From the III. Zoologisches Institut-Entwicklungsbiologie, Universität Göttingen, Humboldtallee 34A, 37073 Göttingen, Germany
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ABSTRACT |
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The high mobility group proteins I and Y (HMGI/Y)
are abundant components of chromatin. They are thought to derepress
chromatin, affect the assembly and activity of the transcriptional
machinery, and associate with constitutive heterochromatin during
mitosis. HMGI/Y protein molecules contain three potential DNA-binding
motifs (AT-hooks), but the extent of contacts between DNA
and the entire protein has not been determined. We have used a
protein-footprinting procedure to map regions of the
Chironomus HMGI protein molecule that are involved in
contacts with DNA. We find that in the presence of double-stranded DNA
all AT-hook motifs are protected against hydroxyl radical
proteolysis. In contrast, only two motifs were protected in the
presence of four-way junction DNA. Large regions that flank the
AT-hook motifs were found to be strongly protected against
proteolysis in complexes with interferon- promoter DNA, suggesting
amino acid residues outside the AT-hooks considerably contribute to DNA binding.
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INTRODUCTION |
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The family of high mobility group proteins I/Y (HMGI/Y)1 comprises four structurally related proteins, the mammalian HMGI, HMGY (1), HMGI-C (2), and the Chironomus HMGI protein (3). Moreover, proteins with some extent of similarity to HMGI have been found in plants (for review see Ref. 4), bacteria (5), and Drosophila (6).
The properties of the insect cHMGI protein resemble the major structural features of the mammalian HMGI and Y proteins. It contains three DNA-binding motifs (AT-hooks) (7) and a negatively charged COOH-terminal domain (3) and have similar charge distribution in the regions flanking the AT-hooks (8). Both human and Chironomus HMGI/Y proteins bind preferentially to four-way junction DNA (3) and to AT-tracts of double-stranded DNA (3, 9). Their binding alters the DNA conformation (10-12) and unbends intrinsically bent DNA (13). Moreover, these proteins from evolutionarily distant organisms are substrates of Cdc-2 kinase (8, 14), mitogen-activated protein kinase (8), and protein kinase C (8, 15). Phosphorylation reduces their DNA-binding affinity (14-16) and alters the mode of binding to DNA (8).
Diverse biological functions for the mammalian proteins have been
suggested. Initially the HMGI/Y proteins were considered as specific
components of constitutive chromatin (17, 18). Further studies revealed
that they are involved in the modulation of transcription of specific
genes (for review see Ref. 19). Studies on the human interferon-
(IFN-
) gene and the gene encoding the
-subunit of the interleukin
2 receptor showed transcriptional activation upon binding of HMGI(Y)
proteins to positive regulatory domains (PRD), which
facilitate binding of transcription factors (20-22). More recently,
HMGI/Y proteins were identified as components of a repressor complex
that inactivates the promoter of the T cell receptor
-chain gene
(23) and as crucial host proteins in the HIV-1 preintegration complex
(24). Cytological studies of insect polytene chromosomes have
demonstrated that the cHMGI protein is present in many
transcriptionally active loci and in nucleoli, suggesting that HMGI/Y
proteins are involved in polymerase I and II transcription (25). They
are highly abundant in undifferentiated and rapidly dividing cells (25,
26). Elevated levels of HMGI/Y in differentiated tissues have been
found to be correlated with progressive and neoplastic transformations
(27, 28). Disruption or rearrangements of their genes lead to
tumorigenesis (29, 30).
Whereas different biological effects of HMGI/Y proteins have been
described, the molecular and biochemical mechanism of affecting DNA and
chromatin structure is not well defined. The spatial organization of
HMGI/Y·DNA complex remains only partially understood. NMR data of a
complex of a truncated form of the protein and a DNA dodecamer show
that the central part of the AT-hook domain, the Arg-Gly-Arg motif, interacts with the bases and the sugar within the minor groove
of the DNA double helix (31). In addition, several residues flanking
this motif interact with the sugar-phosphate backbone and are
responsible for the strength of the overall protein DNA binding (31).
Moreover, the central AT-hook motif mediates specific DNA
binding and cooperates the other two AT-hooks (32). Here we
report the mapping of the regions of the cHMGI protein involved in
contacts with various types of DNA, including linear synthetic poly(dA-dT)·poly(dA-dT), four-way junction DNA, and a region of the
promoter of the IFN- gene. The mapping was performed by means of
protein hydroxyl radical-footprinting technique (33). The data
presented show that the interaction of cHMGI protein with DNA involves
residues of two or three AT-hook motifs dependent on the DNA
type and/or the protein to DNA ratio. Large regions flanking these
motifs also contribute to the binding of cHMGI to DNA.
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EXPERIMENTAL PROCEDURES |
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Preparation of Bacterially Expressed cHMGI and Protein Determination-- The cHMGI protein was overexpressed in Escherichia coli (12) and purified on high performance liquid chromatography columns as described previously (34, 35). The eluted protein was vacuum concentrated and lyophilized. The dried cHMGI was quantified gravimetrically.
32P-Labeling of cHMGI on Ser3--
100
µg (9.64 nmol) of the cHMGI protein were phosphorylated at
Ser3 (8) at 30 °C with 8 units of recombinant human
Cdc2-kinase (New England Biolabs Inc.) for 4 h in the presence of
3.5 mM ATP and 100-150 µCi [ 32P]ATP in
8 µl Cdc-2 kinase buffer containing 50 mM Tris/HCl, 10 mM MgCl2, 1 mM dithiothreitol, 1 mM EGTA, pH 7.5. The reaction was stopped by precipitation
of the proteins with 30% (w/v) CCl3COOH for 30 min at
0 °C. The pellet was washed with 30% CCl3COOH, 0.2% HCl in acetone, twice with pure acetone and dried.
DNA and Oligonucleotides--
The synthetic linear
poly(dA-dT)·poly(dA-dT) DNA was from Amersham Pharmacia Biotech. The
approximate average length of this DNA was 5000 bp. The 34-bp fragment
of the promoter of the IFN- gene containing the PRDII/NRDI sites was
prepared from synthetic oligonucleotides. The sequence of the top
strand was 5'-GAAGTGAAAGTGGGAAATTCCTCTGAATAGAGAG-3' (PRDII site is underlined). Four-way junction c was prepared
according to Bianchi (36).
Hydroxyl Radical Footprinting--
100 pmol of the radioactively
end-labeled protein (15,000-30,000 cpm) were incubated in the presence
or absence of DNA in 257 mM NaCl and 14.3 mM
MOPS, pH 7.2, buffer at room temperature for 15 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
(NH2)2Fe(II)(SO4)2;
(ii) 0.2 M sodium ascorbate; and (iii) 0.375%
H2O2. If not specified otherwise, the reactions
were stopped after 40 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%
-mercaptoethanol, and 0.01% bromphenol blue). The reaction products
were separated on 16.5% polyacrylamide gels using the
Tricine-glycine-SDS buffer system (37). The gels were dried and scanned
by a PhosphorImager (Molecular Dynamics).
Size Markers and Assignment of the Hydroxyl Radical Cleavage
Sites--
Size markers were obtained by limited digestions of the
end-labeled cHMGI protein with trypsin or proteinase Glu-C (V8). 100 pmol of end-labeled cHMGI were digested with 17 ng of trypsin in 10 µl of 180 mM NaCl, 20 mM Tris/HCl, pH 7.5, at
0 °C for 5 min. Reactions were stopped by addition of 1 µl of 0.14 mM N-p-tosyl-L-lysine chloromethyl ketone (TLCK). The cleavage with proteinase Glu-C (V8) was
carried out in the presence of 50 ng of enzyme in 25 mM
sodium phosphate, pH 7.8, and 180 mM NaCl at 0 °C for 2 min. Reactions were stopped by addition of SDS sample buffer and
immediate boiling of the probe. The end-labeled peptides 1-48 and 1-6
were obtained by cleavage of the cHMGI protein with hydroxylamine (38) and trypsin (8), respectively. The peptide 1-32 was synthesized as
described previously (8). The assignment of the hydroxyl radical
fragments was accomplished using a standard curve.
Data Analysis--
The phosphorimages were essentially analyzed
according to Heyduk et al. (39) and Baichoo and Heyduk (40).
Briefly, phosphorimages of the full lanes width were scanned and the
intensities were plotted versus mobility (ImageQuant
Software, Molecular Dynamics). The intensity plots were aligned to
correct distortions between different lanes using ALIGN software (gift
from Dr. T. Heyduk, St. Louis, MO). The aligned intensity plots were
imported into EXCEL (Microsoft), 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 position was 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 the 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. Cutting frequencies within tryptic protein
footprints were calculated by integrating intensities of the bands
subtracted from background (ImageQuant). During electrophoresis,
digestion products shorter than 7 or longer than 90 residues were not
resolved. Therefore, the difference plots were calculated excluding
these regions (39).
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RESULTS |
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Experimental Strategy-- Electrophoretic analysis of the products of a limited digestion of protein labeled at its NH2 terminus yields a characteristic pattern. With DNA-binding proteins, changes in the electrophoretic pattern can be observed. In the presence of DNA, disappearing or fading of bands in defined regions (footprints) can be related to a protection of the protein at sites contacting the DNA. To map on the Chironomus HMGI protein regions of DNA binding it was (i) radioactively phosphorylated at Ser3, (ii) partially digested with sequence specific proteinases or hydroxyl radicals in the presence or absence of DNA, and (iii) the digestion products were separated on polyacrylamide gels. (iv) Finally the gels were subjected to quantitative scanning and objective data analysis, i.e. corrections for gel loading, cleavage efficiency, and the transformation of the electrophoretic mobility of the bands into residue numbers (39, 40).
Cleavage Conditions and Assignment of the Bands-- Phosphorylation of the cHMGI protein at Ser3 could be used as an end-labeling procedure because this is a unique target of Cdc-2 kinase, and because its modification does not change the DNA binding properties of the protein (8). Limited digestion of the labeled cHMGI by proteinase Glu-C or trypsin followed by electrophoresis yielded patterns in which the individual bands could be assigned to peptides of defined lengths (Fig. 1, A, Glu-C and trypsin, and B). Application of end-labeled peptides 1-48, 1-32, and 1-6 facilitated this assignment. Nonlinear regression enabled transformation of the relative mobilities of the cleavage products into residue sites within the protein (Fig. 1B).
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In the Presence of poly(dA-dT)·poly(dA-dT) DNA, Three AT-hook Domains Are Protected from Digestion-- Earlier work has revealed that mammalian HMGI protein binds strongly to poly(dA-dT)·poly(dA-dT) (9). Because this synthetic DNA is also a good ligand of cHMGI, we chose it for our experiments. cHMGI was footprinted in the presence of concentrations of poly(dA-dT)·poly(dA-dT) ranging from 16 to 120 bp per protein molecule (Fig. 3, A and B). At lower ratios (16 bp:1 and 32 bp:1) protection was observed at amino acid residues 10-22 and 54-60. In addition, protection at amino acid residues 30-40 was detected. The region between residues 10 and 20 corresponds to part of the first AT-hook sequence motif and adjacent NH2-terminal stretch. The region 53-60 constitutes the second AT-hook motif. At protein to DNA ratios of 60 bp:1 (Fig. 3B) and 120 bp:1 (not shown) an additional region, amino acid residues 75-81, which comprises the third AT-hook, was protected. Under these conditions, protection of region 30-40 was no longer observed, and in contrast to lower DNA to protein ratios, a large portion of the protein (residues 29 to 50) was found to be highly susceptible to digestion and should therefore be exposed to the solvent. At all concentrations of DNA used an enhanced susceptibility to digestion was observed for the negatively charged COOH terminus of the protein.
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Binding of cHMGI to Oligonucleotide with Binding Site of Human HMGI
Involves the Three AT-hooks and Regions Flanking AT-hooks Two and
Three--
In vivo binding sites of the insect HMGI on the
DNA are not known. Because most properties of this protein are
identical or similar to those of mammalian HMGI, we analyzed the
binding determinants of cHMGI in complexes with a DNA sequence
comprising a mammalian ligand. A 34-bp DNA carrying PRDII/NRDI elements
of the promoter region of the human IFN- gene was selected for the
experiments. The stoichiometry of cHMGI binding to this DNA is
1:1.2 Footprinting revealed
protection of residues 46-82 and 8-10 (Fig. 3, C and
D). The NH2-terminal located region corresponds
to part of the first AT-hook motif, whereas the large
protected region comprises the other two AT-hooks and areas
flanking these motifs. Regions of cHMGI rich in negatively charged
residues (20-44 and 85-90), as in the complex with
poly(dA-dT)·poly(dA-dT) were found to be cleaved more frequently than
without DNA and therefore probably exposed to solvent in complex with
the DNA.
cHMGI Binding to Four-way Junction DNA Does Not Involve the Third AT-hook Motif-- Chironomus and human HMGI proteins specifically recognize cruciform DNA (3). cHMGI was footprinted at different concentration ratios of DNA to protein (1:4, 1:2, 1:1, and 2:1) (Fig. 3E). The results were almost identical at all four conditions and showed protection of residues 9-15 and 51-74. The first region corresponds to part of the first AT-hook motif, whereas the second one comprises the second AT-hook motif and residues between the second and third AT-hooks (Fig. 3F). Interestingly, the third AT-hook was not protected in the presence of the cruciform DNA. The regions of amino acid residues 26-34, 38-48, and >82 were found to be more susceptible in the presence of four-way junction DNA and are thus probably exposed within the complex.
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DISCUSSION |
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Histones and the HMG proteins are the most abundant components of chromatin (for review see Refs. 43 and 44). In the structure of the histones and HMG1/2 proteins, well characterized folded domains are found in central positions. Another characteristic structural feature shared by these proteins is the presence of large structurally undefined regions, termini, bristles, or tails. Two groups of the HMG proteins, the families HMGI/Y and HMG14/17, are thought to be composed mainly of flexible regions of undefined structure. Binding to DNA induces a spatial ordering of regions containing residues involved in contacting DNA. Binding of the HMGI protein to DNA simultaneously leads to specific ordering of the protein structure (31) and induces changes in the conformation of the DNA (10-13). The contacts of the entire HMGI molecule to DNA in protein-DNA complexes have been unknown. Here we have mapped for the first time the regions of an HMGI protein that are involved in the binding to DNAs of various types.
Our protein footprinting experiments revealed some general and some DNA
structure-specific features of the cHMGI·DNA complex (Fig.
4). Binding of cHMGI to
poly(dA-dT)·poly(dA-dT), to cruciform DNA and to HMGI binding site in
interferon- promoter DNA (PRII/NRDI) involved contacts by the first
and second AT-hook motifs and led to exposition to the
solvent of large parts of the region joining these two motifs and the
COOH-terminal acidic tail of the protein. At ratios higher
than 30 bp of the poly(dA-dT)·poly(dA-dT) per protein molecule, the
third AT-hook motif was also protected. This suggests a
lower DNA-binding affinity of the third motif as compared with the
other two motifs and also that the entire protein occupies more than 30 bp on this DNA. This is reminiscent of the situation observed in human
HMGI, where the third AT-hook exhibited a several orders of
magnitude lower DNA-binding affinity in comparison to the second motif
(31). Also, an 18-bp-long DNA molecule was found not to accommodate the
entire HMGI protein (31). Furthermore, a 27-bp PRDII/NRDI-DNA fragment
and a 40-bp PRDI/PRDII/NRDI-DNA fragment were able to bind only one
molecule of an HMGI-C (45) or HMGI (32), respectively.
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Binding of cHMGI protein to the 34-bp PRDII/NRDI-DNA appears to involve all three AT-hook motifs and also residues in front of the second motif and between the second and the third motif. These data suggest that the protein contacts with this DNA are more extensive in comparison to those found in poly(dA-dT)·poly(dA-dT) and probably reflect a capability of the cHMGI to recognize specific sequence contexts and/or a specific conformation of this DNA, which are absent in the poly(dA-dT)·poly(dA-dT). This DNA with alternating A/T residues has a widened minor groove and is conformationally flexible in solution (46), resulting in a weaker binding of the HMGI protein as compared with binding to rigid or intrinsically curved AT-rich DNA (47). The PRDII/NRDI-DNA is intrinsically pre-bent by about 20°, and binding of human HMGI to this DNA induces a partial reversal bending (13). A similar unbending of the PRDII/NRD-DNA was also exhibited by the insect HMGI protein.3 Our finding that the insect HMGI protein makes extensive contacts with this specific DNA is in accordance with data from recent NMR studies that suggest extensive contacts between HMGI and PRDII-DNA and have revealed that 19 of the 42-residues-long HMGI(2/3) peptide are directly involved in the interaction with a PRDII-dodecamer (31).
In contrast, in complexes with cruciform DNA the third motif of the cHMGI protein remained unprotected, suggesting that this DNA cannot accommodate the entire protein or that the protein has a specific conformation in a complex with the cruciform DNA. Because this type of DNA is able to accommodate up to two HMGI molecules (48), the second possibility appears to be more probable. Interestingly, in this complex the region joining the second and the third motif was protected. It contains another sequence motif, PKRP (Fig. 4), which occurs not only in the HMGI/Y proteins but is also characteristic for proteins of the families HMG14/17 and HMG1/2. Peptides carrying this motif interact specifically with the minor groove of the DNA (35). Because all of these proteins exhibit preferential binding to cruciform DNA, it is possible that this motif plays a role in the recognition of this type of DNA.
Four-way junction DNA has been suggested as a model for DNA in chromatin at the site where it enters and exits the nucleosome (49, 50). The binding of HMGI to this DNA may reflect a constitutive function of this protein in the organization of chromatin, in contrast to specific functions as organization of promoter complexes of particular genes. We have recently shown that phosphorylation at various residues may be involved in the adaptation of members of the HMGI/Y family to fulfill different cellular functions (8). In further studies, this possibility could be checked by investigating the binding of various phosphoforms of HMGI/Y proteins to DNA as well as to nucleosomes. By means of the protein-footprinting technique applied in this study it would also be possible to map contacts of the cHMGI protein within reconstituted chromatin or its selected components.
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ACKNOWLEDGEMENTS |
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We thank Dr. U. Grossbach for the continuous support and interest in this work, Dr. T. Heyduk (St. Louis University) for providing the ALIGN software for the quantitative analysis of the phosphorimages and stimulating discussion.
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FOOTNOTES |
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* This work was supported by Grant Wi-1210/2-1 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.
These authors contributed equally to this work.
§ To whom correspondence should be addressed. Fax: 49-551-395416; E-mail: jwisnie{at}gwdg.de.
The abbreviations used are:
HMGI/Y, high
mobility group protein I or Y; HMG, high mobility group protein; cHMGI, Chironomus HMG protein IPRDII/NRDI, positive regulatory
domain II/negative regulatory domain IIFN-, interferon
bp, base pair(s)MOPS, 4-morpholinepropanesulfonic acidTricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine.
2
M. Gymnopoulos and J. R. Winiewski,
unpublished results.
3
T. Heyduk and J. R. Winiewski,
unpublished results.
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REFERENCES |
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