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© The Rockefeller University Press,
0021-9525/1997//19 $5.00
The Journal of Cell Biology, Volume 137, Number 1,
, 1997 19-26
Article |
High Mobility Group 1 Protein Is Not Stably Associated with the Chromosomes of Somatic Cells
DIBIT, San Raffaele Scientific Institute, 20132 Milano, Italy; and Deutsches Krebsforschungszentrum, 69120 Heidelberg, Germany
High mobility group 1 (HMG1) protein is an abundant and conserved component of vertebrate nuclei and has been proposed to play a structural role in chromatin organization, possibly similar to that of histone H1. However, a high abundance of HMG1 had also been reported in the cytoplasm and on the surface of mammalian cells. We conclusively show that HMG1 is a nuclear protein, since several different anti-HMG1 antibodies stain the nucleoplasm of cultured cells, and epitope-tagged HMG1 is localized in the nucleus only. The protein is excluded from nucleoli and is not associated to specific nuclear structures but rather appears to be uniformly distributed. HMG1 can bind in vitro to reconstituted core nucleosomes but is not stably associated to chromatin in live cells. At metaphase, HMG1 is detached from condensed chromosomes, contrary to histone H1. During interphase, HMG1 readily diffuses out of nuclei after permeabilization of the nuclear membranes with detergents, whereas histone H1 remains associated to chromatin. These properties exclude a shared function for HMG1 and H1 in differentiated cells, in spite of their similar biochemical properties. HMG1 may be stably associated only to a very minor population of nucleosomes or may interact transiently with nucleosomes during dynamic processes of chromatin remodeling.
Abbreviations used in this paper: HMG1, high mobility group 1 protein; LDH, lactate dehydrogenase.
High mobility group 1 protein (HMG1)1 is a very abundant and highly conserved component of chromatin which is present in all mammalian tissues and cells. Moreover, HMG1-like proteins also exist in yeast, protozoa, and plants (for reviews see Bustin et al., 1990 and Bianchi, 1995).
HMG1 contains two DNA-binding domains of the HMG box class: they bind with low affinity to single-stranded, linear duplex and supercoiled DNA (Sheflin and Spaulding, 1989; Stros et al., 1994) and with high affinity and specificity to DNA containing sharp bends or kinks, such as four-way junctions or DNA covalently modified with the antitumor drug cisplatin (Bianchi et al., 1989, 1992; Pil and Lippard, 1992). More generally, HMG1 has the ability to transiently introduce bends or kinks into linear DNA and therefore is functionally (but not structurally) similar to the prokaryotic proteins HU and IHF, which it can substitute in several in vitro reactions (for review see Bianchi, 1994).
The evolutionary conservation of HMG1 suggests that it serves an indispensable function. Roles have been suggested in DNA replication, chromatin assembly and disassembly (Bonne-Andrea et al., 1984; Waga et al., 1990; Travers et al., 1994), and transcription (Tremethick and Molloy, 1988; Singh and Dixon, 1990; Ge and Roeder, 1994; Stelzer et al., 1994; Shykind et al., 1995); however, none of these hypotheses has been confirmed unequivocally. More recently it has been proposed that HMG1 plays a role similar to that of histone H1 in the organization and/or maintenance of chromatin. Both HMG1 and histone H1 bind to bent DNA structures (Bianchi et al., 1989; Varga-Weisz et al., 1993), and both appear to interact with linker DNA sequences (Schröter and Bode, 1982; van Holde, 1988). Moreover, Xenopus HMG1 binds to nucleosomes in vitro in much the same way as histone H1 and appears to replace histone H1 during early embryogenesis (Dimitrov et al., 1993, 1994; Nightingale et al., 1996). Likewise, HMG-D, a Drosophila homolog of HMG1, associates with condensed chromatin during embryonal development and is gradually replaced by histone H1 after the midblastula transition (Ner and Travers, 1994).
The present study focuses on the subcellular localization of mammalian HMG1 and its association with chromosomes and chromatin during interphase and metaphase. We show with different antibodies that in nondividing fibroblasts HMG1 is localized exclusively within the nucleus. During metaphase, HMG1, like many transcription factors, detaches from condensed chromosomes and diffuses to the cytoplasm. Histone H1, on the other hand, remains bound to mitotic chromosomes. Moreover, HMG1 is released from interphase nuclei if the membranes are permeabilized with detergents. Thus, the association of mammalian HMG1 with chromatin is much less stable than that of linker histone H1. We suggest that histone H1 prevents HMG1 from binding to nucleosomes and that HMG1 can have a role as a bulk structural protein of chromatin only when histone H1 is absent.
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Other Antibodies
The mouse monoclonal IgM HBC-7, which specifically recognizes the NH2-terminal region of histone H2B (Whitfield et al., 1986), was a gift from B.M. Turner (University of Birmingham, UK). The chicken anti- recAtn antibody was kindly provided by H. Rauvala (Biotekniiken Instituutti, Helsinki, Finland), the rabbit anti–rat H1 antibody by M. Bustin (National Cancer Institute, Bethesda, MD), and the rabbit anti-HMGI(Y) antiserum by D. Thanos (Columbia University, New York, NY). The biotinylated anti-HA mouse mAb 12CA5, recognizing the nonapeptide YPYDVPDYA, was purchased from Boehringer Mannheim Corp. (Mannheim, Germany).
Dot Blots, Immunoprecipitations, and Western Blots
For dot blots, purified proteins were spotted onto wet Immobilon filters (Millipore Corp.). For Western blots, samples were applied to 10% tricine–SDS–polyacrylamide gels (Schägger and von Jagow, 1987) and then electroblotted onto Immobilon filters. Filters were blocked by incubation for 1 h in TBST (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, and 0.1% Triton X-100) containing 10% skim milk and probed with antibodies diluted in TBST containing 0.1% BSA.
For immunoprecipitations, 400 ng of purified proteins dissolved in 200 µl of IP buffer (20 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.1% NP-40, 1 mM EDTA, 1% BSA, 1 mM DTT, and 0.2 mM PMSF) were mixed with 25 µl chIP-AB and 20 µl (packed volume) of swollen protein A-Sepharose (Pharmacia Biotech) presaturated with rabbit anti–chicken Ig (Zymed Labs, Inc., S. San Francisco, CA). The suspension was incubated for 1 h at room temperature with mild agitation. The beads were centrifuged at 800 rpm in a refrigerated Eppendorf microfuge and washed three times with ice-cold IP buffer. The bound protein was eluted using 50 µl of SDSPAGE loading buffer and analyzed by Western blotting.
For detection of chWB-AB, we used a rabbit anti–chicken IgY secondary antibody conjugated to alkaline phosphatase (Chemicon Intl., Inc., Temecula, CA), which was revealed with either BCIP/NBT for color reactions or CDP-Star (Boehringer Mannheim Corp.) for chemiluminescence. The mouse 12CA5 monoclonal antibody was detected by donkey anti– mouse Ig secondary antibody conjugated to HRP (Amersham Corp., Arlington Heights, IL). For histone H1 and HMG-I(Y) detection, incubation with the cognate rabbit primary antibodies was followed by incubation with donkey anti–rabbit Ig conjugated with HRP (Amersham Corp.). For lactate dehydrogenase (LDH) detection, the goat primary antibody was followed by incubation with rabbit anti–goat Ig conjugated with HRP (Southern Biotechnology, Birmingham, AL). HRP was revealed using a Western blotting system (ECL; Amersham Corp.).
Immunofluorescence
All cell lines were cultured as monolayers on glass coverslips. Cells were fixed in 4% paraformaldehyde in PBS for 10 min at room temperature. After rinsing, the coverslips were stored in PBS containing 0.02% sodium azide, no longer than 1 wk at 4°C before use. Different permeabilizing agents were tested: 0.1% NP-40, 0.1% SDS, 0.1% Triton X-100 (all for 5 min at room temperature) and methanol for 5 min at –20°C. Results were always comparable. The coverslips were then incubated with blocking solution (PBS containing 1% BSA and 0.05% Tween-20) followed by primary and secondary antibodies (FITC-conjugated goat anti–chicken Ig antibodies; Southern Biotechnology; and FITC- or rhodamine-conjugated goat anti–mouse Ig antibodies; Sigma Chemical Co., St. Louis, MO) and finally mounted on glass slides with PBS containing 25% glycerol, 100 mg/ ml DABCO as antifading reagent, 0.02% sodium azide, and 100 µg/ml Hoechst 33258. Every incubation was followed by three rinses in PBS containing 0.05% Tween-20. Specimens were examined using an Olympus BH-2 fluorescence microscope with the standard filters for FITC, rhodamine, and Hoechst 33258 emission. Photomicrographs were taken with Ektachrome 400 HC films (Kodak) and either printed directly or scanned and processed using Adobe Photoshop software. Confocal laser scanning microscopy was performed with a Zeiss laser fluorescence microscope equipped with argon and helium lasers. Double fluorescence signals were separated by a two-band filter. The emitted signal was digitalized by Kallman filter collection, and each section was scanned eight times.
Plasmids
Plasmid p-mHMG1 consists of a 9-kb EcoRI–NsiI fragment containing the entire mouse Hmg1 gene, cloned in pBlueScript. To obtain plasmid pmHMG1tag, the sequence 5'-TACCCATACGACGTCCCAGACTACGCT-3', coding for the nonapeptide YPYDVPDYA (HA epitope), was inserted by mutagenic PCR into exon 2 of the Hmg1 gene, between amino acids 1 and 2. This insertion was the only modification to the genomic fragment. The construct was completely sequenced to verify its identity. All oligonucleotides were purchased from Genset (Paris, France). DNA modification and restriction enzymes were from Boehringer Mannheim, Promega Biotech (Madison, WI), and New England Biolabs (Beverly, MA).
Nucleosome Electrophoresis Mobility Shift Assays
A 5' labeled fragment, encompassing mouse rDNA sequences from –160 to +16, was generated by PCR and used for nucleosome assembly by salt dialysis. 5 femtomoles of rDNA was incubated with 200 ng of carrier DNA (phage 
DNA cut with HaeIII) and 100 ng of purified chicken histones in a final volume of 20 µl in a buffer containing 2 M NaCl, 10 mM Tris-HCl, pH 7.6, 1 mM EDTA, 0.1% NP-40, 1 mM β-mercaptoethanol. The preparations were dialyzed overnight against the same buffer containing 50 mM NaCl. Nucleosomes were then incubated with 0–500 ng of HMG1 for 15 min at room temperature. The samples were analyzed by electrophoresis on 0.7% agarose gels in 0.5x TBE. Gels were dried on paper and autoradiographed.
Cell Culture and Transfection
All cell lines were grown in DME supplemented with 10% newborn calf serum (NCF; GIBCO BRL, Gaithersburg, MD). Transfections were performed by the calcium phosphate method. NIH 3T3 clones stably transformed with pmHMG1tag were obtained by transfecting 5 x 105 cells with 20 µg of pmHMG1tag plasmid and 1 µg of pRSV-neo plasmid, followed by selection with 900 µg/ml G418 for 11 d.
Sucrose Gradient Fractionation of Chromatin
About 50 million NIH 3T3 fibroblasts grown to near confluence were washed with ice-cold RSB buffer (10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl2) and resuspended in 25 ml of RSB buffer. The cell suspension was then supplemented with 5 ml of RSB buffer containing 1.2 mM PMSF and 6 µg each of leupeptin, pepstatin, and antipain, homogenized on ice by 20 strokes in a glass teflon homogenizer, and centrifuged at 500 g for 5 min at 4°C. Nuclei were resuspended to a final absorbance of 3.8 at 260 nm in buffer M (10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 3 mM CaCl2, 0.1% NP-40, and 6 µg each of leupeptin, pepstatin, and antipain) and digested in 400 µl aliquots for 5 min at 37°C with varying amounts of micrococcal nuclease (MNase; Worthington Biochemical Corp., Freehold, NJ). The results reported were obtained with 40 U/ml of MNase; different extents of digestion gave comparable results (not shown). The digestion was stopped and nuclei were lysed by the addition of 1.6 ml of 1.5 mM EDTA. The suspension was layered onto a 5–28% linear sucrose gradient (30 ml) prepared in 10 mM Tris-HCl, pH 7.4, 10 mM NaCl, and protease inhibitors as above. The gradients were centrifuged at 24,000 rpm for 30 h at 4°C in a SW27 Beckman rotor. Fractions (1 ml) were analyzed for DNA content after phenol–chloroform extraction and for protein content after precipitation in 20% TCA.
Differential Permeabilization of Cells
NIH 3T3 fibroblasts were grown to subconfluence in 24-well plates (Falcon Plastics, Cockeysville, MD). Adherent cells were washed three times with ice-cold transport buffer (TB buffer), containing 20 mM Hepes, pH 7.3, 110 mM K-acetate, 5 mM Na-acetate, 2 mM Mg-acetate, 1 mM EGTA, 2 mM DTT, and pepstatin, antipain, and leupeptin at 1 µg/ml each (Adam et al., 1990). They were then incubated for 5 min on ice in 80 µl of TB buffer or TB buffer with 0.1% NP-40 or 40 µg/ml digitonin (Sigma Chemical Co.). Supernatants were recovered, and cell remnants were incubated for 10 min at 37°C with 80 µl of TB buffer plus 0.1% NP-40, 10 mM MnCl2, and 20 µg/ml DNaseI (Boehringer Mannheim Corp.). SDS-PAGE loading buffer was then added to supernatants and cell remnants, and samples were analyzed by Western blotting.
Mitotic and interphase cells were obtained and processed essentially as described (Martinez-Balbás et al., 1995). NIH 3T3 fibroblasts, grown in 75-cm2 flasks (Costar, Cambridge, MA) to 
70% confluence, were exposed to 50 ng/ml nocodazole (Janssen Chimica, Beerse, Belgium) for 16 h. Cells blocked in mitosis were detached by manual shaking of the culture flasks and recovered by centrifugation at 800 g. Interphase cells remained attached to the flask bottom and were recovered by mild trypsinization and centrifugation. Both cell populations were washed once in PBS, once in ice-cold TB buffer, and finally resuspended in 0.5 ml of ice-cold TB buffer. Small aliquots were stained with Hoechst 33258 and observed by fluorescence microscopy: >95% of the cells detached by shaking were found to have condensed chromosomes, against <2% of the cells that resisted detachment. Mitotic and interphase cells were permeabilized by adding one volume of ice-cold TB buffer containing 0.2% NP-40, 2 µg/ml each of leupeptin, pepstatin, and antipain, and 0.4 mM PMSF. Cells were then centrifuged for 3 min at 13,000 rpm in a refrigerated Eppendorf microfuge (Brinkman Instruments, Westbury, NY). Supernatants were recovered, and the original volume was reconstituted with TB buffer containing 0.1% NP-40, 10 mM MnCl2, and 20 µg/ml DNaseI. The suspensions were incubated for 10 min at 37°C. SDS-PAGE loading buffer was then added to supernatants and cell remnants, and samples were analyzed by Western blotting.
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Because of the high evolutionary conservation of HMG1, antibodies are hard to obtain, and even repeated injection of rabbits and mice with high doses of purified HMG1 yields sera with low antibody titers. For this study we have raised antibodies to two different truncated forms of HMG1, to avoid the production of antibodies reactive against the COOH-terminal stretch of negatively charged amino acids. Antibody mAP-bA was obtained by immunization of mice with HMG1/M1-F89, a truncated protein encompassing HMG1 box A. The second antibody was obtained by immunizing a hen with HMG1/M1-K147, a polypeptide containing both boxes A and B of HMG1. The antibodies were purified from egg yolks by affinity chromatography using either immobilized native HMG1/M1-K147 (chIP-AB) or denatured HMG1/M1-K147 (chWB-AB). Finally, we used the original anti-HMG1 chicken antibody (anti-recAtn) raised against recombinant rat amphoterin, which is molecularly identical to HMG1 (Parkkinen et al., 1993).
The four different antibody preparations have different reactivities against HMG1, as shown in Fig. 1 A. Reactivity against the native antigens was assessed by dot immunoblots or immunoprecipitation experiments (not shown); reactivity against the denatured antigens was assessed with Western blots (Fig. 1 B). In different cell lines (NIH 3T3 fibroblasts, HeLa and Jurkat cells, and mouse Schwann cells) each of the antibodies stained the nucleus (Fig. 1, C–F and results not shown). The result was the same with several fixation regimes and permeabilization agents. Significantly, the distribution of HMG1 and of AT-rich heterochromatin (revealed by Hoechst 33258 as spots of brighter fluorescence) do not correlate (Fig. 1, compare C with D). Confocal microscopy (Fig. 1, E and F) revealed a diffuse, finely punctate pattern of staining, with little or no HMG1 in the nucleoli.
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80 HMG1-related sequences (Ferrari et al., 1994). To exclude the possibility that our antibodies could cross react with proteins closely related to HMG1, we modified the cloned Hmg1 gene by adding a sequence coding for a nonapeptide from influenza hemagglutinin. The modification does not alter the exon–intron organization of the gene nor its 5' untranscribed region (Fig. 2 D). We obtained several NIH 3T3 clones stably transformed with the tagged gene, one of which (c47) expressed similar amounts of HMG1 and HMG1tag (Fig. 2 E). The anti-HA mAb does not stain wild-type NIH 3T3 fibroblasts but brightly stains the nucleus of transformed cells (Fig. 2 C).
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Confocal microscopy shows that HMG1 is fairly uniformly distributed in the interphase nucleus, with the notable exclusion from nucleoli. We did not see any specific association with (or exclusion from) heterochromatin or speckles containing transcription and splicing factors. This distribution is compatible with a function of HMG1 as a bulk component of chromatin like histone H1, as originally postulated (for review see van Holde, 1988). However, the properties of HMG1 and histone H1 differ in two important respects: displacement from condensed chromosomes and association to chromatin in permeabilized nuclei.
HMG1 is not a component of metaphase chromosomes and diffuses to the cytoplasm when the nuclear membrane is broken down at prometaphase (Fig. 4). By partially solubilizing the membranes of metaphase cells, we can show that condensed chromatin is almost completely devoid of HMG1 but retains core and linker histones as well as the high mobility group protein HMG-I(Y) (Figs. 7 and 8). There is no loss or degradation of HMG1 at metaphase (Fig. 8, compare lanes 1 and 3), and we are unable to detect any M phase–specific posttranslational modification of the protein (results not shown). Thus, HMG1 is displaced from condensed chromosomes like most transcription factors (Martinez-Balbás et al., 1995), in stark contrast to the Drosophila HMG1-like protein HMG-D during embryogenesis (Ner and Travers, 1994).
We have also shown a quantitatively minor association of HMG1 to chromatin even during interphase. HMG1 is almost completely absent from nucleosomal particles obtained by partial digestion of whole nuclei (from Fig. 6 we estimate that a maximum of 2% of the total amount of HMG1 is associated with polynucleosomes) and readily leaks out from detergent-permeabilized interphase cells (Figs. 7 and 8), again in contrast to histone H1 and HMG-I(Y). However, in vitro mammalian HMG1 forms stable complexes with reconstituted nucleosomes (Fig. 3), as previously shown for Xenopus HMG1 (Nightingale et al., 1996). This paradox may be only apparent, however. Ura et al. (1996) showed that Xenopus HMG1 and histone H1 associate to nucleosomal particles in a way that is qualitatively similar but quantitatively different. Both histone H1 and HMG1 bind to linker DNA, have 1:1 stoichiometry to core nucleosomes, protect chromatosomes from micrococcal nuclease digestion, restrict nucleosome mobility, and repress transcription. In short, there is strong evidence that HMG1 and histone H1 compete for the very same sites in chromatin. However, the relative affinity for these sites is almost 20 times higher for histone H1 than for HMG1 (Ura et al., 1996). Taking into account that in differentiated mammalian cells the molar concentration of histone H1 is at least 10 times higher than that of HMG1 (Einck and Bustin, 1985), one can expect that histone H1 will effectively outcompete HMG1 from linker DNA, as we have shown experimentally.
The outcome of the competition between HMG1-like molecules and histone H1 may be completely reversed during early embryogenesis: Xenopus and Drosophila embryos have a large stock of maternally inherited HMG1 and HMG-D, respectively (Kleinschmidt et al., 1983; Dimitrov et al., 1993, 1994; Ner and Travers, 1994). Moreover, histone H1 is absent until after the midblastula transition, which occurs late in both organisms. Thus, HMG1 and HMG-D may "play linker histone" until H1 is expressed and takes over. Whether the same applies to mammalian embryogenesis is not obvious, since midblastula transition occurs at the four-cell stage in the mouse (Hogan et al., 1986), and the concentrations of HMG1 and H1 before that stage have not been measured precisely yet.
In conclusion, our results suggest that histone H1 and HMG1 do not play equivalent roles in differentiated mammalian cells like fibroblasts but do not yet exclude that HMG1 might vicariate H1 during mammalian early embryogenesis. The phenomenon of mitotic displacement and the weak association to chromatin disprove a structural role for HMG1 in the packaging of bulk DNA in differentiated cells but are fully compatible with the stable association of HMG1 with a very minor population of nucleosomes or the involvement of HMG1 in transient interactions with chromatin or individual nucleosomes. HMG1 and 2 can ply and mould DNA and have been shown to cooperate with the progesterone receptor, Oct proteins, and HOX gene products in the control of gene expression (Oñate et al., 1994; Zwilling et al., 1995; Zappavigna et al., 1996). We hold the view that the abundance of HMG1 and 2 simply reflects their versatility and usefulness in the construction of a multitude of transient and specialized nucleoprotein complexes, in defiance of the structural rigidity of naked DNA.
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Acknowledgments |
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This work was supported by grants from Associazione Italiana Ricerca sul Cancro, Telethon (number A.07), and the Human Capital and Mobility programme (CHRX-CT94-0482).
Submitted: 16 September 1996
Revised: 21 October 1996
Please address all correspondence to Marco Bianchi, DIBIT, via Olgettina 58, 4° piano A1, 20132 Milano, Italy. Tel.: 39-2-264-34780; Fax.: 39-2-26434767; E-mail: bianchm{at}dibit.hsr.it
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