and R. David Cole. From the Department of Biochemistry, ..... Cole, R. D. (1977) in The Molecular Biology of the Mammalian. Genetic Apparatus (T'so, P., ed) pp.
Communication
THEJOURNALOF BIOLOGICAL CHEMISTRY Vol. 262 No. 2 Issue of January 15 pp. 524-526 1987 0 1987 by Thekmerican Society of Biolo&al ChemisG, Inc. Printed in U.S.A.
Non-histone Chromosomal Protein HMGl Modulates the Histone H1induced Condensationof DNA* (Received for publication, September 2, 1986) Lori A. Kohlstaedt, Eric C. Sung, Amy Fujishige, and R. David Cole From the Department of Biochemistry, University of California, Berkeley, California 94720
Circular dichroic spectra revealed that the previously known regular, asymmetric condensation of DNA by H1 histone was modulated by HMGl, a nonhistone chromosomal protein. Under approximately physiological salt and pH conditions (150 mM NaCl, pH 7), ellipticities at 270 nm were observed as follows: DNA, 9 X los degree, cm2/dmol nucleotide; D N A * H l histone complex (1:0.4, w/w), -37 X lo3 degree, cm2/ dmol nucleotide, and D N A - H l *HMG1 complex (1:0.4:0.4 w/w/w), -52 x lo3degree, cm2/dmol.HMGl by itself did not distort the spectrum of DNA, showing that the effect of HMGl on the D N A - H l complex was not simply the summation of individual effects of HMGl and H1 on the DNA spectrum. The effect of added HMGl on the spectrum of the preformed DNA-HI complex depended on the amount of HMGl added and developed slowly (a day) as if a structure required annealing. The ternary complex, DNAHMGl *H1, seemed to represent a specific structure, since itsformation depended on the reduced sulfhydryl state of HMG1; the disulfide form ofHMG1, which was shown by circular dichroism to contain more random coil than did the reduced form, had no effect on the circular dichroic spectrum of the D N A * H l complex.
(4, 16, 17) that revealed a parallel between the relative power of different H1 variants in their condensation of DNA by complex formation and the distribution of thosevariants between aggregation-prone and aggregation-resistant classes of chromatin. This parallel suggests that H1-DNA complex formation might, after all, provide a reasonable model for one aspect of chromatin structure, that of gross aggregation, perhaps asoccurs in heterochromatization. With this support(4, 16, 17) for the view that H1.DNA complexes might be meaningfulmodels (see also Ref. 18), we have now reopened a question we first posed a decade ago (19), when the nonhistone chromosomal protein HMGl was shown to complex with H1 histone (20-23). We wondered if HMGl might modulate the ability of H1 to condense DNA and chromatin. As shown below, HMGl does modulate H1-induced DNA condensation. Moreover, the HMGl must be in its native state to do so. MATERIALS ANDMETHODS
HMGl waspurifiedfrom steer thymus as described previously (24). H1 was purified from steer thymus by extraction with 0.74 M perchloric acid followed by precipitation with 78% acidified acetone, yielding a preparationfree of HMG proteins and >98% pure, as judged by gel electrophoresis in sodium dodecyl sulfate (24). H1. DNA complexes were prepared by step gradient dialysis essentially as described previously (14), except that no urea was used and NaCl replaced NaF. Final dialysis was against 10 mM Tris, 150 mM NaC1, 1 mM dithiothreitol, pH 7.0. HMGl was added directly to preformed H1.DNA complexes and incubated for 24 h (4 “ C )before measurement of the spectrum. Circular dichroic spectra were measured on a Jasco model J-5OOC spectropolarimeter equipped with a model DP500N data processor. RESULTSANDDISCUSSION
When a complex of H1 histone and DNA is formed under carefully controlled conditions, the DNA fibers are condensed into some regular asymmetric structure that can be detected (14) by circular dichroism. The condensed nucleohistone is characterized by a spectrum that is negative at 270 nm rather Recently interest has been renewed in the notion that H1 than the positive spectrum characteristic of naked DNA. histone acts as a generalized regulator of gene transcription Therefore, we formed H1-DNA complexes (0.4:l.O charge or replication (1-3). Four factors could lead to controlled ratio) by slow dialysis and observed the expected spectrum modulations of chromatin dynamics: differences in amounts (Fig. 1B). When purified HMGl was added subsequently and of H1 (4) or in sequence variation (5-9) of HI, as well as the mixture was incubated for a day (4 “C),the circular differences in modes of binding (10) or in post-translational dichroic spectrum (Fig. 1C) wasevenmore negatively dismodification (11-13). The circular dichroism studies of Fas- torted from that of naked DNA (Fig. 1A) than in the case of man et al. (14) on H1-induced DNA condensation led us to the H1.DNA binary complex (Fig. 1B). The enhanced distorstudy differences among H1 variants in their ability to con- tion of the circular dichroic spectrum when HMGl was added dense DNA. The variantsof H1 found within a single animal to the H1 .DNA complex thus seems to have produced a differ among themselves in their ability to condense linear ( 5 , further condensation of the DNA fibers or a different mode 6) or supercoiled DNA (6, 7) or, for that matter, their ability of condensation. When DNA and HMGl were mixed under to condense dinucieosomes (9) or chromatin fragments (15). conditions of slow dialysis identical to those used in the formation of H1. DNA complexes, no distortion of the DNA Although we were reluctant (6) to consider theH1.DNA complexes used in some of these experiments as models of spectrum occurred. Therefore, the circular dichroic spectrum chromatin structure, we were struck by recent experiments of the H1.J-IMGl .DNA ternary complex was not simply the sum of the spectra of the two proteins complexed to DNA * This study was funded by National Institutes of Health Grant independently. GMS20338, National Institute of Environmental Health Sciences The enhancement of the negative ellipticity of the H1. Grants ES07075 and ES01896, and by the Agricultural Research DNA complex by HMGl was demonstrated to be dependent Station. The costsof publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby on the amount of HMGl, as shown in Fig. 2. This was not marked “advertisement” in accordance with 18 U.S.C. Section 1734 true, however, when the HMGl was denatured. HMGl in its native, reduced state can become oxidized(24,25) very readily solely to indicate this fact.
524
525
DNA Condensation by HMGl .H1 Complex
- -
-30 200
220
240
2 io
Wavelength (nm) FIG.3. Circular dichroic spectra of reduced (-) and oxidized (- - -) HMG1. Oxidized and reduced HMGl were prepared and authenticated as described previously(24). Protein concentration was 25 pg/ml in 10 mM Tris 1 mM EDTA, 1 mM dithiothreitol, 150 mM
I " " " " " " 230
320
290
260
Wavelenoth hm) FIG. 1. Circular dichroic spectra of DNA and complexes. A, DNA; E , DNA.H1 complex formed at 1.00.4 (w/w; the w/w ratio is equivalent to the charge ratio); C,DNA.H1 complex plus HMGl at 1.00.4:0.4 (w/w/w) DNAH1:HMGl. DNA concentration was 15 pg/ ml. Spectra of complexesbetween DNA and HMGl onlydidnot differ from the spectrum of naked DNA ( A ) .
m al
'0
T0 .x
20 30
1
NaC1, pH 7.0, for both spectra.
under ordinary laboratory conditions with the formation of a disulfide bond (24). Fig. 3 reveals that theoxidation caused a substantial conformational change in the protein. The circular dichroic spectraof oxidized and reduced HMGl are compared in Fig. 3, and it can be seen that they are alike except that the oxidized form has an additionalnegative peak a t 210 nm, whichgenerally is associated with random coils (26). The oxidation of HMGl changedthestructure of theprotein substantially. The strong effect of native HMGl on the circular dichroic spectrum of the H1. DNA complex, in view of the failure of denatured HMGl to affect the spectrum, is a powerful argument for the specificity of the ternary interaction of HI, HMGl,and DNA. Specificity in the ternary interaction was further supported by the kineticsof the complex formation. Theeffect of HMGl on the spectrumof the H1 .DNA complex was not immediate. It was detectable soon after the addition of HMGl to the complex, but the effect increased during a day of incubation; the ellipticity reading was then stable, within experimental error, for at least a week. The slow rate of ternary complex formation is consistent with the apparently complicated kinetics of formation in uitro of the H1 .DNA complex itself (59, 14),as well as the similar situationnucleosome in formation (27, 28). The binary interactionof HMGl and H1 histone has been described previously (20-23), but the specificity of the interaction has been questioned (23). There was the possibility that HMGl and H1 bound each other merely by nonspecific, electrostatic forces between the large number of positively charged lysine residues inH1 and a 40-residue, polycarboxylic amino acid region of HMGl (29). The present data support the view that the interaction isspecific. First, if the HMG1H1 binding was nonspecific, it would be expected thatHMGl would tend to negate the effect of H1 on the circulardichroic spectrum of DNA; instead, HMGl enhanced theeffect of H1. Second, if the H1-HMG1 binding was nonspecific, electrostatic in nature, denaturation of the HMGl would not be expected to eliminate the effect of the HMG1, as was observed. We conclude that the HMG1-H1 interaction is specific. In addition to addressing that controversy, the present report is the first for the ternary complex of HMGl-Hl.DNA, although H1-DNA (5-9, 14, 15),H1-HMG1 (20-23), and
1
0 0.0
0.5
1 .o
1.5
HMG/DNA (wIw) FIG.2. Ellipticity ofDNA at 270 n m as a function of HMGl added to the preformed H1 .DNA complex. E- - a, reduced HMG1; U, oxidized HMG1. Oxidized and reduced HMGl were prepared as previously described (24).
526 H ~ G l - D (30-33) ~ A binary interactions have been described before. The significance of the ternaryinteraction, in light of the parallels between HI-induced DNA condensation and the condensation of chmmatin by H1, is that HMGlmight modulate the ability of H1 to condense chromatin in localized regions, perhaps effecting generalized changes in gene function. Acknowledgment-We thank Kenneth Raymond for the use of the spectropolarimeter. REFERENCES Lennox, R. W. (1984) J. Biol. Chem. 269,669-672 Weintraub, H. (1985) Cell 42,705-711 Jackson, D. A. (1986) Trends Biochem. Sci. 11,249-252 Huang, H.-C., and Cole, R. D. (1984) J.Biol. Chem. 259,1423714242 5. Welch, S. L., and Cole, R. D. (1979) J. Biol. Chem. 254,662-665 6. Welch, S. L., and Cole, R. D. (1980) J. Biol. Chem. 255, 45164518 7. Liao, L. W., and Cole, R. D. (1981) J. Biol. Chem. 256, 67516755 8. Liao, L. W., and Cole, R. D. (1981) J. Biol. Chem. 256, 1114511150 9. Liao, L. W., and Cole, R. D. (1981) J. Bid. Chem. 256,1012410128 10, Weintraub, H. (1984) CeU 38,17-27 11, Adler, A. J., Langan, T. A., and Fasman, G. D. (1972)Arch. Biochem. Biophys. 153,769-777 12. Gurley, L. R., Walthers, R. A., and Tobey, R. A. (1974) J. Cell Biol. 60,356-364 13. Ajiro, K., Borun, T. W., and Cohen, L. (1981) Biochemistry 20, 1454-1464 14. Fasman, G. D., Schaffhausen, B., Goldsmith, L., and Adler, A. 1. 2. 3. 4.
(1970) B ~ h e m ~ t9,2814-2822 ry 15. Biard-Roche, J., Gorka, C., and Lawrence, J.-J. (1982) EMBO J. 1,1487-1492 16. Jin, Y.-J., and Cole, R. D. (1985) FEBS Lett. 182,455-458 17. Jin, Y.-J., and Cole, R. D. (1986) J. Biol. Chem. 261,3420-3427 18. DeBernardini, W., Losa, R., and Koller, T. (1986) J. Mol. Biol. 189,503-517 19. Cole, R. D. (1977) in The Molecular Biology of the Mammalian Genetic Apparatus (T’so, P., ed) pp. 93-104, Elsevier/NorthHolland Biomedical Press, Amsterdam 20. Shooter, K. V., Goodwin, G. H., and Johns, E. W. (1974) Eur, J. Biochem. 47,263-270 21. Smerdon, M. J., and Isenberg, I. (1976) Biochemistry 15,42424247 22. Yu, S. S., and Spring, T. G. (1977) B i ~ h ~ m . B i oActa ~ ~ 492, ys. 20-28 23. Cary, P. D., Shooter, K. V., Goodwin, G. H., Johns, E. W., Olayemi, J. Y., Hartman, P. G., and Bradbury, E. M. (1979) Biochem. J. 183,657-662 24. Kohlstaedt, L. A., King, D. S., and Cole, R. D. (1986)Biochemistry 25,4563-4565 25. Elton, T. S., and Reeves, R. (1985)Anal. Biochem. 149,316-321 26. Greenfield, N. J., and Fasman, G. D. (1969) Biochemistry 8 , 4108-4115 27. Stein, A., Whitlock, J. P., Jr., and Bina, M. (1979) Proc. Nutl. Acnd. Sci. U.S. A. 76,5000-5004 28. Allan, J., Fey, S. J., Cowling, G. J., Gould, H. J., and Maryanka, D. (1979) J. Biol. Chem. 254,11061-11065 29. Walker, J*M., Hastings, J. R. B., and Johns, E. W. (1978) Nature 271,281 30. Boone, C., Sautiere, P., Duguet, M., and d e k o n d o , A.-M. (1982) J. Biol. Chem. 257,2722-2725 31. Javaherian, K., Sadeghi, M., and Liu, L. F. (1983) Nucleic Acids Res. 11,3569-3579 32. Hamada, H., and Bustin, M. (1985) Biochemistry 24,1428-1433 33. Isackson, P. J., Chow, L. G., and Reeck, G. R. (1981) FEBS Lett. 125,30-34
