anti-cancer drugs (Pegg et al., 1982). These agents have also been used to ... National Fund for Scientific Research (Belgium). Present address: Department of ...
Plant Physiol. (1994) 106: 559-566
A Group of Chromosomal Proteins I s Specifically Released by Spermine and loses DNA-Binding Activity upon Phosphorylation' Dirk Van den Broeck, Dominique Van Der Straeten, Marc Van Montagu*, and Allan Caplan' Laboratorium voor Genetica, Universiteit Gent, B-9000 Gent, Belgium Tobacco mutants resistant to the inhibitor methylglyoxal bis(guanylhydrazone), which blocks synthesis of spermidine and spermine, all showed changed polyamine levels as well as a multitude of floral abnormalities (Malmberg and Rose, 1987), whereas a floral developmental mutant in Petunia exhibits changed polyamine levels (Gerats et al., 1988). In yeast, it has been shown that when the Om decarboxylase gene is inactivated, cell division stops but cell expansion continues (Whitney and Moms, 1978; Cohn et al., 1980). On the other hand, a mutant unable to synthesize spermidine still grows, albeit very slowly, but can no longer sporulate (Cohn et al., 1978). Because interpretation of these in vivo effects is difficult, we decided to look for clear in vitro effects at a molecular level. One can expect that altered chromatin organization and gene expression in general are involved in such processes as cell division and developmental programming. Therefore, we focused on possible effects within the nucleus, where two main actions of polyamines can be anticipated. Polyamines, being cations, show a high affinity for DNA, and approximately 50% of the cell's total content is thought to reside in the nucleus (McCormick, 1978; Snyder, 1989). Moreover, in plants at least one isoform of Om decarboxylase is located in the nucleus, tightly bound to chromatin (Foudouli and Kyriakidis, 1989). Spermine and spermidine but not putrescine readily change the B-DNA to Z-DNA equilibrium (Behe and Felsenfeld, 1981) and can also cause DNA bending (Feuerstein et al., 1990). The interactions between Z-DNA and spermine have been resolved by x-ray crystallography (Egli et al., 1991). It is tempting to speculate that spermine can lead either to binding or detachment of nuclear factors by competition or by conformational changes. In fact, Pfeifer and Riggs (1991) showed that the genomic footprint of the PGK-1 promoter can be affected by the loss of particular factors resulting from the isolation of nuclei in buffers containing spermine and spermidine. Spermine also exerts an effect on the activity of nucleusassociated CKII (Hathaway and Traugh, 1984; Datta et al., 1987). When factors like Myb (Liischer et al., 1990) or the plant factor AT-1 (Datta and Cashmore, 1989) are phosphorylated by CKII, they lose their DNA-binding ability. Other proteins, such as the plant factor GBF-1, gain DNA binding upon phosphorylation (Klimczak et al., 1992).
Biologically relevant concentrations as low as 500 PM spermine led to the specific release of chromatin-associated proteins from nuclei of rice (Oryza sativa) seedlings. Using a southwestern technique, it was shown that several of these proteins bind DNA. This affinity was lost upon in organello phosphorylation by an endogenous kinase. The effect of spermine was very specific. Spermidine was far less effective and putrescine was essentially ineffective in releasing these proteins. The most abundant spermine-released protein was shown to be homologous to the maize HMC1 protein. Our results suggest that spermine induces the release of sperminereleased proteins by changing DNA conformation. Binding of these proteins might be sensitive to long-range changes in chromosome structure caused by torsional stress.
Polyamines are important modulators of biological processes such as cell division, stress responses, and development (Smith, 1985; Galston and Kaur-Sawhney, 1990; Koetje et al., 1993), but how they are involved in such diverse phenomena is not clear. Most reports correlate the level of indwidual polyamines with different physiological and developmental states but have been unable to clarify whether these molecules are determinants. Controlled manipulation of polyamines is dfficult because their direct uptake is slow and often depends on specific camers, whereas their concentration is highly regulated (for a review on regulation, see Heby and Persson, 1990). The concentration of a given polyamine can also be altered by the combined use of inhibitors of putrescine-synthesizing and spermidine-synthesizing enzymes. Both types of inhibitors have been developed as anti-cancer drugs (Pegg et al., 1982). These agents have also been used to establish the involvement of polyamines in mediating auxin- and ethylene-induced effects on plant growth (Friedman et al., 1982; Lee and Chu, 1992). Causal relationships were further established by mutating genes involved in polyamine synthesis and/or regulation.
' This work was supported by grants from the Rockefeller Foundation (RF 86058 No. 59), the Belgian Programme on Interuniversity Poles of Attraction (Prime Minister's Office, Science Policy Programming No. 38), and the Commission of the European Communities TS2-0053-8 (GDF). D.V.D.S. is a Senior Research Associate of the National Fund for Scientific Research (Belgium). Present address: Department of Bacteriology and Biochemistry, University of Idaho, Moscow, ID 83843. * Corresponding author; fax 32-9-264-5349.
Abbreviations: CKII, casein kinase 11; HMG, high-mobility group; SRP, spermine-released protein. 559
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Our results show that changes in polyamine levels and phosphorylation can coordinately affect the DNA-binding ability of the same set of proteins. We describe some characteristics of the affected proteins including specific release from chromatin by 500 p~ spermine but not putrescine, subsequent phosphorylation by a nuclear kinase, and loss of DNA-binding ability upon phosphorylation. One protein was microsequenced and turned out to be similar to the HMGl homolog of maize. MATERIALS AND METHODS Preparation of Nuclei and Chromatin
Nuclei were isolated from leaf tissue of 10-d-old rice seedlings (Oryza sativa var Taichung native 1) essentially as described by Hamilton et al. (1972). Leaves were cut into 5mm pieces with a razor blade and subsequently submerged in diethyl ether for 1 min. The material was then filtered. To remove traces of ether, the residue was washed once in Hamilton buffer (H buffer: 1.14 M SUC,5 mM MgCI2, 5 mM /3-mercaptoethanol, 10 mM Tris, pH 7.6). All subsequent manipulations were done at 4OC. Nuclei were squeezed out serially by grinding small aliquots (about 5 g of wet material) with mortar (30 cm diameter) and pestle. The homogenate was collected and stirred gently in H buffer for 1 h to further increase separation between nuclei and cell debris. Upon gentle filtration through two layers of Miracloth (Calbiochem, San Diego, CA), the residue was resuspended in H buffer. The collected filtrates were centrifuged at low speed (1OOOg) to prevent disruption of nuclear structures. The pellet (starch grains, chloroplasts, and nuclei) was resuspended slowly in H buffer, enriched with 0.15% Triton, and incubated on ice for 30 min. Centrifugation and resuspension of the pellet in the same buffer for three more times was usually enough to obtain an essentially chloroplast-free pellet. For all further manipulations, this pellet was washed once in H buffer without Triton to remove the detergent. Starting material (50 g) normally yielded about 7 X lo9 rice nuclei. These were taken up in H buffer to a final volume of 3 mL. This nuclear preparation could be stored for months at -2OOC without any detectable alteration of the characteristics tested. Chromatin was obtained upon lysis of this nuclear preparation by osmotic shock. Nuclei (5 X lo'), obtained as a pellet by centrifugation, were rapidly resuspended in a hypotonic lysis buffer (10 mL: 20 m~ EDTA, 80 m~ NaCI, 10 m Na2S205,10 m~ DTT, pH 6.3). Upon incubation for 30 min on ice, chromatin was collected as the fraction that pelleted by a low-speed centrifugation (2500g). This preparation was always used immediately.
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redishibution of proteins at the respective salt concentration, the nuclei were put on ice and centrifuged at low speed (1OOOg). Characterization of Released Proteins
All proteins that remained in solution after centrifugation at the respective salt concentrations, in the presence or absence of polyamines or after being phosphcrylated, were collected by TCA precipitation. Pellets were taken up in solubilization buffer (0.06 M Tris-HC1, pH 6.8, 2% SDS, 100 m DTT, 10% glycerol, 0.002% bromphenol blue), and after adjustment of pH by Tris-base, loaded on a 1-nim-thick SDSPAGE gel. After electrophoresis, proteins were visualized by silver staining (Ansorge, 1985).Phosphorylated.proteins were detected by autoradiography (XR-Kodak)upon drying the gel on Whatman 3MM paper. DNA-Binding Assays
The DNA-binding activity of released proteins was assayed using a southwestern technique essentially a!; described by Bowen et al. (1980). After renaturation within the polyacrylamide gel, the proteins were relocated to a p,iir of nitrocellulose membranes by diffusion for 48 h. Once located on the membrane, DNA-binding polypeptides were detected by binding to a labeled DNA probe. Genomic rice DNA, prepared according to Dellaporta et al. (1983), was multiprime labeled using a kit from Boehringer (Mannheh, Germany). Preparation of Histones and H M G
HMG proteins were isolated as described t)y Goodwin et al. (1973). The chromatin pellet obtained from 5 X lo7 nuclei was resuspended in 100 pL of 5% perchloric acid with 50 m~ DTT by shaking for 30 min. To diminish contamination of the extracted HMG proteins with histone H2, which is partially solubilized by 5 % perchloric acid, 1 he suspension was kept on ice for another 30 min without agitation. Chromatin was then pelleted at 1500g for 15 min. Soluble HMG proteins were subsequently precipitated in 20 % TCA. For the isolation of the histone fraction, the same amount of chromatin was taken up in 100 pL of 0 9 % NaCl and incubated for 15 min on ice. After centrifugation (1500g for 15 min), the pellet was resuspended in 100 pL of 0.9%NaCl, and 11 pL of 2.5 N HCI was added in a drop-wise fashion. The solution was then incubated for 4 h on ice and subsequently centrifuged at 1500g for 15 min. The resulting chromatin pellet was washed once again with 0.215 N HCl. Both supernatants were collected and the histone fraction was precipitated by adding 5 volumes of acetone.
Differential Salt Wash and Phosphorylation
To perform an in organello phosphorylation assay, 5 X lo7 nuclei were collected by centrifugation (1OOOg) and resuspended in 50 pL of kination buffer (10 m~ MgC12, 1.14 M SUC, 10 m Tris-HCI, pH 7.5, 50 p~ ATP, and 10 pCi [T-~~PIATP) with different salt concentrations (50, 100, or 150 m~ NaCl) and with or without polyamines. Suc was added to prevent osmotic shock. After incubation at room temperature for 20 min to allow phosphorylation and release/
Microsequencing
Starting from 60 g of 10-d-old rice seedlini;~, 7 X lo9 rice nuclei were obtained, providing some 8 pg of the 23-kD protein. This was loaded in two lanes of a :!-"-thick gel and separated by a one-dimensional SDS-PAGE. Further manipulations such as blotting, proteolytic cleavage, recovery, and separation of oligopeptides were performed as described by Bauw et al. (1990).
Spermine-Specific Release of DNA-Binding Proteins RESULTS Previously, it was shown that the addition of spermine to a preparation of nuclei strongly enhanced the level of endogenous phosphorylation. The phosphorylation of at least three protein species (23, 35, and 55 kD) was entirely spermine dependent (Van den Broeck et al., 1991). We developed a simple assay to analyze the consequences of changes in the phosphorylated state of these proteins on their affinity for chromatin and/or DNA by a differential solubilization in the presence of increasing amounts of salt. Differential Salt Wash on Nuclei Nuclei were prepared as described in "Materials and Methods' in the presence of high Sue concentrations. In organello phosphorylation was performed in 50, 80, or 150 mM NaCl buffers in the presence and absence of 2 mM spermine. The Sue concentration of all buffers was kept constant to prevent lysis of nuclei by osmotic shock. The integrity of the nuclei was verified by microscopy after staining with 4',6-diamidino-2-phenylindole (Huang et al., 1986). Low-speed centrifugation allowed fractionation into sedimentable and soluble components. The supernatant was carefully removed and soluble proteins were subsequently precipitated in 20% TCA and separated by SDS-PAGE. Few proteins precipitated from the supernatant when nuclei had been treated with 150 mM salt without any spermine (Fig. 1, lanes 1 and 2). On the other hand, when phosphorylation was performed in the
561
presence of 2 HIM spermine, a discrete number of proteins could be precipitated from the supernatant (Fig. 1, lanes 3 and 4). Three main bands of 23, 26, and 35 kD were reproducibly detected by silver staining. Lanes 3 and 4 also show 55- and 100-kD polypeptides that were less reproducibly detected. To determine whether the appearance of these proteins in the supernatant was phosphorylation dependent or spermine dependent, a control reaction was included in which phosphorylation was restrained by leaving out exogenous ATP. Both the amount and size of released proteins remained the same (Fig. 1, lane 4): the presence of 2 mM spermine appeared to be sufficient for protein release. Moreover, the endogenous kinase activity itself could be recovered quantitatively from the nuclei by a triple 150-mM NaCl wash. No phosphorylation was detectable in the residual nuclei (data not shown). Nevertheless, the same proteins were readily released from these nuclei by 2 mM spermine (data not shown). Decreasing the initial NaCl concentration of 150 to 50 HIM did not change the pattern of proteins released (Fig. 1, lanes 5 and 6). Thus, the protein release from nuclei appears to depend primarily on the addition of spermine and not on their state of phosphorylation. Differential Salt Wash on Chromatin Nuclei were lysed by pelleting and quick resuspension in a hypotonic lysis buffer (see "Materials and Methods"). When chromatin obtained as a low-speed centrifugation fraction was treated as nuclei, the same set of polypeptides was released upon addition of 2 HIM spermine (Fig. 1, lanes 5 and 6). The main difference was that all detectable kinase activity was lost during chromatin preparation (data not shown), confirming that the proteins are directly released by spermine without first being phosphorylated. We will refer to these proteins as spermine-released proteins (SRPs). The Concentration of Spermine Influences the Release of SRPs Qualitatively and Quantitatively
21-
U-
Figure 1. SDS-PAC E analysis showing the set of proteins specifically released by spermine. Proteins were separated in the presence of 1 % SDS on a 12% acrylamide gel. The protein pattern was visualized by silver staining. Lanes 1 and 2, Proteins released from nuclei with 150 mM NaCl without spermine (control). Lanes 3 and 4, Proteins released from nuclei with 150 mM NaCl and 2 mM spermine. Lanes 5 and 6, Proteins released from chromatin with 50 HIM NaCl and 2 mM spermine (lane 5) or 10 mM spermine (lane 6). Lanes 1 and 3 represent proteins phosphorylated during the extraction by adding ATP (20 MM final concentration). Lanes 2 and 4 contain proteins prepared in the absence of ATP. The five polypeptides that are specifically released by spermine are marked on the right by their apparent molecular mass. Migration distances of molecular mass markers are shown on the left.
All previous extractions were done in 2 or 10 mM spermine. Figure 2 shows the amount of proteins released from chromatin with concentrations ranging from 100 MM to 20 mM. A transition occurred between 100 and 500 HM spermine. With 100 fiM spermine, no SRPs were visibly released, whereas the released amounts of 26- and 35-kD proteins increased only slightly when spermine concentrations were raised from 500 HM to 20 mM. Simultaneous with this transition, some low molecular mass proteins (10-20 kD) as well as one 30-kD protein that were readily extracted at low spermine concentrations were no longer detectable at higher concentrations. This was also seen when whole nuclei were extracted (Fig. 2, lanes 1 and 2). The decreased recovery of these proteins in the presence of 500 ^M spermine or higher concentrations might reflect the mechanism through which spermine exerts its effect. It seems as if the conformational state of the chromatin is changed dramatically between concentrations of 100 and 500 ^M spermine.
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Van den Broeck et al.
Figure 2. The effect of spermine concentration on the release of SRPs. Proteins were separated in the presence of 1% SDS on a 12% acrylamide gel. The protein patterns were visualized by silver staining. Lanes 1 and 2 are control lanes and represent a 150-mM NaCI extraction on total nuclei in the absence (lane 1) and the presence (lane 2) of 2 HIM spermine. Lanes 3 to 8 show the proteins released from chromatin with 50 mM NaCI (lane 3) enriched with 100 MM (lane 4), 500 MM (lane 5), 2 mM (lane 6), 10 mM (lane 7), and 20 mM spermine (lane 8). The 23-, 26-, and 35-kD SRPs are indicated by their apparent molecular mass.
bind DNA was tested using a southwestern technique, essentially as described by Bowen et al. (1980). Total genomic rice DNA was used as a probe for DNA binding. This technique enabled us to visualize DNA-binding proteins after a 20-min exposure (Fig. 3B). The binding and subsequent washing procedures appeared to be stringent enough to be specific. Lysozyme, which binds DNA nonspecifically (Cattan and Bourgoin, 1968), was included as a molecular mass marker in 10-fold higher amounts and did not bind DNA at all in our procedure. The proteins released from chromatin with 150 mM NaCI did not bind any genomic DNA (Fig. 4B, first three lanes). Only proteins extracted with 2 mM spermine exhibited DNA-binding activity. All five proteins visualized in this assay (23, 26, 35, 55, and 100 kD) coincided with those that were visualized by silver staining. DNA binding of the 55-kD polypeptide could be visualized only after a 60min exposure. Although southwestern blots tend to swell upon hybridization, all bands could still be correlated by superimposing four different patterns: the silver-stained gel, the autoradiogram of the dried gel, and two autoradiograms
035kDa Q26kDa • 23kDa
kDa I 80
The Release of SRPs Is Spermine Specific
To determine whether the effect of spermine depends on its cationic nature or on more specific structural properties, its capacity to release proteins was compared to that of two other polyamines, spermidine and putrescine, and to magnesium. The released amounts of the 23-, 26-, and 35-kD SRPs were measured indirectly by performing a southwestern blot on each extract (see below). After a 2-h exposure, protein bands were cut out, regardless of whether they were visible or not, and radioactivity of the bound DNA was measured in a scintillation counter. The results are summarized graphically in Figure 3. The amounts of SRPs released by purrescine and 20 mM MgCl2 fell within background levels. Spermidine did release SRPs, but far less effectively than spermine. Two millimolar spermine released more of each SRP than 10 HIM spermidine. This comparison also revealed a difference among the SRPs. When the amounts of the 23-, 26-, and 35kD proteins released by spermine were compared with the amounts released by spermidine, the release of the 26-kD protein seemed the most specific. About 30-fold more of this protein was released by 10 mM spermine than by 10 HIM spermidine, compared with a 5- and a 15-fold difference for the 23- and the 35-kD proteins, respectively. Similarly, 2 mM spermine released about 20-fold more 26-kD protein than 2 mM spermidine, whereas the 23- and 35-kD proteins differed only 5- and 9-fold, respectively. At Least Five SRPs Show DNA-Binding Ability
We considered it possible that the SRPs would be chromosomal proteins capable of binding DNA. The ability to
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Figure 3. Graphic representation of the protein-releasing capacity of spermine, spermidine, putrescine, and magnesium. A, The amounts of the 23-, 26-, and 35-kD SRPs that were released by 2 and 10 mM spermine (Spm) were compared to the amounts that were released by 2 and 10 mM spermidine (Spd) and putrescine (Put) and by 20 and 50 mM MgCI2 using the standard extraction procedure (150 mM NaCI on nuclei). The protein samples obtained with each extraction were separated by SDS-PACE and subjected to a southwestern procedure as shown in B. Membrane pieces containing the 23-, 26-, and 35-kD polypeptides were cut out and counted separately in a scintillation counter. The counting results are shown as the percentage relative to the highest counting result (amount of the 26-kD SRP released by 10 mM spermine). B, Southwestern blot demonstrating DNA binding of SRPs. Total genomic rice DNA was used as a probe (see "Materials and Methods"). Binding with four proteins (23, 26, 35, and 100 kD) is detected after a 20-min exposure.
563
Spermine-Specific Release of DMA-Binding Proteins
NUCLEI
CHR.
NUCLEI
that were released from nuclei by 150 mM NaCl without spermine. None of them showed any DNA binding (Fig. 4B). Proteins released from nuclei by 2 mM spermine were separated in the next lanes. DNA binding was detected for the 23-, 26-, 35-, and 100-kD polypeprides (Fig. 4B, fourth lane). Upon incubation with ATP to allow phosphorylation, they lost their DNA-binding ability (Fig. 4B, fifth and sixth lanes). As such, the assay could not distinguish between those proteins that were labeled heavily during phosphorylation and those binding the labeled DNA. Note, however, that the southwestern blot was exposed for 20 min to visualize the DNA-binding pattern, whereas an overnight autoradiography was necessary to detect the phosphorylated proteins. Abundance of the SRPs
Figure 4. Loss of DNA-binding activity of SRPs upon phosphorylation. A and B result from the same SDS-PAGE gel. Proteins separated in the different lanes were extracted from nuclei (lanes 1-6) with 150 HIM NaCl (lanes 1 -3) or with 150 rriM NaCl and 2 FTIM spermine (lanes 4-6), or from chromatin with 50 mM NaCl and 2 HIM spermine (lane 7). Proteins were phosphorylated by incubation with 20 HM ATP (K) or with 20 MM ATP and [-y-32P]ATP (K*). Upon separation, proteins were renatured and then blotted onto two membranes. A shows the autoradiogram of one membrane after a 24-h exposure and represents the pattern of phosphorylated proteins. B represents the pattern of DNA-binding proteins. For this, the second membrane was hybridized with 32P-labeled total genomic rice DNA following the southwestern procedure as described in "Materials and Methods." An exposure time of 20 min was sufficient to result in an autoradiogram as shown in this panel. The 23-, 26-, 35-, and 100-kD SRPs are indicated on the right by their apparent molecular masses.
of nitrocellulose-bound proteins, one showing the labeled phosphoproteins (24-h exposure) and one showing the DNAbinding proteins after a 20-min exposure (for a schematic representation, see Fig. 5). Addition of 50 HIM spermine during the hybridization and washes of the southwestern blot did not decrease the amount of DNA bound to these proteins (data not shown), although a 100-fold lower spermine concentration (500 /UM) is sufficient to release the SRPs from the chromatin (Fig. 2). These data suggest that the in vivo association of SRPs is different, or that spermine can exert an effect only on chromatin and not on free linear DNA as used in the southwestern blot. Phosphorylation Decreases the DNA-Binding Ability of SRPs
We examined whether phosphorylation could decrease the DNA-binding ability of these proteins. Phosphorylation was allowed to take place during the salt wash by adding cold ATP and was visualized by incorporating [7-32P]ATP (see 'Materials and Methods'). DNA binding was assessed by a southwestern blot with total genomic rice DNA as a probe. The results are presented in Figure 4: panel A shows the phosphorylated proteins, panel B displays the proteins binding DNA. The first three lanes in each panel contain proteins
The relative abundance of the released SRPs was evaluated by comparison with the abundance of the HMG proteins and of histones that could be semi-quantitatively recovered by chemical means. HMG proteins were specifically extracted with 5% perchloric acid, whereas histones were isolated with a 0.25-N HC1 extraction (see "Materials and Methods'). The abundance of each protein group was estimated by comparing the intensities of the silver-stained bands after SDS-PAGE (Fig. 6). As expected, 10-fold more material of HMG had to be loaded onto a gel to reach the same abundance that was reached for the histone fraction. In turn, 10- to 20-fold more nuclei were needed to extract an amount of SRPs comparable to that of HMG proteins.
cut gel in two
Diffusion blotting
Silver-staining
Drying of gel + autoradlography
run
Protein-pattern Autoradiography 1st P32-pattern of phosphorylated proteins
Hybridisation with labe|ed DNA
pattern of 2nd P32-pattern of phosphorylated proteins
Figure 5. Procedure for correlation of the silver-stained protein pattern with the corresponding phosphorylation and DNA-binding pattern.
Van den Broeck et al.
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1-31
m -21 -U Figure 6. An estimation of the abundance of the SRPs. SRP, HMC, and histone extractions were performed on chromatin as described in "Materials and Methods," starting from 5 X 107 rice nuclei. Different amounts of each extract were loaded onto the gel to obtain comparable abundances: 100% of SRP (lane 1), 10% of HMC (lanes 2 and 3), and 1% of histone extraction (lane 4). HMC was extracted immediately after shaking for 30 min (lane 3) or after an additional 30-min incubation without shaking, to decrease H3 and H1 contamination (lane 2). Lane 5 shows a total nuclear extract obtained by adding 5 /iL of solubilization buffer to 10s rice nuclei.
The 23-kD SRP Is the HMG1 Homolog of Rice
The 23-kD SRP was sufficiently abundant for microsequencing protease-derived oligopeptides after scaling up the extraction (see 'Materials and Methods"). Two separate digests were performed: a trypsin digest yielded three and a chymotrypsin digest yielded two peptides that could be sequenced. When combined, the obtained amino acid sequences showed 93% similarity with the HMG1 cDNA from maize (Fig. 7) (Crasser and Feix, 1991). This similarity can be extended by taking into account the recognition sites of the used proteases: trypsin cleaves after Arg or Lys and chymotrypsin cleaves after Phe, Trp, or Tyr. Of the 29 sequenced amino acids, only two differed from the maize protein. One was a conservative Ser-to-Thr substitution. Besides several CKH target sites located in the acidic C-terminal region, there are also two perfect target sites located in the sequence KSLTEAD, which is bordered by two o-helices and comprises the DNA-binding domain. As shown above, the amount of the 23-kD SRP released by spermine was about 10-fold lower than a semi-quantitative extraction of HMG1. From this we infer that only a fraction (about 10%) of the 23-kD HMG homolog is released by spermine in the concentration range from 0.5 to 20 mM.
Plant Physiol. Vol. 106, 1994
such as stress (Flores and Galston, 1984), development (Koetje et al., 1993), or progress through the cell cycle (Torrigiani et al., 1987), this releasing concentration of 500 JIM presumably has biological significance. Different polyamines can exhibit different physiological effects (Galston and KaurSawhney, 1990). Recently, it was shown (Snyder, 1989) that some characteristics of chromatin, isolated from polyaminedepleted HeLa cells (slower sedimentation, higher DNase sensitivity, and Mg solubility), could be restored by addition of spermine but not of spermidine or putrescine. Our results also show a differential effect of spermine when compared to the other polyamines. Spermidine was much less effective, whereas putrescine was ineffective. We assume that the specific release of SRPs from chromatin is not an artifact of our in vitro manipulation but actually reflects an in vivo effect that occurs whenever the local spermine concentration reaches a specific threshold level. Five of the SRPs were shown to bind DNA. One of the SRPs is now proven to be the rice HMG1 homolog. A consensus DNA-binding motif was recognized in several transcription factors (Jantzen et al., 1990; Sinclair et al., 1990), which was named the HMG-box motif because it was similar to the DNA-binding domain of HMG1 and HMG2. All of these transcription factors contact DNA via the minor groove that leads to a bent DNA duplex (Bianchi et al., 1992). One might expect that within this group of factors more than just one (HMG1) will be released by spermine. Currently, we are scaling up our extraction procedure to microsequence the other SRPs. Extracted SRPs were phosphorylated by an endogenous nuclear kinase(s) resulting in a slightly increased recovery in the supernatant, as if they had lost their DNA-binding activity. Indeed, the DNA-binding activity of all five SRPs was
M K G A K S K G A A K A D A K L A V K S K G A E K P A K G
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-Tr2-
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DISCUSSION
Low levels of spermine (500 ^M) specifically release a set of DNA-binding proteins (SRPs) from chromatin (Figs. 1 and 2). The intracellular concentration of spermine in bovine lymphocytes and rat liver has been estimated at around 1 mM (Watanabe et al., 1991). Because one can expect polyamine amounts to vary depending on physiological conditions
E D E E E K S D K S K S E V N D E D D E E G S E E D E D D D E Figure 7. Alignment of the microsequenced part of the 23-kD SRP with the aminoacid sequence of HMC1 of Zea mays. The peptides Trl, Tr2, and Tr3 were isolated after a trypsin digest; Chl and Ch2 were isolated after a chymotrypsin digest. Identical amino acids are boxed.
Spermine-Specific Release of DNA-Binding Proteins decreased upon phosphorylation. It could be speculated that when a transient rise in spermine concentration occurs within the nucleus, the resulting dissociation of SRPs from chromatin will be prolonged by this phosphorylation. In a previous report it was shown that at least one SRP was phosphorylated by a CKII-like activity (Van den Broeck et al., 1991). This 23-kD protein has now been shown to be the HMGl homolog of rice. The HMGl protein of maize (Grasser and Feix, 1991) also contains consensus sequences phosphorylated by CKII. It was shown that in vitro phosphorylation of HMGl by CKII (Grasser et al., 1991) does not affect any of the tested DNA-binding activities. Therefore, it remains to be seen which kinase phosphorylates the rice SRPs in organello, such that their DNA-binding activity is influenced. How spermine can exert such a specific effect remains to be elucidated. One particular effect of spermine is that it can induce the formation of left-handed Z-DNA of altemating purine pyrimidine tracts (Behe and Felsenfeld, 1981). The fact that spermidine is less effective at inducing Z-DNA and putrescine is ineffective correlates with our results. A potential site of action could be one of the repetitive sequences [d(TG)n] that is found in intergenic regions and that readily forms Z-DNA in vitro under appropriate conditions (Singleton et al., 1982), and possibly also in vivo (Lancillotti et al., 1987). Such a transition of right-handed B-DNA to lefthanded Z-DNA will create positive supercoilingalong native or chromatinized DNA. This, in tum, could lead to the release of proteins that are inherently storing negative super-helicity by binding cruciform structures or by trapping them in metastable interconversionintermediates (Sheflin and Spaulding, 1989; Pontiggia et al., 1993). Since HMGl specifically recognizes cruciform structures (Bianchi et al., 1989), it could be expected that spermine would release the rice HMGl homolog, as found. Analogous effects are seen with various DNA intercalators. They too cause a specific release of HMG proteins from chromatin (Schroter et al., 1985) by creating positive supercoiling upon intercalation. The DNA probe, once bound to the SRPs on the southwestem blot, could not be washed away by spermine (data not shown). If spermine works indirectly by increasing positive supercoiling, such a negative result could be anticipated. Since the DNA probe is linear with free ends, these molecules do not have any topological constraint and the formation of left-handed ZDNA will not lead to positive supercoiling. Spermine will induce stress only near DNA sequences that are sensitive to a B- to Z-DNA transition. As a result, only proteins bound in the vicinity of such sequences are expected to be released by spermine. Indeed, by comparing the amount of the 23-kD SRP released by spermine to the total amount of HMGl protein in the nucleus, we estimated that approximately 10% of all HMGl is susceptible to spermine treatment. We propose that spermine causes the release of SRPs by changing torsional stress via a B- to Z-DNA transition. First, the spermine-induced release resembles the specific release seen with DNA intercalators that cause positive supercoiling. Second, spermine does not decrease the amount of DNA bound to SRPs on a southwestem blot: this DNA is linear and therefore does not have a topological constraint. Third, the ability of spermine, spermidine, and putrescine to induce
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a B- to Z-DNA transition (Behe and Felsenfeld, 1981) coincides exactly with their respective capacity to release SRPs. ACKNO WLEDCMENTS
The authors thank Guy Bauw and José Van Damme for microsequencing, Rudy Dekeyser for advice and concem, Tom Gerats for critical reading of the manuscript, Karel Spruyt and Vera Vermaercke for figures and photographs, and Martine De Cock for help with the manuscript. Received March 4, 1994; accepted June 28, 1994. Copyright Clearance Center: 0032-0889/94/l06/0559/08. LITERATURE CITED
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