Eur. J . Biochem. 57. 97V 105 (1975)

Studies on the Role and Mode of Operation of the Very-Lysine-Rich Histone H1 (Fl) in Eukaryote Chromatin Histone H1 in Chromatin and in H1 . DNA Complexes E. Morton BRADBURY, Shirley E. DANBY, Henry W. E. RATTLE, and Vincenzo GIANCOTTI

biophysics Laboratory, Portsmouth Polytechnic (Received February 13, 1975)

The nuclear magnetic resonance (NMR) spectrum of chromatin at ionic strengths below about 0.5 M may be attributed solely to its histone H1 component. The effect of various ions and urea on the complex has been investigated using NMR and confirm that the contraction of the complex on increase of ionic strength is largely due to electrostatic interactions. A detailed study of the H1 . DNA complex has also been undertaken. The behaviour of H1 in the two cases is virtually identical, implying that in chromatin the HI is complexed with the D N A rather than with the other histones. Microcalorimetric measurements reveal that the binding of H I to D N A is athermic or involves a heat of reaction which is very small indeed.

This is thc second in a series of papers in which the role of the very-lysine-rich histone H1 in Chromatin structure is investigated by N M R and other methods. The first of the series [ l ]was devoted to the structure of the isolated histone. Chromatin can easily be extracted from calf thymus by isolation of clean nuclei, followed by gentle breakage of nuclear membrane to release the chromatin. The standard product of this process is a uniform gel having a DNA concentration in the region of 6 mgjg chromatin, together with the five histone fractions and a few percent of non-histone proteins. As has been reported previously [2] the gel may be caused to contract to about 15 ?< of its original volume by dialysing against sodium chloride solutions in the region of 0.05 - 0.45 molar, while higher concentrations of sodium chloride remove the H1 and cause the contracted gel to relax to its original volume. The contraction of chromatin is dependent on the presence of H I , and the nuclear magnetic resonance (NMR) spectra of the gel may be attributed entirely to the HI component of the complex, the other histones apparently being bound much more tightly, so that their spectra are broadened and are unobservable under the conditions used. A more detailed study of the effects of various ions and of urea on chromatin gel Ahhreviurions. NMK, nuclear magnetic resonance; HI, hislone H1 (FI).

Eur. J. Biochem. 57 (1975)

has now been undertaken, and the results are presented in this communication. In addition studies have been made of the interaction between H1 and DNA alone, with the interesting result that both the N M R spectra and the macroscopic behaviour of the complex exactly parallel those in chromatin. This may be taken as an indication that the operation of HI in chromatin is largely or completely independent of the other histones. The formation of the H1 . DNA complex has been subjected to microcalorimetric measurements.

EXPERIMENTAL PROCEDURE Calf thymus chromatin was prepared by the method of Panyim et al. [3] using fresh, cleanly welldissected tissue. Owing to the larger amounts of material required, a Waring blender was used (instead of a Potter Elvehjem homogeniser) to wash the nuclear pellet after sucrose purification. The final pellets were suspended in 100- 150 ml distilled water by stirring for 1 h with a magnetic bar. After overnight dialysis against distilled water, a clear viscous gel was obtained with D N A concentration 5-6 mg per g of = 20 for 1 mg/ml DNA). chromatin (using Material was also made by the method of Zubay and Doty [4]. The final pellets obtained were stirred with 100 ml of distilled water using a magnetic bar,

Histone HI in Chromatin

98

until a translucent viscous gel was obtained. After exhaustive dialysis against distilled water the chromatin was spun at 130000 x g for 2-4 h to give a DNA concentration of 6 mg/g of chromatin. Nuclear Magnetic Resonance of Chromatin Dialysis tubing (6.35-mm diameter) was boiled in 5 mM EDTA and washed extensively with distilled water before use. Samples of 1- 2 g of chromatin were placed in this tubing and ddysed against three changes of ten times the sample volume of the appropriate salt solution in deuterium oxide (99.7 deuterated, Prochem Ltd, and 99.8 deuterated, Ryvan Chemical Co.,) to produce a gel of 99.9% of its nominal ionic strength and deuteration. The gel was then placed in a standard 5-mm N M R sample tube and spun to the bottom of the tube in a small hand centrifuge which also removed bubbles. The length of NMR tube occupied by the sample was then measured: since the diameter of such tubes is highly uniform, the volume occupied by the gel could be calculated, and its contraction found by reference to the amount originally placed in the dialysis tubing, as determined by weighing. NMR spectra were run at 270 MHz on the Bruker WH 270 spectrometer in this laboratory; normally 1000 pulses were accumulated. A 12-ps pulse length was employed, followed by an accumulation time of 0.5 s giving a theoretical minimum linewidth of 2 Hz. Contraction of the gel led to large changes in concentration and hence in signal strength in the NMR. Careful measurements of the total peak areas in the spectra revealed that all the protons of the H1 molecules in the sample were visible at the ionic strengths studied.

x

with glass cells at 25 "C. In a typical experiment a sample (2-4 ml) of a DNA solution (type 1, Sigma Chemical Co.) was placed in the largest compartment of the microcalorimetric cell. 1- 2 ml of histone solution were placed in the other compartment. After thermal equilibration the cell was rotated and the heat exchange recorded. The total heat recorded was the sum of two different terms, the heat of dilution of DNA and the heat of histone-DNA interaction. The heat of dilution of the protein was not taken into account as it was found to be practically zero at any concentration. Using AH values previously determined for the DNA solution, the AH for the histone- DNA interaction was obtained. All measurements were carried out in Tris buffer at pH 7, 1- 10 mM and 0.01 or 0.14 M NaC1. The concentration of histone solution (1 - 4 mmol monomer/l) and of DNA (0.7 - 4 nmol nucleotide/l) were determined by means of spectral absorption measurements using a value of 1600 M-'cm-' at 210 nm for the absorption coefficient of the histone and a value of 6800 M-' cm-' at 259 nm for the absorption coefficient of the DNA. In order to remove bivalent cations occasionally present in the products, some DNA and histone solutions were dialysed first against EDTAiNaCl 10 nM solution, then against Tris buffer/NaCl 10mM. At higher DNA and histone concentration it was necessary to carry out the measurements at low HI/DNA ratio to prevent precipitation. The error on AH values is 0.05 - 0.10 kcal (0.21 - 0.42 kJ)/mol histone, monomer.

RESULTS CHROMATIN GEL

Recombination qf H I and DNA HI was recombined with DNA (type 1, Sigma Chemical Co.) by thoroughly mixing solutions of the two in 1.0 M NaCI, 10 mM EDTA, pH 7 and then dialysing the mixture against a number of NaC1/ 10 mM EDTA solutions of diminishing salt concentration until pure water was reached. The solutions were prepared for NMR by taking about 0.5- 1 ml of recombined material, at a concentration of 10 mg of DNA/ml, and dialysing against ten times its own volume of the required sodium chloride or other salt in deuterium oxide (99.7 Prochem Ltd and 99.8 Ryvan Chemical Co.). The dialysis was repeated twice.

x

Microcalorimetric Measurements Microcalorimetric measurements were carried out with an LKB 10700 batch-type microcalorimeter

Effect of Monovalent Cations on Chromatin Gel The effects of sodium chloride on the NMR spectra of chromatin prepared by the method of Panyim et al. [ 3 ] are shown in Fig.1 and are similar to those described previously [2]. In 2 H 2 0virtually all the H1 spectrum can be seen but signals are broader than those for the isolated histone. Increase of salt to 0.05 M sodium chloride causes contraction to 50% of the original volume, but the spectrum seen is identical to that in 'H20 apart from the change in peak height caused by the concentration change : increase to 0.15 M caused further contraction to between 10 and 20% of the original volume, with considerable broadening of the resonance peaks. At 0 . 4 M the spectrum began to sharpen up, and by 0.5-0.55 M the HI spectrum was fully developed with a marked increase in the height of the lysine Eur. J . Biochem. 57 (1975)

E. M. Bradbury, S. k. Danby, H. W. E. Rattle, and V. Giancotti

OOl-

i.1

>

99

0.7

0:2

0:3 0:4 NaCl (M)

0:5

0'5

\

0 I

-0-

&U.-L

5

4

3

2

1

0.b2 O.b4

0

6' (PPm)

O.b6

0.b8 CaC12 (MI

0:l"

Fig. 1, 270-MHr (high-fkldj spectru of calf thymus chromatin in NaCI. (A) 'H,O; (B) 0.05 M NaCl; (C) 0.15 M NaCl; (D) 0.4 M NaC1; (E) 0.5 M NaCl; (F) 0.6 M NaCl

Fig 2 Contruction of t u l f thjnius thiomutrn ( B ) CaCl,. 5 mg DNA per g chromatin

residues. At the same time the gel cxpands to 80% of its original volume. At 0.6 M and above, the appearance of a peak at - 3.2 ppm corresponding to arginine indicates that histones other than H1 are beginning to be released from the chromatin. Neither the observed gel contractions nor the NMR spcctra are observed with partial chromatin from which H1 has been removed. The pattern of contraction for chromatin gel is shown graphically in Fig.2; the N M R spectra and contraction obtaining using potassium chloride are indistinguishable from those in sodium chloride. The effects on this chromatin preparation produced by sodium and potassium ions with various anions are shown in Table 1. It appears that the maximum degree of contraction, and the broad NMR spectrum which occurs with it is independent of the nature and concentration of the anion; in all cases, the greatest contraction occurred when the sodium or potassium ion concentration was 0.15 M . The release of HI from chromatin and concurrent loss of shrinkage does appear to depend on the nature

Table 1. Effrct of ions on chromatin

Eur. J. Biochem. 57 (1975)

Salt

0:15

riitli

012

( A J NaCl and

Concentration for

Cationic strength on

maximum H1 release concentra- o n N M R tion

release of H1

first sign of release of HI

0.5-0.6 0.5-0.6 0.5-0.6

0.4 0.4 0.4

M MgCI, CaCI, MgSO, MnCI, La(NOd3 pH 4.5

0.002 0.002 0.002 0.001

0.125 0.150 0.125 - 0.150 0.125- 0.150

0,001

NaCl KCI Na,SO, NaCIO, KI NaOAc Na,HPO, pH 4.5

0.150 0.150 0.075 0.150 0.150 0.150

remains contracted 0.5 -0.6 0.5 -0.6 0.25 0.4 0.3 -0.4 0.5 -0.6

0.075

-

-

remains contracted

0.5-0.55 0.4 0.5- 0.55 0.4 0.5 0.4 0.4 0.3 0.4 0.5- 0.6 -

Histone HI in Chromatin

100

4

3

2 6 (PPm)

1

0

Fig. 3.270-MHz (liigh,firldJ spectra oJ'HI . D N A complex in 0.15 M NuCI. (A) 2 mg Hl/inl, 10 mg DNA/ml. (B) 3.3 mg Hljml, 10 mg DNA/ml. (C) 5 mg Hl;'ml, 10 mg DNA/ml

of the anion. Salts with the anion chloride, sulphate or acetate release H1 at ionic strength 0.5-0.55 M whereas iodide, chlorate and nitrate release H1 at 0.3-0.4 M. This shows the ability of the latter group of anions to act as 'structure-breakers' and reflects their position in the Hofmeister series [5,6]. NMR spectra identical to those shown in Fig.1 were obtained for all chromatin preparations prepared by the method of Panyim et al. Comparison of these spectra with those given previously [2] shows that in the state of maximum concentration the latter exhibit a much more prominent alanine resonance. Since these earlier preparations had been prepared by the method of Zubay and Doty, a series of experiments were carried out to find the source of the difference. These resulted in the conclusion that gels made by the Zubay-Doty method differed from one another according to the condition of the thymus used. Preparations from very fresh thymus obtained from young animals gave spectra identical to those from Panyim et al. preparations, while those from older animals or from thymus not used until 2 - 3 h after slaughter gave the prominent alanine resonance. An interesting correlation with these results was found when investigating the effect of varying H l :DNA ratio on the spectra of H1 . DNA complexes. High H1:DNA ratios (1:2 ratio of H1:DNA by weight) gave the same spectrum as the Panyim et al. or the best Zubay-Doty chromatin, while lower ratios (1:s) gave spectra like those of the poorer Zubay-Doty material (Fig. 3). We may conclude that the latter spectra are influenced by loss or degradation of H1. Polyacrylamide gel electrophoresis confirms that the H1 content of such gels has fallen and reveals numerous degradation bands (P. G. Boseley, unpublished data). Effects of Divalent Cations with Various Anions on Chromatin Gel

The main difference observed between monovalent and divalent cations is that the latter are much more

i

,

4

3

2

2 8 (PPm)

1

0

Fig. 4. 270-MHz (high-field) spectra of calf thymus chromatin with MgCI,. (A) 'H'O; (B) 2-25 mM MgClz; (C) 0.05 M MgCI,; (D) 0.1 M MgCI,: (E) 0.15 M MgC1,

effective in causing contraction. Details are given in Table 1 where it may be seen that 2 mM MgC1, is sufficient to cause full contraction, compared with 0.15 M NaCI. Identical results and spectra were observed with magnesium chloride, calcium chloride, and magnesium sulphate. The NMR spectra of chromatin in MgCl, are shown in Fig.4 and the contraction shown in Fig2(B). In each case the spectral changes produced in the presence of divalent cations are identical to those produced by monovalent cations, but at different ionic strengths. The low-field spectrum of chromatin in magnesium sulphate is shown in Fig. 5. In 1 mM MgSO, when the chromatin has contracted to about 40 % of its original volume the spectrum from the aromatic residues is very broad, indicating the central portion of H1 is held rigidly in the complex. At 0.15 M MgS0, peaks are visible due to the phenylalanine (residue 106) and tyrosine (residue 72) in the molecule, showing that the region of the H1 around these residues is relatively free. When H1 has been removed from chromatin, no aromatic spectrum can be seen under these solutions and accumulation conditions. As with the monovalent cations, the onset of contraction and the broad NMR spectrum associated with it is governed only by the cation concentration, the values of which are shown in Table 1. The cation concentrations required to produce contraction are similar to those reported by several authors [7-91. As can be seen from Table 1 for the chlorides and sulphates of both mono and divalent cations, the release of H1 is governed by the effective ionic strength of the cation (ionic strength calculated as cation molarity~charge~). In all cases H1 is released at a cationic strength of 0.5 - 0.6 M. Eur. J. Biochem. 57 (1975)

E. M. Bradbury, S. E. Danby, H. W. E. Rattle, and V. Giancotti

101

Phe Tyr Tyr

I

l

l

1 0

--

120

m

g._

.‘ “ a,

5

100 80 60

2

4

6 8 Urea (M)

1

0

i‘

40 20

1

a

I

I

7

6

6 (ppm)

Urea (M)

Fig. 5. 270-MHz (lowfield) spectrum of Lalf thymus chromatin. (A) H1-depleted chromatin in 0.4 M NaC1; (B) 1 mM MgSO,; (C) 0.15 M MgSO,

Fig. 7. Ef@ct of’ urea on ( A ) the ratio ofapolar,lolanine peak heights and ( B ) the contraction of chromatin. ( x ) No salt present; (0) 0.15 M NaC1; (0)0.4 M NaCl

n

I

4

I

3

2

1

0

6 (PPm)

Fig. 6 . 270-MHz (high-field) spectra oj cay thymus chromatin with urea. (A) ’H,O; (B) 2 M urea; (C) 4 M urea; (D) 6-8 M urea

The Effect of Urea on Chromatin No NuCl Present. Addition of urea up to 8 M causes a slight expansion of the gel and a slight change in “elasticity”. NMR spectra reveal a broad sprectrum with visible alanine and proline peaks at low concentration of urea and are shown in Fig.6. The ‘apolar’ resonance corresponding to methyl groups of valine, leucine and isoleucine gradually increases in height Eur. J. Biochem. 57 (1975)

with increasing urea concentration. At 8 M urea, it has more than doubled in height indicating that apolar groups from histones other than Hl are freed. At 6 - 8 M urea, the lysine and arginine peaks have also increased in height and it is likely that whole regions of histones are freed in urea. However, no histones are completely released in 8 M urea when there is no salt present. (B. G. Carpenter, unpublished results.) The graph in Fig. 7A shows the apolarlalanine peak height ratio which may be used as a measure of how strongly apolar residues are involved in interactions. The use of this ratio as an indicator of structure was described previously [l]. With 0.15 M NuCl. Addition of the urea to fully contracted chromatin in 0.15 M NaCI, or dialysis of a solution of chromatin against a solution of 0.15 M NaCl and urea in H 2 0 , produce exactly the same effects; the NMR spectra are shown in Fig. 8 and the contraction of the gel as a fraction of its original volume in Fig.7B. Contraction is hardly affected until above 6 M, and the increase in peak height begins at 4 M urea, again reaching values larger than those for H1 alone, so that the apolar residues of other histones are also becoming mobile. In the absence of H1, it may be shown that the other histones are almost completely free at 0.15 M NaCl, 8 M urea (P. G. Boseley and S. E. Danby, unpublished results).

102

Histone HI in Chromatin

Fig. 8. 270-M Hz (higli-fic.ld) sprctru of culf tIz~r71usckrornutin with 0.15 A4 A'uCI and uwu. (A) 0.15 M NaCI; (B) 0.15 M NaC1. 2 M urea; (C) 0.15 M NaCl, 4 M urea; (D) 0.15 M NaC1, 6 M urea; (E)0.15 M NaC1, 8 M urea

With 0.4 A4 NaCl. On increasing the ionic strength, the trend started in 0.15 M is continued, contraction becoming less and the growth of apolar, lysine and arginine peaks showing removal of H1 and other histones from the complex. The results are summarised in the graphs in Fig. 7A and 7B. By 6 M urea 0.4 M NaCl, the N M R spectrum is identical to that of total calf thymus histone under the same conditions. T H E HI . D N A COMPLEX

_ _ I ( .

4

~

3

2

~

~

.L-L

1

6 (PPml Fig. 9. 270-MHr (high,firld) spcctm of H I

0

. D N A c o i ? ~ p k rwith

( a ) NuCl und ( h ) CaC1,. 5 mg Hl/ml, 10 mg DNAlml. (A) 'H,O;

(B) 0.05 M NaCl; (C) 0.15 M NaCl; (D) 0.4 M NaC1; (E)0.5 M NaCl; (F) 'H,O; (G) 2 - 2 5 m M CaCI,; (H) 0.05 M CaC1,: ( I ) 0.1 M CaCI,; (J) 0.15 M CaCI,

Efiect of Sodium Chloride on HI ' D N A Complex Fig. 9a shows the NMR spectra at 270 MHz of an H1 . DNA complex at a histone:DNA ratio of 1 :2 (w/w) with increasing sodium chloride concentration. These spectra are in nearly all respects identical with those obtained from chromatin gel under the same solution conditions. The only visible difference between these spectra and those of chromatin is seen in Fig. 9a(A) in H,O, where the spectrum is not as broad as that of chromatin and shows strong evidence of more free lysine residues than appear in chromatin. Otherwise the changes are as in chromatin; in 0.05 M NaCl the alanine peak is most prominent, with at least 40 of the 60 lysines in the molecule immobilised, presumably by interaction with DNA phosphate groups, and evidence in the height of the apolar peak (methyl resonances of valine, leucine and isoleucine at -0.9 ppm) that the apolar-rich region of H1 (approx. residues 40- 100) is involved in a structure as found in H1 solutions [1]. By 0.2 M NaCl the sample is condensed into a small volume and the whole spectrum is considerably broadened as seen in chroinalin spectra. From 0.4 to 0.5M NaCl the contrac-

tion relaxes and the spectrum sharpens up, with the appearance of sharp lysine resonances as the lysine sidechain is released from the complex. By 0.6 M NaCl the resonances of all the protic groups in the HI molecule are sharp. The apolar region of H1 is still in the structured state as might be expected in 0 . 6 M NaC1. Effrct of Calcium Chloride

Fig. 9 b shows the N M R spectra of the HI . DNA (1 :2 by weight) complex at various concentration of calcium chloride ; the pattern is again identical to that observed in chromatin. The volume of the sample is plotted in Fig. 10 showing that the maximum contraction is reached by 0.02 M CaC1, and is about 8 % of the original volume of the sample. In complete agreement with the behaviour of chromatin, the complex does not return to its original volume in the presence of large concentrations of divalent cation, presumably because of the reduction in phosphatephosphate repulsive forces in the DNA. Eur. J. Biochem. 57 (1975)

E. M. Bradhury, S. E Danby, H. W. E. Rattle, and V. Giancotti

0.b2

0.b4

O.b6

0:08 0'.1 " O l l S

CaCI2 (M)

Fig. 10. Contraction of HI jml, 10 mg DNA/ml

012

'

H I . D N A complex with CaC1,.

103

J

4

3

2

Elfeet of Ureu on H1 ' D N A Complex Fig. 12 shows the effect of 8 M urea on the spectrum of the H1 . DNA complex (ratio 1 :2, w/w) with varying sodium chloride concentration. Although the sample remains contracted in 0.05 and 0.2 M NaC1, the increased area of the apolar resonance indicates that interactions involving the methyl groups of the H 1 (presumably hydrophobic interactions) are being broken down by the urea. The implication of this result is that while structures involving the central apolar-rich regions of H1 are certainly present in the contracted complex, other interactions are also involved in the maintenance of the contraction, possibly salt bridges; there are 7 acidic and 14 basic residues in Eur. J. Biochem. 57 (1975)

0

8 (PPm)

Fig. 11. 270-MHz (high-field) spectra of' HI . DNA conzplex with NaCI. 2 mg Hl/ml, 10 mg DNA/ml. (A) 'H,O; (B) 0.025 M NaCl; (C) 0.05 M NaCl; (D) 0.15 M NaCl; (E) 0.35 M NaCl; (F) 0.4 M NaCl

n

Ejjects of H I : D N A Ratio

One of the problems discussed in the section on chromatin was the fact that some chromatin preparations made by Zubay-Doty method never attained the fullest degree of contraction and spectrum broadening, seen in all Panyim et al. preparations. The spectrum at 0.15 M NaCl remained the same as that found for 0.05 M NaC1, with a very prominent alanine resonance. During the investigation of H1 . DNA complexes similar behaviour was found for H1 :DNA ratios of less than 1 : 2 by weight although at this or higher ratios the spectra and contraction were directly comparable to those of the best chromatin preparations. This effect has already been summarised in Fig. 3 ; a set of spectra for a complex with an HI . DNA ratio of 1 : 5 (w/w) is given as Fig. 11, which may be compared with Fig. 9a.

1

I

4

I I ' 1 -

3

2

1

0

8 ippm)

Fig. 12. 270-MH: (high-fieldj spectra of H1 . D N A complex with 8 M urea and NaCI. 5 mg Hl/ml, 10 mg DNA/ml. (A) 8 M urea; (B) 0.15 M NaCl, 8 M urea; (C) 0.4 M NaCI, 8 M urea; (D) 0.5 M

NaCI, 8 M urea

the central segment. Further experiments in which the whole recombination was carried out in the presence of 8 M urea, which was then dialysed out, showed identical NMR and contraction results to those obtained with normal recombination conditions. The spectrum of HI ' D N A in 2 H 2 0

As mentioned earlier, the spectrum of the H1

. DNA complex in 'H20 is sharper than that of chromatin and in particular shows considerably more area in the lysine resonances. Using peak heights as a rough indication of line broadening through complex formation, the height of the lysine E-CH, resonance is

Histone H I i n Chromatin

104 Table 2. Hiw/ of' r'euction on nlixitlg H I mid D N A ut differetzr c'onc~el~tr'a/io~~s untl mtios DNA concn

H1 coiicn

H1 :DNA (w w)

NaCl

Tris (pH 7)

rnrnol nucleotidd

:n!nol monomer 'I

M

:ii

3.63 3.62 3.90 4.04 2.06

3.81 3.81 3.85 2.81

0.30 0.28 0.57 0.21

0.01

10

0.92

0.15

6.8

1-2

0.5 0.7 0.9 1.35 1.75

0.01

1.0

0.7

1-2

0.35 0.5 0.55 0.7 0.76 0.85 1 .0

0.14

1.0

M

AH

kcallmol (kJ 'mol) +0.05 (+0.21) - 0.01 (~ 0.04) +0.01 (+0.04) +0.03 (+0.12) +0.04 (+0.17)

-

0.022" (0.092) 0.022" (0.092) 0.064" (0.268) 0.01 1 (0.046) 0.00.' (0.00) 0.01 ''

(0.04)

- 0.01 ''

(~ 0.04) (+O.OX) (0)

f0.02"

0"

+0.03" (+0.12) f0.02" (+0.08) f0.04.' (+0.17)

k0.l

0.3- 0.4 times the height of the alanine resonance, as compared with 0.15 for either the complex or chromatin in 0.05 M NaCI. This large lysine peak height indicates that in 'H,O only a few (