Proc. Nati. Acad. Sci. USA Vol. 74, No. 11, pp. 4852-4856, November 1977

Biochemistry

Structure of histone H1-DNA complex: Effect of histone H1 on DNA condensation (histone H5/nucleoprotein complexes/electron microscopy/DNA rigidity/core histones)

MYRTLE W. HSIANG AND R. DAVID COLE Department of Biochemistry, University of California, Berkeley, California 94720

Communicated by H. A. Barker, August 15,1977

ABSTRACT Large doughnut-shaped complexes were formed when histone Hi was mixed with DNA in amounts that extensively neutralized it. The doughnut shape is the most prevalent form observed by electron microscopy for linear double-stranded DNA independent of the molecular weight of the DNA in the range 1.2 X 100 to 25 X 106 at an HI/DNA input weight ratio of 1.3, at ionic strength 0.17. Doughnuts were not observed for single-stranded DNA-Hi complexes; instead, the complexes were globular. The circumference of the doughnutshaped particles indicates that much of the rigidity of duplex DNA in the complex has remained. Evidently, e condensation of the nucleohistone is constrained by the rigidity of duplex DNA and, under this constraint, surface contact with water is minimized by adopting a doughnut shape. Histone H5 causes a type of DNA condensation similar to that of HI at comparable charge ratios. Core histones H2A-H2b, H3 and H4 complex with DNA to form globular aggregates of suck small diameter that the duplex DNA in them must be much more tightly folded than is the case with the doughnut-haped complexes. Because these histones are designed to-fold DNA into nucleosomes 100 A wide, they must destroy the rigidity of free duplex DNA, perhaps by forming kinks in the chain. The structural role of four of the five major classes of histones appears to be the formation of protein cores (1) around which (2) DNA is folded and condensed (3) to form nucleosomes (v bodies) (4), which are the subunits in chromatin structure. The function of H1, the other major class of histone, is not understood even at this rudimentary level. Although H1 has the highest positive charge density (5) among the five histones, it is the first to be displaced from chromatin by acid (6), salt (7), or tRNA (8), and H1 is the most susceptible among the histones to protease degradation when it is still bound in chromatin (9). These observations have been interpreted as indications of an exterior location of H1 on the

20). They generally suffered from the problem of aggregation and precipitation; the observed properties were frequently highly dependent on the way the complex was prepared. In this communication we report two methods of complex preparation at much lower DNA concentrations than previously used; the lower concentration of DNA avoided the problem of precipitation. In determining the effect of various amounts of H1 on the formation of complexes it was discovered that, at a threshold ratio of histone H1 to DNA, a cooperative condensation produced doughnut-shaped complexes.

MATERIALS AND METHODS H1 of rabbit thymus gland (Pel-Preeze Biologicals Co., Rogers, AR) was prepared by the method of De Nooij and Westenbrink (21) from isolated nuclei (22) and further purified by gel filtration in 10 mM HCl through Sephadex G-100 (2.0 X 195 cm). Chicken erythrocyte H5 was prepared by T. Spring (23) and further purified on Sephadex G-100. Steer H2A-H2B, H3, and H4 were extracted from steer liver chromatin and fractionated on Bio-Gel P-30 (24). Calf thymus DNA was purchased from Sigma. The average molecular weight was estimated by sedimentation velocity to be 4.2 X 106. The molecular weights of the majority of the DNAs fall in the range of 3 to 5 X 106 as indicated by agarose gel electrophoresis (25). T7 DNA and the L strand of T7 DNA were generous gifts from George Kassavetis and M. J. Chamberlin. Linear pVH51 DNA (25) and pBR3S3 DNA,* prepared by EcoRI endonuclease digestion of the corresponding superhelical DNAs, were kindly provided by Pat Greene. ColEl DNA was prepared from Escherichla colf K-12 strain JC411. Linear ColEl DNA was prepared by EcoRI endonuclease digestion of the superhelical form. Preparation of Histone-DNA Complexes. H1-DNA complex was prepared by slowly pipetting various concentrations of H1 solution in 0.15 M NaCl/0.015 M Na citrate, pH 7.0 (SSC) into a test tube containing an equal volume of DNA solution in the same buffer. The tip of the pipet was made to touch the wall of the test tube and, after delivery, the test tube was gently tapped to mix the contents. As judged by turbidity measurement and electron microscopy, reproducible H1-DNA complexes were prepared in this manner in a final volume of 0.1-1.0 ml. This method will be referred to as the "slow mixing method." Another technique involving vigorous mixing of the two solutions with a vortex mixer was also used. This "rapid mixing" resulted in complexes that were somewhat irreproducible and heterogeneous in shape although the turbidities were quite reproducible.

surface of the chromatin. Chemical crosslinking experiments have failed to find evidence that H1 is part of the nucleosomal protein core (10). Some observations suggest that it is located in the internucleosome space (11-13) and may play the role of determining the internucleosome spacing (14, 15). Studies on the function of H1 are numerous in the literature. Olins (16) and Johns and Forrester (17) found that, at fairly high concentrations of DNA, Hi as well as other histones precipitate DNA effectively before the charge of DNA is fully neutralized. Olins and Olins (18) studied the physicochemical properties of H1-DNA complexes at several charge ratios but generally at Hi concentrations that did not fully neutralize the DNA. The structures of such complexes were very heterogeneous; extensive networks, bundles, and a few doughnut-shaped entities were observed by electron microscopy. Numerous spectrophotometric studies of the binary complex have been done (19, The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.

Abbreviations: SSC, 0.15 M NaCI/0.015 M Na citrate, pH 7.0; Mr, molecular weight. * F. Bolivar, personal communication.

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FIG. 2. Electron micrographs of H1-DNA (calf thymus) complexes prepared by slow mixing at H1/DNA weight ratios of 0.36 (a) and 1.3 (b) in SSC ata DNA concentration of 31 ,g/ml. The sample was stained with 0.5% aqueous uranyl acetate.

Histone HI/DNA Weight Ratio FIG. 1. Turbidity of Hi/calf thymus DNA mixtures at a DNA concentration of 5 ,g/ml in SSC at 250 prepared by slow mixing. A320 was read within 2 hr after mixing. nm

The turbidity of the complex-containing solutions was measured by a Cary 118 or a Zeiss spectrophotometer at 320 nm. Prior to mixing, the DNA and HI solutions were filtered through 0.45-,gm pore size Millipore filters. All the test tubes used were rinsed six times with distilled water and dried immediately before use. Electron microscopy was done by Alice Taylor with a Siemens Elmiskop IA or a Philips EM300 electron microscope. Parlodion-coated copper grids (400 mesh) were coated with carbon and glow-discharged for 20 sec at 50Mzm Hg. The grids were used within 1 hr after the discharge operation. A drop of sample solution was placed on the grids, blotted with filter paper, and stained with 0.5% aqueous uranyl acetate in the same way. The grids were photographed at magnifications of X8000 to X60,000.

RESULTS Turbidity of HI-DNA Complexes. When solutions of Hi and DNA were combined to form complexes, a turbidity developed. The relationships between the turbidity and the HI/DNA weight ratio, at a final DNA concentration of 5 i/ml (in SSC) is shown in Fig. 1. The turbidity measurement of the complex-containing solutions at such a low DNA concentration was made possible by using a Cary 118 and also by using a wavelength of 320 nm, at which the sensitivity for turbidity is about 2.4 times that at 400 nm. The increased sensitivity allowed the use of such low concentrations of DNA that artifacts due to precipitation (16, 17) were avoided. The turbidity increased linearly when the amount of HI was increased up to an H1/DNA ratio of 1.0. When the ratio went above 1.0, however, the change of turbidity became cooperative and eventually leveled off at HI/DNA ratios >2.0. This part of the turbidity curve is very sensitive to the method of complex preparation. When the complexes

were

prepared by vigorous

mixing with a vortex mixer, the turbidity reading was reproducible for a given H1/DNA ratio, but the range of cooperative change of turbidity was shifted to a higher H1/DNA ratio and the shapes of the complexes were irregular as observed in the electron microscope. When the complex was prepared by the

slow mixing method, both the turbidity and the shape of the complex were reproducible. Contrary to numerous previous reports of HI-DNA complexes at this ionic strength (18-20), the turbidity of the very dilute solutions prepared by our slow mixing method was not evident to the eye at HI1/DNA ratios 1.4) slowly precipitated at 250, as indicated by turbidity at 320 nm and absorption at 260 nm after standing for 24 hr. Therefore, a ratio of 1.3 was used throughout this study. Shape of Histone H1-DNA Complexes. Before the cooperative change in turbidity, no dominant structure was observed; instead, many free DNA fibers, irregular bundles, and networks and few doughnut-shaped particles were observed. The representative micrograph shown in Fig. 2a agrees reasonably well with previous observations by Olins and Olins (18). However, during the cooperative change in turbidity, doughnut-shaped particles of varying sizes were the predominant form (Fig. 2b). The doughnut-shaped structures do not seem to be artifacts produced during preparation of samples for electron microscopy. Olins and Olins (18) shadowed the alcohol-dehydrated complexes with palladium-gold after formaldehyde fixation. We stained the complexes with dilute aqueous uranyl acetate, and no dehydration was used except for the vacuum in the electron miscroscope. Because two such different procedures yielded the same doughnut-shaped particles, we believe that neither staining with uranyl acetate nor dehydration with alcohol created the doughnuts as artifacts. Effect of the Length of Double-Stranded Linear DNA. Several linear double-stranded DNAs were used to form complexes with Hi: pBR333 DNA, molecular weight (Mr) 1.2 X 106;* pVH51 DNA, Mr 2.1 X 106 (25); CoWEI DNA, Mr 4.2 X 106; T7 DNA, Mr 25 X 106. The common feature of all these complexes was that doughnut-shaped particles were observed in every preparation. The sizes of such particles varied, even for DNAs of uniform size (Fig. 3 a and b), but the diameters of the majority of the doughnut-shaped particles still fell in the range 500-2000 A and were never smaller than 500 A. In fact, when DNA was as large as T7 DNA (Mr 25 X 106), well-separated particles, which were the main form for smaller DNAs, were relatively rare; instead, there appeared to be connections between doughnut-shaped forms as if a single piece of DNA might have joined together several such particles (Fig. 3c). Complexes of Calf Thymus DNA with Other Histones. Complexes of calf thymus DNA and H2A-H2B, H3, and H4

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Structure of histone H1-DNA complex: effect of histone H1 on DNA condensation.

Proc. Nati. Acad. Sci. USA Vol. 74, No. 11, pp. 4852-4856, November 1977 Biochemistry Structure of histone H1-DNA complex: Effect of histone H1 on D...
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