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Originally published In Press as doi:10.1074/jbc.M110233200 on December 17, 2001

J. Biol. Chem., Vol. 277, Issue 9, 7157-7164, March 1, 2002
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HMGB1 and HMGB2 Cell-specifically Down-regulate the p53- and p73-dependent Sequence-specific Transactivation from the Human Bax Gene Promoter*

Michal StrosDagger §, Toshinori Ozaki, Alena BacíkováDagger , Hajime Kageyama, and Akira Nakagawara||

From the Dagger  Institute of Biophysics, Academy of Sciences of the Czech Republic, 612 65 Brno, Czech Republic, and the  Division of Biochemistry, Chiba Cancer Center Research Institute, Chiba 260-8717, Japan

Received for publication, October 24, 2001, and in revised form, December 17, 2001

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The recently cloned gene p73 is a close homologue of p53, which is a crucial tumor suppressor gene for preventing the malignant transformation of cells by inducing cell cycle arrest and apoptosis. Previous reports have shown that architectural DNA-bending/looping chromosomal proteins HMGB1 and HMGB2 (formerly known as HMG1 and HMG2), which function in a number of biological processes including transcription and DNA repair, interact in vitro with p53 and stimulate p53 binding to DNA containing p53 consensus sites. Here, we report that HMGB1 physically interacts with two splicing variants of p73, alpha  and beta  (pull-down assay), and enhances binding of p73 to specific cognate DNA sites (gel-shift assay). Both HMG box domains of HMGB1, A and B, interact with p73alpha . Association of HMGB1 with p73, like the demonstrated ability of HMGB1 to stimulate p73 binding to different p53-responsive elements, requires the oligomerization region and/or region between DNA-binding domain and oligomerization domain of p73 (residues 312-381). Transient transfections revealed that ectopically expressed or endogenous HMGB1 and HMGB2 (antisense strategy) significantly inhibit in vivo both p73alpha /beta - and p53-dependent transactivation from the Bax gene promoter (and much less from Mdm2 and p21waf1 promoters) in p53-deficient SAOS-2 cells. In contrast, HMGB1 and HGMB2 stimulate p73- or p53-dependent transactivation in p53-deficient H1299 cells, irrespective of the promoter used. Our results suggest that ubiquitously expressed HMGB1 and HMGB2 have potential to cell- and promoter-specifically down- or up-regulate in vivo transcriptional activity of different members of the p53 family. A possible mechanism of HMGB1-mediated modulation of p73- and p53-dependent transactivation is discussed.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The HMGB11 and HMGB2 proteins (formerly known as HMG1 and HMG2) are the most abundant members of a large HMG family of chromosomal proteins (1, 2). Vertebrate HMGB1 and HMGB2 proteins contain two similar, but distinct "HMG boxes" (domains A and B), and a long acidic C-terminal "tail." HMGB1 has been numerously implicated in a host of biologically important processes including transcription, DNA repair, recombination, differentiation, development, and extracellular signaling (1, 3). HMGB1 can interact in vitro both with DNA (with a selective preference to distorted DNA structures such as Holliday junctions and DNA modified with anticancer drug cisplatin (see Refs. 4-7)) and a number of biologically important proteins. The latter include transcription factors such as the TATA-binding protein TBP (8), Oct-1/2 (9) and HoxD9 (10), steroid hormone receptors (11), Rel proteins (12), and the tumor suppressor protein p53 (13).

p53 is one of the most extensively studied genes. It is now generally accepted that p53 is a crucial tumor suppressor gene for preventing the malignant transformation of cells. This concept is supported by the fact that loss of p53 functions by genetic alternations represents the most common genetic lesions in human cancers occurring in over than 50% of all the tumors (14). p53 gives rise to a variety of cellular outcomes, most notably cell cycle arrest and apoptosis (14). These activities are due, at least in part, to the ability of p53 to form homotetramers that bind to specific DNA sequences and activate transcription of a great number of its downstream genes, such as the Mdm2 gene (the product of which, the Mdm2 protein, is a key player in the regulation of stability of p53), a cell cycle-control gene p21 (also known as WAF1), and a apoptosis-inducing gene Bax (Bcl2-associated protein X).

p53 has three functional domains: 1) the amino-terminal region involved in transactivation (TA), 2) the central region (the "core domain") involved in specific DNA-binding (DBD), and 3) the carboxyl-terminal region involved in homooligomerization (oligomerization domain (OD)) and regulation of DNA binding. A p53-related gene, p73 (15), encodes six spliced variants (alpha , beta , gamma , delta , epsilon , and zeta ; Ref. 16). The p73 isoforms possess all the functional domains found in p53. Further analyses of p73 showed that not only the primary amino acid sequence (63% identity to p53 within the core domain) but also its function resembles that of p53 (14). p73 diverges from p53 most prominently in the COOH terminus. p73alpha (but not other isoforms) contains a potential protein-protein interaction module, the SAM domain, that is frequently found in proteins involved in developmental regulation. Although the core domain of p53 is the most frequent target for genetic alternations (mainly single point mutations in half of all tumors), very rare mutations in p73 have been found so far despite extensive efforts (16-18). p73, like p53, is induced by treatment of the cells with DNA damaging agents such as ionizing irradiation and anticancer drug cisplatin (19). In addition, p73 can transactivate in vivo genes containing p53-responsive promoters.

It was shown previously that HMGB1 could significantly stimulate in vivo p53-mediated transactivation in p53-deficient H1299 cells (13). The latter finding was explained as a consequence of an HMGB1-mediated enhancement of p53 binding to p53-responsive elements as a result of interactions of HMGB1 with the p53 "core domain" (12). Other authors reported that HMGB1 could bind to p53 exclusively via the extreme basic C-terminal domain (20).

In this paper, we demonstrate that HMGB1 physically interacts in vitro with two splicing variants of p73, alpha  and beta . Both HMG boxes of HMGB1, A and B, interact with p73alpha . Association of HMGB1 with p73, like the demonstrated ability of HMGB1 to stimulate p73 binding to the Bax and Mdm2 promoters by gel-shift assays, requires the oligomerization region and/or region between DNA-binding domain and oligomerization domain of p73 (amino acids 312-381). Transient transfections revealed that ectopically expressed or endogenous HMGB1 and HMGB2 (antisense strategy), significantly inhibit in vivo p73alpha /beta - or p53-dependent transactivation from the Bax gene promoter (and much less from Mdm2 and p21waf1 promoters) in p53-deficient SAOS-2 cells. In contrast, HMGB1 and HMGB2 stimulate p73- or p53-dependent transactivation in p53-deficient H1299 cells, irrespective of the used promoter. Our results suggest that ubiquitously expressed HMGB1 and HMGB2 have potential to cell- and promoter-specifically down- or up-regulate in vivo transcriptional activity of several members of the p53 family. A possible mechanism of HMGB1-mediated modulation of p73- and p53-dependent transactivation is discussed.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Antibodies-- A polyclonal HMGB1 antiserum, generated against calf thymus HMG1, was purified by affinity chromatography on a Sepharose column with covalently linked bacterially expressed rat HMGB1. Polyclonal anti-Sp1 antibody (PEP-2X) were from Santa Cruz Biotechnology. Monoclonal p53 (Ab-6 or DO-1) and p73 (Ab-1, Ab-2, and Ab-3) antibodies were from Calbiochem.

Plasmid Construction-- Human p53 and p73 (plus truncated forms) were amplified from the corresponding cDNAs by PCR using the Pfu DNA polymerase and specific oligonucleotide primers. Sense and antisense HMGB1 and HMGB2 were prepared by PCR from the corresponding human cDNAs as above. The amplified DNA samples were gel-purified, and cloned in-frame into the mammalian expression vector pcDNA3 (Invitrogen). All plasmid constructs were dideoxy-sequenced on both strands.

In Vitro Translation-- HMGB1, p53, p73 and truncated forms were synthesized in vitro from the corresponding cDNAs in the presence of either L-[35S]methionine (Amersham catalog no. AG1094; >37 TBq/mmol) or unlabeled L-Methionine in reticulocyte lysates using the TNT® T7 polymerase quick-coupled transcription/translation system (Promega).

GST Pull-down Assay-- The wild-type p53 and p73 as well as the different p73 deletion mutants were in vitro transcribed/translated with the TNT reticulocyte lysate kit (Promega) in the presence of [35S]methionine. The lysate with labeled proteins was pre-cleared with glutathione-Sepharose beads, followed by addition of GST-HMGB1 or truncated forms of GST-HMGB1 and incubation by rotation for at least 2 h at 4 °C in PD buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.5% Nonidet P-40, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, protease inhibitor mixture (Sigma)). The glutathione-Sepharose beads were then added, and the samples were rotated for at least 1 h at 4 °C. The beads were washed five times with the PD buffer and mixed with 40 µl of 4× concentrated Laemmli buffer, followed by boiling for 5 min. The bound proteins were then resolved by electrophoresis on SDS, 10% polyacrylamide gels. After electrophoresis, the gels were stained in Coomassie Blue R-250, destained, and soaked in Amplify solution (Amersham Biosciences, Inc.) for 30 min. The dried gels were finally exposed to Fuji RX-U films using two intensifying screens at -80 °C.

Isolation of HMGB1-- HMGB1 was isolated under nondenaturing conditions from calf thymus and highly purified to near homogeneity on fast protein liquid chromatography as described previously (6, 24).

Preparation of Cell Lysates-- Cellular lysates were prepared from SAOS-2 and H1299 cells as detailed previously (25), and total protein concentration was determined by protein assay (Bio-Rad).

Electrophoretic Mobility Shift Assay (EMSA)-- p73 and the truncated polypeptides were synthesized in vitro using the TNT reticulocyte lysate kit (Promega) in the presence of unlabeled L-methionine. DNA for EMSA was directly 32P-labeled by PCR of the human Bax (from -138 to -19 from the start site of transcription; Ref. 20) or Mdm2 (intronic, from 109 to 224; Ref. 22) gene promoters using Taq DNA polymerase with [alpha -32P]dATP, and the amplified DNA fragments were purified on 1% agarose gels. Reaction mixtures contained 1× EMSA buffer (20 mM Hepes, pH 7.9, 25 mM KCl, 0.1 mM EDTA, 10% glycerol, 2 mM MgCl2, 2 mM spermidine, 0.5 mM dithiothreitol, 0.025% Nonidet P-40) and 0.1 mg/ml acetylated bovine serum albumin as described (13). Double-stranded poly(dI-dC), approximately 0.2-1 µg/reaction, was present as a competitor DNA. The protein-DNA complexes were loaded onto native 4 or 5% polyacrylamide gels (29:1, acrylamide/N,N'-methylene-bis(acrylamide)) containing 0.5× TBE (45 mM Tris, 45 mM borate, 1 mM EDTA, pH 8.3) and 0.05% Nonidet P-40. The electrophoresis buffer was in 0.5× TBE containing 0.05% Nonidet P-40. The samples were loaded while the gel was running at 50 V, and the gel was then run at 250 V for 3-4 h at ~4-8 °C, followed by vacuum drying onto Whatman no. 3MM chromatography paper. Gels were exposed to Fuji RX-U films using two intensifying screens at -80 °C. Quantification of the bands intensity was performed on a PhosphorImager Storm (Molecular Dynamics) using ImageQuant 4.1 software for data processing. For the permanent record, the gels were scanned and subsequently adjusted for contrast/brightness using Adobe Photoshop.

Luciferase Reporter Gene Assay-- p53-deficient SAOS-2 and H1299 cells were maintained in Dulbecco's modified Eagle's medium and RPMI 1640 medium, respectively, supplemented with 10% heat-inactivated fetal calf serum and antibiotics. The SAOS-2 or H1299 cells (2.5-5 × 104 cells/well of a 12-well plate) were transiently transfected using LipofectAMINE (Invitrogen) or FuGENE 6 (Roche Molecular Biochemicals), respectively. Transfection mixtures contained one or two expression vectors (encoding human p53, p73, HMGB1/HMGB2, or antisense HMGB1/HMGB2 in pcDNA3 plasmid; typically 250 ng each) and two different reporter constructs, the pRL family Renilla luciferase control reporter vector with the cDNA encoding Renilla luciferase under the control of the herpes simplex virus thymidine kinase promoter (pRL-TK vector; 40 ng) and a construct of firefly (Photinus pyralis) cDNA under the control of a promoter containing p53-responsive elements (pGL3-p21waf1-luc, kindly provided by K. Watanabe; pGL2-NA(hMdm2)-luc, Ref. 21; pGL3-Bax-luc, Ref. 23). The luciferase activity was measured 48 h after transfection using a dual-luciferase reporter gene assay system, according to the procedures provided by the manufacturer (Promega).

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Direct Interactions between HMGB1 and p73alpha as Revealed by Pull-down Assay-- It was reported earlier that HMGB1 physically associates with p53 (13, 20). To investigate whether HMGB1 (Fig. 1, panel A) can also interact with other members of p53 family, pull-down assays were carried out with HMGB1 and in vitro synthesized (reticulocyte lysate) isoforms of p73, alpha  and beta . Lysates were incubated with GST-HMGB1 or domains, followed by incubation with glutathione-Sepharose beads and subsequent washing of the beads. Proteins that were associated with glutathione beads were then subjected to SDS-polyacrylamide gel electrophoresis. As seen in Fig. 1 (panels C and D), HMGB1, like its isolated A domain, could clearly bind both isoforms of p73, alpha  and beta  (binding of c-Abl SH2/3 domains to p73alpha , a positive control in panel C, was very weak in the pull-down assay as compared with the previously reported in vivo interactions (27), possibly because of the lack of post-translational modifications). The B domain of HMGB1 could bind p73alpha only when it contained a seven-residue NH2-terminal extension (85TKKKFKD91) (Fig. 1, compare B domain polypeptides designated as B and B7 in panels A and C). Similar results were obtained using the pull-down assay with p53,2 explaining the previously reported inability of the B domain to bind p53 (20). These finding provided the first evidence that the NH2-terminal 85TKKKFKD91 sequence of the HMGB1 B domain is essential not only for binding to DNA (7, 24) but also for interaction with proteins.


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  		Fig. 1.   Direct interactions between HMGB1 and p73. A, a schematic structure of HMGB1 protein and design of HMGB1 domains used for the pull-down assay. B, Coomassie Blue staining of an SDS-polyacrylamide gel with resolved GST or GST-fused HMG proteins used for the pull-down assay. C, 35S-labeled p73alpha that was pull-downed by equal amounts of GST (negative control), GST-HMGB1, and GST-HMGB1 domains. SH2/3 domains of c-Abl were used as a reported positive control for binding to p73 (27). D, 35S-labeled p73beta that was pull-downed by equal amounts of GST or GST-HMGB1.

To determine which region(s) of p73 is involved in binding of HMGB1, pull-down assays were carried out with lysates containing in vitro synthesized 35S-labeled truncated forms of p73alpha . As shown in Fig. 2 (panel A), binding of p73alpha lacking the extreme C-terminal region (peptide 1-550) to HMGB1 was significantly weaker (~10-fold) relative to the full-length p73alpha . On the other hand, binding of p73 lacking both the extreme C-terminal and the SAM domain (peptide 1-484), or p73 truncated up to the middle of the TA2 domain (peptide 1-427), to HMGB1 was similar to the full-length p73alpha . These results suggest that, in the absence of the extreme C-terminal region, the SAM domain only slightly reduces (<2-fold) the ability of HMGB1 to bind p73 in free solution. Removal of the extreme C-terminal region, SAM, and TA2 domains significantly decreased binding (>20-fold) of the p73 peptide (residues 1-381) to HMGB1, with no binding observed with the p73 peptide containing only the TA and DBD (peptide 1-311). The above results indicate that the amino acid residues 312-381 (OD and residues between DBD and OD) are essential for interaction of p73 with HMGB1, with residues 382-426 (TA2) enhancing p73 binding to HMGB1 (Fig. 2).


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  		Fig. 2.   Interactions of HMGB1 with truncated forms of p73. 35S-Labeled p73alpha or its truncated forms were incubated with GST or GST-HMGB1, followed by immobilization on glutathione-Sepharose beads, washing, and SDS, 10% polyacrylamide gel electrophoresis. A, autoradiograms of gels with 35S-labeled p73alpha and truncated forms that were pull-downed by equal amounts of GST or GST-HMGB1. B, p73 and its truncated forms used for the pull-down assay, and a relative assessment of results of p73 binding to HMG1 as indicated by pull-down assay (PD) or HMGB1-mediated enhancement of p73 binding to DNA fragments containing p53-responsive elements as detected by EMSA in Fig. 3. TAD, transactivation domain.

HMGB1 Stimulates Binding of p73 to the Bax and Mdm2 Promoters in Vitro-- It was shown previously that HMGB1 could stimulate p53 binding to DNA fragments containing the p53 consensus sites (13). Here we have investigated the possibility whether HMGB1 could also enhance specific DNA binding of p73. As shown in Fig. 3, binding of in vitro synthesized p73alpha to the Bax or Mdm2 promoters was barely detectable (lanes 4), in agreement with the reported inhibitory role of the extreme COOH-terminal region of p73alpha on specific DNA binding in vivo and in vitro (25). Nevertheless, HMGB1 could slightly enhance binding of p73alpha to DNA (Fig. 3, lanes 5 in panels A and B). Partial deletion of the COOH terminus (peptides 1-550 or 1-381; see Fig. 2B for the design of the used truncated p73 peptides) resulted in a significantly increased binding of the truncated p73alpha to the Mdm2 promoter (lanes 6 and 8 in Fig. 3B), with no visible binding to the Bax promoter (lanes 6 and 8 in Fig. 3A; higher affinity of truncated p73 for Mdm2 promoter was likely the result of the presence of two consensus sequences, unlike a single and imperfect consensus sequence within the Bax promoter, Ref. 28). However, binding of the latter p73 peptides to both promoters was markedly enhanced in the presence of HMGB1 (lanes 7 and 9 in Fig. 3; the absence of free DNA probe in panel B (lanes 7 and 9), is a result of binding of excessive amount of the p73 peptides to DNA probe, whereas the absence of most of the free DNA probe in panel A (lane 9) is caused by a prolonged electrophoresis for better resolution of the complexes). Interestingly, very little, if any, DNA binding was observed with p73 peptide containing only transactivation domain and DBD (peptide 1-311), irrespective of the presence of HMGB1 (Fig. 3, lanes 10 and 11). Our results suggest that amino acid residues 312-381 are required for binding of p73 to both HMGB1 and specific DNA sites that are otherwise recognized by the "core domain" of p73 (Fig. 2B).


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  		Fig. 3.   HMGB1 enhances binding of p73 to the Bax and Mdm2 promoters in vitro. EMSA of 32P-labeled DNA duplexes derived from human Bax (panel A) or Mdm2 promoters (panel B) with 6 µl of p73alpha or its truncated forms synthesized in vitro from the TNT lysates (Promega). C, control lysate with empty vector only. The amount of HMGB1 was 400 ng. FL, full-length p73alpha . F, free probe. Asterisks indicate mobility of the p73(FL)-DNA complexes. All binding mixtures contained 400 ng of poly(dI-dC) as a nonspecific competitor DNA.

HMGB1 Stimulates the p53- and p73-dependent Transcriptional Activation in H1299 Cells as Revealed by Transient Transfections-- Previously it was demonstrated that HMGB1 could stimulate the p53-dependent transactivation from the cyclin G promoter in p53-deficient H1299 cells (12). To find out whether HMGB1 could also stimulate p73-dependent transcriptional activation and whether the effect of HMGB1 is distinct on different promoters, transient transfections were carried out with reporter plasmids containing the Bax, Mdm2, or p21waf1 promoters in H1299 cells (endogenous p73 protein is undetectable in H1299 cell extracts by direct Western blot analysis (Ref. 29), but a weak signal corresponding to p73 m-RNA is to be detected after 25 cycles of reverse transcription-PCR).3 The latter promoters, when placed upstream of the luciferase gene, were previously shown to be activated to varying degree by p73 (25, 26). As shown in Fig. 4, cotransfection of plasmids encoding p73alpha significantly stimulated transcription from all the studied reporter plasmids. Cotransfection of plasmids encoding p73alpha and HMGB1 into H1299 cells resulted in up to ~2-fold enhancement of the p73-dependent transactivation from the Bax, Mdm2, or p21waf1 promoters, with no clear differences among the tested promoters (Fig. 4). Similarly, HMGB1 could stimulate the p53-dependent transactivation from all the promoters studied,2 as also reported preciously for the cyclin G promoter (13). Our results indicate that HMGB1 can up-regulate both the p53- and p73-dependent transactivation in H1299 cells.


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  		Fig. 4.   HMGB1 stimulates the p73-dependent transactivation in H1299 cells. H1299 cells were transfected with 100 ng of one of the Bax, Mdm2, or p21waf1-luciferase reporter plasmids, 10 ng of second reporter plasmid pRL-TK, 36 ng of plasmid constructs encoding p73alpha (HA-p73alpha -pCDNA3), and/or plasmid encoding HMGB1 (HMGB1- pCDNA3) to final 400 ng. Empty vector was used to compensate for equal DNA amounts. Mass-to-mass ratio of p73/HMGB1 was 1:10. All transfections were performed in triplicate. Approximately 48 h after transfection, the cells were lysed and luciferase assays performed. Luciferase activity measured in cell lysates transfected with reporter plasmids and vector only was arbitrarily set as 1.

Ectopically Expressed HMGB1 Significantly Inhibits the p53- or p73-dependent Transcriptional Activation in SAOS-2 Cells as Revealed by Transient Transfections-- It was previously demonstrated that the extent of p73-dependent transactivation in SAOS-2 cells was proportional to the amount of plasmids encoding p73alpha and beta  (30). To find out whether the effect of HMGB1 on the p73- or p53-dependent transactivation is cell-specific, transfection experiments were carried out with p53-deficient osteosarcoma (SAOS-2) cells exhibiting only very low levels of endogenous p73 protein or p73 m-RNA (31). Interestingly, cotransfection of plasmids encoding p73alpha (or the alternatively spliced p73beta ) or p53 with plasmid encoding HMGB1 into SAOS-2 cells resulted in significant (up to ~4-fold) inhibition of p73/p53-dependent transactivation from the Bax promoter (Fig. 5). The observed inhibition was proportional to the amount of the plasmid encoding HMGB1 (Fig. 5). The HMGB1-mediated inhibition of transcriptional activation from the Bax promoter was also observed using p73 truncated up to the OD, peptide 1-381 in Fig. 5C (nuclear localization of the p73-(1-381) peptide was confirmed by confocal microscopy).2 Our results may indicate that the region encompassing the OD, and/or the region between the OD and DBD (amino acids 312-381), is required for HMGB1-mediated inhibition of transactivation. This conclusion is also supported by the fact that truncated p73 containing only TA and DBD (peptide 1-311) was unable to bind HMGB1 (pull-down assay; Fig. 3), explaining the inability of HMGB1 to enhance binding of the p73 peptide to specific DNA sites (EMSA; Fig. 3).


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  		Fig. 5.   Ectopically expressed HMGB1 inhibits the p53- and p73-dependent transactivation from the Bax promoter in SAOS-2 cells. SAOS-2 cells were transfected with 150 ng of Bax-luciferase reporter plasmid, 27 ng of second reporter plasmid pRL-TK, 33 ng of plasmid constructs encoding p73alpha (HA-p73alpha -pCDNA3, panel A), p53 (HA-p53-pCDNA3, panel B), p73beta or p73-(1-381) (HA-p73beta -pCDNA3 or HA-p73-(1-381)-pCDNA3, panel C), and either empty vector (pCDNA3) or different amounts of plasmid encoding HMGB1 (HMGB1-pCDNA3). Empty vector was used to compensate for equal DNA amounts. Mass-to-mass ratios of p73/HMGB1 and p53/HMGB1 were 1:1 and 1:4 (panels A and B, left to right), and 1:10 (panel C). All transfections were performed in triplicate. Approximately 48 h after transfection, the cells were lysed and luciferase assays performed. Luciferase activity measured in cell lysates transfected with reporter plasmids and vector was arbitrarily set as 1.

In contrast to transient transfections with the Bax promoter, cotransfections of reporter plasmids containing either the Mdm2 promoter (Fig. 6, panel A) or the p21waf1 promoter2 with plasmids encoding p53 or p73alpha and HMGB1 resulted in only a slight (up to ~20%) inhibition of the p53- and p73-dependent transactivation in SAOS-2 cells. These results gave evidence that HMGB1 can specifically inhibit p53- and p73-dependent transactivation from the Bax promoter in SAOS-2 cells.


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  		Fig. 6.   Endogenous HMGB1 down-regulates the p53- and p73-dependent transactivation from the Bax promoter as revealed by antisense strategy. SAOS-2 cells were transfected with 150 ng of Mdm2-luciferase (Mdm2-luc, panel A) or Bax-luciferase (Bax-luc, panel B) reporter plasmids, 27 ng of second reporter plasmid pRL-TK, 33 ng of plasmid constructs encoding p73alpha , p73beta , or p53, and either empty vector (pCDNA3) or different amounts of plasmid encoding HMGB1 (HMGB1-pCDNA3) or antisense HMGB1 (asHMGB1-pCDNA3) plus empty vector to compensate for equal DNA amounts (p73/HMGB1 or p53/HMGB1 mass ratios were 1:10). All transfections were performed in triplicate. Luciferase activity measured in cell lysates transfected with reporter plasmids and empty vector was arbitrarily set as 1. asHMGB1, antisense HMGB1.

Endogenous HMGB1 and HMGB2 Down-regulate the p53- or p73-dependent Transcriptional Activity in SAOS-2 Cells as Revealed by Antisense Strategy-- HMGB1 is a relatively abundant architectural nuclear protein, and it is possible that not all HMGB1 molecules are engaged in chromatin structure and may also serve other functions in the cell. To test whether endogenous HMGB1 could affect in vivo transcriptional activity of p53 or p73, plasmids encoding p73alpha /beta or p53 were transiently transfected into SAOS-2 cells together with reporter constructs and plasmid encoding human antisense HMGB1. Expression of antisense (or sense) HMGB1 had little effect on p53- or p73alpha /beta -mediated transactivation from the human Mdm2 (Fig. 6, panel A) or p21waf1 promoters.2 However, similar experiments with the Bax-luciferase reporter plasmid revealed a reproducibly enhanced (>2-fold) p53 or p73alpha /beta -dependent transactivation when the SAOS-2 cells were co-transfected with a construct producing antisense HMGB1 (Fig. 6, panel B). These results clearly demonstrated that endogenous HMGB1 suppresses in vivo transcriptional activity of p53 and two splicing variants of p73, alpha  and beta .

HMGB1 is not the only abundant HMG protein in the nucleus. A closely related HMGB2 protein is present in the nucleus in comparable amounts relative to HMGB1. We have therefore asked whether ectopically expressed HMGB2 could also affect the p53- or p73-dependent transcriptional activation from the Bax promoter. As shown in Fig. 7, HMGB2 also significantly suppresses transcriptional activity of p53 and p73alpha /beta . Effect of ectopically expressed HMGB1 and HMGB2 on transactivation was reproducibly more apparent on p53- (up to ~6-fold) than on p73alpha -dependent transactivation (up to ~4-fold), with HMGB2 being more potent than HMGB1 (Fig. 7). Using the antisense strategy, we have demonstrated that antisense HMGB2 could stimulate (~2-fold) both p53- and p73alpha /beta -dependent transactivation from the Bax promoter. These results suggest that endogenous HMGB1 and HMGB2 down-regulate the p53- and p73alpha /beta -dependent transactivation from the Bax promoter in SAOS-2 cells (Fig. 7). Because the transcriptional activities of all studied promoters were not affected by HMGB1 or HMGB2 (irrespective of the cell line used; see Figs. 4-7), it is very likely that HMGB1/2 can directly target in vivo several members of the p53 family.


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  		Fig. 7.   Both HMGB1 and HMGB2 repress the p53- and p73-dependent transactivation from the Bax promoter in SAOS-2 cells. SAOS-2 cells were transiently transfected with 150 ng of Bax-luciferase reporter plasmid, 27 ng of second reporter plasmid pRL-TK, 33 ng of plasmid constructs encoding p53 (p53-pCDNA3), p73alpha (HA-p73alpha -pCDNA3), or p73beta (HA-p73beta -pCDNA3), and different amounts of plasmids encoding HMGB1 or HMGB2 (HMGB1-pCDNA3 or HMGB2-pCDNA3) or antisense HMGB1 or HMGB2 (asHMGB1-pCDNA3 or asHMGB2-pCDNA3) plus empty vector to compensate for equal DNA amounts (p73/HMGB1 or p53/HMGB1 mass ratios were 1:10). Transcriptional activation was plotted as positive (transcriptional stimulation) or negative (transcriptional repression) relative to the transactivation obtained with reporter plasmids and plasmids expressing p53 or p73 only (arbitrarily set as 1, dashed lines). Results with antisense or sense HMGB1 and HMGB2 are indicated on the upper or lower part of the figure, respectively.

HMGB1 Promotes Binding of Sp1 or Sp1-like Transcriptional Factor to the Bax Promoter-- In contrast to other p53 target genes, like Mdm2 and p21waf1, in which p53-dependent transactivation is mediated by a response element containing two consensus sites, activation of the Bax promoter by p53 is mediated by a cooperative interaction of three adjacent half-sites (32). However, only two of the p53 half-sites represent the minimal Bax response element capable of mediating p53-dependent transactivation (33). The adjacent 6 base pairs 5'-GGGCGTG-3' (the GC box) are required for p53-dependent transactivation, likely by mediating sequence-specific binding to the Sp1 transcription factor and conferring Sp1-dependent transcriptional activation on a minimal Bax promoter (33). This suggests that p53 requires the co-operation of Sp1 (or Sp1-like factor) to mediate transactivation of the human Bax promoter (33).

Identification of factors that bind with a sequence specificity to the Bax promoter could help to unveil a possible mechanism for the differential transactivation of the Bax promoter in H1299 and SAOS-2 cells. To find out whether the H1299 and SAOS-2 cells contain distinct proteins that may differentially bind to the Bax promoter in the presence or absence of HMGB1, EMSA was carried out with cell lysates from both cell lines and the radiolabeled Bax promoter. Although both SAOS-2 and H1299 extracts generated similar gel-shifts with the Bax probe (Fig. 8), additional complexes were detected with the SAOS-2 extract (asterisks). Addition of purified HMGB1 to the SAOS-2 or H1299 cell extracts significantly enhanced binding of several proteins to the Bax promoter (in brackets), including the Sp1 transcription factor (Fig. 8, lanes 2 and 3 and lanes 6 and 7). The identity of Sp1 (or Sp1-like) transcription factor was verified by the appearance of a "supershifted" complex when an anti-Sp1 antibody was added to the lysates (Fig. 8, lanes 4 and 8; addition of control antibody (the whole rabbit IgG) to the cell lysates, or addition of HMGB1 to the probe alone, did not produce any supershift2). These results clearly demonstrated that HMGB1 enhanced binding of Sp1 (or Sp1-like) transcription factor from both SAOS-2 and H1299 cell extracts to the Bax promoter, suggesting than factors other than Sp1 (or Sp1-like) are likely responsible for the differential effect of HMGB1 on modulation of p53- or p73-dependent transactivation from the Bax gene promoter in SAOS-2 or H1299 cells.


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  		Fig. 8.   HMGB1 enhances binding of Sp1 or Sp1-like factor to the Bax promoter in vitro. A DNA duplex corresponding to the -138/-19 sequence from the human Bax promoter was radiolabeled (probe) and used in EMSA. Approximately 16 µg of total proteins from cell lysates from SAOS-2 or H1299 cells were incubated with ~5 ng of the probe alone (lanes 1 and 5) or in the presence of 400 or 800 ng of calf thymus HMGB1 (lanes 2 and 3 and lanes 6 and 7) or with 4 µl of polyclonal anti-Sp1 antibody (Ab) (lanes 4 and 8). The protein-DNA complexes, the intensity of which was markedly enhanced by HMGB1, are in brackets, and complexes that were visibly more intense in SAOS-2 cell lysate are marked by asterisks. The position of the Sp1-DNA complex and the supershifted complex containing Sp1, antibody and DNA, is marked by arrows. All binding mixtures contained 1 µg of poly(dI-dC) as a nonspecific competitor DNA.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In this paper we have shown that HMGB1 can interact with two splicing variants of a candidate tumor suppressor protein p73, alpha  and beta . The existence of p53/p73-HMGB1 interactions may explain the in vitro observed enhancement of recruitment of both p53 and p73 to the Bax and Mdm2 promoters. However, it is possible that protein-protein interactions between p53/p73 and HMGB1/2 serve mainly the purpose to target HMGB1/2 to genes with p53-responsive promoters to facilitate p53/p73 binding by DNA bending. There are numerous published papers demonstrating the ability of HMGB1 and HMGB2 to overcome the axial rigidity of DNA by bending (1, 24, 34, 35). HMGB1 and HMGB2 were shown previously to interact with a number of transcription factors (8-12, 36, 37) including the tumor suppressor protein p53 (13, 20), and in some cases it was demonstrated that the mechanism of enhanced binding of transcription factors to specific DNA sites involves DNA flexure of the target DNA (11, 36, 38, 39). In addition, HMGB1 stimulates specific DNA binding of not only p53 but also p53Delta 30 lacking the extreme COOH-terminal 30 amino acids (13). However, the latter residues (amino acids 363-376) were reported to be necessary for interaction with HMGB1 (20). Thus, the HMGB1-mediated enhancement of p53 binding to DNA, which occurs via the "core" domain of p53 (13), may not require direct interactions between p53 and HMGB1. On the other hand, the stimulatory effect of HMGB1 on specific DNA binding of p73 in vitro (that is manifested, like in the case of p53, via both HMG domains of HMGB1) requires amino acid residues outside of the "core" domain of p73 (amino acids 312-381), which are also essential for interaction with HMGB1 in vitro. The regulatory consequences of p73/p53-HMGB1 interactions are evident from our transfection experiments suggesting differential promoter- and cell-specific effects of HMGB1 on p73/p53-dependent transcriptional activation in vivo.

Many questions regarding the involvement of HMGB1 and HMGB2 in regulation of p53/p73-dependent transactivation remain yet unanswered, such as how HMGB1 encounters in the nucleus proteins of p53 family. p53 is an important factor in determining cellular sensitivity to anticancer agents. Treatment of cells with anticancer drug cisplatin results in increased levels of p53 in the nucleus (19). HMGB1 was recently reported to be significantly overexpressed in cisplatin-resistant human cancer cells (40), and DNA regions damaged by cisplatin or UV irradiation are high affinity binding sites for both p53 and HMGB1 (5, 7, 41-43). Thus, we can speculate that HMGB1 can meet and bind p53 (and also p73) at the sites of DNA damage from which the p53/p73-HMGB1 complexes are delivered to p53-responsive promoters where HMGB1 enhances binding of p53/p73 by increasing DNA flexure. The enhanced binding of p53 (and likely also p73) to pre-bent DNA would be in agreement with the observed enhanced stability of p53-DNA complexes by increasing the DNA-bending angle (44). However, DNA bending by HMGB1 might not only recruit p53/p73 and other transcription factors (such as Sp1), it could also adversely prevent binding of certain transcription factors and/or inhibit their binding by interference with the DNA-bound HMGB1 (as demonstrated by shielding the cisplatin DNA lesions from the nucleotide excision repair or by interference of HMGB1 with recruitment of transcription factor TFIIB within the pre-initiation complex leading to inhibition of transcription; Refs. 45-48). p53 was reported previously to cooperate with Sp1 or Sp1-like factor to mediate transcriptional activation of human Bax (33) and cyclin-dependent kinase inhibitor p21waf1gene promoters (49). However, a differential effect of HMGB1 on p53/p73-dependent transactivation from the Bax and p21waf1 promoters in SAOS-2 cells argues against the involvement of Sp1 in this regulatory function by HMGB1 (this report). In support of this, HMGB1 could enhance binding of endogenous Sp1 to the Bax promoter from both H1299 and SAOS-2 cell lysates. A possible mechanism that could explain the observed modulation of p53/p73-dependent transactivation by HMGB1 in vivo could be a differential interaction of HMGB1 with cell-specific endogenous co-activators and/or co-repressors as well as with other nuclear factors, directly or indirectly affecting transcription of genes containing p53-responsive elements.

The ability of the proapoptotic Bax to function as an important mediator of p53-dependent apoptosis and a suppressor of oncogenic transformation is supported by a number of studies (50-52). Several tumor-derived p53 mutants fail to activate transcription through the Bax promoter, which leads to a failure to induce apoptosis (21, 29). The resistance of certain tumor cell lines to therapy by irradiation was shown to be associated with inability of a wild-type p53 to induce Bax expression (51, 53). In addition, identification of Bax as an obligatory downstream effector of p53 in the suppression of tumor growth suggests that the ability of p53 to activate transcription from the Bax promoter is important for the functioning of p53 as a tumor suppressor (54). Our finding that HMGB1 and HMGB2 proteins can modulate in vivo transcriptional activity of p53 and p73 from the Bax gene promoter raises a number of questions regarding a possible involvement of HMGB1 and HMGB2 in modulation of the Bax gene expression and apoptosis.

In conclusion, we have discovered that ubiquitously expressed chromosomal proteins HMGB1 and HMGB2 have potential to cell-specifically down- or up-regulate in vivo transcriptional activity of different members of the p53 family from the Bax promoter. These findings cannot be explained solely by the observed HMGB1-mediated in vitro enhancement of specific DNA binding of p73/p53 and Sp1 (or Sp1-like) transcription factor, suggesting an involvement of other cellular factors that can modulate in vivo the ability of HMGB1 and HMGB2 to affect the transcriptional activity of genes containing p53-responsive promoters.

    ACKNOWLEDGEMENTS

We thank Dr. S. Sakiyama for interest in the project, continuous support, and critical reading of the manuscript, and Dr. K. Watanabe for advice on transient transfections.

    FOOTNOTES

* This work was supported in part by Grants A7004902/1999 and IAA5004105 from the Internal Grant Agency of the Academy of Sciences of the Czech Republic and Grants 301/99/0691 and 301/02/0952 from the Grant Agency of the Czech Republic (to M. S.)., and by a grant-in-aid from the Ministry of Health and Welfare of Japan for a New 10-year Strategy of Cancer Control (to A. N.).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.

§ Supported by the Foundation for Promotion of Cancer Research. To whom correspondence may be addressed: Inst. of Biophysics, Academy of Sciences of the Czech Republic, Královopolská 135, 612 65 Brno, Czech Republic. Tel.: 420-5-41517183; Fax: 420-5-41211293; E-mail: stros@ibp.cz.

|| To whom correspondence may be addressed: Division of Biochemistry, Chiba Cancer Center Research Inst., 666-2 Nitona, Chuoh-ku, Chiba 260-8717, Japan. Tel.: 81-43-264-5431; Fax: 81-43-265-4459; E-mail: akiranak@chiba-ccri.chuo.chiba.jp.

Published, JBC Papers in Press, December 17, 2001, DOI 10.1074/jbc.M110233200

2 M. Stros, unpublished results.

3 J. Shimbo, unpublished results.

    ABBREVIATIONS

The abbreviations used are: HMG, high mobility group; EMSA, electrophoretic mobility shift assay; GST, glutathione S-transferase; Ab, antibody; FL, full-length; HA, hemagglutinin; TA, transactivation domain; DBD, DNA binding domain; OD, oligomerization domain; SAM, alpha sterile motif.

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RESULTS
DISCUSSION
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