.
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. As expected, p53 inhibited and activated the AFP and p21WAF1 promoters, respectively (Fig. 5A)
, whereas hSIR2 had the opposite effects (Fig. 5C)
. Furthermore, HDAC1, which deacetylates histone protein, repressed both the AFP and p21WAF1 promoter (Fig. 5B)
. To determine the effect of the ING proteins on hSIR2-modulated p53 activity, hSIR2 and combinations of ING and hSIR2 were cotransfected into Hep G2 (p53 wt) cells with an AFP promoter-driven reporter construct. As shown in Fig. 5D
, ING proteins inhibited the AFP promoter in the presence hSIR2 in a dose-dependent manner, suggesting that the ING proteins antagonize the effects of hSIR2 upon p53 activity.
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showed that ING1 IPs but not control IPs contained hSIR2 and that the amounts of hSIR2 detected was increased considerably by transfection of p33ING1b and hSIR2. Similarly, hSIR2 IPs but not control IPs contained p33ING1b protein (Fig. 6B)
. Co-IP was not evident between hSIR2 and p33ING2 (data not shown). This result is consistent with p33ING1b blocking the inhibitory effect of hSIR2 on p53 by binding to and inhibiting the deacetylation of p53 by hSIR2.
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. As shown in Fig 7A
, Lane 2, ING1b clearly induced acetylation of p53 at Lys373 and/or Lys382. Control blots using the p53-specific DO-1 antibody showed that total amounts of p53 expressed in each cell transfection were similar (Fig. 7B)
.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, which may be attributable to p47ING1a interacting with HDAC1 avidly (37)
, perhaps inhibiting local histone acetylation. This is consistent with results showing that HDAC1 also repressed both promoters (Fig. 5B)
. Because little is known regarding how p47ING3 acts, it is unclear why it is able to stimulate p21WAF1 promoter activity but has no effect on the AFP promoter. hSIR2 mediates chromatin silencing through NAD-dependent HDAC activity (22
, 23)
, and it also binds to p53 and reduces its transcriptional activity through deacetylation at Lys382 (24, 25, 26)
. Consistent with this mechanism, overexpression of hSIR2 increased AFP promoter activity (Fig. 5C)
, suggesting that the inhibition of p53 by hSIR2 through deacetylation may be more effective for AFP-transcriptional regulation than chromatin silencing through histone deacetylation by hSIR2. If chromatin silencing was the major effect, overexpressed hSIR2 would be expected to repress AFP promoter activity like overexpressed HDAC1 (Fig. 5B)
. These results are consistent with the possibility that the binding of p33ING1b to hSIR2 inhibits its ability to induce p53 deacetylation, thereby helping to maintain the inhibitory activity of p53.
The HDAC inhibitor TSA blocked the inhibitory effects of p53 (Fig. 4)
. p53 is known to recruit HDAC1 via mSin3A or PID, and these complexes are necessary for p53 to transcriptionally repress at least two of its targets, MAP4 and stathmin (38
, 39) . These observations suggest that the repressive effect of p53 on AFP was only partially because of an effect of HDAC1 because the effect of p53 (Fig. 5A)
was much stronger than that of HDAC1 (Fig. 5B)
. Consistent with this idea, TSA also neutralized the inhibitory effects of the p53, p33ING1b, and p33ING2 proteins (Fig. 4)
. As p33ING1b associates with HAT complexes (CBP, p300, PCAF, TRRAP) and HDAC1 (9
, 13)
, TSA might enhance HAT complex activity, in part, by inhibiting HDAC1.
The precise mechanisms of p53 activation by p33ING1b and p33ING2 remain unclear. A recent study suggested that p33ING1b could stabilize p53 by disrupting the regulation of p53 by MDM2 (14)
, and a previous study showed that p33ING2 promotes acetylation of p53 (7)
, possibly through inhibition of hSIR2 (24)
. There is some controversy regarding the role that acetylation plays in the regulation of p53 ability to function as a transcriptional activator (40)
. If acetylation does activate p53, there is the possibility that p33ING1b stimulates p53 by inducing acetylation of its Lys residues (Lys373 and/or Lys382) through HAT activity because p33ING1b associates with and activates components of HAT complexes (13
, 40)
. In our study, p33ING1b bound to hSIR2 (Fig. 6)
, but p33ING2 did not (data not shown). Similarly, a previous report showed that p33ING1b could bind to p53 when overexpressed but p33ING2 could not and that p33ING2 led to increased acetylation of p53 at Lys382 (7)
. Similarly, our data show that p33ING1b also induces acetylation of p53 on Lys residue 373 and/or 382 (Fig. 7)
, suggesting that both p33ING1b and p33ING2 proteins that affect reporters similarly may operate through some common mechanisms. Interaction of ING with other posttranslational modification pathways such as those affecting phosphorylation (41)
, ubiquitination (42)
, ribosylation (43)
, or sumolation (44)
might also play roles in regulating p53-transcriptional activity.
EMSA using nuclear extracts from Hep G2 cells showed that ING1 binds to DNA, in particular to the AT-motif (Fig. 3)
, which is a novel observation because ING1 has been previously reported to act as a cofactor but not as a DNA binding protein. ING proteins contain a highly conserved plant homeodomain, which is found in many proteins involved in chromatin-mediated transcriptional regulation (45)
, but ING does not have an obvious DNA binding domain. Therefore, there is the possibility that ING1 binds to AT-motifs via other DNA binding proteins such as HNF1 and ATBF1. Because EMSA showed that different bands were shifted by anti-ING1 and anti-HNF1 antibodies and IP-Western analysis could not detect interactions between these proteins (data not shown), we believe that ING1 binds to the AT-motif independently of HNF1. However, there still remains the possibility that ING1 binds to AT-motifs via other DNA binding proteins such as ATBF1 or p53. We are currently testing this possibility.
AFP is a sensitive and reliable marker of hepatocellular carcinomas, and p53 mutation is commonly found in a variety of human malignancies, including hepatocellular carcinoma (46, 47, 48)
. Clinicopathological investigations have suggested that higher AFP serum levels are associated with mutant p53 overexpression in hepatocellular carcinoma tissue (17
, 49)
. Our study helps to explain the basis of this clinicopathological result because loss of wild-type p53 function in hepatocarcinoma cells would be expected to increase AFP production and secretion into serum. Although mutations of ING are infrequent, decreased expression levels of ING1 in various types of cancer cells has been reported (50
, 51)
, which would have the effect of altering local HAT and HDAC levels, as well as decreasing the inhibition of hSIR2 as shown in the model presented in Fig. 8
. Consistent with a role in the emergence of this cancer type, it was reported recently that expression levels of ING1b are reduced in hepatocellular carcinoma, particularly those that are at advanced stages and that are poorly differentiated (52)
. In light of these numerous reports of ING1 down-regulation, epigenetic changes such as methylation of the ING1 promoter might, therefore, be important events for AFP production in hepatocellular carcinoma, contributing to their transformed pheno-type (9)
.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This investigation was supported by grants from the Canadian Institutes of Health Research (CIHR) (to K. R.) and the Mochida Memorial Foundation for Medical and Pharmaceutical Research, Japan (to H. K.). K. R. is a Scientist of the CIHR and the Alberta Heritage Foundation For Medical Research (AHFMR), H. K. was the recipient of fellowships from the AHFMR and Alberta Cancer Board (ACB), D. V. holds studentship from the CIHR, AHFMR, and ACB, and H. V. was supported by the Joes Carreras Leukemia Foundation. 
2 To whom requests for reprints should be addressed, at #370 HMRB, 3330 Hospital Drive Northwest, Calgary, Alberta, T2N 4N1 Canada.
3 The abbreviations used are: HAT, histone acetyltransferase; HDAC, histone deacetylase; AFP, -fetoprotein; HNF1, hepatocyte nuclear factor 1; ATBF1, AT-motif binding factor 1; SIR, silent information regulator; TSA, trichostatin A; IP, immunoprecipitation; EMSA, electrophoretic mobility shift assay.
Received 12/13/02. Revised 6/23/03. Accepted 7/16/03.
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