Cell, Vol. 12, 101-107,

Influence

September

1977, Copyright

of Histone

F. Thoma and Th. Kolier lnstitut fur Zellbioiogie Eidgenossisch Technische Hdnggerberg Zurich, Switzerland

0 1977 by MIT

HI on Chromatin

Hochschule

Summary Removal of histone Hl produces a transition in the structure of chromatin fibers as observed by electron microscopy. Chromatin containing ail histone proteins appears as fibers with a diameter of about 250 A. The nucieosomes within these fibers are closely packed. If histone Hl is seiectively removed with 50-100 mM NaCi in 50 mM sodium phosphate buffer (pH 7.0) in the presence of the ion-exchange resin AG 50 W - X2, chromatin appears as “beads-on-a-string” with the nucieosomes separated from each other by distances of about 150-200 A. if chromatin is treated in the presence of the resin with NaCi at concentrations of 650 mM or more, the structural organization of the chromatin is decreased, yielding fibers of irregular appearance. introduction The discovery of a chromatin subunit (Hewish and Burgoyne, 1973; Sahasrabuddhe and Van Holde, 1974) has had a great impact on the concept of the structure of chromatin (for reviews, see Feisenfeld, 1975; Li, 1976). Evidence for this subunit, which has also been termed v body or nucleosome, rests on results obtained from nuclease digestion of nuclei and subsequent isolation of subunit monomers, dimers and trimers by Noli (1974), from direct visualization in the electron microscope by Oiins and Oiins (1974), and from neutron diffraction studies by Baldwin et al. (1975). DNA seems to be wrapped around a globular histone core which has been proposed to consist of two of each of the histones H2 A, H2 B, H3 and H4 (Kornberg, 1974). Histone Hl appears not to be involved in this core (Baldwin et al., 1975). Some investigators report that chromatin containing all the histone proteins appears in the electron microscope as a linear array of separated nucieosomes connected by thin filaments about 2040 A in diameter (“bead-and-bridge” structure) (Woodcock, 1973; Olins and Olins, 1974; Langmore and Wooley, 1975; Oudet, Gross-Bellard and Chambon, 1975; Kiryanov et al., 1976; Woodcock, Safer and Stanchfield, 1976). Others describe nucleosomes in close contact (Finch, Nell and Kornberg, 1975; Griffith, 1975; Compton et al., 1976; Finch and Klug, 1976) forming a filament of about

Structure

100 A in diameter, similar to the one observed earlier (Ris, 1966; Bram and Ris, 1971). When nuclei are thin-sectioned (Brasch, 1976) or lysed directly on electron microscope supports (Oudet et al., 1975), or when chromatin is spread in the presence of divalent cations (Gall, 1963; Lampert, 1971; Pooley, Pardon and Richards, 1974; Finch and Klug, 1976; Kiryanov et al., 1976), thicker filaments on the order of 200-300 A in diameter are found, implying a higher order of structural organization (Dupraw, 1970; Ris and Kubai, 1970; Finch and Kiug, 1976). The role of histone Hl still remains fairly unclear. Baldwin et al. (1975) and Shaw et al. (1976) have suggested from nuclear magnetic resonance and biochemical studies that histone Hl binds to the outside of chromatin subunits. Removal of histone Hl exposes a 30-50 base pair DNA segment between nucleosomes as judged from nuclease digestion experiments (Varshavsky, Bakayev and Georgiev, 1976; Whitlock and Simpson, 1976). Based on electron microscopy, Bram (1975) and Finch et al. (1975) believe that the internucleosomal distance is not influenced by histone HI, whereas Oudet et al. (1975) Bellard et al. (1976) and Finch and Klug (1976) believe that removal of histone Hl is related to the appearance of the “bead-and-bridge” structure. In the present paper, we describe the morphological changes of chromatin upon removal of histone Hl . The elimination of histone Hl is performed at the lowest possible sodium concentration in the presence of an ion-exchange resin (Bolund and John, 1973) to minimize redistribution of chromosomai proteins (Varshavsky, 1974; Germond et al., 1976). Samples are prepared for electron microscopy under controlled conditions which allow, in the absence of mechanical stretching, good adsorption of the chromatin fibers to the carbon support films. The chromatin samples are analyzed simultaneously for protein content and with respect to the ultrastructural appearance. These are technical prerequisites to clarify conflicting observations described in the literature. Results Chromatin freshly extracted from rat liver by the nuclease method of Noll (1974) contains all the histone proteins (Figure 1, scan 1). When this chromatin is fixed in 0.1% glutaraldehyde, 10 mM sodium acetate and 5 mM triethanolamine acetate at pH 7.9 and prepared for electron microscopy by the BAC method (Vollenweider, Sogo and Koiler, 1975), it appears as filaments with a width of approximately 250 A (Figure 2a). These filaments show a granular substructure with the individual

Cdl 102

HI X

HI X

Hi X

HI X

2

3

4

Figure 1. Protein Analysis of Chromatin Samples against 0.2 mM EDTA by SDS Gel Electrophoresis The gels are stained with brilliant blue and the scans of this figure are from the same Scan 1: chromatin in 0.2 mM EDTA. Scan 2: same chromatin as in scan 1 after 0.2 mM EDTA (pH 7.0) for 150 min. Scan 3: same chromatin as in scan 1 after 100 mM NaCl and 50 mM sodium phosphate Scan 4: same sample as in scan 3, except was performed for 90 min. HI represents histone Hl; x represents-from tones H3, H2b, H2a and H4.

after

Dialysis

scanned at 560 nm. All experiment. being

resin-treated

in

being resin-treated in (pH 7.0) for 30 min. that resin treatment left to right-his-

particles in close contact. In analogy to published data (for example, Oudet et al., 1975; Finch and Klug, 1976), we call these subunits nucleosomes. A similar morphology is observed when chromatin in 0.2 mM EDTA (pH 7.0) is gently stirred for 150 min at 0°C in the presence of the ion-exchange resin AG 50 W - X 2 (Figure 2b). The histone pattern revealed by gel electrophoresis remains unchanged under these conditions with respect to the sample not treated with the resin (Figure 1, scan 2). To remove histone Hi selectively, the ion-exchange resin AG 50 W - X 2 is added to chromatin samples gently stirred at 0°C in the presence of 50 mM sodium phosphate buffer at pH 7.0 and increasing NaCl concentrations. After separating the resin from the chromatin by centrifugation, the samples are dialyzed against 0.2 mM EDTA (pH 7.0). The protein analysis by gel electrophoresis shows that the extraction of Hl with a constant amount of resin depends upon the time and the sodium concentration used. Sodium phosphate without NaCl partially removes histone Hi during the 150 min tested. With 50 mM NaCI, the removal Figure

2. Electron

Micrographs

of Chromatin

Samples

after

Dialysis

of Hl is about 90% after stirring for 150 min with the resin. Scans 3 and 4 of Figure 1 show the results obtained with 100 mM NaCI. Whereas after 30 min histone HI is only partly eliminated, it is completely absent after 90 min without any obvious loss of the other histone proteins. With 450 mM and 650 mM NaCI, the removal of Hi is complete after 20 min (data not shown). Electron micrographs of samples stirred with resin in 50 mM sodium phosphate (pH 7.0) and 100 mM NaCl (same as Figure 1, scans 3 and 4) are shown in Figure 2. After partial removal of histone Hl (Figure 2c), relaxed filaments are seen with their nucleosomes arranged one behind each other separated by a small bridge of about 150-200 A. Basically the same morphology is observed after complete elimination of histone Hl (Figure 2d). Parallel aliquots, however, in which resin is omitted (Figure 2f) show thick filaments with closely packed subunits similar to the starting material demonstrated in Figure 2a. Electron micrographs like the ones in Figures 2c and 2d are obtained from resin-treated samples from 50-450 mM NaCI, and the corresponding controls without resin are indistinguishable from Figures 2a, 2b and 2f. Electron micrographs of resin-treated samples with 50 mM sodium phosphate (pH 7.0) and NaCl concentrations between 650 mM (Figure 2e) and 1000 mM show with increasing salt an increasingly irregular morphology with variable internucleosomal distances (arrow). The corresponding controls without resin appear again as thick fibers similar to Figure 2f, however, with a more irregular morphology with frequent long and thin filaments which we believe to be more or less deproteinized DNA. When NaCl is added to chromatin samples in the absence of resin, the increase in salt concentration is accompanied by the appearance of a turbidity of the solution with a maximum at NaCl concentrations between 100 and 150 mM. After dialysis against 0.2 mM EDTA, the turbidity disappears. To test the removal of histone proteins from chromatin without resin, we have separated the chromatin from the solvent by centrifugation (250,000 g for 2 hr) after a 60 min of exposure to 50 mM sodium phosphate and increasing amounts of NaCI. The pellet is resuspended in the same volume and in the same buffer as the supernatant. Supernatant and pellet solution are analyzed by gel electrophoresis against

0.2 mM EDTA

(pH 7.0)

(a) Chromatin in 0.2 mM EDTA, corresponding to Figure 1, scan 1. (b) Same chromatin as in (a) after being resin-treated in 0.2 mM EDTA (pH 7.0) for 1.50 min, corresponding to Figure 1, scan 2. (c) Same chromatin as in (a) after being resin-treated in 100 mM N&l and 50 mM sodium phosphate (pH 7.0) for 30 min. corresponding to Figure 1, scan 3. (d) Same preparation as in (c), except that the resin and salt treatment were performed for 90 min, corresponding to Figure 1, scan 4. (e) Same preparation as in (c) and (d), except that the resin and salt treatment were performed for 20 min in 650 mM NaCI. Gel electrophoresis of this sample (not shown) reveals complete removal of histone Hl, in agreement with Bolund and John (1973). (f) Chromatin exposed without resin for 60 min to 100 mM NaCl in the presence of 50 mM sodium phosphate (pH 7.0) and 0.1 mM EDTA. Gel electrophoresis of this sample (not shown) reveals the presence of all the histone proteins.

iii3tone

HI and Chromatin

Structure

0.5 pm

Cell 104

A6

A6

1

2

Figure

3. Protein

HI X

Hl X

2A

3A

samples are fixed prior to electron microscope specimen preparation to minimize effects of possible mechanical forces occurring during adsorption to the supporting films. The conditions applied for the electron microscope specimen preparation are based on the data of Vollenweider et al. (1976). The lowest possible BAC (benzyldimethylalkylammonium chloride) concentration for reproducible adsorption of nucleic acid filaments to carbon supports is used. The chromatin solution contains IO mM sodium acetate and 5 mM triethanolamine acetate at pH 7.9, as well as EDTA to remove divalent cations. Under these conditions, deproteinized DNA is fully unfolded and no superstructure (Vollenweider et al., 1976) is seen. This is an important technical prerequisite for the achievement of the best analyzability of the protein-DNA complexes, and it allows the assumption that folded complexes under these conditions are likely to be folded as a consequence of the protein-nucleic acid interaction and not as a consequence of a superstructure primarily present in DNA. Furthermore, special care is taken to avoid stretching of chromatin fibers during adsorption to the electron microscope supports, because stretching is known to increase the internucleosomal distance (Compton et al., 1976). A detailed study of the effects of specimen preparation upon the appearance of chromatin is in preparation (G. de Murcia, personal communication). Although with the protein analysis used no statement can be made with regard to the nonhistone proteins, the method described for the removal of histone Hl appears to be rather selective with respect to the other histone proteins. Treatment of the chromatin with resin at low salt leads to a disappearance of the HI band only. The high speed centrifugation experiments without resin clearly show that with increasing salt concentrations, HI is the first histone protein to be dissociated. This is in agreement with the results of Billett and Barry (1974) and Bradbury et al. (1975). In these experiments, no detectable Hl is removed at 100 mM NaCI; however, under the same conditions in the presence of the resin, a complete elimination of HI is observed. Since resin treatment of chromatin without salt does not lead to a loss of Hl, we conclude that the presence of resin shifts the sodium-mediated dissociation of histone HI to lower salt concentrations. Whether the salt-induced dissociation of the other histone proteins is also shifted cannot be determined from the present experiments. The resin binds histone Hi very tightly. In agreement with Bolund and John (1973), we have not been able to recover this protein even after heating the resin to 95°C for 4 min in a solution containing 2% SDS (sodium dodecylsulfate)

L-.ia Analysis

by SDS Gel Electrophoresis

The chromatin samples after being exposed without resin for 60 min to NaCl concentrations between 50 up to 650 mM were centrifuged at 250,000 g for 2 hr. The pellet was resuspended in the same volume and the same solvent as the supernatant. To the supernatant and the resuspended pellet solution we added one fifth their volume of a 5 times concentrated electrophoresis sample buffer to give 2% SDS, 2 M urea, 2% glycerol and 0.12 M Trisglycine at pH 9. Of this mixture, 10 /LI were applied per electrophoresis tube. Gel 1: chromatin exposed to 100 mM NaCl in 50 mM sodium phosphate at pH 7.0. (A) supernatant with an ApcO = 0.4. (B) pellet solution with an A,, = 7. Gel 2: same experiment as in gel 1, except that 450 mM NaCl were used. (A) supernatant with an AzM) = 0.8. (B) pellet solution with an &So = 6.6. Gel 3: same experiment as in gel 1, except that 650 mM NaCl were used. (A) supernatant with an qao = 0.6. (B) pellet solution with an & = 8.0. Scan 2A: scan at 580 nm of gel 2A. Note the predominance of histone Hl. Scan 3A: scan at 580 nm of gel 3A.

as shown in Figure 3 (for technical details, see legend to Figure 3). At 50 and 100 mM NaCI, no proteins can be detected in the supernatant and all the histone proteins are present in the pellet. In the case of 450 mM NaCI, histone HI appears in the supernatant and is absent in the pellet (Figure 3, gels 2 and scan 2A). At 650 mM NaCI, not only Hl but also part of the other histone proteins are removed, as shown on the gel of the supernatant (Figure 3, gels 3 and scan 3A). Discussion The morphology of the chromatin fibers observed in the present study is the result of a number of factors such as salt concentrations, glutaraldehyde fixation, BAC-chromatin interactions, adsorption of the chromatin to carbon support films, and drying and shadow casting of the specimens. The

Histone 105

HI and Chromatin

Structure

and 2 M urea, or 2 M NaCI. Taking all these observations into account, it appears that at low salt, a partial dissociation of HI from chromatin takes place, and that due to the high affinity of the resin for HI, a slow transfer of Hl from chromatin to the resin takes place. It is highly improbable that the removal of histone HI described in this paper is due to a protease effect. In all experiments, a parallel control is made without resin (see Experimental Procedures). In no case do we see any detectable loss of histone proteins, particularly HI, in these controls. Furthermore, the sharpness of the HI bands (see Figure 1) in resin-treated controls (0.2 mM EDTA without sodium phosphate and NaCI) as well as in controls without resin makes a possible action of disturbing proteases improbable. In the electron microscope, we observe that with the removal of histone Hl, a structural transition takes place from a fiber with a diameter of about 250 A to a filament with the appearance of “beadson-a-string.” Since the removal of histone Hl in the experiments described is quantitatively the most pronounced effect of the salt and resin treatment, we believe that the structural transition observed is mainly due to the removal of this protein. A possible effect of spermine and spermidine used for the isolation of chromatin has been ruled out in experiments in which these compounds have been omitted (F. Thoma, unpublished results). A similar transition upon increasing salt concentrations has already been observed by Jones and Beer (1970)not, however, in connection with the subunit structure of chromatin-and very recently by H. Ris (personal communication). Our electron micrographs of HI-depleted chromatin show that the nucleosomes are connected by a thin fiber about 150-200 A in length. If this thin fiber represents deproteinized DNA, a suggestive comparison could be made to the data of Varshavsky et al. (1976) and Whitlock and Simpson (1976), who report that upon removal of histone Hl, 30-50 base pairs of DNA become accessible to the action of micrococcal nuclease. With respect to structural details, the approximately 250 A thick, histone Hl-containing fibers cannot be compared with morphologically similar observations made under a wide variety of experimental approaches (Gall, 1963; Lampert, 1971; Pooley et al., 1974; Ris, 1975; Brasch, 1976.; Bustin, Goldblatt and Sperling, 1976; Compton et al., 1976; Finch and Klug, 1976; Kiryanov et al., 1976). The relaxed Hl-depleted fibrils with regularly spaced nucleosomes are compatible with findings reported by Olins and Olins (1974), Finch et al. (1975), Griffith (1975), Oudet et al. (1975), Bellard et al. (1976), Finch and Klug (1976), and Woodcock et al. (1976) using different specimen preparation

methods and chromatin samples with and without histone HI. Our data, however, give clear evidence that in chromatin containing HI, the nucleosomes are in close contact, forming a fiber of a higher structural organization even in the absence of divalent cations and at low salt concentration. It is tempting to speculate that the 250 A thick fiber observed in this paper is an intermediate form between the 100 A thick filament in the absence of salt and the compact forms induced by MgTT described by Finch and Klug (1976). When chromatin is treated with high NaCl concentrations (650-1000 mM) in the presence of the resin, the ultrastructure of the “beads-on-a-string” is more irregular than after the removal of histone HI at low salt. The higher the salt concentration, the more frequently segments of filaments with variable thickness and without nucleosomes are observed. This structural deorganization might be related to the fact that in this range of sodium concentrations, other histone proteins are also dissociated (Billett and Barry, 1974; Bradbury et al., 1975), as indicated in the experiment of Figure 3, and rearranged along the chromatin fiber (Varshavsky, 1974; Germond et al., 1976). The method described in this paper for the extraction of histone Hl promises to be extremely selective for this particular protein. The data suggest that using these extraction conditions, the interactions of the residual histone proteins with DNA are probably less affected than under conditions previously used by Bolund and John (1973). Experimental

Procedures

Materials BAC [benzyldimethylalkylammonium chloride (n-alkyl mixture: C&H,, 60% and CIaH,, 40%)] was a gift from Bayer (Leverkusen, Germany). Micrococcus nuclease was purchased from Worthington. Acrylamide, N,N,N’,N’-tetramethylethylenediamine, ammonium persulfate and AG 50 W - X 2 were obtained from Biorad. Brilliant blue R was bought from Sigma, and all other chemicals (analytical grade) were from Merck. Buffer A contained 15 mM Tris (tris-hydroxymethylaminomethane)-HCI at pH 7.4,15 mM NaCI, 60 mM KCI, 0.5 mM spermidine and 0.15 mM spermine. Extraction of Chromatin and Removal of Histone Hl Nuclei were isolated from rat liver as described by Hewish and Burgoyne (1973). For the extraction of chromatin, a procedure similar to that of Nell, Thomas and Kornberg (1975) was followed. To 1 ml of a nuclei suspension (derived from about 2 g of liver) in 0.34 M sucrose in buffer A were added 10 &I of a solution containing 0.1 M CaCI,. After preheating the sample for 5 min to 37”C, 30 units of micrococcus nuciease were added. The digestion was terminated after 60 set by the addition of 10 ~1 of a solution containing 0.2 M EDTA (ethylenediaminetetraacetic acid disodium salt at pH 7.0) and quenching in ice. The nuclei were then pelleted (4000 g for 5 min) and lysed during 30 min at 0°C in 1 ml of 0.2 mM EDTA at pH 7.0. The sample was centrifuged again for 5 min at 1000 g. The chromatin-containing supernatant had an AXE0

Cell 106

(absorbance at 260 nm) of about 20. For the removal of histone HI, an equal amount of a solution containing 100 mM sodium phosphate at pH 7.0 and varying concentrations of NaCl was slowly added. Each sample with a given NaCl concentration was divided into two aliquots. To one of them we added 0.2 ml sedimented AG 50 W - X 2 [prepared as described by Bolund and John (1973) and equilibrated with 50 mM sodium phosphate and the appropriate NaCl concentration] per ml of chromatin solution. After gently stirring for 20-150 min at O’C, both aliquots (resintreated and control) were centrifuged at 500 g for 5 min. The supernatants were then dialyzed overnight at 4°C against 100-200 vol of 0.2 mM EDTA at pH 7.0, and then analyzed by gel electrophoresis and electron microscopy. Controls in 0.2 mM EDTA with and without resin were treated strictly in parallel. The above procedures were performed within 24 hr, and the subsequent analyses described below were started immediately.

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Electron Microscopy For fixation, chromatin in 0.2 mM EDTA was diluted about IO times to an Asso of about one in a solution containing 0.1% glutaraldehyde. IO mM sodium acetate and 5 mM triethanolamine acetate at pH 7.9. The sample was kept for 60 min at room temperature and then for more than 12 hr at 4°C. It was diluted 50 times in the above glutaraldehyde solution, and BAC (Vollenweider et al., 1975) was added to a final concentration of 2 x IO+% from a 0.2% stock solution in formamide. Droplets of 5 (.d were applied to electron microscope grids covered with a carbon film. Adsorption of the chromatin fibers to the carbon supports was allowed to take place during 5-10 min. The droplet was then washed off the grid by floating the specimen on distilled water for 10 min. The grids were dehydrated during 2-3 set in ethanol and then blotted dry on filter paper. For contrast enhancement, the grids were rotaryshadowed at an angle of 7” using carbon-platinum evaporated from an electron gun. Samples were examined in a Siemens electron microscope 101 at 20,000x magnification. For magnification calibration, a carbon grating replica grid from Balzers Union (Lichtenstein) was used. Acknowledgments

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revised

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We thank G. de Murcia for stimulating discussions and for communicating unpublished results. We are grateful to Drs. M. Lezzi, D. Turner and K. Downing for critical readings of the manuscript, and to Mrs. H. Mayer-Rosa for excellent technical assistance. This work was supported by the Schweizerischer Nationalfonds zur Fdrderung der wissenschaftlichen Forschung.

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Bustin, Gel Electrophoresis The method used for the analysis of the histone proteins is based on the procedure of MacGillivray et al. (1972). The running gel contained 12% (w/v) acrylamide and 0.24% (w/v) N,N’-methylenebisacrylamide. The stacking gel was made of 4% (w/v) acrylamide and 0.06% (w/v) N,N’-methylenebisacrylamide. For polymerization, 0.1% N,N,N’,N’-tetramethylethylenediamine and 0.025% ammonium persulfate were used. The gel buffer contained 0.12 M Tris-glycine at pH 9, 4 M urea and 0.1% SDS. 1 liter of electrode buffer was made up of 1.2 g Tris-HCI at pH 9,25 g glycine and 1 g SDS. The chromatin samples were mixed with a sample buffer solution giving an As60 of 6-6 in 0.12 M Tris-glycine at pH 9, 2% SDS, 2 M urea, 2% glycerol and bromophenol blue. The samples were then heated for 2 min at 95°C. IO ~1 of such a solution were applied per tube. The electrophoresis was performed at 150 V and 9 mAmp for 3 hr at room temperature using 12 glass tubes with a diameter of 3 mm. The gels were fixed for 30 min in 10% trichloroacetic acid, stained for 2 hr in 0.25% brilliant blue in 50% methanol and 10% acetic acid, destained in 25% methanol, and scanned at 560 nm in a Gilford spectrophotometer 2400. The histone protein bands are identified according to Hayashi, Matsukera and Ohba (1974).

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Influence of histone H1 on chromatin structure.

Cell, Vol. 12, 101-107, Influence September 1977, Copyright of Histone F. Thoma and Th. Kolier lnstitut fur Zellbioiogie Eidgenossisch Technische...
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