MOLECULAR AND CELLULAR BIOLOGY, Feb. 1994, p. 1410-1418
Vol. 14, No. 2
0270-7306/94/$04.00+0 Copyright X) 1994, American Society for Microbiology
suGF1 Binds in the Major Groove of Its Oligo(dG). Oligo(dC) Recognition Sequence and Is Excluded by a Positioned Nucleosome Core DANIELLE PAT ERTONt AND JANET HAPGOOD* Research Centre for Molecular Biology, Department of Biochemistry, University of Cape Town, Rondebosch 7700, South Africa
Received 7 June 1993/Returned for modification 24 August 1993/Accepted 15 November 1993
We have elsewhere reported the purification of a poly(dG) poly(dC)-binding nuclear protein (suGFl) from urchin embryos (J. Hapgood and D. Patterton, Mol. Cell. Biol. 14:this issue, 1994). We proposed that suGFl may be a member of a family of G-string factors involved in developmental gene regulation, possibly via alterations in chromatin structure. In this article, we characterize the binding of purified suGFl to 11 contiguous Gs in the H1-H4 intergenic region of a sea urchin early histone gene battery in vitro. It is shown that suGFl-DNA binding is dependent on ionic strength and requires divalent cations. Purified suGFl forms discrete protein-DNA multimers, consistent with suGFl-suGFl interactions. In a model for the suGFl-DNA complex derived from our footprinting and methylation interference data, suGFl contacts the Gs in the major groove as well as one of the bordering phosphate backbones. The data are consistent with the direction of curvature of the DNA in the suGFl-DNA complex being the same as that preferred by the free DNA and exhibited by the DNA when bent around a positioned nucleosome core in vitro. However, on the basis of steric considerations, the binding of suGFl and that of the histone octamer are predicted to be mutually exclusive. We show that suGFl is indeed unable to bind to the G string when occupied by a histone octamer located in the major in vitro positioning frame in the H1-H4 intergenic region. sea
urchin embryo (13, 44). suGF1 binds with high affinity and specificity in vitro to poly(dG) poly(dC) and to oligo(dG). oligo(dC)-containing sequences, including a G string present in the H1-H4 intergenic region of a sea urchin early histone gene battery (13). This region contains a string of 11 contiguous Gs flanked by 16 GA repeats and has been shown to adopt a triple-helix DNA structure under conditions of negative superhelical stress and/or low pH in vitro (16, 27, 31, 38). The suGF1 recognition site is located close to the dyad of a strongly positioned nucleosome core in both linear and supercoiled plasmids containing the H1-H4 intergenic region (31, 32). suGF1 shares many properties with BGP1 (13), including an identical DNase I footprint on the G string in the pA_globin promoter in vitro (9, 13, 33). We have suggested that suGF1 may play a role in gene regulation of the sea urchin early histone gene battery similar to the role proposed for BGP1 in regulation of chicken erythrocyte globin gene expression (13). As a first step towards elucidating the mechanism whereby a G-string factor may affect gene regulation, we investigated the biochemical and structural features of the suGF1-DNA interaction. Our results allowed us to construct a model of the complex of suGF1 with the G string in the H1-H4 intergenic region. On the basis of predictions from this model, we discuss the possibility of the involvement of suGF1 in alterations in chromatin structure. Finally, we use an electrophoretic mobility assay to directly investigate whether suGF1 binds to the G string when it is wrapped around a histone octamer.
Long runs of contiguous Gs frequently occur upstream of eukaryotic genes and have been implicated in gene regulation in several studies (4, 22). The ability of these G strings to form unusual DNA structures such as triple-helix DNA in vitro has been proposed to play a role in this process (4, 22, 23, 25). Such unusual structures may be stabilized by the binding of transcription factors or, alternatively, hinder factor binding in vivo. Current experimental evidence for the proposed role of factors binding to G strings is, however, indirect. The chicken erythrocyte protein BGP1 is a well-studied example of a poly(dG) poly(dC)-binding factor thought to be involved in gene regulation (4, 25). In this case, there is strong correlative evidence that binding of BGP1 to a string of 16 to 18 Gs in the chicken pA_globin promoter destabilizes or excludes a positioned nucleosome, allowing access of transcription factors to their cognate sites in vivo (4, 25). The nuclease susceptibility of the G-string region in the 3A_globin promoter in nuclei is consistent with the presence of a positioned nucleosome when the gene is not expressed and the absence of a nucleosome as well as the presence of BGP1 when the gene is actively transcribed (18, 19). We have reported elsewhere the purification of a 59.5-kDa protein (suGF1) from embryonic sea urchin nuclei (13). suGF1 is implicated as a transcription factor involved in the regulation of expression of several unrelated genes in the sea * Corresponding author. Present address: Regulatory Peptides Research Unit, Department of Chemical Pathology, Medical School, University of Cape Town, Observatory 7925, South Africa. Phone: (021) 406 6354. Fax: (021) 406 6153. Electronic mail address:
[email protected]. t Present address: Laboratory of Molecular Embryology, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892.
MATERIALS AND METHODS DNA fragments and protein preparations. Plasmids were purified and gene fragments were isolated as described elsewhere (13). The 335-bp EcoRI-HindIII fragment (probe 1410
suGFl BINDING TO OLIGO(dG)- OLIGO(dC)
VOL. 14, 1994
1) and 216-bp Asp 718-XbaI fragment (probe 2) both contain part of the H1-H4 intergenic region including the
(GA)16G11
sequence of the Psammechinus milians early histone gene battery cloned into pHP2 (30). Probe 1 or 2 was chosen to allow selective labeling of the Watson or Crick strand, respectively. Fragments were 3' end labeled by a Klenow fill-in reaction (13). suGF1 was purified from Parechinus angulosus 14-h embryos (13). EMSA and DNase I footprinting. Electrophoretic mobility shift assays (EMSAs) and standard DNase I footprinting were carried out essentially as described elsewhere (13). For analysis by EMSA of the effects of EDTA on suGFl DNA binding, purified suGF1 was extensively dialyzed at 4°C against two changes of buffer (20 mM HEPES [N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid] [pH 8.0], 100 mM KCl, 1.0 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 20% [vol/vol] glycerol, 0.1% [vol/vol] Nonidet P-40) in the absence or presence of 50 mM EDTA prior to EMSA. For analysis of specific complexes by DNase I footprinting, end-labeled DNA was incubated for 30 min at 4°C with purified suGF1 and 6 Fg of poly(dI-dC) (pdIdC) in a final volume of 100 pl of EMSA incubation buffer containing 153 mM KCl and 72 p,g of bovine serum albumin (BSA) per ml. Reaction mixtures were placed on ice for 1 min, adjusted to 15 mM MgCl2 and 15 mM CaCl2, and DNase I was added to final concentrations of 0.37 to 3 pLg/ml. The digestions were stopped after exactly 1 min by the addition of 16.8 ,ul of stop solution B (80 ,ul of 0.5 M EDTA, 254 pl of EMSA incubation buffer) and loaded onto an EMSA gel. Complexes were excised from the wet gel after autoradiography, and DNA was isolated and electrophoresed on sequencing gels together with Maxam and Gilbert G-sequencing standards (1, 29). Methylation interference. Methylated, end-labeled DNA (12 ng) was incubated with 2.8 ng of purified suGFl and 9 pg of pdIdC in a final volume of 120 ,ul in EMSA incubation buffer (13) containing 175 mM KCl and 60 p,g of BSA per ml, and the samples were electrophoresed on EMSA gels (13). Free and complexed DNA was isolated and purified after wet autoradiography, cleaved with piperidine, lyophilized, and electrophoresed on sequencing gels together with unselected methylated and piperidine-cleaved DNA (1). Hydroxyl radical footprinting. Hydroxyl radical footprinting was based on published methods (40, 41). An iron (II)-EDTA stock solution containing 13 mM ferrous ammonium sulfate ([NH4]2Fe[SO4]2- 6H20; Aldrich) and 26 mM EDTA was stored in aliquots under nitrogen at -70°C in the dark. H202 (30% [vol/vol]; BDH) was stored at 4°C, and a 130 mM stock solution of ascorbic acid (pH 7; Merck) was stored at -20°C. These reagents did not have any significant effect on suGF1 DNA binding at the concentrations used, as assessed by EMSA and DNase I footprinting. End-labeled DNA (2 ng) was incubated with purified suGF1 (2.8 ng) and 2 p,g of pdIdC in a final volume of 77 pl for 30 min at 4 or 37°C in 20.8 mM HEPES-225 mM KCl-1.3 mM MgCl2-1.1 mM dithiothreitol-0.52 mM phenylmethylsulfonyl fluoride104 p,g of BSA per ml. Iron(II)-EDTA, ascorbic acid, and H202 were mixed and added to final concentrations of 1 mM iron(II), 2 mM EDTA, 2.3% (vol/vol) ascorbic acid and 1 mM H202. The reaction was allowed to proceed for 2 min at the appropriate temperature and stopped by the addition of 10 pl of a stop solution (750 pl of 80% [vol/vol] glycerol, 87.5 pl of 2 M KCl, 162.5 pl of H20). Cleaved DNAs in the free and suGF1-DNA complexes were separated on preparative EMSA gels and analyzed on sequencing gels as described for DNase I footprinting.
1411
Difference probability plots. A file of datum points of a one-dimensional densitometric trace was obtained by scanning autoradiographs on a densitometer. By using the computer program DENS_TOOLS, ln(difference probability) was calculated for every position in the sequence and plotted as a three-point running average against the sequence position to yield a difference probability plot (8, 26, 34). A negative value of ln(difference probability) indicates protection of the DNA from the probe, whereas a positive value indicates hypersensitivity towards the probe in the presence of protein. Interaction of nucleosome cores with suGFI. Nucleosome cores were reconstituted onto the end-labeled 216-bp probe 2 by exchange of histone octamers from Hi-stripped chromatin from chicken erythrocytes (34). Cores were purified on 5 to 20% sucrose gradients in 10 mM Tris-HCl (pH 8.0)-i mM EDTA-5 mM ,B-mercaptoethanol-0.2 mM phenylmethylsulfonyl fluoride and then dialyzed at 4°C overnight against the same buffer without sucrose. The integrity and stoichiometry of the core histones in the reconstituted particle were confirmed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (data not shown). Reconstituted cores were incubated with suGF1 for 30 min at 4°C in EMSA incubation buffer (13) containing 150 mM KCl and supplemented with 0.01% Nonidet P-40, 2.0 ,ug of BSA, and 1.5 ,ug of pdIdC. The complexes were separated on 4% nondenaturing polyacrylamide gels (acrylamide/bisacrylamide ratio, 40:1) containing 5% glycerol in TAE buffer (1) at 40 mA at room temperature for 2 h.
RESULTS Biochemical properties of the suGFl-DNA interaction. Two G-string-specific suGF1-DNA complexes (Bi and B2) were reproducibly observed in EMSAs after incubation of purified suGF1 with gene fragments from the H1-H4 intergenic region (13). The DNAs in the two complexes were intact as determined by selective labeling of either the Watson or the Crick strand by the Klenow fill-in reaction, isolation of the DNAs from complexes Bi and B2 after EMSA, and electrophoresis on sequencing gels (data not shown). The formation of suGF1-DNA complexes Bi and B2 was strongly dependent on ionic strength, with optimal protein-DNA binding taking place at 175 mM KCl or NaCl (data not shown). Variation of the ratio of the concentration of exogenously added divalent cation Mg2" to that of monovalent cation K+ at the same ionic strength had no effect on suGF1-DNA binding (data not shown). Preincubation of suGF1 with up to 50 mM divalent cation chelator EDTA had no effect on subsequent suGF1-DNA binding (data not shown). However, dialysis of purified suGF1 against a buffer containing 50 mM EDTA resulted in abolishment of more than 95% of the DNA-binding activity in comparison with a control sample dialyzed in the absence of EDTA (data not shown). Binding was not restored by subsequent dialysis into the same buffer containing 0.2 mM EDTA. Although these results strongly suggest that divalent cations are essential to suGF1 DNAbinding activity, the activity could not be reconstituted by the addition of ZnCl2 (1 to 500 ,uM) or MgCl2 (0.01 to 2 mM) (data not shown). When the ratio of purified suGFl to the DNA probe is increased, the amounts of suGF1-DNA complexes Bi and B2 decrease, with the concomitant appearance of complexes B3 and B4, which are in turn replaced by even larger complexes at higher suGF1 concentrations (Fig. 1 and 6). The appearance of these discrete complexes was dependent
PATTERTON AND HAPGOOD
1412
-
iH WL4 w
4
Hi. 14'2
Ij ~~~~~~~~~ [ 1- B
~
A
1 t!
[i;
Iv
Lii
-
_
4I'
I
6d :1 1A
MOL. CELL. BIOL.
il
il.
I( 1 ).i,-'!..
I 1.
I 6 (
'j
.;._
;
1]. ,
1.1
.
1
x._
IgS 6\
FIG. 1. suGF1 forms multimers. Probe 1 (1 ng) was incubated with increasing volumes (lanes 4 to 9) of purified, concentrated suGF1 (14 ng/,ul), and the samples were subjected to EMSA and autoradiography. Lane 1, purified suGF1 before concentration on Centricon devices (1.4 ng of protein per ,ul); lanes 2 and 3, absence of suGF1 in the filtrate after concentration. Incubations were in the absence (-) or presence (+) of pdIdC (20 p,g/ml) and BSA (60 ,ug/ml) as indicated. Lane 13, a lighter photographic exposure of lane 9. F, free probe 1; Bi to B4, B5-B6, B7-B8, B9-B10, and B11-B12, suGF1-probe 1 complexes.
on the presence of BSA and pdldC in the incubation mixtures (Fig. 1; compare lane 6 with lanes 10 to 12). To directly investigate the protein-DNA interactions in the individual complexes, we isolated end-labeled, DNase I-digested probe 1 from complexes Bi, B2, Bi and B2 combined (lower complexes), and B3 and B4 combined (upper complexes) after analysis by EMSA. Identical DNase I footprints were
obtained for the individual complexes (Fig. 2), suggesting that the more slowly migrating complexes are due to suGF1suGF1 interactions. DNase I footprinting of suGFl on the H1-H4 intergenic fragment. The DNase I cleavage products of the Watson and Crick strands of the purified suGF1-DNA complex are shown in Fig. 2 and 3A, respectively. In order to compensate for the sequence specificity of DNase I cleavage, the cutting data were interpreted as probabilities of cleavage of free and protein-associated DNAs. The resulting difference probability plots are shown in Fig. 3B. The regions protected from DNase I digestion by suGF1 are clearly visible as minima in Fig. 3B. The borders of the footprint were assigned to positions -345 and -317 on the Watson strand and positions -346 and -315 on the Crick strand. suGF1 thus protects 29 and 32 bp on the Watson and Crick strands, respectively, including the 11 contiguous Gs (-329 to -339). The actual region of proximity of the protein to the DNA is expected to be a few base pairs smaller on either side because of steric hinderance caused by the large size of the DNase I molecule
(7). An interesting feature of the DNase I footprint on the Watson strand is a modulation in the ln(difference probability) of cleavage within the borders of the footprint; i.e., local maxima and minima appear within the global minimum (Fig. 3B, upper trace). Local minima are present at positions -339, -327, and -319, while local maxima are observed at position -323 and between positions -335 and -330. These local maxima and minima appear at a period similar to the helical repeat of a DNA double helix (10 to 11 bp), suggesting that the protein-DNA complex is more accessible to DNase
w4Em~
*~-WImp.I (4~~~~~~-
B-
1.i FIG. 2. DNase I footprints on the Watson strand of suGF1-DNA complexes Bi, B2, B1-B2, and B3-B4 are identical. Probe 1 (6 ng) was incubated with purified suGF1 (8.4 ng), and samples were digested with various concentrations of DNase I. Complexes were separated by EMSA and recovered from EMSA gel slices, and the digestion products were analyzed on sequencing gels. (A) Results for suGF1-DNA complexes Bi (lanes 5 and 6) and B2 (lanes 3 and 4). In panel B, lanes L and U show the results for complexes Bl-B2 and B3-B4, respectively. Lanes 1 and 2 in panel A and lanes F in panel B contain digestion products in the absence of suGF1. DNase I final concentrations (in micrograms/milliliter) during digestion were 0.75 (lanes 1 and 5), 1.5 (lanes 2, 3, and 6), and 3 (lane 4) for panel A and 0.75 (lanes 4 and 6) and 1.5 (lanes 1 to 3 and 5) for panel B. The DNase I footprint is indicated (brackets). Lane G in panel A, G-sequencing standards. Sequence positions are indicated on the left in panel A.
I from one side of the helix and more protected by suGF1 on the other side. It thus seems probable that the bulk of the suGF1 molecule approaches the helix from one side only. A similar modulation, however, cannot be detected on the Crick strand, which is protected more strongly than the Watson strand in the center of the footprint (Fig. 3B, lower trace). Methylation interference with suGFl binding to the H1-H4 intergenic region. Fragments resulting from methylation of the central Gs of the G string (positions -333, -334, -335, and -336) are more abundant in the population of free fragments than in the naked unselected DNA but depleted in the populations of bound fragments obtained from complex Bi or B2 (Fig. 4). The results are consistent with the presence of suGF1 contacts to the Gs located approximately in the center of the G string in the major groove in both complexes, Bi and B2. Gs located on the 5' side (positions
suGF1 BINDING TO OLIGO(dG). OLIGO(dC)
VOL. 14, 1994 A
1413
B 0 -1
31 -286
-2
-3
-296 .00
-4
L. r-L
-5
u
a
317 -4 .* I
l.
c0
...........
-390
CD
-
r,,, .....................
..........
t
380 -370 -360 -350 -340 -330 -320 -310 -300 -290
-28S0
-5
r-
-4 .R
-3
z
-2
-i
T.
-41.6m
-345
g~~~~~~~~~~~~~~.....
....
-i 0
2 3 4 5 6 7 8 9 10 G
lane
suGFI 1tl)
0
0.25 0.5
1
2.5
-337, -338, and -339) of the G string interfere with suGF1-DNA binding to a lesser extent than the central Gs. Hydroxyl radical footprinting of suGFl on the H1-H4 intergenic region. suGF1-DNA incubations and hydroxyl radical cleavage reactions were performed at 4, 25, or 37°C. The different conditions were designed to probe for possible temperature-dependent structural perturbations in the phosphate backbone. Figure 5A shows the cleavage products after isolation of the free DNA and complexes Bi and B2 from a preparative EMSA gel followed by DNA purification and electrophoresis on a sequencing gel. The results were analyzed by densitometric scanning and the construction of difference probability plots. No significant temperature-dependent differences in cleavage products resulting from incubations and/or cleavage at the different temperatures could be detected for free DNA or complex Bi or B2 (Fig. 5A and B and data not shown). In addition, complexes Bi and B2 showed essentially identical hydroxyl radical cleavage patterns (Fig. 5A and data not shown). Figure SB shows the densitometric traces obtained from lanes in Fig. 5A containing free DNA or complex Bi after incubation at 4 or 37°C and cleavage at 4°C. The difference probability plot of hydroxyl radical cleavage of the Watson and Crick strands for complex B1 is shown in Fig. SC. A striking feature of our results is the unexpected periodic modulation in the extent of hydroxyl radical cleavage which is visible on both strands of the free DNA (Fig. SB, free traces). The small hydroxyl radical probe is known to attack DNA from the minor-groove side, cleaving the sugar phosphate backbone with very little sequence specificity (41). The variable cleavage pattern of free probe 2 is therefore indicative of pronounced sequence-dependent perturbations in the conformation of the minor groove and/or phosphate backbone. Consecutive local maxima (indicated by asterisks on both the Watson strand and the Crick strand in Fig. 5B) and minima in the extent of hydroxyl radical cleavage of the free DNA appear at a period matching that of the double helix (approximately 10.5 bp per turn [35, 42]). This is consistent with periodic narrowing and widening of the minor groove, suggesting that a section of the DNA molecule
FIG. 3. (A) DNase I footprint of suGF1 on the Crick strand of the H1-H4 intergenic region. An autoradiograph of a sequencing gel on which the cleavage products of DNase I digestion of various amounts of purified suGF1 (14 ng/,ul) incubated with probe 2 (1 ng) were separated is shown. Sequence positions and the DNase I footprint (bracket) are indicated on the right. DNase I final concentrations (in micrograms/milliliter) during digestion were 0.75 (lanes 1, 2, 4, 6, 8, and 10) and 1.5 (lanes 3, 5, 7, and 9). (B) Difference probability plot of the suGF1 DNase I footprint. ln(difference probability) of DNase I cleavage in the absence or presence of suGF1 was calculated and plotted for the Watson (upper panel) and Crick (lower panel) strands as a three-point running average against the sequence position (x axis).
FREE
FIG. 4. Methylation interference with suGF1-DNA binding. A densitometric trace of the portion of an autoradiograph where differences between the free, bound, and unselected populations of fragments were visible is shown. Results for partially methylated and piperidine-cleaved naked unselected probe 1, probe 1 isolated from suGF1-DNA complexes Bi and B2, and free probe 1 (isolated from the same lane as B1 and B2) are shown. The sequence positions of the borders of the G1l string are indicated at the top.
PATTERTON AND HAPGOOD
1414
MOL. CELL. BIOL.
Crick
A
radical cleavage products of probe 2 resolved on a sequencing gel are shown. Free DNA (F) or DNA bound to protein in complex B1, B2, or B1-B2 (L) was isolated from an EMSA gel. EMSA incubations were carried out at 4, 25, or 37°C as indicated before subjection -'{v6-to hydroxyl radical cleavage at the temperatures (degrees Celsius) indicated. Lanes G, G-sequencing standards. Sequence positions are indicated. (B) Densitometric traces of cleavage patterns shown in panel A. Traces 1 to 4 for the Watson strand correspond to lanes -3171, 3, 7, and 9, respectively, of the left-hand autoradiograph in panel A. Traces 1 to 4 for the Crick strand correspond to lanes 3, 1, 6, and 4, respectively, of the right-hand autoradiograph in panel B. suGFlDNA binding incubations took place at the indicated temperatures and were followed by hydroxyl radical cleavage at 4°C. The extent of the DNase I footprint (bracket), the DNA sequence, and sequence positions are indicated. Asterisks, approximate positions of local maxima of hydroxyl radical cleavage. (C) The difference probability plot for complex B1 with incubation at 37°C and hydroxyl radical cleavage at 4°C is shown. ln(difference probability) of hydroxyl radical cleavage in the absence and presence of suGFl was plotted for the Watson (upper panel) and Crick (lower panel) strands as for Fig. 3B.
-405 -i
-379 -
-' 59
- L
X
FIG. 5. Hydroxyl radical footprinting of suGF1-DNA complexes on the H1-H4 intergenic region. (A) Autoradiographs of hydroxyl
w
..z
....
-306
-387 9
9
111 12 SpeLles tem7 ) hiicubuaiolliiii
Chtlcaag inp
4
320FBB-7 F I. F FRI 113 -3B 23 3
4
37
-1
341
4
[cillc ~ ~ ( ~ ~ i789(;I^4i( ~ ~ ~ ~ ~ ~
B
Bl
Crick
Watson
3
-7 -326 v
36 v
-9
-350
1'~~~~~~~~racelI
v 336
37'C
~
-
Trace 37C
-326
-318
-316
-306
A-337 30-6 -360 3s
'rrace 2
Trace2
380
Free
Free 37"C
17"C
GAtsoC
cc
G
ACA A A6 A-3,6 -2, TA AlTc C2C TvCC-T5 'Trace 3 T TTIJIAI iC B ~~~~~~~~~~~~~ I\ 37"C
C
:
cj
C
Trace 3 TTc~CC CCTA TGT T1CTCTAAB CTC
C
CCT A4TC
CC
CC1~ l
-
Alh1
GG
TTT
C T
A TC
T
T
T
TraTT GTATrace 4 ATrace TAFree 4
AA WIAA6AAA6TILRJII.
~Free 4C
5
UJpstream -r
3' Downstream
49C
5' Downistream
3' Upstrealli r-. -
suGF1 BINDING TO OLIGO(dG)- OLIGO(dC)
VOL. 14, 1994
1415
C i
0 -4 -4
_0
_l _l
B4-
-350
u ID
.4
n^3.
-1 -340
-330
-320
-310
-300
-290
83 1B4
N
1(-
-1 ~~
1-
z
0
I . 4
1
FIG. 5-Continued.
is curved in solution (3, 11, 37). It has previously been reported that this fragment migrates anomalously in a polyacrylamide gel, and the fragment was suggested to be curved in solution (31). The effect of suGF1 binding to the H1-H4 intergenic fragment can be most clearly seen in a difference probability plot (Fig. SC). Interestingly, a striking modulation in ln(difference probability) is visible for both strands. The sequence positions of local maxima and minima correspond well to those for the modulation in extent of hydroxyl radical cleavage observed in the densitometric traces for free DNA (Fig. SB). The modulation in the extent of hydroxyl radical cleavage of free DNA appears to be enhanced upon suGF1DNA binding. Such an increase in the amplitude of the modulation would be consistent with enhancement of curvature of the DNA in the complex in the same direction as that in the free DNA molecule (i.e., suGF1-induced bending). However, suGF1-induced bending cannot explain the extension of the modulation in ln(difference probability) beyond the borders of the DNase I footprint (Fig. 3 and 5C). The hydroxyl radical cleavage pattern gives an indication of the average conformation in the population of molecules. A more likely explanation for the apparent increase in curvature is thus the restriction of the degree of movement of the DNA molecule due to the proximity of suGF1. This would limit the population of DNA molecules to a range of structures closer to that of a curved molecule than would be assumed for the same population of DNA molecules free in solution. Although suGF1 bending of the DNA within the borders of the DNase I footprint cannot be excluded by our data, we propose that this statistical explanation accounts at least in part for the modulation in ln(difference probability) of hydroxyl radical cleavage. The modulation in the extent of hydroxyl radical cleavage extends across the G string in the Watson strand of bound DNA but clearly does not extend across the G string in the free DNA (Fig. SB). Instead, a local maximum is found at position -330 in the free DNA. In contrast, the modulation includes the C string in the Crick strand of both the bound DNA and the free DNA. The offset of 5 bp between the maxima in the Watson and Crick strands of the free DNA is too large to be explained by staggered cleavage across the minor groove in a curved DNA molecule (8). Curvature is thus probably disrupted towards the G string in the free DNA fragment but not in the suGF1-DNA complex. suGF1 does not bind to the G string in the presence of a
'
-
9
)
11
12
14
1'
It
FIG. 6. Incubation of suGF1 with a positioned nucleosome core particle. Increasing concentrations of purified suGF1 (0.1, 0.2, 0.4, 0.8, 1.6, and 3.2 ng) were incubated with probe 2 (0.5 ng) in the absence (lanes 2 to 7, respectively) or presence (lanes 9 to 14, respectively) of reconstituted nucleosome cores (0.5 ng of DNA). For controls, the migration of free probe 2 in the absence of protein (lane 1) and the migration of nucleosome cores alone (lane 16) and in the presence of free probe 2 (lane 8) are shown. All samples were incubated as described in Materials and Methods except for lane 15, which shows nucleosome cores in Tris-EDTA buffer with 20% glycerol. The position of the nucleosome core is indicated. F, free probe; Bi to B4, suGF1-probe 2 complexes.
positioned nucleosome core in vitro. The H1-H4 intergenic region has previously been shown to position a nucleosome core such that the G string is located near the dyad (13, 31, 32). We have reconstituted a nucleosome core onto the 216-bp H1-H4 gene fragment (probe 2) and investigated whether purified suGF1 is able to bind to the G string in the presence of the histone octamer located in the major in vitro positioning frame. No binding of suGF1 to the nucleosome core was observed, even in the presence of a large molar excess of suGF1 over the G string present in the nucleosome core (Fig. 6). Free probe 2 was added to the nucleosome incubations as an internal control, showing that suGF1 binds efficiently to free DNA in the presence of nucleosome cores. DISCUSSION
suGF1-DNA binding is dependent on ionic strength and divalent cations. suGF1 was shown to bind optimally to the G string in the H1-H4 intergenic fragment at 175 mM KCl. Optimal DNA binding at relatively high ionic strength has also been shown for other DNA-binding proteins (44, 45) and may reflect the high affinity and specificity of these proteinDNA interactions necessary for the recognition of cognate sequences in vivo. Our data suggest that suGF1 requires a divalent cation for DNA binding. Such a cation is expected to be complexed very tightly by suGF1, since suGF1 DNAbinding activity is insensitive to concentrations of o-phenanthroline which are sufficient to result in the removal of all DNA-binding activity from the Zn2+-containing factor TFIIIA (12, 14). Tight complexing of a divalent cation by suGF1 is supported by the fact that buffers do not need to be supplemented with Zn2+ or other divalent cations during or after the purification (14). The treatment required for removal of DNA-binding activity from suGF1 was similar to that resulting in a loss of DNA-binding activity of transcription factor Spl due to removal of Zn + from the Zn fingers (21). Both Spl (21) and suGF1 (14) DNA-binding activities are unaffected by 1 mM o-phenanthroline but are abolished by dialysis against 50 mM EDTA. Only 10 to 20% of Spl
1416
PATTERTON AND HAPGOOD
DNA-binding activity could be restored by addition of 500 ,uM ZnCl2 to the EDTA-treated sample (21), whereas addition of ZnCl2 up to 500 ,uM did not restore suGFl DNAbinding activity. It is possible that suGF1 contains a Zn finger. If suGF1 indeed requires a divalent cation such as Zn2+, the failure to reconstitute DNA binding of suGF1 after removal of such a divalent cation by dialysis against EDTA can most probably be ascribed to oxidation of cysteine residues required to complex the metal. suGF1 forms discrete multimeric complexes with DNA. The two sequence-specific suGF1-DNA complexes Bi and B2 both contain suGF1, since both activities could be renatured from the single 59.5-kDa band (suGF1) in an SDS gel (13). In addition, complexes Bi and B2 exhibited the same hydroxyl radical and DNase I footprints (Fig. 2A and 5A) and interference with suGF1 binding by the same methylated Gs (Fig. 4). Several transcription factors have been shown to generate multiple complexes on EMSA due to posttranslational modifications or the presence of truncated forms (4, 5, 20). We found no evidence for differential phosphorylation or RNA association in Bi and B2 (13). Furthermore, the DNAs in the two complexes were intact, indicating that G-specific endonuclease activity was not associated with suGF1 (36). The most likely explanations for the presence of the two suGF1-DNA complexes are posttranslational modifications other than phosphorylation or the presence of a slightly truncated form of suGF1 not resolved on our SDS-polyacrylamide gels. We have shown that purified suGF1 forms discrete multimers in EMSAs in the presence of excess protein. These multimers are sequence-specific suGF1-G-string complexes which can be blocked by specific but not by nonspecific DNA competitors in EMSAs (15). The DNase I footprint remained unaltered in complexes B1-B2 and B3-B4, indicating the presence of suGF1-suGF1 contacts (Fig. 2B). It is interesting that suGF1 in nuclear extracts eluted in the void volume from a Sephacryl S300 gel filtration column (14), implying the existence of multimeric species at relatively dilute protein concentrations. Similar behavior was reported for the transcription factor Spl, which is known to form large complexes consisting of tetramers stacked in register (2, 28, 30, 39). These complexes were shown to involve Spl molecules bound far apart on the DNA, causing the intervening DNA to be looped (28). Looping may play an integral role in regulation of transcription (17). By implication, multimerization may be involved in the function(s) of suGF1. A model for binding of suGFl to the G string in the H1-H4 intergenic region. We have summarized our footprinting and methylation interference results in Fig. 7. Local minima of hydroxyl radical cleavage (open squares) are located on the phosphate backbone of the Watson and Crick strands, opposite each other across the minor groove at the "back" of the helix (the lower plane). Corresponding local maxima in hydroxyl radical cleavage (open triangles) are located across the minor groove at the "front" of the helix. These results suggest periodic narrowing of the minor groove at the back of the helix and widening at the front, consistent with DNA curvature, in both the free molecule and the bound molecule. The direction of curvature in the region of the suGF1 DNA-binding site is probably related to the occurrence of A-rich sequences at a period matching the helical repeat (3, 31). Our DNase I footprinting data are consistent with a model of the suGF1-DNA interaction in which suGFl approaches the helix from one side (i.e., from the left in Fig. 7) and
MOL. CELL. BIOL.
Watson5S
A'
3'Crick
FIG. 7. Summary of nuclease and chemical protection and methylation interference data. The sugar phosphate backbones of a double-stranded DNA helix (B type) are schematically represented by ribbons. Horizontal lines represent base pairs. The minor groove and major groove are indicated (m and M, respectively). The sequence of the Watson strand is given. Numbers indicate the sequence positions of base pairs directly underneath. Open squares and triangles, local minima and maxima, respectively, of hydroxyl radical cleavage; crosshatched rectangle, extended local inaccessibility to hydroxyl radical cleavage; closed squares and triangles, local minima and maxima, respectively, of DNase I cleavage (small closed triangles indicate small local maxima in DNase I cleavage); arrows, borders of the DNase I footprint. The entire region between these borders is protected from DNase I cleavage on both strands. Stars, Gs which interfere with suGF1-DNA binding when methylated at the N-7 position (large stars indicate the most interference). The radius of curvature of the DNA discussed in the text, but not shown here for simplicity, is in a plane perpendicular to the plane of the page, with the imaginary midpoint of the circle located below the plane of the page.
extrudes a bulky structure into the major groove in the region of -335. Contacts are made to the central Gs in this major groove (marked by asterisks) and to the phosphate backbone "above" these Gs (indicated by a crosshatched rectangle). This major-groove structure is supported by our methylation interference data as well as the inaccessibility of the backbone to hydroxyl radical cleavage on the Crick strand in the region of -328. This inaccessibility is in stark contrast to the occurrence of local maxima of hydroxyl radical cleavage at the front of the helix at other positions (open triangles), implying direct suGF1 contacts with the sugar phosphate backbone at this position. In our model, suGFl is in close proximity to the DNA over a region of approximately 1½2 helical turns (-340 to -324) (10). We
VOL. 14, 1994
cannot rule out the possibility of a small protrusion into the major groove at -324. It is very unlikely that suGF1 binds to the H1-H4 intergenic fragment as a rotationally symmetric dimer, since no obvious rotational symmetry can be detected in the suGF1 nuclease and chemical probing data or in the sequence of the binding site. The methylation interference results presented here are consistent with our proposal that suGF1 is structurally related to the chicken poly(dG) poly(dC)-binding factor BGP1 (4, 13, 25). Whereas all Gs interfered with BGP1 binding to a G string containing only 7 contiguous Gs, the Gs in the center of a string of 12 or 16 contiguous Gs interfered the most with BGP1 DNA binding when methylated (4). This could be explained statistically by the central Gs being present in the majority of possible binding frames consisting of seven Gs each (4). Similarly, methylation of the central seven or eight Gs of an 11-bp G string was shown to interfere the most with suGFl binding (Fig. 4). This result may reflect binding in different frames, although the clear modulation in DNase I cleavage in the suGF1-DNA complex suggests a single preferential frame. We could not find any evidence from DNase I and hydroxyl radical probing for formation or stabilization of a triple-helix DNA structure in the suGF1 complex with linear DNA fragments containing the sequence (GA)16G11. The lack of suGF1 binding to single-stranded DNA (13) suggests that suGFl differs from factors proposed to stabilize unusual structures by binding to single-stranded regions (6, 24). Although it is possible that a triple-helix structure is assumed by the region of the G string in vivo under localized superhelical stress in the absence of a nucleosome and plays a regulatory role by excluding suGFl from binding, we have not investigated this experimentally. A positioned nucleosome excludes suGFl. Binding of suGFl to its cognate site in vitro stabilized the curved conformation present in the population of free DNA fragments. The direction of curvature of free and suGFl-bound DNAs presented in this article is the same as the direction in which the H1-H4 intergenic region is bent around the nucleosome core positioned on linear and supercoiled plasmids (32), i.e., with the radius of curvature of the DNA in a plane perpendicular to the plane of the page in Fig. 7. In our model of the suGFl-DNA complex, the bulk of the large 59.5-kDa suGF1 protein approaches the DNA from the left in Fig. 7. It thus appears that DNA binding of suGFl and placement of the nucleosome core in this rotational frame over the G string would be mutually exclusive because of steric hindrance between suGF1 and the histone octamer. We have shown that suGF1 is unable to bind in vitro to its G string when a nucleosome is positioned in the major in vitro frame on the H1-H4 intergenic fragment. We found no evidence for nucleosome displacement or destabilization by suGFl under our in vitro conditions. Our data are consistent with a regulatory mechanism whereby suGFl may gain access to its cognate sites shortly after replication and before assembly of the newly replicated DNA into chromatin (43). It is possible that bound suGFl could exclude a nucleosome core from the DNA and play a role in maintaining a nucleosome-free region during transcription. A more detailed analysis of the interaction of suGFl with chromatin will be reported elsewhere (15). ACKNOWLEDGMENTS This research was supported by grants from the Foundation for Research Development, Republic of South Africa, and the UCT Research Committee to J.H. D.P. was supported by a postgraduate research fellowship from AECI.
suGFl BINDING TO OLIGO(dG) * OLIGO(dC)
1417
We thank C. von Holt for support and encouragement. We thank H.-G. Patterton for the use of the computer program DENS_ TOOLS, for advice on chromatin reconstitution methodology, and for critical reading of the manuscript. We thank B. T. Sewell and A. Roseman for computer software used to collect densitometric data.
ADDENDUM IN PROOF In a recent study, published after submission of our manuscript, it was found that BGP1 is not able to derepress nucleosome-repressed P-globin templates in vitro (M. C. Barton, N. Madani, and B. M. Emerson, Genes Dev.
7:1796-1809, 1993). REFERENCES 1. Ausubel, F. M., R. Brent, R. E. Kingston, D. D. Moore, J. G. Seidman, J. A. Smith, and K. Struhl. 1987. Current protocols in molecular biology. John Wiley and Sons, New York. 2. Briggs, M. R., J. T. Kadanoga, S. P. Bell, and R. Tjian. 1986. Purification and biochemical characterization of the promoterspecific transcription factor Spl. Science 234:47-52. 3. Burkhoff, A. M., and T. D. Tullius. 1987. The unusual conformation adopted by the adenine tracts in kinetoplast DNA. Cell 48:935-943. 4. Clark, S. P., C. D. Lewis, and G. Felsenfeld. 1990. Properties of BGP1, a poly(dG)-binding protein from chicken erythrocytes. Nucleic Acids Res. 18:5119-5126. 5. Dailey, L., S. B. Roberts, and N. Heintz. 1988. Purification of the human histone H4 gene-specific transcription factors H4TF-1 and H4TF-2. Genes Dev. 2:1700-1712. 6. Davis, T. L., A. B. Firulli, and A. J. Kinniburgh. 1989. Ribonucleoprotein and protein factors bind to an H-DNA forming c-myc DNA element. Possible regulators of the c-myc gene. Proc. Natl. Acad. Sci. USA 86:9682-9686. 7. Drew, H. R. 1984. Structural specificities of five commonly used DNA nucleases. J. Mol. Biol. 176:535-557. 8. Drew, H. R., and A. A. Travers. 1984. DNA structural variations in the E. coli tyrT promoter. Cell 37:491-502. 9. Emerson, B. M., C. D. Lewis, and G. Felsenfeld. 1985. Interaction of specific nuclear factors with the nuclease-hypersensitive region of the chicken adult 1-globin gene: nature of the binding domain. Cell 41:21-30. 10. Fairall, L., and D. Rhodes. 1992. A new approach to the analysis of DNase I footprinting data and its application to the TFIIIA/5S DNA complex. Nucleic Acids Res. 20:4727-4731. 11. Hagerman, P. J. 1985. Sequence dependence of the curvature of DNA: a test of the phasing hypothesis. Biochemistry 24:70337037. 12. Hanas, J. S., D. J. Hazuda, D. F. Bugenhagen, F. Y.-H. Wu, and C.-W. Wu. 1983. Xenopus transcription factor A requires zinc for binding to the 5S RNA gene. J. Biol. Chem. 258:1412014125. 13. Hapgood, J., and D. Patterton. 1994. Purification of an oligo(dG). oligo(dC)-binding sea urchin nuclear protein, suGF1: a family of G-string factors involved in gene regulation during development. Mol. Cell. Biol. 14:1402-1409. 14. Hapgood, J., and D. Patterton. Unpublished data. 15. Hapgood, J., and H.-G. Patterton. Unpublished data. 16. Hentschel, C. C. 1982. Homocopolymer sequences in the spacer of the sea urchin histone gene repeat are sensitive to S1 nuclease. Nature (London) 295:714-716. 17. Hochschild, A. 1990. Protein-protein interactions and DNA loop formation, p. 107-138. In N. R. Cozzarelli and J. C. Wang (ed.), DNA topology and its biological effects. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. 18. Jackson, P. D. 1986. Ph.D. thesis. Johns Hopkins University, Baltimore. 19. Jackson, P. D., and G. Felsenfeld. 1985. A method for mapping intranuclear protein-DNA interactions and its application to a nuclease hypersensitive site. Proc. Natl. Acad. Sci. USA 82:
2296-2300. 20. Jackson, S. P., J. J. MacDonald, S. Lees-Miller, and R. Tjian.
1418
21.
22. 23. 24. 25.
26.
27.
28.
29.
30.
31.
32.
PATITERTON AND HAPGOOD
1990. GC box binding induces phosphorylation of Spl by a DNA-dependent protein kinase. Cell 63:155-165. Kadonaga, J. T., K. R. Carner, F. R. Masiarz, and R. Tjian. 1987. Isolation of cDNA encoding transcription factor Spl and functional analysis of the DNA binding domain. Cell 51:10791090. Kohwi, Y., and T. Kohwi-Shigematsu. 1991. Altered gene expression correlates with DNA structure. Genes Dev. 5:25472554. Kohwi, Y., S. R. Malkhosyan, and T. Kohwi-Shigematsu. 1992. Intramolecular dG dG dC triplex detected in Eschenchia coli cells. J. Mol. Biol. 223:817-822. Kolluri, R., and A. J. Kinniburgh. 1992. Full length cDNA sequence encoding a nuclease-sensitive element DNA binding protein. Nucleic Acids Res. 19:4771. Lewis, C. D., S. P. Clark, G. Felsenfeld, and H. Gould. 1988. An erythroid-specific protein that binds to the poly(dG) region of the chicken 3-globin gene promoter. Genes Dev. 2:863-873. Lutter, L. C. 1978. Kinetic analysis of deoxyribonuclease I cleavages in the nucleosome core: evidence for a DNA superhelix. J. Mol. Biol. 124:391-420. Lyamichev, V. I., S. M. Mirkin, and M. D. Frank-Kamenetskii. 1985. A pH-dependent structural transition in the homopurinehomopyrimidine tract in superhelical DNA. J. Biomol. Struct. Dyn. 3:327-338. Mastrangelo, I. A., A. J. Courney, J. S. Wall, S. P. Jackson, and P. V. C. Hough. 1991. DNA looping and Spl multimer links: a mechanism for transcriptional synergism and enhancement. Proc. Natl. Acad. Sci. USA 88:5670-5674. Maxam, A. M., and W. Gilbert. 1980. Sequencing end-labeled DNA with base-specific chemical cleavages. Methods Enzymol. 65:499-560. Pascal, E., and R. Tjian. 1991. Different activating domains of Spl govern formation of multimers and mediate transcriptional synergism. Genes Dev. 5:1646-1656. Patterton, H.-G., and C. von Holt. 1993. Negative supercoiling and nucleosome cores. I. The effect of negative supercoiling on the efficiency of nucleosome core formation in vitro. J. Mol. Biol. 229:623-636. Patterton, H.-G., and C. von Holt. 1993. Negative supercoiling and nucleosome cores. II. The effect of negative supercoiling on the positioning of nucleosome cores in vitro. J. Mol. Biol. 229:637-655.
MOL. CELL. BIOL. 33. Plumb, M. A., R. H. Nicolas, C. A. Wright, and G. H. Goodwin. 1985. Multiple sequence-specific DNA binding activities are eluted from chicken nuclei at low ionic strengths. Nucleic Acids Res. 13:4047-4065. 34. Rhodes, D. 1985. Structural analysis of a triple-complex between the histone octamer, a Xenopus gene for 5S RNA and transcription factor IIIA. EMBO J. 4:3473-3482. 35. Rhodes, D., and A. Klug. 1986. An underlying repeat in some transcriptional control sequences corresponding to half a double helical turn of DNA. Cell 46:123-132. 36. Ruiz-Carrillo, A., and J. Renaud. 1987. Endonuclease G: a (dG)n (dC)n-specific DNase from higher eukaryotes. EMBO J. 6:401-407. 37. Stellwagen, N. C. 1983. Anomalous electrophoresis of deoxyribonucleic acid restriction fragments on polyacrylamide gels. Biochemistry 22:6186-6193. 38. Stokrova, J., M. Vojtiskov6, and E. Palecek. 1989. Electron microscopy of supercoiled pEJ4 DNA containing homopurine. homopyrimidine sequences. J. Biomol. Struct. Dyn. 6:893-897. 39. Su, W., S. Jackson, R. Tjian, and H. Echols. 1991. DNA looping between sites for transcriptional activation: self-association of DNA-bound Spl. Genes Dev. 5:820-826. 40. Tullius, T. D., and B. A. Dombroski. 1986. Hydroxyl radical "footprinting": high-resolution information about DNA-protein contacts and application to 2 repressor and Cro protein. Proc. Natl. Acad. Sci. USA 83:5469-5473. 41. Tullius, T. D., B. A. Dombroski, M. E. A. Churchill, and L. Kam. 1987. Hydroxyl radical footprinting: a high resolution method for mapping protein-DNA contacts. Methods Enzymol. 155:537-558. 42. Wang, J. C. 1979. Helical repeat of DNA in solution. Proc. Natl. Acad. Sci. USA 76:200-203. 43. Wolffe, A. P. 1991. Implications of DNA replication for eukaryotic gene expression. J. Cell Sci. 99:201-206. 44. Xiang, M., S.-Y. Lu, M. Musso, G. Karsenty, and W. H. Klein. 1991. A G-string positive cis-regulatory element in the LpSl promoter binds two distinct nuclear factors distributed nonuniformly in Lytechinus pictus embryos. Development 113: 1345-1355. 45. Zabel, U., R. Schreck, and P. A. Baeuerle. 1991. DNA binding of purified transcription factor NF-KB. Affinity, specificity, Zn2+ dependence and differential half-site recognition. J. Biol. Chem. 266:252-260.
