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Vol. 14, Issue 8, 3414-3426, August 2003
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* Unité de Virologie, Service de Bactériologie-Virologie,
Hôpital de Bicêtre, Assistance Publique/Hôpitaux de Paris,
94275 Le Kremlin Bicêtre, France;
San Raffaele Scientific Institute, 2032 Milan, Italy; and
Université Pierre et Marie Curie, 75252 Paris, France
Submitted September 10, 2002;
Revised January 10, 2003;
Accepted February 24, 2003
Monitoring Editor: Douglas Koshland
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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), are two highly
abundant nuclear nonhistone proteins that are almost identical in all mammals
and have counterparts in all eukaryotes
(Bianchi, 1995;
Bustin and Reeves, 1996). They
have been implicated in transcription regulation, DNA repair, recombination,
differentiation, and extracellular signaling
(Muller et al.,
2001b; Thomas,
2001; Scaffidi et
al., 2002). Despite their high sequence similarities,
tertiary structure, and biochemical activities
(Bustin, 1999;
Bianchi and Beltrame, 2000;
Thomas, 2001), HMGB1 and 2 are
not functionally completely redundant
(Calogero et al.,
1999; Ronfani et al.,
2001).
HMGB1/2 bind in a sequence-nonspecific manner and with a low affinity to
single-stranded, linear duplex and supercoiled DNA, and exhibit a higher
affinity for unusual DNA structures such as four-way junctions, cruciform DNA
and cisplatin-modified DNA. In turn, these proteins induce structural
modifications in the linear DNA they are bound to, such as bending and looping
(Thomas, 2001).
Two nonhomologous but structurally related domains called HMG boxes A and B
are responsible for HMGB DNA binding properties. Both DNA boxes bind and bend
DNA, although they exhibit differences in their DNA binding and bending
activities (Teo et al.,
1995; Payet et al.,
1999; Webb and Thomas,
1999). These activities are modulated, at least in vitro, by
sequences that flank the HMG boxes
(Sheflin et al.,
1993; Stros et al.,
1994; Payet and Travers,
1997; Ritt et al.,
1998; Stros, 1998;
Muller et al.,
2001a).
HMGB1/2 interact with cellular (RAG1, p53, Oct, and Hox proteins, some
steroid receptors, and TATA binding protein) and viral proteins (Rep78, Rep68
of the adeno-associated virus, and ZEBRA of the Epstein-Barr virus)
(Ge and Roeder, 1994;
Onate et al., 1994;
Zwilling et al.,
1995; Zappavigna et
al., 1996; Costello
et al., 1997;
Boonyaratanakornkit et al.,
1998; Jayaraman et
al., 1998; Aidinis et
al., 1999; Sutrias-Grau
et al., 1999;
Butteroni et al.,
2000; Lu et al.,
2000) and usually increases the ability of their partners to
interact with DNA. Together, these observations lead to the proposal that
HMGBs act as architectural facilitators in the assembly of nucleoprotein
complexes.
HMGBs have been proposed to be important component of chromatin and to
share some functions with histone H1. Notably, they have been purified in
association with chromatin (Johns,
1982), and both H1 and HMGB bind to linker DNA
(Schroter and Bode, 1982), to
four-way junctions (Hill and Reeves,
1997) and to cis-platin–modified DNA
(Yaneva et al.,
1997). However, HMGB1 binding affinity is much lower than H1's,
suggesting that HMGB may interact with the linker region only when histone H1
is absent or weakly expressed (Ura et
al., 1996), such as during Xenopus
(Dimitrov and Wolffe, 1996;
Nightingale et al.,
1996) or Drosophila development
(Ner and Travers, 1994). In
mammalian cells, HMGB1 attaches only loosely to chromatin
(Falciola et al.,
1997).
EBNA1, a nuclear protein encoded by the Epstein-Barr virus, interacts with
cellular chromosomes during mitosis, most likely by the intermediate of one or
several cellular protein(s) (Petti et
al., 1990; Marechal
et al., 1999; Shire
et al., 1999). Preliminary results in one of our
laboratories suggested that HMGB2 could recruit or be recruited by EBNA1 onto
mitotic chromosomes. However, previous studies based on immunofluorescence
analysis indicated that HMGB1, which is closely related to HMGB2, dissociates
from mitotic chromosomes (Falciola et
al., 1997).
In the present work, the use of enhanced green fluorescent protein (EGFP)- and DsRed-tagged proteins allowed to reinvestigate HMGB1/2 localization in the context of living cell, while preserving some of their biological properties. It is shown herein that HMBG1/2 interact with chromosomes in a highly dynamic manner in mitotic living cells and that the binding is mediated by two regions encompassing HMG boxes A and B. This interaction is abrogated in permeabilized or chemically fixed cells. A comparable behavior was also observed for two proteins of the HMG-nucleosome binding (HMGN) group, namely, HMGN1 and 2.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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Recombinant Plasmids
Plasmids pEGFP-N1, pEGFP-C1, pEGFP-C2, pDsRed-N1, and pDsRed1-C1 (BD
Biosciences Clontech, Palo Alto, CA) encoded a variant of the green
fluorescent protein with enhanced fluorescence (EGFP) and a red fluorescent
protein (DsRed1). Human HMGB2 was expressed as a fusion protein to the N and
the C termini of EGFP, the N and the C termini of DsRed, respectively, in
pHMGB2-EGFP, pEGFP-HMGB2, pHMGB2-DsRed, and pDsRed-HMGB2. HMGB2 cDNA was
cloned from HeLa cells. For this purpose, total RNA was extracted with High
Pure RNA isolation kit (Roche Diagnostics, Mannheim, Germany) and subjected to
a reverse transcription in the presence of oligo(dT) primers by using the
Ready To Go You Prime First Strand Beads kit following the manufacturer's
recommendations (Amersham Biosciences UK, Little Chalfont, Buckinghamshire,
United Kingdom). The region encoding human HMGB2 was subsequently amplified
from the cDNA by polymerase chain reaction (PCR) with HMGB2 upstream primer
5'-CCCAAGCTTGGGCCACCATGGGTAAAGGAGACCCCAACAAG-3' and HMGB2
downstream primer 5'-CGCGGATCCCGTTCTTCATCTTCATCCTCTTCCTCC-3'. The
resulting PCR products was digested by HindIII and BamHI,
gel purified, and cloned into pEGFP-N1 and pDsRed-N1. Then, the
HindIII/BamHI insert from pHMGB2-DsRed was subcloned into
pDsRed-C1 and pEGFP-C1.
Deletion mutants were generated from pHMGB2-DsRed by PCR by using the indicated primers: A, 5'-CGCGGATCCCGACCTTTGGGAGGAACGTAATTTTTC-3'; AN, 5'-CGCGGATCCCGCTTTTTCTTCCCCTTCTTATCACC-3'; NB, 5'-CCCAAGCTTGGGCCACCATGGATAAGAAGGGAAAGAAAAAGGAC-3'; B1, 5'-GCTCAAGCTTCACCATGGGTGACCCCAATGCTCCTAAAAGG-3'; and B2, 5'-CGCGGATCCCGGCCCTTGGCACGATATGCAGC-3'. These primers were designed to contain HindIII or BamHI restriction sites to allow cloning in pEGFP-N1. Mutant A was generated with primers HMGB2 upstream and A; mutant AN, with primers HMGB2 upstream and AN; mutant B, with primers B1 and B2; mutant NB, with primers NB and B2; mutant ANB, with primers HMGB2 upstream and B2; mutant BC, with primers B1 and HMGB2 downstream; mutant NBC, with primers NB and HMGB2 downstream. Mutant N was obtained by cloning the HindIII-BamHI linker obtained by hybridizing oligonucleotides Nfor 5'-AGCTTGGGCCACCATGGATAAGAAGGGGAAGAAAAAGCGG-3' and Nrev 5'-GATCCCGCTTTTTCTTCCCCTTCTTATCCATGGTGGCCCA-3'.
pHMGB1-EGFP and pHMGB1-DsRed encode human HMGB1 fused to the N terminus of EGFP and DsRed, respectively. The region encoding human HMGB1 was generated by PCR from reverse transcribed polyadenylated RNA by using HMGB1 upstream primer 5'-GGAAGATCTTCGCCACCATGGGCAAAGGAGATCCTAAGAAGC-3' and HMGB1 downstream primer 5'-CCGGAATTCCTTCATCATCATCATCTTCTTCTTCATC-3'. The PCR product was digested by BglII and EcoRI, gel purified and inserted into pEGFP-N1 and pDsRed-N1 between the BglII and EcoRI sites. Using HMGB1.2 upstream primer 5'-GGAAGATCTTTCGCCACCATGGGCAAAGGAGATCCTAAGAAGC-3' and HMGB1.2 downstream primer 5'-CCGGAATTCCTTATTCATCATCATCATCTTCTTCTTCATC-3', a PCR product containing the HMGB1 coding region was generated from pHMGB1-DsRed, digested with BglII and EcoRI, and inserted within pDsRed-C1 and pEGFP-C1 between the BglII and EcoRI restriction sites.
The regions encoding HMGN1 and HMGN2 were generated by PCR from purified human fibroblast DNA by using primers HMG14/17for 5'-GGGGGAAGCTTCCGCCGCCACCATGCCCAAGAG-3' and HMG14rev 5'-GGGGGATCCGACTTGGCTTCTTTCTCTCC-3', and HMG14/17for and HMG17rev 5'-GGGGGATCCTTGGCATCTCCAGCACCTTC-3', respectively. The PCR products digested by HindIII and BamHI were cloned into pEGFP-N1 and pDsRed-N1, which resulted in vectors pHMGB14-EGFP, pHMGB14-DsRed, pHMGB17-EGFP, and pHMGB17-DsRed, respectively.
The cDNA encoding human histone H1 was obtained by reverse transcription on
total human RNA extracted from Raji cells by using primer H1.F3C
5'-GGGAATTCTCACTTTTTCTTCGGAGCTGCCTTCTTTGC-3'. The region encoding
H1 was produced from the cDNA by PCR by using primers H1.F3N
5'-GGCGGGATCCTGTCGGAGACTGCTCCACTTGCTCCTAC-3' and H1.F3C. The PCR
product was gel purified, digested with BamHI and EcoRI, and
cloned into pEGFP-C2 (BD Biosciences Clontech) digested by BglII and
EcoRI. The resulting plasmid was named pEGFP-H1. Plasmid DNA was
purified using the QIAGEN-plasmid maxi kit (QIAGEN, Hilden, Germany). DNA
sequencing was performed by automated sequencing using the dideoxynucleotide
chain termination method
accordingtothemanufacturer'srecommendations(ABIPrismdRhodamine Terminator
Cycle Sequencing Ready Mix; Applied Biosystems, Foster City, CA). Plasmids
pTHCR, pSGD9, and pHMG1 were described previously
(Zappavigna et al.,
1996).
Transfections
HeLa cells were grown in six-well plates until they reached 
80%
confluence. Plasmids were transfected with the FuGENE 6 transfection reagent
(Roche Diagnostics) according to manufacturer's recommendations. Briefly, 1
µg of purified plasmid DNA was mixed with 100 µl of antibiotic-free
culture medium complemented with 10% fetal calf serum and 4 µl of FuGENE 6.
The DNA–FuGENE complex was incubated for 30 min at room temperature and
then added dropwise into the wells containing 2 ml of antibiotic-free DMEM
complemented with 10% fetal calf serum. The cells were incubated at 37°C
for 16 h.
For stable cell lines, one-fifth of the cells from a well were plated on a
78-cm2 culture dish 16 h after transfection, and grown in the
presence of 400 µg/ml geneticin (Sigma-Aldrich, St. Louis, MO) for 3 wk.
Fluorescent foci were then cloned, plated in a six-well plate, and grown in
the absence of selection until 80% confluence. Subcloning was repeated as
described above from three to up to five times.
Fluorescence Microscopy
Unless otherwise indicated, fluorescent microscopy was performed on living
cells 24–48 h after transfection. Briefly, one-fourth of the transfected
cells were plated in a single-well chamber culture slide (Falcon Plastics,
Oxnard, CA) and incubated for 48 h. The cells were incubated for an additional
10 min at 37°C in culture medium containing 0.2 µg/ml Hoechst 33342,
washed once, and observed by fluorescence microscopy in the presence of
prewarmed culture medium either at 365 nm (Hoechst), at 488 nm (EGFP and
enhanced yellow fluorescent protein), or at 558 nm (DsRed).
For the preparation of chromosome spreadings, transfected HeLa cells were washed once in prewarmed culture medium, incubated for an additional 16 h in the presence of 0.1 µg/ml colcemid (Sigma-Aldrich), and then stained for 10 min with Hoechst 33342 (0.2 µg/ml) at 37°C. Mitotic cells were then collected by gentle pipetting, washed once, and resuspended in 75 mM KCl. After 10 min at room temperature, swelling cells were cytocentrifuged on slides (3 min at 500 rpm; Cytospin 3; Shandon Scientific, Cheshire, England, United Kingdom). The slides were then observed by epifluorescence microscopy either immediately or after the addition of phosphate-buffered saline (PBS) containing 20% glycerol.
For the analysis of fixed cells, transfected HeLa cells were transferred on glass coverslides 16 h after transfection and grown for an additional 24–48 h. Then, the cells were washed once in prewarmed PBS and treated at room temperature either by 4% paraformaldehyde in PBS or by 1% formaldehyde in PBS for 10 min.
Fluorescence Loss in Photobleaching (FLIP) Experiments
FLIP experiments were carried out on a TCS-SP confocal microscope (Leica,
Wetzlar, Germany) by using the 488-nm line of an Ar laser (20 mW nominal
output, beam width at specimen 0.2 µm, detection 500–575 nm) as
described previously (Phair and Misteli,
2000). In brief, five single scans were acquired, followed by a
series of bleach pulses of 200–500 ms by using a spot of 1 µm in
radius followed by imaging scanning. For imaging, the laser power was
attenuated to 1% of the bleach intensity. The bleach/scanning iterations were
separated by 6-s intervals. Relative loss of fluorescence was determined as
described previously (Phair and Misteli,
2000).
Western Blot Analysis
Western blot analyses were performed 24–48 h after transfection as
described previously (Marechal et
al., 1999). EGFP fused proteins were detected with a 1:1000
dilution of the mouse JL-8 monoclonal antibody (BD Biosciences Clontech).
Human HMGB1 was detected with a 1:3000 dilution of a rabbit antiserum
(Falciola et al.,
1997). Detection of the primary antibodies was performed with a
1:10,000 dilution of a peroxidase-conjugated anti mouse or anti rabbit IgG
polyclonal antibody (Amersham Biosciences UK). Proteins were detected by
chemiluminescence according to the manufacturer's recommendations (ECL Western
blotting detection reagents; Amersham Biosciences UK).
Cell Permeabilization Assay
Cells were cultured on a glass coverslip in a 6-cm dish; the coverslip was
inserted into a coverslip dish assembly (Harvard/Medical Systems, Holliston,
MA) containing 500 µl of PBS. Cells were permeabilized by adding NP-40 to a
final concentration of 0.15% while imaged on an inverted microscope (Axiovert
135 M; Carl Zeiss), and sequential images were collected.
For Western blot analysis of the soluble and insoluble fractions, the cells
were transfected in a 6-cm dish. Twenty-four hours after the transfection, the
cells were washed once in PBS and incubated for 5 min in 500 µl of PBS
containing 0.1% NP-40. The soluble and insoluble fractions were separated by
centrifugation (20,000 x g; 10 min; 4°C). The pellet was
resuspended in 500 µl of denaturing solution (50 mM Tris-HCl, 2% SDS, 2%
-mercaptoethanol) and incubated at 90°C for 3 min. Ten micrograms of
soluble proteins and a comparable volume of the insoluble fraction were
subjected to SDS-PAGE and Western blot analysis. The membrane was first probed
with the antibody directed against EGFP. After detection, the primary and
secondary antibodies were completely removed by incubating the membrane in a
stripping solution (100 mM -mercaptoethanol, 2% SDS, 62.3 mM Tris-HCl,
pH 6.7) for 30 min at 50°C. The membrane was washed in PBS, blocked for 1
h, and reprobed with a rabbit antiserum directed against human HMGB1.
HoxD9 Transactivation Assay
Cells (200,000) were transfected with 1.5 µg of reporter plasmid
(pTHCR), 1 µg of pSGD9, increasing amounts (0–2 µg) of constructs
expressing HMGB1 or HMGB1-GFP (pHMGB1 or pEGFP-HMGB1), and 300 ng of pRLnull
as an internal control. Transfection was carried out in triplicate batches.
Forty-eight hours after transfection, cells were harvested and luciferase
activities were measured using the dual-luciferase reporter assay system
(Promega, Madison, WI) and Lumino luminometer (Stratec Biomedical Systems,
Birkenfeld, Germany).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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). Importantly, its chromosome binding domains
(CBDs) are independent from the DNA binding domain (DBD), which suggests that
EBNA1 may be recruited onto chromosomes by one or several cellular proteins
(Marechal et al.,
1999; Shire et al.,
1999). Preliminary results from a double-hybrid screening
identified HMGB2 as a potential cellular partner of EBNA1, raising the
possibility that HMGB2 could recruit EBNA1 onto mitotic chromatin. However,
the association of HMGB2 itself with mitotic chromosomes was unknown, and
results from one of our laboratory indicated that the closely related protein
HMGB1 was not associated with mitotic chromosomes
(Falciola et al.,
1997). To examine this issue in greater detail, we analyzed the
localization of HMGB proteins fused to EGFP in living HeLa cells. For this purpose, cDNAs encompassing human HMGB2 were generated by reverse transcription from polyadenylated RNAs and amplified by PCR. Two cDNAs were amplified, cloned into pEGFP-N1 and sequenced (Figure 1). Sequence analysis indicated that hmgb2.1 resulted from a mature mRNA, whereas hmgb2.2 contained an intron between exons 3 and 4. After transfection in HeLa cells, both expression vectors gave rise to fusion proteins of comparable fluorescence, cellular localization and mobility on SDS-PAGE (our unpublished data), indicating that the intron had been spliced from primary transcripts originating from the longer expression construct. Therefore, only the construct containing hmgb2.1 was used for further experiments.
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The subcellular localization of HMGB2 fused to EGFP was assessed by
fluorescence microscopy in living cells from 48–72 h after transfection.
In interphase cells, HMGB2-EGFP localized in the nucleus (Figures
2 and
3). In mitotic cells,
HMGB2-EGFP colocalizes with the condensed chromatin, although some
fluorescence was also visible in the cytoplasm
(Figure 2). Western blot
analysis indicated that the cytoplasmic fluorescence was not attributable to
free EGFP (Figure 6C). This
localization contrasts with that of unfused EGFP, which did not interact with
mitotic chromosomes, and with that of histone H1-EGFP, which was exclusively
localized to the chromatin both in interphase and in mitotic cells
(Figure 2A). To evaluate the
effect of the fluorescent tag and of its position in the fusion protein,
additional vectors were constructed that encoded HMGB2 fused to the C termini
of EGFP and to the C and N terminus of the DsRed protein. As shown on
Figure 2A, HMGB2 binding to
mitotic chromosomes was not affected by the presence of EGFP at its N
terminus. Similarly, DsRed-tagged HMGB2 was also capable of interacting with
mitotic chromosomes. However, DsRed protein is known to oligomerize to form
tetramers, which may be not compatible with the intrinsic properties of the
proteins to which it is fused (Baird et
al., 2000). For this reason, subsequent experiments were
mainly done with EGFP-fused proteins.
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HMGB1 is highly homologous to HMGB2 but may not be completely redundant to
it functionally (Calogero et al.,
1999; Ronfani et al.,
2001). It was therefore important to test whether binding to
condensed chromosomes was a common property of HMGB proteins. A unique cDNA,
whose sequence corresponded to the expected mature HMGB1 mRNA, was obtained
from polyadenylated RNA by reverse PCR
(Figure 1) and cloned into EGFP
and DsRed expression vectors. As shown on
Figure 2, transfections of
vectors encoding HMGB1 fused either to EGFP or to DsRed confirmed that HMGB1
associates to condensed chromosomes as well; this property was independent of
the position and of the nature of the fluorophore. Importantly, HMGB1 and
HMBG2 interact with condensed chromosomes from early to late phases of mitosis
(Figure 2B; our unpublished
data). Experiments were also performed in mouse 3T3 cells, where it is
possible to analyze subnuclear compartments in more detail.
Figure 2C shows mouse fibroblast fixed with PFA (right): HMGB1-GFP is almost excluded from heterochromatic blocks close to nucleoli and to the nuclear membrane (red arrowheads). In living cells (left), the distribution of HMGB1 overlaps rather completely the Hoechst staining, confirming that the protein roams the whole nucleus, including heterochromatic AT-rich regions (yellow arrowheads). In contrast, HMGB1 seems to be less concentrated in nucleoli of living cells, compared with fixed ones. These results would suggest that PFA is able to fix better HMGB1 on chromatin where it is less compacted, such as euchromatin. However, in vivo HMGB1 seems to be distributed both in euchromatin and heterochromatin.
HMGB Proteins Fused to EGFP or to DsRed Associate with Mitotic
Chromosomes in Stably Transfected Cell Lines
The present findings conflict in part with the previous observation that
HMGB1 could not be detected on condensed chromosomes during mitosis
(Falciola et al.,
1997). However, the present study was performed in living cells
that were transiently transfected with HMGB proteins fused to fluorescent
proteins, whereas the previous study was performed on endogenous HMGB1 in
fixed cells.
In a first attempt to solve this discrepancy, we wondered whether transient transfections might induce an abnormal interaction of HMGB proteins with mitotic chromosomes. For example, if the binding of endogenous HMGB proteins to mitotic chromosomes is normally prevented by posttranslational modification(s), transient transfections may result in the rapid accumulation of an unproperly modified protein that, in contrast to its endogenous counterpart, might be able to interact with mitotic chromosomes. If true, binding of HMGB2 (or HMGB1) should not be observed in stable cell lines, or when the proteins are expressed at a low level.
To test this hypothesis, HeLa cells were transfected with pHMGB2-EGFP or with pHMGB2-DsRed and grown for 3 wk in the presence of geneticin. Fluorescent foci were then cloned and grown for five additional weeks in the absence of geneticin. Although the positive foci were repeatedly subcloned, a large cell-to-cell variation in the fluorescence level was noted, which likely reflects cell-to-cell fluctuation in the expression of the fusion protein (Figure 3). Because stable clones expressing EGFP homogeneously were obtained in the same experiment, this suggests that the cells contain integrated vectors, but that the expression of the fusion proteins may be modulated by epigenetic events and/or counterselected. Nonetheless, a specific association of HMGB2 with mitotic chromosomes was still observed, and this interaction was not dependent on the expression level of the fusion protein. Similar results were obtained for HeLa clones expressing EGFP- and DsRed-tagged HMGB1 for >2 mo (our unpublished data). Therefore, we concluded that the interaction of HMGB proteins with mitotic chromosomes is not linked to their transient expression.
EGFP Does Not Significantly Alter Known Biological Properties of HMGB
Proteins
We next examined whether EGFP-tagged HMGB proteins behave like their
endogenous counterparts. Previous work had shown that endogenous HMGB1
interacts only weakly with mitotic and interphase chromatin. As a consequence,
HMGB1 rapidly diffuses in the extracellular environment when the cellular
membranes are permeabilized (Falciola
et al., 1997). HeLa cells transfected with an expression
vector encoding EGFP-fused HMGB2 or HMGB1 were exposed to a low level of
NP-40. After the addition of detergent, both EGFP-tagged HMGB1
(Figure 4A) and HMGB2 (our
unpublished data) rapidly diffuse out of interphase and mitotic cells, as
previously observed for endogenous HMGB1.
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This was further confirmed by comparing the diffusion of the EGFP-tagged and endogenous HMGB in a permeabilization assay. Briefly, living HeLa cells expressing HMGB1-EGFP were incubated in the presence of NP-40 for 5 min. Then the soluble and insoluble fractions were separated by centrifugation and their protein content was analyzed by Western blot (Figure 4B). In contrast to EGFP-H1, which was detected in the insoluble fraction only, HMGB1-EGFP as well as the endogenous HMGB1 was exclusively detected in the soluble fraction. Therefore, HMGB1 and HMGB1-EGFP diffuse similarly in a permeabilization assay. Complementary experiments indicated that HMGB2 and HMGB2-EGFP behaved similarly and that this was not dependent of the salt concentration in the permeabilization buffer (our unpublished data).
Another experiment was performed in order to test whether EGFP affected the ability of HMGB1 to enhance HOXD9-controlled transcriptional activation. Figure 4C shows that EGFP-HMGB1 can enhance the transcriptional activity from an HOXD9-dependent promoter like the untagged HMGB1 protein.
Together, these experiments provide evidences that EGFP does not significantly alter known biological properties of HMGB proteins.
HMGB Proteins Rapidly Exchange between Chromosome and Cytoplasm
during Mitosis
A significant amount of fluorescence was detected in the cytoplasm of
mitotic cells expressing EGFP-fused HMGB1 or HMGB2. This could be observed
both after short- and long-term expression of the fusion protein. Western
blots did not indicate the presence of major EGFP-tagged breakdown products,
and we concluded that during mitosis HMGB1 and HMGB2 exchange between a free
and a chromosome-associated form. To prove this, we performed FLIP
experiments.
Photobleaching techniques are noninvasive microscopy methods that reveal
the dynamics underlying the steady-state distribution of a fluorescently
tagged protein in living cells. A fluorophore within a small volume of the
cell is irreversibly destroyed with a high-intensity laser pulse. After
bleaching, the labeled protein is photochemically altered, so that it no
longer fluoresces, but otherwise retains completely its biological activity
(Tsien and Waggoner, 1995).
The exchange between the bleached and unbleached populations of fluorophores
is then monitored and used as an indicator of the overall mobility of the
protein.
In FLIP, a region of interest is repeatedly bleached, and the loss of fluorescence from outside the bleached region is monitored by imaging after each bleach pulse. FLIP studies are particularly useful when the protein is not uniformly distributed but concentrated in defined intracellular sites, because it visualizes the flux between populations of fluorophores localized in different regions. In mitotic cells, HMGB1 is mostly associated with chromosomes, but it is also clearly visible in the cytoplasm. In this situation, it is possible to discriminate between soluble and chromatin-bound molecules. We performed FLIP experiments by targeting either the condensed chromosomes, or the cytoplasm. In both cases, we recorded images of the whole cell (Figure 5A) and estimated the residual amount of fluorescence remaining both on chromosomes and in the cytoplasm (Figure 5B). We found that in both cases (chromosomes and cytoplasm) repeated photobleaching led to a rapid and complete loss of fluorescence both from condensed chromosomes and from the cytoplasm, with comparable kinetics. This result indicates that all HMGB1 molecules are highly dynamic and rapidly shuttle between the cytoplasm and chromosomes. The concentration of HMGB1 on chromosomes therefore is not due to a static binding, but rather is the result of a steady state in which HMGB1 rapidly and continuously associates and dissociates on DNA.
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Control experiments using H1-EGFP confirmed that histone H1 is stably associated to condensed mitotic chromosomes, and exchanges very slowly with the surrounding cytoplasm.
The Chromosome Binding Regions of HMGB2 Encompass HMG Boxes A and
B
In a first attempt to investigate the molecular details of HMGB interaction
with mitotic chromatin, we evaluated the ability of various truncated forms of
HMGB2 to address EGFP onto mitotic chromosomes. A schematic representation of
these proteins is shown in Figure
6A. Western blot analysis indicated that these proteins were
expressed in HeLa cells with minor amounts of breakdown products
(Figure 6C). Therefore, an
apparent absence of binding was not attributable to the presence of free
EGFP.
Recent work in one of our laboratories suggested that some proteins need to
reach the nucleus during interphase to be able to interact with chromatin
during mitosis (Piolot et al.,
2001). As shown on Figure
6B, HMGB2-EGFP–truncated forms were either strictly or
partly nuclear and therefore competent to bind to chromosomes during
mitosis.
In a first set of experiments, the chromosome binding properties of these proteins were assessed in living HeLa cells 48–72 h after transient transfection. As indicated in Figure 6A and illustrated on Figure 7A, binding was observed for truncation A, which encompasses the HMG box A. Binding was notably increased by the addition of regions N and B and truncation ANB exhibited a chromosome binding activity that was even stronger than that of the wild-type protein, as estimated from the relative level of fluorescence on the chromosomes and in the cytoplasm. Surprisingly, HMG box B did not significantly target EGFP onto mitotic chromosomes, except when the N region was also present. Therefore, although amino acids 84–90 did not relocate EGFP to the mitotic chromosomes, they seemed 1) to be necessary for the binding of box B and 2) to increase binding of box A. The apparent lack of binding observed for mutant NBC argued for a negative effect of the acidic tail, at least in the context of EGFP-fused proteins.
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This assay was convenient to assess the possible interaction of HMGB2 mutants with mitotic chromosomes. However, it may not be able to detect weak binding, such as in the case of mutants B, BC, NBC, and N. To circumvent this possible limit, complementary studies were performed on mitotic cells subjected to a mild chromosome spreading that does not involve fixation. Briefly, HeLa cells were transfected as described above and incubated for 16 h in the presence of colcemid. Mitotic cells were collected by gentle pipetting, subjected to a hypotonic shock, and centrifuged onto glass slides. Chromosome binding was then immediately assessed either in the absence of any mounting medium or in the presence of PBS containing 20% glycerol. Figure 7B shows that this treatment did not affect H1–EGFP interaction with metaphase chromosomes. EGFP was detected in the cytoplasm of the transfected cells in the absence of mounting medium, but rapidly diffused away from most cells when the mounting medium was added. Overall, the observations made with the various truncated forms of HMGB2 were in agreement with the results obtained in living cells. Notably, it was confirmed that mutants N, B, NBC, and BC did not significantly interact with chromatin in mitotic cells.
As a conclusion, HMGB2 binding to mitotic chromosomes is mediated by two regions that encompass but are not strictly identical to HMG box A and B. Because HMGB1 and HMGB2 are highly homologous, it is very likely that corresponding regions are responsible for HMGB1 chromosome binding activity.
HMGB Protein Interaction with Mitotic Chromosomes Is Abrogated by
Cross-linking Fixatives
Because the previous experiments established that HMGB1 and HMGB2 interact
with mitotic chromosomes in living cells, we suspected that the previously
reported release of HMGB1 from mitotic chromosomes might be due to the
procedure that was used, i.e., immunofluorescence on paraformaldehyde-treated
cells. Because paraformaldehyde does not abrogate EGFP fluorescence, its
effect on HMGB protein binding could be investigated. HeLa cells were
transfected by vectors encoding HMGB1- or HMGB2-EGFP, grown on glass
coverslides for 48 h, and incubated in the presence of PBS containing 4%
paraformaldehyde while being observed under the microscope. As early as 1 min
after paraformaldehyde addition, HMGB1- and HMGB2-EGFP mostly diffused away
from chromosomes of mitotic cells. This resulted in the absence of any
detectable signal on the chromosomes after a 10-min incubation
(Figure 8). Nevertheless, it
should be noticed that HMGB were detected in the vicinity of condensed
chromosomes in late phases of mitosis, such as in telophase
(Figure 8). In contrast,
H1-EGFP binding was remarkably stable. Similar results were obtained when the
cells were treated with 1% formaldehyde (FA) (our unpublished data), a
cross-linking fixative that is broadly used to investigate
protein–protein and protein–DNA interactions. Paraformaldehyde was
also shown to alter the distribution of HMGB proteins in interphase cells,
where it induces nucleolar concentration and loss of colocalization with some
heterochromatin rich regions (Figure
2C).
|
Thus, we concluded from these experiments that 1) HMGB proteins interact with mitotic chromosomes in living cells and 2) this interaction is highly sensitive to cross-linking agents commonly used to investigate the subcellular localization of proteins or their interaction with DNA.
Other HMG Proteins Interact with Mitotic Chromosomes in Living Cells
but Not in Paraformaldehyde-fixed Cells
The HMGA group of proteins do interact with metaphase chromosomes
(Disney et al., 1989;
Saitoh and Laemmli, 1994).
Conversely, proteins of the HMGN group have not been detected on mitotic
chromosomes (Hock et al.,
1998). Because these studies were also done by immunofluorescence
on paraformaldehyde-fixed cells, we wondered whether fusion to EGFP or DsRed
might indeed reveal that HMGN proteins are associated with mitotic chromosomes
in living cells. As illustrated by Figure
9, HMGN1-EGFP was detected in association with the chromosomes at
every stage of mitosis. This behavior was also observed for HMGN2-EGFP and did
not depend either on the position or on the nature of the fluorophore (our
unpublished data). In contrast to HMGB proteins, only minor amounts of protein
were detected in the cytoplasm during mitosis. The effect of paraformaldehyde
on HMGN proteins was also investigated and proved to induce the release of
most if not all the protein from the chromosomes
(Figure 9B).
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|
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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During interphase, HMGB1- and HMGB2-EGFP fusions were fairly homogeneously
distributed in the cell nucleus of human cells, including the nucleolus, and
occasionally colocalized with DNA-rich regions. In contrast with human cells
where the border between euchromatin and heterochromatin is not very clear,
heterochromatin occurs in the form of well defined chromatin blobs with a high
concentration of DNA in mouse cells. The use of an EGFP-tagged protein in
living mouse fibroblasts indeed confirmed that HMGB protein localizes with
both euchromatin and heterochromatin. Whether binding to certain
heterochromatic regions reflects the ability of some HMGB proteins to form a
complex with SP100B and HP-1, a protein that is also found predominantly in
heterochromatin, has still to be determined
(Lehming et al.,
1998). During mitosis, both human HMGB1 and 2 associate with
condensed chromosomes in living HeLa cells. In contrast to histone H1, which
is stably and exclusively associated with chromosomes, HMGB1 and 2 were also
detected in the cytoplasm, and evidence is provided herein that the bound and
unbound forms of HMGB1/2 can rapidly exchange. This high mobility of HMGB1 has
also recently been observed in interphase cells
(Scaffidi et al.,
2002)
Two CBDs have been identified in HMGB2. The first one encompasses HMG box A
(aa 1–83), but amino acids 84–90 are required to confer a
chromosome binding activity comparable with the entire protein. The second CBD
mapped between amino acids 84–166. It comprises HMG box B (aa
91–166) but was strictly dependent on the presence of amino acids
84–90 for binding. The HMG boxes are defined as the DBDs of HMGB
proteins; therefore, DBDs and CBDs do not coincide exactly. In vitro, the
regions immediately C-terminal to the HMG boxes have been shown to increase
the DNA-binding activity to some forms of DNA, including supercoiled DNA
(Stros, 2001). Importantly, we
also noticed that the C-terminal acidic tail of HMGB2 exerts a negative effect
on chromosome binding, either in association with box B or in the context of
the full-length protein (compare NB with NBC and ANB to full-length protein on
Figure 6). Together, these
results are reminiscent of a recent investigation of HMGB1 interaction with
linear duplex DNA where it was shown that 1) box A has a higher affinity than
box B for double-stranded linear DNA in solution, 2) box A and B behave as
independent domains, and 3) the acidic tail reduces DNA binding affinity,
possibly by interacting with box B (Muller
et al., 2001a).
Together, these results indicate that HMGB proteins directly interact with
chromatin in mitotic chromosomes. Although the nature of this interaction is
still elusive, indirect evidence suggests that HMGB and histone H1 may share a
common role in organizing higher order chromatin structure
(Nightingale et al.,
1996; Bustin,
1999). Notably, both proteins bind to linker DNA
(Ura et al., 1996),
four-way junctions or cis-platined DNA in vitro
(Hill and Reeves, 1997;
Yaneva et al., 1997).
However, the affinity of histone H1 for these structures is much higher than
HMGB in vitro, suggesting that the binding of HMGB proteins may occur in vivo
only where histone H1 is absent. Accordingly, HMGB proteins have been shown to
interact with mitotic chromosomes during Xenopus laevis early
embryogenesis (Dimitrov et al.,
1993; Dimitrov et
al., 1994) until histone H1 accumulates. A related protein,
HMG-D, is also expressed early in Drosophilia melanogaster
development (Ner and Travers,
1994) and associates with mitotic chromosomes only until histone
H1 first accumulates in the dividing cells. In differentiated mammalian cells
the concentration of histone H1 is 10 times higher than that of HMGB1
(Einck and Bustin, 1985).
Therefore, H1 should efficiently outcompete HMGB for binding to linker DNA,
and our results might seem surprising. Two nonmutually exclusive hypotheses
can be proposed: 1) HMGBs interact with linker DNA regions that remain free
after all histone H1 has occupied its preferred binding sites, and 2) HMGB
interaction with mitotic chromosomes is mediated through other proteins bound
to nonlinker DNA. The first hypothesis rests on the observation that histone
H1 is substantially substoichiometric in relation to core histones, so that a
large amount of nucleosomes remain H1-free. The second possibility is
supported by the fact that HMGB proteins interact with several
chromatin-associated proteins, including the TATA binding protein (TBP)
(Ge and Roeder, 1994;
Sutrias-Grau et al.,
1999). TBP is associated with condensed chromosomes during mitosis
(Chen et al., 2002;
Christova and Oelgeschlager,
2002), and might "bookmark" previously active genes
and promote their rapid reactivation after mitosis. We currently do not have
evidence for a direct binding between HMGB proteins and TBP on mitotic
chromosomes, but this assumption is compatible with our current knowledge of
TBP–HMGB interaction. Indeed, HMGB1 interacts with the core domain of
TBP through multiple regions, including HMG box A, and this results in a
HMGB1/TBP/TATA complex that can modulate RNA pol II transcription
(Sutrias-Grau et al.,
1999).
The discrepancy between the present work and previous ones with regard to
the presence of HMGB proteins on mitotic chromosomes is most likely due to the
paradoxical effect of paraformaldehyde and formaldehyde. Second, we could
observe chromosome associated EGFP-tagged HMGB for >10 min in cells
incubated in a methanol/acetone mixture. However, the fluorescence was lost
when the cells were dried and incubated in PBS, suggesting that HMGB proteins
were not properly fixed by this procedure. Paraformaldehyde is extensively
used to cross-link proteins to chromatin. Because formaldehyde can efficiently
cross-link HMGB1/2 on nucleosomes reconstituted in vitro
(Stros et al., 1985),
this treatment is unlikely to directly alter preformed complexes. Rather, PFA
or FA may alter the accessibility of HMGB to their target(s) by modifying the
overall structure of the mitotic chromosomes, and/or inactivate the free form
of HMGB1/2. HMGBs contain >40 lysine residues, and a lot of these are
expected to interact directly with the charged phosphate backbone of DNA. PFA
reacts with the 
amino group of lysines, and the reaction product is a
Schiff base. This is still charged, but both hydrogen bonding and van der
Waals contacts of the lysine residue will be disrupted.
The deleterious effect of PFA has also been demonstrated for other members
of the HMG protein family. Indeed, it was initially considered that HMGN1 and
2 were released from mitotic chromosomes when cells enter mitosis
(Hock et al., 1998).
Actually, EGFP-HMGN1 and 2 do not dissociate from condensed chromosomes in
living cells, but they are rapidly released in the cytoplasm upon treatment by
PFA and FA. Altogether, our results indicate that many members of the HMG
protein family, including HMGB, HMGN, and HMGI/Y, interact with chromatin
during interphase and mitosis. Our results justify a careful reexamination of
nuclear protein interactions with mitotic chromosomes.
|
ACKNOWLEDGMENTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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|
Footnotes |
|---|
Corresponding author. E-mail address:
vincent.marechal{at}snv.j'ussieu.fr.
|
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