ABSTRACT
High mobility group I proteins (HMGI, HMGY and HMGI-C) are a family of low molecular mass non-histone nuclear proteins which constitute an important component of the active chromatin structure. Two members of this family, HMGI and HMGY, have been demonstrated to contribute to the transcriptional regulation of several promoters by interacting with the DNA and with different transcription factors. On the contrary, very little is known about the third member, HMGI-C, which plays an important role during embryonic growth and in the process of cell transformation, its gene being rearranged in a large number of mesenchimal tumors. In this paper we show for the first time that HMGI-C is also able to function as architectural factor, enhancing the activity of a transcription factor, NF-[kappa]B, through the PRDII element of the [beta]-interferon enhancer. Moreover we show that this enhancement is absolutely dependent on the binding of HMGI-C to its target sequence. The demonstration that HMGI-C is able to modulate transcription is thus an important initial step in the identification of genes regulated by this factor.
The high mobility group proteins HMGI-C, HMGI and HMGY belong to a family of non-histone nuclear proteins collectively termed as HMGI proteins. These architectural factors are able to modulate DNA conformation and are involved in fundamental processes such as transcription and recombination (1). Although encoded by a different gene (2), HMGI-C shares some structural homologies with HMGI and HMGY, which are produced by differential splicing from a single gene, and therefore also referred as HMGI(Y) (3). All three proteins in fact have three short basic domains termed AT-hooks which recognise AT-rich DNA sequences via the minor groove, and highly acidic C-terminal tails (1).
HMGI expression levels are very low or undetectable in adult tissues, but after oncogenic transformation their expression is up-regulated reaching levels comparable to those observed during early embryo development (4,5). The existence of a correlation between the expression levels of HMGI proteins and the degree of malignant transformation has suggested their use as tools for tumor diagnosis (6,7).
Direct evidence that HMGI-C participates in the oncogenic process was first provided when the expression of antisense HMGI-C RNA was shown to prevent retrovirally induced neoplastic transformation of rat thyroid cells (8). Moreover, the human HMGI-C gene, mapped to a `multiple aberration region' (MAR) on chromosome 12, has been reported to be a target for translocations in eight different benign solid tumor types (9-11) and to be amplified and rearranged in a number of malignant sarcomas (12). Because of the extremely high incidence of these tumors, this rearrangement can be considered one of the most frequent aberrations in human tumors (13).
It has been shown that disruption of the Hmgi-c gene in mice results in growth retardation and pygmy phenotype characterized by a marked inhibition of adipose tissue development (4). Since HMGI(Y) expression remains unaltered in these mice, this implies that HMGI(Y) cannot compensate for the lack of HMGI-C, suggesting that their functions are not redundant and therefore implying a specific role for each of the HMGI proteins. It could therefore be possible that HMGI-C regulates a different set of genes than HMGI(Y).
Although increasing evidence is accumulating on the biological relevance of the HMGI-C protein in proliferation and development, very little is known about its biochemical properties, such as its DNA-binding specificity and potentiality to modulate transcription. Its homology to HMGI(Y) suggests that it could have a similar function as architectural transcription factor, but despite the increasing number of gene regulatory elements where HMGI(Y) have been shown to cooperate with other proteins to modulate transcription (positively or negatively) (14-20), there are still no data showing that HMGI-C is able to act in a similar way.
In this paper we start to address this point by asking whether HMGI-C is able to modulate transcription. To this aim we have used a simple system consisting of the well characterised PRDII element of the [beta]-interferon promoter (14,21,22). Previous work by Thanos and co-workers has shown that the HMGY protein binds to this element together with the p50/p65 NF-[kappa]B heterodimer to activate transcription from the promoter. In this paper we demonstrate that HMGI-C is able to bind the PRDII element with similar affinity as HMGY and that it can enhance the activating properties of the transcription factor NF-[kappa]B. Moreover, we demonstrate that this enhancement is dependent on the binding of the protein to its target sequence.
The following oligonucleotides were used in gel-retardation assays and for reporter constructs (only the upper strand sequence is shown): PRDII, 5'-gggaaattccgtgggaaattccgagct-3'; mPRDII, 5'-gggagattccgtgggagattccgagct-3'; Ig[kappa]B, 5'-gggactttccgtgggactttccgagct-3'. For the band-shift analysis of the effect of HMGI-C on the binding of the NF-[kappa]B to the PRDII element, a shorter oligonucleotide (16mer), containing only one PRDII element, was used.
Recombinant HMGI proteins were expressed as previously described (20) using the murine HMGI-C and HMGY cDNAs cloned in the vector pAR3038, expression being under control of the bacteriophage T7 promoter. Briefly, proteins were expressed using the BL21 (DE3) Escherichia coli strain which contains the T7 RNA polymerase under lacUV5 promoter control. Cultures were grown to OD600 = 0.6, induced with 0.5 mM isopropyl [beta]-d-thiogalactopyranoside, grown overnight and harvested. HMGI proteins were selectively extracted from bacterial cells with 5% (v/v) perchloric acid and precipitated with acetone-HCl (23). The proteins were purified by reverse-phase HPLC on a BioRad RP304 column using a Waters apparatus as described elsewhere (24). The purified recombinant HMGI proteins were analyzed by mass spectrometry (Perkin-Elmer API 1 spectrometer) to confirm that they had the correct molecular masses. The NF-[kappa]B subunits, p50 and p65, were provided by Dr Ron Hay (University of St Andrews). These subunits were expressed as GST fusion proteins. After purification the proteins were cleaved with thrombin and further purified on a DNA-Sepharose column. The p50/p65 heterodimer was formed by mixing the subunits in an equimolar ratio and incubating for 30 min at 37°C, followed by the addition of 20 mM DTT and incubation on ice for 15 min. Protein concentrations were determined by the Bradford assay using a known protein mixture as a standard.
Double-stranded oligonucleotides were labeled at the 5'-end with [[gamma]-32P]ATP and 10 U of T4 polynucleotide kinase (Epicentre Technologies) or by filling in the 5' overhang with Klenow enzyme and [[alpha]-32P]dCTP. Unincorporated nucleotides were removed by chromatography through a Sephadex G-50 (Pharmacia) spun-column equilibrated in TEN buffer (10 mM Tris-HCl, pH 7.5, 1 mM EDTA, 100 mM NaCl). One hundred fmol of each probe (30 000 c.p.m.) were then separately incubated with increasing concentrations of HMGY or HMGI-C protein (5-200 ng, as indicated) in 20 µl reactions containing 20 mM Tris-HCl, pH 7.5, 75 mM KCl, 5 µg/µl BSA, 1 mM DTT, 13% glycerol for 20 min at room temperature. After incubation, protein-bound DNA and free DNA were separated on native 7% polyacrylamide gel run in 0.5× TBE, at 15 V/cm at 4°C. Gels were then dried and exposed to X-ray films (Hyperfilm MP, Amersham). For band-shifts of NF-[kappa]B and HMGI-C, 12.5 ng of HMGI-C and a range from 50 to 0.5 ng of NF-[kappa]B were used.
Basic TKluc reporter vector was constructed as follows. A BamHI-XbaI fragment, containing the Herpes Simplex Virus (HSV) Thymidine Kinase (TK) promoter, was obtained from pT109luc plasmid (25) and inserted into pGL2basic luciferase reporter vector (Promega), cleaved with BglII-XbaI. PRDII TKluc, mPRDII TKluc and Ig[kappa]B TKluc reporter plasmids were constructed by inserting the corresponding double-stranded (ds) oligonucleotides listed above into the SmaI-SacI sites of BasicTKluc, upstream of the TK promoter.
The pSVHMGY expression construct containing the full-length murine HMGY cDNA under the control of SV40 promoter has been described previously (20). pSVHMGI-C was constructed by inserting into the pGDSV7 expression vector (26) a 1.8 kb EcoRI-HindIII fragment containing the full-length murine HMGI-C cDNA (2). cDNAs for p50 and p65 NF-[kappa]B components, kindly provided by Dr Neil Perkins (Dundee University), were under control of the RSV promoter (27).
NIH-3T3 cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum at 37°C in a humidified 5% CO2 incubator. PC Cl3 and FRTL-5 Cl2 cells were maintained in Coon's modified Ham's F12 medium supplemented with 5% fetal calf serum and six growth factors: the thyrotropic hormone (TSH), insulin, transferrin, somatostatin, hydrocortisone and the tripeptide glycyil-hystidyl-lysine as described (28). Cells were plated at a density of 0.5 × 106 cells per 60 mm diameter culture dish and transfected by the calcium phosphate co-precipitation procedure. For NIH-3T3 transfection, precipitates containing 2 µg of reporter plasmid, 5.2 µg of expression constructs and 0.4 µg of pSV [beta]-galactosidase expression vector (Promega) as internal control, were applied to subconfluent cells 16 h before changing medium. Total amounts of each expression plasmid were kept constant in all the experiments by adding the corresponding empy vectors. For PC Cl3 and FRTL-5 Cl2 transfection, precipitates were left on cells for 1 h, followed by glycerol shock treatment and medium replacement. Due to the lower transfection efficiency of thyroid cells, internal control was provided by adding 0.25 µg of pRL-TK Renilla luciferase expression vector (Promega), which allows a more sensitive detection of enzyme activity.
After transfection (48 h), cells were harvested and lysed with lysis buffer (Promega). For NIH-3T3 cells, firefly luciferase enzyme assays were performed using the Luciferase Assay System (Promega) in a luminometer (Lumat LB 9501, EG&G Berthold) according to the manufacturer's instructions. Colorimetric [beta]-galactosidase assays were performed using o-nitrophenol-[beta]-d-galactoside (ONPG-Sigma) as substrate. Luciferase activity of the reporter vector was then normalized for transfection efficiency using [beta]-galactosidase activity. For PC Cl3 and FRTL-5 Cl2 cells, reporter (firefly luciferase) and control (Renilla luciferase) enzyme assays were performed using the Dual Luciferase Reporter Assay System (Promega).
To investigate if HMGI-C could play a similar role as HMGY in the assembly of a complex able to activate transcription from a combinatorial promoter, the well characterized model of the human [beta]-interferon enhancer was chosen. Previous work has shown that HMGY plays a structural role by facilitating the binding of different transcription factors to their consensus sequences and this contributes to the viral inducibility of the entire enhancer (14). Part of the enhancer consists of the PRDII element, which binds NF-[kappa]B in the major groove and HMGY in the minor groove of the central AT-rich region (14). Binding of the HMGY protein has been shown to potentiate the transcriptional activation by the NF-[kappa]B protein and we have sought to investigate whether the HMGI-C protein will function in a similar manner.
Eukaryotic cells are able to respond to a large number of different extracellular signals and environmental stresses, leading to the activation of specific sets of genes. The understanding of this process is complicated by the fact that many transcription factors like homeodomain-containing proteins and members of the Rel family, once activated can bind specifically to regulatory sequences upstream of a large number of different genes (33), but only a subset of such genes are activated in response to a given signal. One mechanism through which this specificity is achieved is the interaction with accessory factors that can bind nearby, altering the DNA conformation and consequently facilitating or inhibiting the interaction of a transcription factor with DNA. HMGI proteins are a class of non-histone chromosomal proteins that have been shown to fulfill this role, acting as ancillary or architectural factors. By doing so they can increase the affinity (14-18) or can compete for the binding (19,20) of the transcription factor to the DNA. Probably the best characterized example is the virus-inducible enhancer of the human interferon-[beta] (IFN-[beta]) gene (14,21,22) where four positive regulatory domains (PRDI, PRDII, PRDIII and PRDIV) are present. HMGI(Y) are required for the transcriptional activities of both NF-[kappa]B and ATF-2/c-Jun, binding respectively to the PRDII and PRDIV elements of the promoter. For this reason we chose the PRDII element to investigate whether another member of the HMGI family, HMGI-C, is able to function as architectural factor facilitating the transactivating ability of NF-[kappa]B.
HMGI(Y) proteins contain a DNA-binding motif, the AT-hook, which is repeated three times in the protein sequence. It has been shown that this motif is able to specifically bind to the minor groove of AT-rich DNA sequences (1). HMGI-C also contains three AT-hooks suggesting that this protein binds to similar sequences to those bound by HMGI(Y), but since the proteins differ in amino acid sequence outside the AT-hooks and the acidic C-terminals, it is possible that HMGI-C functions differently to HMGI(Y) in the regulation of transcription. There are in fact many examples of nuclear factors sharing the same type of DNA binding domain which, however, display quite different transacting capabilities.
In this report we show that HMGI-C is able to bind to the PRDII element with the same affinity as HMGY. The integrity of the AT-rich core present in the PRDII element is necessary for the binding of both HMGY and HMGI-C since a single base pair substitution is sufficient to drastically decrease their affinity for the DNA. We also demonstrate that HMGI-C is able to enhance the binding of NF-[kappa]B to this element. Furthermore like HMGY, HMGI-C enhances the NF-[kappa]B mediated transcriptional activation of the PRDII element in different cell lines. Since it was not able to affect the basal activity of the PRDII TKluc reporter in the absence of NF-[kappa]B, we can conclude that all the HMGI proteins do not possess transactivation capabilities on their own, but rather act by influencing the activities of classical transcription factors. It is apparent that the HMGI proteins need to bind to DNA in order to exert their effect since HMGY and HMGI-C are not able to enhance NF-[kappa]B mediated transactivation when transfected with the reporter vectors mPRDII TKluc and Ig[kappa]B TKluc which, although able to bind NF-[kappa]B, do not bind HMGY and HMGI-C strongly. It has been shown that HMGI(Y) are able to interact with NF-[kappa]B in the absence of DNA and our data would suggest that such protein-protein interactions might stabilize the ternary complex. This mechanism is thus different to that described for other nuclear proteins like the viral tax protein that can facilitate the binding of transcription factors without binding to the DNA (34).
Thanos and Maniatis originally demonstrated that the HMGI(Y) protein was required for the virally-induced and NF-[kappa]B-dependent promoter activity of the PRDII element, by using an antisense HMGI(Y) construct in transient transfection experiments (14). Transfection of the antisense plasmid into HeLa and Jurkat T cells inhibited the PRDII element, demonstrating the requirement for HMGI(Y), but furthermore may suggest that HMGI-C cannot substitute for HMGI(Y). However T cells do not express HMGI-C (35) and HeLa express very low levels, as judged by northern analysis of HeLa cell RNA (unpublished data). Also, in experiments with antisense HMGI-C we found that the expression of HMGI(Y) as well as HMGI-C is inhibited (8) and so it is possible that an HMGI(Y) antisense would inhibit the expression of HMGI-C as well. Thus, the antisense experiments of Thanos and Maniatis do not exclude the possibility that HMGI-C can bind to and activate the PRDII element in the similar manner as HMGI(Y).
HMGI(Y) act as accessory factors on a number of other inducible promoter elements, interacting with the transcription factors ATF-2 (36), IRF-1 (21), Elf-1 (16) and Oct-2A (17), but it is not known whether HMGI-C interacts with any of these proteins. Whilst it is possible that the three HMGI proteins interact with some common partners, such as NF-[kappa]B, it is likely that the functional specificity of these proteins resides in their interactions with different subsets of transcription factors. Thus, since an altered HMGI-C protein is involved in the abnormal growth and development of fat cells resulting in lipomas (9-11), and that HMGI-C knock-out is responsible in the pygmy mouse for a disproportionate reduction in body fat (14) which is not compensated by HMGI(Y), it is feasible that HMGI-C might specifically interact with transcription factors involved in adipocyte growth and development.
Promoter structure may also play a role in determining whether HMGI(Y) or HMGI-C is recruited. In fact, regulatory elements which contain multiple AT sites interact with HMGI proteins with higher affinity, this cooperative binding is possible since two or three AT-hooks of the same molecule are simultaneously employed in the interaction with DNA (22,29). In this context the different lengths of the spacer regions between the AT-hooks of the HMGI proteins might be relevant (2); AT elements that are differently spaced within promoters might preferentially recruit only one of the HMGI. Thus, an important line of future research will be the identification of target genes involved in cell growth and development that are activated by HMGI-C and the characterisation of the promoters and the transcription factors that it interacts with.
This study was supported by Associazione Italiana per la Ricerca sul Cancro (AIRC), Milano, Italy, Ministero dell'Universit e della Ricerca Scientifica e Tecnologica, Roma, Italy and Universit degli Studi di Trieste, Italy. F.M. was a recipient of a fellowship from Fondazione Italiana per la Ricerca sul Cancro (FIRC). G.H.G. is supported by the Cancer Research Campaign. We thank V.Giancotti for continuous encouragement and advice during this work.
Nucleic Acids Research
Pages
Introduction
Materials And Methods
Oligonucleotides
Protein expression and purification
Band-shift analyses
Plasmid construction
Cell culture and transfections
[beta]-Galactosidase and luciferase assays
Results
HMGI-C binds to the PRDII element in vitro with the same affinity as HMGY
Disruption of the AT-rich core of PRDII abolishes the binding of both HMGY and HMGI-C
HMGI-C enhances the ability of NF-[kappa]B to bind to the PRDII element in vitro
Both HMGY and HMGI-C are able to enhance NF-[kappa]B mediated transcriptional activation through the PRDII element
Mutations that decrease the binding affinity of HMGY and HMGI-C to the PRDII sequence in vitro abolish HMGY/HMGI-C-dependent transcription enhancement in vivo
Discussion
Acknowledgements
References
REFERENCES
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