From the III. Zoologisches
Institut-Entwicklungsbiologie, Universität Göttingen,
D-37073 Göttingen, Germany, the ¶ Zentrum für
Humangenetik und Genetische Beratung, Universität Bremen, ZHG,
D-28359 Bremen, Germany, and the
Edward A. Doisy Department
of Biochemistry and Molecular Biology, St. Louis University Medical
School, St. Louis, Missouri 63104
Received for publication, May 12, 2000, and in revised form, October 13, 2000
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ABSTRACT |
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High mobility group (HMG) proteins HMGI, HMGY,
HMGI-C, and Chironomus HMGI are DNA-binding proteins
thought to modulate the assembly and the function of transcriptional
complexes. Each of these proteins contains three DNA-binding domains
(DBD), properties of which appear to be regulated by phosphorylation.
High levels of these proteins are characteristic for rapidly dividing
cells in embryonic tissues and tumors. On the basis of their
occurrence, specific functions for each of these proteins have been
postulated. In this study we demonstrate differences in the nature of
contacts of these proteins with promoter region of the interferon- The family of high mobility group I/Y chromosomal
(HMGI/Y)1 nonhistone proteins
comprises different structurally related proteins found in evolutionary
distant organisms, including bacteria, insects, plants, and mammals
(1). The mammalian HMGI, HMGY (HMGI(Y)), and HMGI-C and insect cHMGI
belong to the best characterized proteins of this family (1, 2). HMGI/Y
play an important role in regulation of transcriptional activity of
many genes (3), including those of the Despite continuously increasing interest in the function(s) of this
group of proteins, the nature of the interaction of these proteins with
DNA is still not sufficiently understood. The proteins of the HMGI/Y
family are 10-11 kDa in size, are highly charged, are rich both in
acidic and basic residues, are proline-rich, and contain only few
residues with bulky hydrophobic side chains. This unusual amino acid
composition inhibits folding of the polypeptide backbone of these
proteins into any defined secondary structure. Common for HMGI/Y
proteins is the presence of three putative DNA-binding domains (DBD),
so called AT-hooks (26). NMR analysis of a complex of a peptide derived
from HMGI(Y) bound to a short DNA fragment revealed that the centrally
located RGR residues are essentially for binding and responsible for
contacts of the protein with the bases and phosphate-sugar backbone
(27). Alternative approaches, which used deletion and point-mutated
proteins, revealed that two or three DBDs of the protein bind to DNA in
a cooperative way (28-30). Application of the protein-footprinting
method for mapping of protein regions interacting with DNA (31) allowed more detailed characterization of the binding of HMGI(Y) proteins to
DNA (32). A combination of this method with other biochemical approaches has recently brought insights into the organization of the
complex of the HMGI-C protein with a promoter region of the
interferon- Preparation of Bacterially Expressed Proteins--
The coding
region of the human HMGY, HMGI, HMGI-C, and cHMGI proteins were cloned
into the expression vector pET3a, and the proteins were overexpressed,
extracted, and purified as described previously (34, 35).
Protein Phosphorylation--
For Cdc2 kinase, 50 µg of the
recombinant HMGY protein were phosphorylated at 30 °C with 10 units
of recombinant human Cdc2 kinase (New England Biolabs Inc.) for 5 h in the presence of 4 mM ATP in 8 µl of Cdc2 kinase
buffer containing: 50 mM Tris/HCl, 10 mM
MgCl2, 1 mM dithiothreitol, 1 mM
EGTA, pH 7.5. For casein kinase 2 (CK2), 50 µg of purified mammalian
HMG protein or Cdc2-phosphorylated HMGY were phosphorylated at 37 °C
with 500 units of recombinant human CK2 (New England Biolabs Inc.) for
5 min in the presence of 200 µM ATP in 50 µl of CK2
buffer containing 20 mM Tris/HCl, 50 mM KCl, 10 mM MgCl2, pH 7.5. Phosphorylation of cHMGI was
extended for 2.5 h. For 32P-end-labeling, 100-150
µCi of [ DNA and Oligonucleotides--
The synthetic linear
poly(dA-dT)·poly(dA-dT) DNA was obtained from Amersham Pharmacia
Biotech. The approximate average length of this DNA was 5000 bp. The
34-bp fragment of the promoter of the IFN Mobility Shift Assay--
Electrophoretic mobility shift assays
were carried out as described previously (34, 35). Briefly, purified
proteins were incubated with less than 1 nM of labeled DNA
in 180 mM NaCl, 1 mM MgCl2, 0.01%
bovine serum albumin, 8% glycerol, 10 mM Tris/HCl, pH 7.9, at 20 °C for 10 min. The DNA and DNA·protein complexes were run on
8% polyacrylamide gels at 37 °C unless otherwise indicated.
Hydroxyl Radical DNA Footprinting--
10,000-15,000 cpm
5'-labeled IFN Methylation and Ethylation Interference Assays--
The
5'-labeled IFN Hydroxyl Radical Protein Footprinting (31, 40)--
10 pmol of
the radioactively end-labeled protein (10,000-20,000 cpm) were
digested in presence or absence of DNA in a total volume of 10 µl of
180 mM NaCl and 10 mM MOPS buffer, pH 7.2, at
room temperature for 30 min. The chemical digestions were started by
sequential addition of 1 µl each of the following freshly prepared solutions: (i) 20 mM EDTA and 10 mM
(NH4)2Fe(II)(SO4)2,
(ii) 0.2 M sodium ascorbate, and (iii) 0.375% (v/v)
H2O2. Reactions were stopped after 30 min by
addition of 3.3 µl of 4-fold SDS sample buffer (4% SDS, 16%
glycerol, 25 mM Tris/HCl, pH 6.8, 6% Fluorochrome-labeled DNA--
The 34-bp IFN Fluorescence Measurements--
Luminescence lifetime
measurements were performed on a laboratory-built two-channel
spectrofluorometer with a pulsed nitrogen laser (LN300, Laser
Photonics, Orlando, FL) as an excitation source (43). Donor emission
was observed at 617 nm, whereas acceptor emission was observed at 670 nm. The details of the luminescence resonance energy transfer (LRET)
measurements and the advantages of using europium chelates for energy
transfer measurements have been discussed previously (43, 46-50). Only
sensitized acceptor decays were analyzed since these decays contain
information pertaining to energy transfer in the absence of any
contamination with the signal of the donor not engaged in
energy transfer (42-45). The sensitized acceptor decay curves
were analyzed according to Equation 1.
The energy transfer (E) between europium chelate and CY5 was
calculated as described previously (50, 51) from measurements of
luminescence lifetime of a donor in the absence
(td) and in the presence of acceptor
(tda).
High Mobility Group Proteins HMGI, HMGY, HMGI-C, and cHMGI Interact
Distinctly with Interferon
During migration through the gel, the protein·DNA complex is under
nonequilibrium conditions and dissociates continuously (Fig.
1D). Analysis of the process allows obtaining information on
the stability of the complex, e.g. its susceptibility to
dissociation in response to changing physiological conditions.
Quantification of the amounts of the labeled DNA in the complex after
various times of electrophoresis (Fig. 1D) allowed
determination of kinetic dissociation constant
(kd) for each protein (Fig. 1, E and
F; Table I). The HMGY protein has the lowest
kd value, whereas the HMGI protein exhibits the
highest one (Table I). These values correspond to half-life times of
the complex of 6.3 and 1.5 h, respectively. For the
HMGI-C·INF Proteins HMGI, HMGY, HMGI-C, and cHMGI Bind Differentially to
IFN
To extend the insights in the organization of the individual HMGI/Y
proteins·DNA complexes DNA-footprinting (Fig.
4) and interference assays (Fig.
5) using methylated and ethylated DNAs
have been performed. Our previous studies revealed that HMGI-C protein
binds to the PRDII and NRDI elements of this DNA. In contrast, binding of the HMGY protein resulted in the protection of three regions of the
DNA, including additionally the PRDIII-1 element (Fig. 4A).
In the presence of HMGI protein, protection of both DNA strands in the
PRDII and NRDI region was observed (Fig. 4), whereas the protection in
the PRDIII-1 element was restricted to the bottom strand (Fig.
4B). Essentially the same results were obtained using up to
10 times higher protein concentrations (data not shown). Ethylation
interference assay showed that modification of majority of phosphate
groups in the DNA fragment interferes with binding. However, strongest
interference in binding of both proteins was observed in region of the
PRDII element (Fig. 5A). Modifications within the
NRDI-element affect the binding of HMGY somewhat more as compared with
HMGI (Fig. 5A). A moderate extent of interference within the
PRDIII-1 was found for both proteins (Fig. 5A). Similar results were obtained analyzing the binding interferences on the bottom
strand (data not shown). Methylation of purines interfered to higher
extent with binding of DNA fragments modified at adenines located
within PRDII and NRDI (Fig. 5B). No interference in binding was observed for fragments methylated at purines within the PRDIII-1 region (Fig. 5B). These data suggest that interaction of
HMGY and HMGI in the PRDIII-1 region is probably mostly nonspecific by
interaction with the phosphate backbone and therefore detectable by
footprinting and ethylation interference techniques but is not affected
by individual modification of the bases.
In the DNA footprinting experiments with HMGI and HMGY, the distances
between the minima of the cutting frequency are 10-11 and 7-8 bases
between NRDI - PRDII and PRDII-PRDIII-1, respectively (Fig. 4). A
similar distance of 7-8 bases between the minima within PRDII and
PRDIII-1 was also observed in ethylation interference experiment (Fig.
5A). Thus, the centers of binding to NRDI-PRDII and
PRDII-PRDIII-1 appear to be separated by one and approximately three-quarters helical turns, respectively. This suggest that the
center of protein binding to the PRDIII-1 is rotated by about 90° in
respect to the binding at PRDII and NRDI.
In the presence of Chironomus cHMGI protein the patterns of
footprinting were distinct from those obtained for the human proteins (Fig. 4). Strong protection within PRDII element was observed on both
strands. Moreover, wide regions of the top and bottom strands,
respectively, comprising NRDI and PRDIII-1, were protected. Essentially
the same protection patterns were found and higher up to 10:1 protein
to DNA ratios (not shown).
Phosphorylation Affects Contacts of HMGY with DNA--
Previous
studies have shown that mammalian and insect HMGI/Y proteins are
in vivo phosphorylated at several positions by CK2 (53-55)
and Cdc2 kinase (56-58). In some reports, a general
phosphorylation-dependent attenuation of binding affinity
of the proteins has been demonstrated (56-60); however, the mechanism
of these changes was not studied. Recently, we have shown that
phosphorylation of the HMGI-C protein by Cdc2 kinase "derails" DBD2
of this protein from the minor groove (33). In this work we have
observed differences in the organization of complexes of human HMGI-C
and HMGI(Y) with IFN
An approximately 3-fold weakening of binding of HMGY to IFN
To map changes resulting from Cdc2-phosphorylation of HMGY, the protein
was subjected to hydroxyl radical footprinting in the presence of
IFN
To obtain more information about the changes in binding of HMGY to the
IFN Phosphorylation Modulates the Extent of Unbending of IFN
An example of a decay curve of europium luminescence in donor-acceptor
labeled DNA is shown in Fig. 9D. Although the observed distance changes were small, they were reproducible and were larger then the error of the measurement. Table
II summarizes the results of distance
measurements. Assuming a model shown in Fig. 9 (A-C) and
using a prediction of the curvature of the DNA (65) between the donor
and acceptor yielded a value of a mean value of ~10° per helical
turn (33). Binding of HMGY unbends the DNA by ~7°, whereas
consecutive phosphorylation of the protein by CK2 and Cdc2 kinase led
to partial restoration of the bend by ~2° and ~3.5°,
respectively (Table II). Interestingly, substantially weaker effect of
HMGI-C on the DNA curvature was observed (unbending by ~3°). This
probably reflects that IFN The presence of three AT-hooks in each of the human and murine
HMGI/Y proteins, as well as similarities in the regions adjacent to the
respective DBDs and the presence of the acidic tails, have usually been
a reason for a consideration that members of this protein family have
similar properties. One of the significant findings emerging from this
study is the demonstration of differences in DNA binding of individual
proteins of the HMGI/Y family. The proteins are distinct in DNA binding
affinities and thermodynamic stabilities of the complexes. Moreover,
the use of only two DBDs by HMGI-C and cHMGI or three DBDs by HMGI and
HMGY (Fig. 10) is probably important
for a creation of an unique conformation of the bound DNA, which may
facilitate recruitment of other proteins involved in assembly of
multiprotein promoter/enhancer complexes as it has been well documented
for the IFN
gene. We show that HMGI and HMGY interact with this DNA via three DBDs, whereas HMGI-C and Chironomus HMGI bind to this DNA using
only two domains. Phosphorylation of HMGY protein by Cdc2 kinase leads to impairing of contacts between the N-terminally located DBD and a
single promoter element. The perturbations in the architecture of the
protein·DNA complexes involve changes in the degree of unbending of
the intrinsically bent IFN
promoter. Our results provide first
insights into the molecular basis of functional specificity of proteins
of the HMGI/Y family and their regulation by phosphorylation.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-interferon (4-6) and the
-subunit of the interleukin 2 receptor (7). During the last 5 years,
the HMGI/Y genes have attracted a lot of interest because
they are causally involved in the genesis of benign tumors. Aberrations
involving the chromosomal regions 12q15 and 6p21.3 affecting the
HMGI-C and HMGI(Y) gene, respectively, have been
shown in a variety of benign tumors, e.g. uterine
leiomyomas, endometrial polyps, pleomorphic adenomas, pulmonary
chondroid hamartomas, and lipomas (8-13). At the molecular level, in
case of intragenic breakpoints, different fusion transcripts have been
detected as, e.g., HMGI-C-ALDH2 (14) and
HMGI-C-LPP (12). In addition, a re-expression of
HMGI-C and high level expression of HMGI(Y) have
been observed in a number of malignant human tumors (15-20). The
oncogenic potential of HMGI(Y) genes has been confirmed by
functional tests including NIH3T3 assay, knock-out technique, and
antisense construct transfection (21-23). Despite structural
similarities between the three mammalian members of the HMGI/Y family,
it appears that each of the proteins have a specific function. In the
pygmy phenotype that arises from the inactivation of the
HMGI-C gene, the gene expression of the HMGI(Y) gene is not affected, thus indicating that HMGI and HMGY proteins are
not able to substitute functionally for HMGI-C protein (23). Neoplastic transformation of the murine JB6 cells by a phorbol ester is
accompanied by synthesis of the HMGY but not HMGI protein, suggesting
that HMGI and HMGY have different functions (24). Moreover, in MCF-7
cells, posttranslational modifications appear to affect differentially
the abilities of the HMGI and HMGY proteins to interact with AT-rich
ligands and nucleosomes (25).
(IFN
) gene (33). That work revealed, that the first
and the second AT-hook of the HMGI-C protein interact with PRDII and
NRDI elements of the promoter (33) and that phosphorylation by Cdc2
kinase of HMGI-C perturbs organization of the protein·DNA complex. In
this work we have extended our studies for other three members of the
HMGI/Y family: the HMGI and HMGY as well as the insect cHMGI protein.
We have found that the proteins differ in their affinities for binding
to the IFN
promoter, stabilities of the complex, and the number of
protein-DNA contacts. Furthermore, we demonstrate that phosphorylation
of the HMGY protein by Cdc2 kinase weakens contacts of the first
AT-hook to the PRDIII-1 element of IFN
promoter. We show that
phosphorylation of the HMGY protein attenuates the unbending activity
on the IFN
promoter. Our data support the experimental evidence that
various members of the HMGI/Y family have distinct functions.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]ATP was added to the reaction mixture.
The reaction products were separated by reversed-phase high performance
liquid chromatography. The identity and phosphorylation extent of the
isolated proteins were confirmed by matrix-assisted laser desorption
ionization/time-of-flight spectroscopy.
gene containing the
PRDIII-1, PRDII, and NRDI elements was prepared from synthetic
oligonucleotides (32, 33). Four-way junction DNA (4H DNA) was
prepared according to Bianchi (36). For DNA footprinting and
mobility shift experiments, the oligonucleotides were
32P-end-labeled with T4 polynucleotide kinase.
-DNA (30 nM) was partially digested with
hydroxyl radicals in a 10-µl reaction volume in presence or absence
of 30 nM HMG protein in 180 mM NaCl, 20 ng/µl bovine serum albumin, and 10 mM MOPS buffer, pH 7.2, at
room temperature for 20 min as described previously (33). The reaction
products were separated on 18% polyacrylamide sequencing gels
containing 7 M urea/TBE. The gels were scanned and the data
analyzed as described previously (33).
-DNA was methylated or ethylated with dimethyl sulfate
(37) or N-ethyl-N-nitrosourea (38), respectively. ~100 nM modified DNA was incubated with 0.3 µM protein, and the protein-DNA complexes were separated
from unbound DNA by gel electrophoresis. The DNAs were eluted from the
gels and cleaved at methylated purines or ethylated phosphates with
10% piperidine or 0.14 M NaOH, respectively. Finally,
equal amounts of radioactivity (~5000 cpm) of the cleavage products
were analyzed on sequencing gels. G+A standard was generated according
to Maxam and Gilbert (39). For data analyses, gels were scanned and gel
loading efficiencies were corrected. Intensities of the bands were
measured for bound and unbound fraction using ImageQuant software
(Molecular Dynamics) and EXCEL (Microsoft). The extent of interference
of modification at single site was defined as a ratio of the
intensities of bound to free DNA.
-mercaptoethanol, and 0.01% bromphenol blue). The reaction products were separated on
16.5% polyacrylamide gels using the Tricine-SDS buffer system (41) and
analyzed as described previously (32). Briefly, phosphorimages of the
full lane widths were scanned and the intensities were plotted
versus mobility (ImageQuant software). The intensity plots were aligned to correct distortions between different lanes using ALIGN
software. The aligned intensity plots were imported into Excel
(Microsoft), and gel-loading efficiencies and the extent of cleavages
were normalized. The electrophoretic mobilities were transformed into
amino acid residue positions, and mean values for each positions were
calculated. Finally, each amino acid residue position was compared in a
difference plot:
norm =
(Iwithout DNA
Iwith DNA)/Iwithout DNA,
where
norm is the normalized difference,
Iwithout DNA is mean value of the corrected PhosphorImager intensity of single residue position measured in the
absence of DNA, and Iwith DNA is the mean value
of corrected PhosphorImager intensity of the same position measured in the presence of DNA. Due to ambiguity of the assignment and poor
resolution, the peptides at the front of the gel and those of near
full-length protein, respectively, were excluded from the analysis.
Size markers were obtained by limited digestions of 10 pmol of
end-labeled HMG protein by trypsin, thermolysin, proteinase Arg-C,
proteinase Glu-C, proteinase Asp-N, or chymotrypsin in a 10-µl
reaction volume. Cleavage in the presence of 10 ng of trypsin,
thermolysin, or chymotrypsin was carried out in 180 mM
NaCl, 20 mM Tris/HCl, pH 7.5, at 0, 20, or 20 °C,
respectively. The reactions with trypsin were stopped by addition of 1 µl of 0.14 mM
N
-p-tosyl-L-lysine
chloromethyl ketone. The cleavage with proteinases Glu-C and Asp-N was
carried out in the presence of 50 ng of enzyme in 25 mM
sodium phosphate, pH 7.8, and 180 mM NaCl at 20 °C.
Digestion of the protein in the presence of 20 ng of Arg-C was
performed in 90 mM Tris/HCl containing 8.5 mM
CaCl2, 5 mM dithiothreitol, and 0.5 mM EDTA, at 20 °C. Finally, the reactions were stopped by addition of 4-fold SDS sample buffer with 20 mM EDTA.
DNA fragment
derivative with the amino-dT residues (Glen Research, Sterling, VA)
incorporated into base pair 7 (bottom strand) and base pair 28 (top
strand) was prepared by automated oligonucleotide synthesis. The bottom
and top strands were labeled with donor and acceptor fluorochrome and
were purified as described previously (42). The luminescent donor used
was (Eu3+)DTPA-AMCA (43, 44). The fluorescent
acceptor was Cy5 (45) purchased from Amersham Pharmacia Biotech. Upon
hybridization of donor- and acceptor-labeled strands, a 34-bp DNA
duplex was obtained with the probes in the major groove of DNA (42), on the opposite face of DNA with respect to protein-DNA interface, and
separated by two turns of DNA helix.
I is the measured intensity at time t
after the excitation pulse, and ai and
ti are the amplitude and the lifetime of the ith decay component, respectively. The decay curves were
fitted to Equation 1 by a nonlinear regression using SCIENTIST
(Micromath Scientific Software, Salt Lake City, UT). All decays could
be fitted to the two exponential decay equation. About 1-2% of the total donor signal (the amplitudes of donors corresponding to observed
lifetimes and amplitudes in sensitized acceptor decays were calculated
as described elsewhere2
decayed with a short lifetime (~100 µs), whereas remaining 98-99% of the signal decayed with a lifetime in the 400-550-µs range. The
latter lifetime was used for energy transfer calculations.
(Eq. 1)
The distances between donor and acceptor were calculated
according to Förster theory (52).
(Eq. 2)
R is a distance between a donor and an acceptor, and
R0 is a distance at which the energy transfer is
0.5. The R0 was calculated assuming a completely
randomized orientations of donor and acceptor. This assumption is
particularly valid in the case of long-lived europium chelate donors
(47-49) and practically eliminates the uncertainty of distance
measurement due to unknown mobility of fluorochromes.
(Eq. 3)
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Promoter--
Studies in which
properties of the all three mammalian HMGI/Y proteins have been
compared are rare. Maher and Nathans (29) were the first who
simultaneously analyzed binding properties of HMGI, HMGY and HMGI-C.
However, the authors focused their work on the role of the length and
the spacing of AT-tracts of the DNA ligand for tight binding revealing
differences between individual proteins in binding to various DNA
fragments. Using the IFN
promoter DNA fragment, we have extended
their work. We compared binding properties of the three human HMGI/Y
proteins and the insect cHMGI protein (Fig.
1). The proteins differ in their
affinities for the studied DNA fragment (Fig. 1B). The
interaction of the HMGI protein was found to be ~5 and ~8 times
stronger in comparison to HMGY and HMGI-C, respectively (Fig. 1,
B and C; Table I). The strong binding of Chironomus protein to this promoter
may suggest that function(s) of cHMGI are similar to that of HMGI and
HMGY in mammals (Fig. 1, B and C; Table I).
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Fig. 1.
The HMGI, HMGY, HMGI-C, and cHMG proteins
interact differentially with the IFN
promoter. A, schematic drawing showing primary
structures of the mammalian HMGI, HMGY, and HMGI-C and
Chironomus cHMGI. DBDs are indicated by boxes.
Closed and open circles indicate phosphorylation
sites of Cdc2 kinase and CK2, respectively. Numbers behind
sequences show length in amino acid residues. B, binding of
HMGI, HMGY, HMGI-C, and cHMGI to 34-bp fragment of the IFN
promoter.
<1 nM 32P-labeled IFN
DNA was incubated
with increasing concentrations of proteins and electrophoresed for
15 min on 8% polyacrylamide gels. The gels were dried, and the
radioactivity was quantified with a PhosphorImager. C,
quantification of the data from B. The percentage of free
DNA was plotted against ligand concentration according to Carrey (71).
The lines are theoretical curves calculated from the relationship
Kd = [free DNA] × [free protein]/[complexes].
The calculated Kd(app) are shown in Table I.
D, analysis of the stability of the protein·IFN
complexes. 300 nM protein were incubated with labeled DNA,
electrophoresed for various times (0.25-2 h), and analyzed as
described in B. This panel is only a representative analysis
for cHMGI (a), HMGY[PCK2PCdc2]
(b), and HMGY (c). Identical experiments with
other studied proteins are not shown. E and F,
analysis of the data from D. The ratio of [bound
DNA]t to [bound DNA]0 was plotted
against time. Time 0 indicate that the measurements starts after 15 min
of electrophoresis, which was necessary for separation of free and
bound DNA. The lines are theoretical curves calculated from the
relationship ln{[bound DNA]t/[bound
DNA]0} =
kdt. The
calculated kd(app) are shown in Table I.
Closed circles, HMGI; open circles, HMGY;
closed triangles, HMGI-C; open triangles, cHMGI;
diamonds, HMGY[PCdc2PCK2].
Properties of binding of the HMGI/Y proteins to the IFN fragment
complex, an intermediate value of 3.9 h was
determined. The complex containing cHMGI dissociated as rapidly as that
one of the HMGI protein (Fig. 1, D-F).
Promoter--
Hydroxyl radical protein footprinting experiments
with HMGI, HMGY, HMGI-C, and cHMGI proteins were performed to identify
regions of the proteins involved in contacts with the fragment of the promoter of the IFN
gene. Phosphorylation with CK2 kinase of the
sites located in the C-terminal region of each protein was used for the
end-labeling of the proteins (Fig. 1A). This phosphorylation reflects a constitutive modification found in native forms of these
proteins (53-55). The free and DNA-bound proteins were digested with
hydroxyl radicals, and the digestion products were separated by
electrophoresis. The gels were calibrated using arrays of peptides generated by limited digestion of the labeled proteins by different proteinases. As an example, digestion patterns of the HMGY protein are
shown in Fig. 2A. Relative
mobilities of the digestion products were transformed by nonlinear
regression into residue positions within the protein, which allowed the
alignment of the hydroxyl radical products (Fig. 2B). Since
the changes in the cleavage pattern are usually small (Fig.
3A), the radioactivity in the lanes was scanned and the intensities were transformed into difference plots (Fig. 3, B-D). This analysis revealed that each of
the proteins has an individual protection/exposition pattern (Fig. 3,
B-D), indicating that each protein-DNA complex has a
specific topology and a characteristic extent of contacts between the
protein and the promoter fragment. The differences between HMGI and
HMGY proteins are essentially moderate and reflect high similarity
between these proteins. In the presence of DNA, the HMGI protein was
protected mainly in the region of the DBD2 and to lower extent within
the other two AT-hooks (Fig. 3B). In contrast, all three
AT-hooks in the HMGY·DNA complex were similarly protected (Fig.
3B). In agreement with our previous data (33), footprinting
of HMGI-C revealed that only DBD1 and DBD2 of this protein are involved in contacts with the DNA fragment and that in the complex a large segment between the DBD2 and DBD3 is strongly exposed to the solvent (Fig. 3C). Binding of cHMGI protein involved the second and
the third DBD of the protein (Fig. 3D).
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Fig. 2.
Size markers and assignment of the bands for
protein footprinting of the HMGI/Y proteins. A, size
markers were generated by site-specific cleavage of
32P-end-labeled HMGY with thermolysin, endoproteinases
Glu-C and Arg-C, and trypsin. Hydroxyl-radical
lanes show peptide patterns of the protein digested with the
chemical proteinase in the absence ( ) or presence (+) of DNA.
B, plot of size of peptide markers versus
relative mobility. Relative mobility of uncleaved HMG protein was
defined as 0, and the most rapidly migrating band of hydroxyl radical
cleavage as 1. Open, black, dark gray,
and gray symbols correspond to peptide markers of
HMGY, HMGI, HMGI-C, and cHMGI, respectively.
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Fig. 3.
Protein footprints of IFN
fragment on HMGY and HMGI (A and
B), HMGI-C (A and
C), and cHMGI (A and
D). Hydroxyl-radical lanes
show peptide patterns of the protein digested with the chemical
proteinase in the absence (
) or presence (+) of IFN
fragment.
Averaged data from four to eight independent experiments were analyzed
and are shown in panels B-D. Positive values mean less
cutting in DNA-bound protein compared with unbound protein at this
particular position. Schematic primary structure of the individual HMGI
proteins with DBD (boxes) are in the panels. Residue
numbering in panel B is for HMGI. HMGI and HMGY are products
of a differentially spliced transcript. The gap
between residue
positions 34-44 reflects the absence of 11 residues in HMGY.
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Fig. 4.
Hydroxyl radical DNA footprints of the HMGI,
HMGY, and cHMGI proteins on the IFN
fragment. End-labeled DNA at the top (A) or
bottom (B) strand was digested with hydroxyl radicals in the
absence or presence of protein. The reaction products were separated on
a sequencing gel and scanned with a PhosphorImager, and bands were
quantified with ImageQuant software. Each bar shows relative
cutting frequency at a single base. 100% cutting frequency corresponds
to digestion of the DNA fragment in the absence of protein, so that
lower values mean protection upon protein binding. The concentrations
of the DNA and proteins were 30 nM. The top and bottom
strand sequences of the IFN
promoter are shown between the
panels.
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Fig. 5.
Ethylation (A) and
methylation (B) interference analyses of binding of
HMGI and HMGY to IFN DNA. The 5'-labeled
top strand of the IFN
-DNA was methylated or ethylated as described
under "Experimental Procedures." ~100 nM modified DNA
was incubated with 0.3 µM protein, and the protein-DNA
complexes were separated from unbound DNA by gel electrophoresis. The
DNAs were eluted from the gels and cleaved at methylated purines or
ethylated phosphates with 10% piperidine or 0.14 M NaOH,
respectively. Finally, equal amounts of radioactivity (~5,000 cpm) of
the cleavage products were analyzed on sequencing gels. The gels were
scanned and the analyzed as described in legend for Fig. 4.
Asterisks indicate the positions of C and T, which are not
methylated. The sequence of the analyzed strand of the IFN
promoter
is shown between the panels.
promoter. Therefore, we decided to study more
detailed how phosphorylation by the Cdc2 kinase affects binding of the
HMGY protein to DNA.
was
found upon phosphorylation of the protein at Ser-87, Ser-90, and Ser-91
by CK2 (Table I). Further phosphorylation by Cdc2 kinase additionally
weakened the interaction of the protein with this DNA (Table I).
Analysis of the binding of the protein to 4H DNA, which is a well
characterized ligand of the HMGI protein (28, 61, 62) revealed at least
10-fold reduction of the affinity of the CK2 phosphorylated protein
(Fig. 6, A and B). Consecutive phosphorylation by Cdc2 kinase further reduced the strength
of the binding (Fig. 6C). Interestingly, phosphorylation by
Cdc2 kinase had influence on the mobility of protein·DNA complex (Fig. 6C). Since the reduction of the mobility of the
complex cannot be directly attributed to the decrease of the charge of the protein, which should increase the mobility of the complex, it
appears that the change in the mobility reflects alterations in the
organization of the complex. Either changes in the complex stoichiometry or changes in conformation of 4H DNA are possible and
require further analysis.
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Fig. 6.
Effect of phosphorylation of HMGY on binding
to 4H DNA. <1 nM 32P-labeled 4H DNA was
incubated with increasing concentrations of nonphosphorylated and
phosphoforms of HMGY, and electrophoresed on 8% polyacrylamide gels at
20 °C. The gels were dried and autoradiographed. Differences in the
mobility of 4H DNA bound to HMGY[PCK2] and
HMGY[PCdc2PCK2] are indicated by
arrows to the left of panel
C. The protein concentrations were: a, 0.3;
b, 1; c, 3; d, 10; e, 30;
f, 100 nM.
and 4H DNA. We also analyzed binding of the protein to
poly(dA-dT)·poly(dA-dT) because earlier work showed that HMGI(Y)
proteins bind strongly to this type of DNA (63). Protein footprinting
of HMGY in the presence of poly(dA-dT)·poly(dA-dT) and IFN
promoter fragment revealed that three regions of the HMGY protein
carrying AT-hooks were protected from hydroxyl radical digestion (Fig.
7, A-C). In both complexes,
phosphorylation of the protein by Cdc2 kinase led to weakening of the
DBD1 binding and an substantial increase of protection in the region
containing DBD3 (Fig. 7, A-C). In the presence of 4H DNA,
regions comprising residues of DBD2 and DBD3 were protected from
digestion (Fig. 7, A and D). This observation
correlates well with a previous demonstration that the HMGI protein
binds to this 4H DNA at two positions on opposite arms of the junction
(61, 62). Phosphorylation by Cdc2 kinase impaired the binding of DBD2,
whereas contacts of the DBD3 appeared somehow strengthened (Fig.
7D).
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Fig. 7.
Hydroxyl radical protein footprinting of
poly(dA-dT)·poly(dA-dT) (A and
B), IFN fragment
(A and C), and 4H DNA (A
and D) on the end-labeled HMGY protein.
A, representative electrophoretic patterns of hydroxyl
radical digestions of the HMGY[PCK2] and
HMGY[PCdc2PCK2] proteins in the absence (
)
or presence (+) of poly(dA-dT)·poly(dA-dT) (64 bp/molecule HMGY),
IFN
fragment (2:1 DNA/protein), or 4H DNA (2:1 DNA/protein).
B-D, difference plots showing averaged data from four
independent experiments. Results of HMGY[PCK2] and
HMGY[PCdc2PCK2] are shown in black
and gray lines, respectively. Schematic primary
structure of the HMGY protein with DBD (boxes) are shown in
the lower part of the panels (B-D).
promoter upon phosphorylation of HMGY by Cdc2 kinase
phosphorylated, we carried out a DNA footprinting analysis. The
end-labeled 34-bp fragment of the promoter was digested with hydroxyl
radicals in the absence or presence of either HMGY[PCK2]
or HMGY[PCdc2PCK2] (Fig.
8). Three regions of the DNA
corresponding to PRDIII-1, PRDII, and NRDI elements were protected.
Phosphorylation of the protein by Cdc2 kinase resulted in a substantial
reduction of the protection at the PRDIII-1 element and a concomitant
increase of protection of the NRDI region (Fig. 8).
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Fig. 8.
Hydroxyl radical DNA footprints of the
HMGY[PCK2] and HMGY[PCdc2PCK2]
proteins on the IFN fragment. End-labeled
DNA (top strand) was digested with hydroxyl radicals in the absence or
presence of HMGY[PCK2] (black bar) or
HMGY[PCdc2PCK2] (gray bar). The
reaction products were separated on a sequencing gel and scanned with a
PhosphorImager, and bands were quantified with ImageQuant software.
Each bar shows relative cutting frequency at a single base.
100% cutting frequency corresponds to digestion of the DNA fragment in
the absence of protein, so that lower values mean protection upon HMGY
binding. The concentrations of the DNA and proteins were 30 nM.
Promoter--
The IFN
promoter is intrinsically curved. The
orientation of the intrinsic curvature of the PRDII element is toward
the minor groove, and a bend angle of 20° has been estimated by means
of phasing analysis (64). Binding of the HMGI protein to this element partially unbends this DNA (64). To measure the unbending potential of
HMGY and its phosphoforms, we have constructed the 34-bp IFN
fragment with Eu3+-DPTA-AMCA donor and CY5 acceptor
molecules separated by 2 helical turns of DNA and located in major
grooves on the opposite face of DNA with respect to where the proteins
bind (Fig. 9A). The distance
between the fluorochromes was monitored by LRET measurements.
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Fig. 9.
Luminescence resonance energy transfer
measurements. A-C, rationale of the LRET-based assay
for measurement of unbending of IFN -fragment. A, the
donor and acceptor moieties are located in the major groove of the
PRDIII-1 and NRD-I. The distance between fluorophores is 20 bp; thus,
in B-DNA, the distance between them would be R0 = 6.8 nm. Assuming that the total bend between the position of the
fluorophores is 20°, the changes in the bend angle upon protein
binding can be calculated using the equation system shown in
C. R, the distance calculated from energy
transfer measurements. D, an example of sensitized acceptor
decay of donor- and acceptor-labeled 34-bp DNA fragment. The curve
shown is for the DNA in the absence of any protein. The
solid line is a nonlinear regression fit to
two-exponential decay model. Inset, comparison of
decay-curves for LRET of the DNA in the absence (solid line)
and presence (broken line) of HMGY. E, the
quality of the fit is demonstrated by random distribution of residuals
and by a value of
2R close to 1. The quality of the fits observed with every protein studied was
comparable to an example shown in this figure.
gene is not a primary target for HMGI-C.
Binding a HMGI/Y-related protein, HMGIY-L1 (66), in which two of the
three AT-hooks are mutated and do not bind tightly to this DNA (67),
has no effect on the bend angle of the DNA.
Summary of luminescence resonance energy transfer measurements
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-enhanceosome (4, 6). This study shows that HMGI binds to
IFN
promoter with the highest affinity, whereas the other two human
proteins, HMGY and HMGI-C, bind less tightly. This appears as a logical
consequence because usually HMGI is more abundant than HMGY and the
occurrence of HMGI-C is restricted to early development or
neoplastically transformed cells. An inverse situation was found for a
promoter that is regulated by HMGI-C. The HMGI-C protein exhibited the highest binding strength to this promoter, whereas binding of HMGI was
less tight.3 Thus, it seems
that each of the HMGI/Y proteins plays a specific role in gene
regulation by activation/inactivation of a specific set of genes. The
findings that HMGI(Y) cannot substitute HMGI-C in the pygmy phenotype
(23) and that HMGI and HMGY translation and/or stability is
differentially regulated (24) are in agreement with this postulate.
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Fig. 10.
Model of DNA binding by HMGI-C and HMGY
within the IFN fragment and its modulations
upon Cdc2 phosphorylation. A, DBD 1 and 2 of the HMGI-C
protein bind to the NRDI and PRDII elements of the IFN
promoter,
respectively. After phosphorylation of the protein at Ser-43 and Ser-58
by Cdc2 kinase multiple contacts of DBDs, especially with the bases,
are impaired and the protein binds to DNA in a different way, extending
the contacts to the sugar-phosphate backbone (33). B, three
DBDs of the HMGY protein interact with AT-rich tracts of the DNA.
Phosphorylation of the protein by Cdc2 kinase at Thr-41 and Thr-67
weakens binding of DBDI to the PRDIII-1 element. Ellipses,
DBDs in proteins that are phosphorylated by CK2 (black) or
by Cdc2 and CK2 (gray). P, sites that are
phosphorylated by Cdc2 kinase.
As DBDs of the human HMGI, HMGY and HMGI-C protein are nearly identical, the differences in the binding of these proteins to particular DNAs appear to result from the other parts of these proteins. In this respect, both the length of the polypeptide chain between the DBDs and its primary structure seem to be important. The differences in the binding properties between HMGI and HMGY must be attributed to the presence of additional 11 apolar residues between the first and the second AT-hook in the HMGI protein. The HMGI-C protein is rich in glutamine (12% versus 5% in HMGI), a residue that can form two hydrogen bonds with adenine and guanine in the major and minor groove, respectively (68). Moreover, the absence or presence of leucine and isoleucine residues in HMGI(Y) proteins versus absence of these residues in HMGI-C, may also influence the nature of how these proteins interact with specific DNA.
The properties and specific functions of majority of HMG proteins are probably regulated by phosphorylation (55). In respect to HMGI/Y family, CK2 and Cdc2 kinase appear to have a pivotal position. Whereas CK2 phosphorylation plays obviously a constitutive role in protein conformation and stability (55), the Cdc2 kinase modulates the properties of these proteins temporarily during late G2 and M phases of the cell cycle (56-58). In contrast to transcription factors and other HMG proteins (HMG1 family), the HMGI/Y proteins remain on the chromosomes during cell division (69, 70). Our binding studies show that the DNA binding affinity of the Cdc2 phosphorylated HMGY and HMGI-C (33) is several times weaker in comparison to their "interphase" forms lacking this modification, but probably sufficiently high to explain their presence in mitotic chromosomes. Changes in the conformation of gene promoters on which HMGI/Y proteins bind, resulting from Cdc2 phosphorylation, may contribute to the process of disassembly of promoter complexes.
The phosphorylation of the HMGY protein at residues adjacent to DBD 2 and DBD3 by Cdc2 kinase affects mainly binding of the DBD1 (Fig. 10). This finding emphasizes the cooperative nature of DNA binding of the AT-hooks in proteins of the HMGI/Y family. Moreover, since this binding domain is located distantly from the phosphorylation sites (in the primary structure scale), our finding stresses the advantage of studying protein-DNA interactions of this type of proteins using entire and native molecules over using peptides derived from the sequence. In HMGI-C the Cdc2 phosphorylation sites flank the DBD2, and the phosphorylation affects mainly contacts of DBD2 with the minor groove of the PRDII element (33).
The native binding sites of the HMGI(Y) proteins in the
promoter/enhancer regions of many genes targets have been described (1,
2). In contrast, target genes for HMGI-C are still not reported. A
number of genes that are up- or down-regulated by a HMGI-C
gene expression have been found recently using the micro-array technique.4 By means of the
methods employed in this study and using appropriate regions of these
putative genes, it could be possible to analyze the organization of a
native HMGI-C complex. Furthermore, studies in which the structural
changes upon binding of mutants of HMGI-C protein that are found in
many tumors could contribute to understanding molecular events
important for the role of this group of HMG proteins in neoplasia.
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ACKNOWLEDGEMENT |
---|
We thank Dr. U. Grossbach for continuous support and interest.
![]() |
FOOTNOTES |
---|
* This work was supported by Grants Wi-1210/2-1 and Wi-1210/3-1 (to J. R. W.) from the Deutsche Forschungsgemeinschaft.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Present affiliation: Inst. of Biochemistry, Warsaw University, 02-096 Warszawa, Poland.
** To whom correspondence should be addressed. Fax: 49-551-395416; E-mail: jwisnie@gwdg.de.
Published, JBC Papers in Press, October 16, 2000, DOI 10.1074/jbc.M004065200
2 T. Heyduk, manuscript in preparation.
3
R. Schwanbeck and J. R. Winiewski,
unpublished results.
4 J. Bullerdiek, unpublished results.
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ABBREVIATIONS |
---|
The abbreviations used are:
HMGI/Y family, diverse proteins countering multiple AT-hooks;
CK2, casein kinase 2;
DBD, DNA-binding domain containing an AT-hook;
HMG, high mobility
group;
HMGI(Y), HMGI and HMGY proteins that are products of a single
gene;
cHMGI, HMGI protein from the midge Chironomus tentans;
IFN, interferon
gene;
LRET, luminescence resonance energy
transfer;
bp, base pair(s);
4H DNA, four-way junction DNA;
Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine;
MOPS, 4-morpholinepropanesulfonic acid.
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