* Dipartimento di Genetica e di Biologia dei Microrganismi, Universitá di Milano, 20133 Milano, Italy; 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.
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 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 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 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.
Preparation of Antibodies against HMG1
HMG1/M1-K89 and HMG1/M1-F147 were expressed and purified in Escherichia coli as previously described (Falciola et al., 1994 Other Antibodies
The mouse monoclonal IgM HBC-7, which specifically recognizes the
NH2-terminal region of histone H2B (Whitfield et al., 1986 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 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 Nucleosome Electrophoresis Mobility Shift Assays
A 5 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 × 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 Mitotic and interphase cells were obtained and processed essentially as
described (Martinez-Balbás et al., 1995 Anti-HMG1 Antibodies Stain the Cell Nucleus
Several previous cell fractionation and immunofluorescence studies indicated that HMG1 is located both in the
cytoplasm and in the nucleus of mammalian cells (Bustin
and Neihart, 1979 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.
Tagged HMG1 Protein Is Accumulated
in the Cell Nucleus
HMG1 is a member of a protein family including HMG2
and ~80 HMG1-related sequences (Ferrari et al., 1994
HMG1 Protein Associates to Nucleosomes In Vitro
but Not in Condensed Chromosomes
Nightingale et al. (1996)
To further confirm the association of HMG1 with nucleosomes, we then probed whether HMG1 is an integral
component of condensed chromosomes, like its proposed
Drosophila homolog, HMG-D (Ner and Travers, 1994
HMG1 Is Weakly Associated with Nucleosomes in
Interphase Chromosomes
The results shown above suggest that the bulk of HMG1 is
not associated with mitotic chromatin. To examine HMG1's
association to chromatin during interphase, nuclei of NIH
3T3 fibroblasts were partially digested with micrococcal
nuclease, and nucleosomal particles were fractionated on
sucrose gradients (Fig. 6). Consistent with previous data,
histone H1 was found associated with mono- and polynucleosomes. Most of HMG1, however, was recovered at the top of the gradient in fractions that do not contain DNA;
only longer exposures of the Western blot revealed a minor amount of HMG1 in the fractions containing polynucleosomes (Fig. 6, lanes 20-26).
Since our results are in contrast to the prevailing view
that HMG1 is a structural component of chromatin (van
Holde, 1988), we studied the interaction of HMG1 with interphase chromatin by a more gentle technique. Monolayers of fibroblasts were treated either with digitonin, which
permeabilizes the plasma membrane exclusively (Diaz and
Stahl, 1989
We also compared directly the detergent-mediated release of HMG1 from metaphase and interphase cells. NIH
3T3 fibroblasts were treated with nocodazole, an inhibitor
of microtubule assembly; mitotic cells were shaken off the
dishes, while nonmitotic cells were detached by mild trypsin digestion (Martinez-Balbás et al., 1995
These results demonstrate that HMG1 is released as
easily from condensed and noncondensed chromosomes.
Since HMG1 is apparently excluded from mitotic chromosomes, its association to interphase chromatin is also very
weak.
By using several purified antibodies, we have clearly established that HMG1 protein is exclusively located in the
nucleus in several cell lines. The discrepancy with previous
results (for review see Einck and Bustin, 1985 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 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 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 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 and Bianchi, 1995
).
; 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
).
; 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
).
Materials and Methods
). Antibody
mAP-bA was raised by injecting BALB/c mice four times with 200 µg of
HMG1/M1-F89 at 2 wk intervals. Chicken antibodies were raised by injecting 200 µg of HMG1/M1-F147 three times at 2 wk intervals. Antibodies mAP-bA and chIP-AB were affinity purified on the cognate native antigen immobilized on CL-4B Sepharose (Pharmacia Biotech, Piscataway,
NJ) at the concentration of 1 mg/ml and eluted with 0.1 M glycine-HCl
(pH 2.5). Antibody chWB-AB was affinity purified using a strip of Immobilon filter (Millipore Corp., Bedford, MA) bearing 2 mg of HMG1/M1F147 transferred from an SDS-PAGE gel.
), 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).
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.
-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).
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
-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 0.5× TBE. Gels were dried on paper and autoradiographed.
). 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.
). 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.
Results
; Isackson et al., 1980
; Einck et al., 1984
;
Kuehl et al., 1984
; Mosevitsky et al., 1989
). Moreover, HMG1
has been localized to the surface of neural and fibroblastoid cells (Parkkinen et al., 1993
). These results are not
necessarily in conflict with HMG1's proposed role(s) as a
DNA-binding protein but warranted a reconsideration of
HMG1's subcellular localization.
).
Fig. 1.
(A) Reactivity of
the various anti-HMG1 antibodies used in this study. For
each antibody preparation
the species of origin is indicated (m, mouse; ch,
chicken), and the ability (+)
or inability () to recognize
boxes A and B of HMG1 by
Western blotting (denatured) or by immunoprecipitation (native) is also shown.
The notation ± indicates that
recovery of the HMG1bA
polypeptide by immunoprecipitation is <5% under the
conditions indicated under
Materials and Methods. (B)
Reactivity of the chWB-AB
antibody in Western blots.
Whole NIH 3T3 cells were
lysed by addition of SDSPAGE loading buffer, and
10 µg of total protein was
loaded on a 10% tricine-SDS-
polyacrylamide gel (lane
3T3), alongside 20 ng of purified recombinant HMG1bA polypeptide (lane bA) or 20 ng of purified recombinant HMG1bB polypeptide (lane bB). (C and D) Anti-HMG1 antibodies stain the cell nucleus. NIH 3T3 cells were grown on glass coverslips, fixed with paraformaldehyde, permeabilized with 0.1% NP-40, and stained with anti-recAtn (C) and Hoechst 33258 (D). (E and F) Localization of HMG1 by confocal microscopy. HeLa cells were fixed with
paraformaldehyde, permeabilized with 0.1% SDS, stained with mAP-bA antibody, and viewed in green fluorescence (E) or by phase
contrast microscopy (F). Bars, 10 µm.
[View Larger Versions of these Images (85 + 15 + 28K GIF file)]
).
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).
Fig. 2.
The product of the
Hmg1 gene localizes to the nucleus. NIH 3T3 fibroblasts stably transfected with the
pHMG1tag plasmid were fixed
and stained simultaneously
with (A) Hoechst 33258, (B)
anti-HMG1 antibody chIPAB, and (C) monoclonal antibody 12CA5 recognizing the
HA epitope. (D) Structure of
the Hmg1-tag gene. The mouse gene Hmg1, which codes for
protein HMG1, is transcribed
under the control of its own
strong promoter/enhancer. Exons are numbered. Black boxes
represent untranslated sequences
and white boxes translated sequences. Plasmid pmHMG1
was modified by the insertion
of 27 bp coding for the HA
epitope (bold and underlined)
immediately after the ATG
codon for the first methionine
of HMG1 and in frame with the
rest of the protein. A stable
clone (c47) expressing HMG1tag
approximately to the same
level of unmodified HMG1 was selected. (E) Western blotting of whole cell extracts from wild-type fibroblasts (lanes 3T3) and from the
stable transfected cells (lanes c47). The anti-HMG1 antibody chWB-AB (left) recognizes in the same way HMG1 and HMG1tag; the
monoclonal antibody 12CA5 recognizes the HA epitope (right). Protein HMG1tag runs slightly slower than wild-type HMG1 in tricine-
SDS-PAGE because of the addition of nine amino acids.
[View Larger Versions of these Images (28 + 13 + 52K GIF file)]
have recently shown that Xenopus HMG1 forms stable complexes with in vitro reconstituted nucleosomes, similar to the complexes formed by
histone H1 and its embryonic variant B4. Both HMG1 and
B4 associate with linker DNA and protect it from micrococcal nuclease digestion. We confirmed these observations with mammalian HMG1: HMG1 binds very weakly to linear DNA not organized in nucleosomes, whereas it forms
complexes with in vitro reconstituted core mononucleosomes (Fig. 3). Nucleosomes lacking linker DNA were unable to bind HMG1 (results not shown).
Fig. 3.
HMG1 binds to reconstituted mononucleosomes. A labeled DNA fragment (176 bp) was assembled into mononucleosomes, incubated with increasing amounts (0, 10, 50, 100, and 500 ng) of HMG1 (lanes 6-10), and electrophoresed on a 0.7% agarose gel. DNA not assembled in nucleosomes was treated similarly for comparison (lanes 1-5). The bands corresponding to free
DNA, to nucleosome particles, and to HMG1-nucleosome complexes are indicated.
[View Larger Version of this Image (39K GIF file)]
),
and histone H1 (Breneman et al., 1993
). Unexpectedly, in
fibroblasts undergoing mitosis our anti-HMG1 antibodies
stained the cytoplasm in a diffuse way (Fig. 4 and results
not shown), while the chromosomes appear as dark areas,
indicating that HMG1 is displaced from condensed chromatin. As a control for the accessibility of mitotic chromosomes to antibodies, the same cells were stained with the
monoclonal IgM antibody HBC-7 directed against histone H2B (Whitfield et al., 1986
) and with a polyclonal rabbit
antibody directed against histone H1. In contrast to the
anti-HMG1 antibodies, the antibodies against core and
linker histones brightly stained the condensed chromosomes (Fig. 5).
Fig. 4.
HMG1 protein is not associated to mitotic condensed chromosomes. Dividing NIH 3T3 fibroblasts were fixed and stained for
HMG1 with antibody chIP-AB (top row, green fluorescence) and for DNA with Hoechst 33258 (bottom row, blue fluorescence). Representative cells at different stages during mitosis: prophase (A), metaphase (B), anaphase (C), and telophase (D). After the breakdown
of the nuclear membrane, HMG1 diffuses throughout the cytoplasm, and the pattern of green fluorescence corresponds to the shape of the cell. However, fluorescence from HMG1 is clearly reduced in correspondence to the volume occupied by condensed chromosomes (compare top and bottom images), indicating that HMG1 is not associated with DNA during mitosis. After cell division and the reformation of nuclear membranes (D), the majority of HMG1 colocalizes with DNA, but some is still found in the cytoplasm, suggesting
that the protein is being concentrated in the nuclei by passage through the nuclear membrane.
[View Larger Version of this Image (36K GIF file)]
Fig. 5.
Core histone H2B and linker histone H1 remain associated to condensed chromosomes throughout mitosis. As a control
for the mitotic displacement observed with HMG1, dividing NIH
3T3 fibroblasts were fixed and stained for DNA with Hoechst
33258 (bottom) and either (A) for histone H2B with the monoclonal IgM HBC-7 (top) or (B) for linker histones with a rabbit
polyclonal antibody against histone H1 (top).
[View Larger Version of this Image (62K GIF file)]
Fig. 6.
A minor fraction of HMG1 cofractionates with polynucleosomes in sucrose gradients. Nuclei of NIH 3T3 fibroblasts
were partially digested with micrococcal nuclease, lysed, and sedimented through a sucrose gradient (see Materials and Methods).
Individual fractions were analyzed by Western blotting for the
presence of HMG1 and histone H1; DNA was also extracted
from the fractions and analyzed on a 2% agarose gel.
[View Larger Version of this Image (96K GIF file)]
), or with NP-40, which permeabilizes all cellular membranes including the nuclear ones. The proteins released in the medium and the cell remnants attached to
the plastic surface were analyzed by Western blotting using anti-HMG1 and anti-histone H1, as well as a control
antibody against LDH. As shown in Fig. 7 A, LDH was almost quantitatively released from the cells by treatment
with digitonin, while HMG1 and histone H1 remained contained within the permeabilized cells. On the other hand,
treatment with NP-40 released the majority of HMG1,
whereas histone H1 remained associated to the cell remnants. The release of HMG1, but not of histone H1, from
NP-40-treated cells was also revealed by immunofluorescence. Histone H1 remained within the nucleus in cells
fixed either before or after permeabilization, whereas
HMG1 was lost from cells permeabilized before fixation (data not shown).
Fig. 7.
HMG1 leaks out from detergent-permeabilized nuclei
of adherent cells, but histone H1 does not. Adherent NIH 3T3 fibroblasts were incubated in buffer with no detergent (lanes 3 and
4), in buffer containing 0.1% NP-40 (lanes 1 and 2), or in buffer
containing 40 µg/ml digitonin (lanes 5 and 6). After incubation,
the buffer bathing the cells (lanes 1, 3, and 5; S, supernatant) and
the remnants of permeabilized cells (lanes 2, 4, and 6; P, pellet) were analyzed by Western blotting with antibodies against LDH, HMG1, and histone H1. Digitonin selectively permeabilizes the plasma membrane and causes the complete leakage of LDH but
not of HMG1 and H1. The faint bands in lanes 2 and 6 do not correspond to LDH, because they have a slightly different molecular
weight. NP-40 causes the disruption of all membranes including
the nuclear ones: LDH is completely released, as well as ~75%
of HMG1 (as determined by densitometric analysis), but H1 remains associated with the DNA. We observed an incomplete release of HMG1 only when we permeabilized cells still attached to
their plastic substrate, possibly because HMG1 sticks avidly to secreted glycoproteins (Falciola, L., and M.E. Bianchi, unpublished
results).
[View Larger Version of this Image (65K GIF file)]
). The two cell populations were then permeabilized with NP-40 and analyzed for protein retention by Western blotting (Fig. 8). In
both mitotic and nonmitotic fibroblasts, histone H1 is retained in the cell pellets; in contrast, the vast majority of
HMG1 is released into the medium. Moreover, HMG-I(Y),
a different high mobility group protein which is a component
of isolated condensed chromosomes (Saito and Laemmli,
1994
) was retained after permeabilization in both mitotic
and nonmitotic cells.
Fig. 8.
HMG1 leaks out in
a similar way from both interphasic and mitotic permeabilized cells. NIH 3T3 fibroblasts were exposed overnight to nocodazole, an inhibitor of
microtubule polymerization.
Cells that had entered M
phase could not proceed further and were detached from
their plastic substrate by
manual shaking (metaphase
cells). Cells that remained
adherent to the substrate after shaking (interphase cells)
were detached by treatment with trypsin. The two cell populations were checked for the presence of condensed chromosomes
(95% for metaphase cells; 2% for interphase cells). The cell suspensions were then exposed to 0.1% NP-40 and immediately centrifuged. Supernatants (lanes 1 and 3, S) and cell pellets (lanes 2 and 4, P) were analyzed by Western blotting with antibodies
against HMG1, histone H1, and protein HMG-I(Y).
[View Larger Version of this Image (54K GIF file)]
Discussion
) may in part
be related to the quality of the antibodies used and to potential cross reaction to HMG1-like molecules. The distribution of epitope-tagged HMG1, however, confirms that
HMG1 normally resides in the nucleus.
), in stark contrast to the
Drosophila HMG1-like protein HMG-D during embryogenesis (Ner and Travers, 1994
).
).
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.
; 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.
; 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.
Received for publication 16 September 1996 and in revised form 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.itWe thank Andrea Pontiggia for performing several preliminary experiments, Maria Carmo-Fonseca for help with confocal microscopy, Michael Bustin, Heikki Rauvala, Dimitri Thanos, and Bryan Turner for sharing their reagents with us, and Peter Becker, Carl Wu, and Alan Wolffe for stimulating discussions.
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).
HMG1, high mobility group 1 protein; LDH, lactate dehydrogenase.