Expansion of chicken erythrocyte nuclei upon limited micrococcal nuclease digestion

Copyright 0 1982 by Academic Press. Inc. All rights of reproduction in any form reserved 00 14-4827/82/070063-08102.00/O

Experimental Cell Research 140 (1982) 63-70

EXPANSION

OF CHICKEN

LIMITED

ERYTHROCYTE

MICROCOCCAL

Correlation

NUCLEASE

with Higher Order Chromatin

NUCLEI

UPON

DIGESTION Structure

JOHN E. HYDE’ Department

of Physiology and Biochemistry, University Whiteknights. Reading RG% ZAJ, UK

of Reading,

SUMMARY A sensitive method for measuring nuclear volumes with a Coulter counter is described. It has been applied to the digestion of chicken erythrocyte nuclei by micrococcal nuclease and DNase I. Early in digestion, micrococcal nuclease induced a 20% increase in the effective spherical volume of the nuclei, followed by a gradual reduction. At the peak of nuclear swelling, about 17% of the chromatin was soluble after lysis and its average chain length was about 18 kilobase pairs (kb). DNase I digestion did not give rise to a corresponding expansion of the nuclei. Several preparation conditions, including the treatment of nuclei with 0.2% Triton X-100, led to a loss of the expansion effect upon subsequent micrococcal nuclease digestion. The results support the domain theory of higher order chromatin structure. In the context of this model, the observed maximum nuclear expansion correlates with an average of one nuclease scission per domain.

Deoxyribonucleases have proved invaluable in the elucidation of chromatin structure, and we now have a detailed picture of the basic 200 base-pair (bp) repeat unit, the nucleosome [ 11. Our understanding of higher orders of chromatin organization is much less developed. Here too, however, experiments employing limited nuclease digestion have played an important part in formulating ideas about supercoiled domains of chromatin fibres attached to a nuclear matrix [2, 31, about transcriptionally active chromatin regions [4, 51 and in attempts to detect medium-range periodicities in nucleosome organisation [6, 7j. Chromatin in the nucleus is found either as condensed, inactive heterochromatin or decondensed active euchromatin. The volume of the nucleus has been shown to be a sensitive function of the degree of chro5-821808

matin condensation. It may be increased, for example, by in vivo or in vitro contact with Xenopus egg cytoplasm [8, 91, by treatment with polyanions such as heparin or dextran sulphate [9, lo] or by reducing local cation concentrations [ 111, all factors which bring about a decondensation of inactive chromatin regions. In this work, the volumes of intact nuclei have been measured with a Coulter counter before and after limited nuclease digestion. By using chicken erythrocyte nuclei, where the chromatin is fully condensed, with low levels of nuclease, it was hoped to shed some light on events occurring long before the chromatin is broken down to the oligonucleosome level. The results suggest that micrococcal ’ Present address: Department of Molecular Biology, Ring’s Buildings, Edinburgh EH9 3JR, Scotland. Exp CdRes

140 (1982)

64

J. E. Hyde Washing was repeated three more times with O.l0.2% Triton X-100 (Sigma) added to the second wash as required (see Results). ‘The final pellet was resuspended in buffer A to a concentration of about 3~ 1oHnuclei/ml (ca 1 mg DNA/ml). For digestion with micrococcal nuclease (EC 3.1.4.7., Worthington), buffer A was supplemented with 1 mM CaCI,. Incubations were at 3PC with 0.2-12 units/ml of enzvme. Digestions with pancreatic DNase I (EC 3.1.4.5., grade DN-EP, Siama) were at 12-24 units/ml, 3PC. The extent of digestions was estimated either from the amount of oligonucleotides produced that was soluble in perchloric acid (PCA) [12], or by measuring the percentage of the total chromatin solubilized after lysis of the nuclei in 0.2 mM EDTA, pH 7.4 at 4°C [13, 141.

6

h-h Volume

of nuclei

JJ&

I. The volume distribution of nuclear preparations: (top) Untreated nuclei; (bottom) nuclei treated with 0.2% Triton X-100. Arrows mark the median volumes (52.8 and 22.7 pm3 respectively).

Fig.

Sizing of nuclei with the Coulter counter A Coulter counter model ZB was calibrated with spherical latex beads having diameters of 2.03, 4.94, 8:79 and 18.0 pm (Coulter-Electronics). All buffers were passed through 0.22 pm membrane filters (Millipore) before adding nuclei to eliminate counting artefacts from foreign particles. Buffers with less than 30 mM ionic strength were found to have insufficient conductivity and gave rise to an artefactuallv high background. Therefore all measurements were made with at least 50 mM NaCl present. The residual low background for blank buffers was subtracted from experimental values. Just before measurement, nuclei were diluted lOOOO-foldinto buffer A to give a concentration of 3xlff/ml in the same ionic strength. 0.5 ml aliquots of this suspension were counted in size intervals of 6 Nrn3. The total number of nuclei counted over the full size range was about 15000. The results are expressed as the effective spherical volume of the nuclei, plus or minus the standard deviation.

nuclease and DNase I differ in their initial mode of attack on the nucleus and that a low concentration of the detergent Triton X-100, often used to produce nuclei of high purity, has a detrimental effect on what are presumably quite fragile higher order Gel electrophoresis from lysed nuclei was separated into structures. The results are also discussed in Chromatin soluble and insoluble fractions f141 and the DNA terms of the domain theory of higher-order extracted and analysed on 0.35 %-ag&ose slab gels in 36 mM Tris-Cl, 30 mM Na phosphate, 1 mM EDTA, chromatin organization [ 31. pH 7.7. Gels were run 18 h at 2 V/cm, stained in

0.005% Stains-all (Eastman Kodak) in 50% aqueous formamide to locate the bands and scanned at 260 nm.

MATERIALS Preparation

AND METHODS

and digestion of nuclei

Blood freshlv collected from the wina vein of one or more chickens was centrifuged at ilO g (10 min, 4°C). The plasma and huffy coat were removed and the.erythrocytes washed 3iimes in an equal volume of buffer A (0.25 M sucrose, 1.5 mM MgCI,, 50 mM NaCl, 10 mM Tris-Cl, pH 7.4). They were frozen slowly at -20°C and then nuclei were obtained by thawinn aliouots for 2-3 min at 37°C with brief vortexing. This caused lysis of more than 95 % of the erythrocytes as judged in the light microscope. Six volumes of buffer A were added to the thawed lysed blood and the nuclei spun at 3300 g (10 min, 4’C). Exp Cell RPS 140 (1982)

RESULTS Measurement of nuclear volume with the Coulter counter

A major advantage in using a Coulter counter to measure changes in nuclear volume is that very large numbers of nuclei can be rapidly measured in physiological buffers and a statistically valid distribution of values obtained. Fig. 1 (top) shows the size

Micrococcal n&ease-induced

Time

hinl

Fig. 2. Nuclear volume as a function of micrococcal nuclease digestion: Curve a 1, Untreated nuclei with enzyme; n2, as a 1, but without enzyme; 6, c, nuclei treated with 0.1 and 0.2% Triton X-100 respectively before digestion. Concentration of nuclei 2.4~ l@/ml, enzyme 12 units/ml. Figures in parentheses under al are the percentages of the total chromatin solubilized after nuclear lysis at each time point.

expansion of erythrocyte nuclei

65

Reduction of the divalent ion concentration to 0.15 mM caused a marked increase in volume, consistent with earlier observations [ 111,although problems with clumping of nuclei under such conditions made it difficult to make reliable measurements. Fig. 1 (bottom) represents nuclei prepared simultaneously from the same batch of erythrocytes as used in fig. 1 (top), but with 0.2% Triton included in the second of the four wash steps. The median volume of these nuclei (22.7 pm”) was less than half that of the untreated nuclei. It is likely that removal of the remnants of the plasma membrane by the detergent, together with the inner and outer nuclear envelopes [16], contribute to some extent of this reduction in size. Whole nuclei and cells behave as high resistance insulators in the Coulter counter. If the complete removal of membrane material caused the conductivity of the residual nucleus to approach that of the surrounding electrolyte, the counter would underestimate to a certain degree the size of Triton-treated nuclei relative to untreated nuclei.

distribution of one preparation of nuclei after the routine four washes in buffer A The effect of micrococcal nuclease without detergent. Measurements on 9 such digestion on nuclear volume preparations gave a median effective spher- Experiments were performed to see if the ical volume of 51.7k3.5 pm3, equivalent to breakdown of chromatin organisation in the a diameter of 4.6 pm. This is larger than the nucleus by nuclease digestion would cause volume measured in the microscope [15], changes in nuclear volume measurable by due at least partially to the fact that mature the Coulter counter. Erythrocyte nuclei erythrocyte nuclei are elongated or even were digested with a low level of microrod-shaped, rather than spherical [ 151. The coccal nuclease and their size distribution adherence of a ghost cell membrane to measured at different time intervals. A typinuclei prepared in this way [ 151 may also cal curve of the change in median effective be a factor. The median size was found to spherical volume with time of digestion is be relatively independent of NaCl concen- shown in fig. 2, al. Note that each point tration from 50 to 150 mM and divalent ion on this curve results from measurements concentrations above 1 mM in buffers used on about 15000 nuclei. Early in the digesfor the preparation and sizing of nuclei. tion, the nuclear volume rose sharply and Exp Cell Res 140 (1982)

66

J. E. Hyde

Table 1. Digestion of nuclei with micrococcal nuclease and DNase I; volume changes and the fraction of nuclei relative to the zero time-point found in successive aliquots as digestion proceeds DNase I

Micrococcal nuclease % PCAsoluble

% of nuclei counted

% size increase

% PCAsoluble

0

(100)

(0)

0

: 5 12 23

96 95 94 93 94

+24.4 +25.3 +21.9 +11.6 - 2.5

62 13 21

then diminished slowly as the reaction proceeded. The same nuclei incubated identically but without enzyme showed a negligible change in size over the same time period (fig. 2, a2). In six experiments with different preparations of nuclei, the average maximum increase in nuclear volume during the early phase of digestion was 20.1+4.4% (SD). The micrococcal nuclease apparently caused very little lysis of the nuclei (see below and table 1); therefore this increase in median volume could not be due to a selective loss of smaller sized nuclei from the overall distribution. Examination of the detailed histograms for each time point confirmed that this was not the case. Fig. 2, b, c shows the time courses for digestion of the same preparation of nuclei, but with 0.1 and 0.2% Triton respectively included in the second of the four wash steps. Pretreatment with 0.1% Triton reduced the relative expansion of the nuclei during early digestion to about 5 %, while 0.2% Triton abolished the effect entirely. Only the slow reduction in size with prolonged digestion was still seen. Control experiments showed that the degree of digestion was parallel for Triton-treated and untreated nuclei. Exp Cell Rcs 140 (1982)

% of nuclei counted (1;) 75 67 39

% size increase (0) +4.8 +os +1.8 +1.1

Other treatments gave nuclei which, while having a normal starting size, also did not expand upon subsequent micrococcal nuclease digestion. This was the case with nuclei prepared from erythrocytes that had been frozen and thawed several times, or initially frozen in liquid Nz, and with nuclei prepared in buffers with a pH above 8.0. This suggested strongly that over-harsh treatments could disrupt some type of higher-order structure that had to be intact at the start of digestion in order for the expansion effect to be seen. The effect of DNase I digestion on nuclear volume Nuclei were also digested with DNase I to see if a similar expansion occurred. To facilitate comparison with the micrococcal nuclease experiments, reaction conditions were adjusted to yield comparable amounts of PCA-soluble nucleotides over the same incubation times. However, DNase Itreated nuclei behaved differently in that progressively fewer nuclei in a given aliquot were recorded by the counter as digestion proceeded (table 1). Compare the situation with micrococcal nuclease where at least 93 % of the starting nuclei were still measurable after extensive digestion. Size meas-

Micrococcal

n&ease-induced

expansion of erythrocyte

nuclei

67

The size of soluble chromatin fractions from micrococcal nuclease digested nuclei; A, Nuclei minimally digested (nuclei 9.6~ lOa/ ml, enzyme 0.2 units/ml, 30 set); B, nuclei digested to yield maximum increase in nuclear volume (nuclei 2.4~ lOVml, enzyme 12 units/ml, 2 min). The weight average DNA chain lengths indicated were calculated from a linear log MW/mobility plot of A and its three largest Hind111 fragments, A’-A”‘. Fig. 3.

236 Chain length

97

6.7

lkbl

urements of DNase I-treated nuclei thus became statistically less meaningful with increasing time of digestion. A comparison could only be validly made in the early stages of digestion where more than 90% of the nuclei were still intact in both cases. After DNase I digestion to about 2% acid solubility, little or no expansion was seen, the maximum observed being less than 5 %. By contrast, the micrococcal nucleasetreated nuclei were at their peak size of over 20% above their starting value after the same degree of digestion (table 1). Thus DNase I, unlike micrococcal nuclease, does not appear to induce a marked increase in nuclear volume. Correlation qf the nuclear expansion caused by micrococcal nuclease with the amount and size of soluble chromatin produced

An analysis of the chromatin fragments produced by micrococcal nuclease was carried out in order to learn more about the molecular basis of the nuclear expansion induced by the enzyme. The extent of nuclease digestion was expressed in table 1 as the

percentage of the total DNA converted to PCA-soluble nucleotides. A more informative measure is the percentage of the total chromatin that is soluble after lysis of the nuclei in low ionic strength [3, 131. This is particularly so for very mild digestion conditions, where only a small amount of acidsoluble material is released. The amount of soluble chromatin obtained from each aliquot of nuclei at the different time points in the digestion of fig. 2, a 1, is shown below the curve in parentheses. Very similar degrees of digestion, as measured both by acid solubility and chromatin solubilized, were observed for the parallel Triton-treated nuclei (data not shown). The suppression of nuclear expansion caused by Triton (fig. 2, b, c) was thus not due to an inhibition of digestion relative to untreated samples. In four experiments with different preparations of nuclei, 13-20% of the total chromatin was released as soluble material from nuclei that had expanded maximally (e.g. nuclei at the 2 min time-point in fig. 2, a 1). The size of the soluble chromatin from such nuclei was measured on agarose gels Exp Cell Res 140 (1982)

68

J. E. Hyde

(fig. 3, B). As expected, no entities of a discrete size were excised by micrococcal nuclease at such a low level of digestion. The weight average length of the hetero disperse material was 18.5 kilobase-pairs (kb) (DNA MW 12.2~10~). A scan of the largest DNA that could be obtained from a soluble chromatin fraction is also shown for comparison (fig. 3, A). This resulted from minimal digestion with micrococcal nuclease, rendering only about 0.1% of the total chromatin soluble. The weight average length of this DNA was approximately twice as great (36 kb; DNA MW 24~10~) with material up to about 80 kb (MW 56~ 106)detectable in the scan. DNA extracted from the corresponding chromatin pellets that remained insoluble after nuclear lysis was so large that it barely entered the gel.

sperm induced, e.g., by sub-fractions of Xenopus egg cytoplasm and different ionic

conditions. The main observation in this work is that mild micrococcal nuclease treatment of erythrocyte nuclei leads to a 20% increase in nuclear volume early in the digestion. When this occurs, the chromatin is still very large; over 80% is insoluble after nuclear lysis, while the soluble fraction averages about 18 kb in length (fig. 3). The average size of the largest soluble chromatin observable after minimal digestion was only about 36 kb. These chain lengths are of the same order of magnitude as those found to constitute discrete domains of supercoiling in several eukaryotes [2, 3, 18, 191. It is thus instructive to consider the present results in terms of the domain theory of higher-order chromatin organization. IgoKemenes & Zachau have given a detailed DISCUSSION quantitative analysis of the domain model The use of a Coulter counter to monitor [3]. In particular, they consider the relationchanges in nuclear volume marks a novel ship between the fraction of the total chroapproach to this type of assay. The ability matin solubilized by nuclease digestion and to rapidly size very large numbers of nuclei the average chain length of that soluble (i.e., lo4 and upwards) makes detection of chromatin. When they digested rat liver small volume changes more reliable and less nuclei with micrococcal nuclease, the averlaborious than is possible using micro- age chain length of the chromatin was about scopy. The technique is simpler and less 12-16 kb when l&20% had been solubiltechnically demanding than using a fluo- ised. These data were best predicted by a rescence-activated cell sorter [ 171, while model where the average domain length giving more information than previous was about 40 kb. In the present work, methods, such as spinning nuclei in a hema- where 13-20 % solubilization corresponded tocrit tube [ll] or measuring their trans- to the maximum expansion of the nuclei, lucence to 600 nm light [lo]. A histogram the length of the corresponding chromatin of the volume distribution with any desired fragments was very similar, about 18 kb. size interval is obtained for each experi- The maximum length of soluble chromatin mental condition, and any loss of nuclei observed in the rat liver case was estimated during the experiment is readily detected, to be about 75 kb; here, too, nothing larger as seen above with the DNase I digestions. than about 80 kb was seen on gel scans. We have used the method in preliminary These facts suggest that domains in erythexperiments to monitor the swelling of rocyte nuclei are likely to be very similar nuclei from erythrocytes and mammalian in size to those of rat liver. Exp Cell Res 140 (1982)

Micrococcal nuclease-induced expansion of erythrocyte nuclei Igo-Kemenes & Zachau also discuss the correlation of the percentage chromatin solubilized with the average number of nuclease cuts per domain. They show that even if the domain sizes vary quite widely, an average of one cut per domain solubilizes about 15% of the chromatin, while an average of two cuts per domain increases this to about 36%. In the present work, nuclei which had expanded maximally yielded between 13 and 20% soluble chromatin. This corresponds to about 0.9-1.2 cuts per domain. A picture thus emerges in which the volume of the nuclei increases sharply as digestion begins and peaks when an average of one cut per domain is made. One cut is, of course, sufficient to relax the supercoiling in any given domain. The slow decrease in nuclear volume (fig. 2), as the number of cuts per domain increases beyond one, might be due to a gradual loss of material as progressively more mononucleosomal-sized fragments pass through the nuclear membrane. A possible physical basis for the correlation between maximum nuclear expansion and one cut per domain is suggested by the data reviewed by Comings [20]. He proposes that chromatin is anchored not only to an intranuclear matrix, but also to the inner nuclear membrane. A total relaxation of supercoiled chromatin domains within the confined space of the nucleus might be expected on this basis to have a direct effect on the nuclear membrane and hence on nuclear volume. Triton could abolish the expansion effect solely by its removal of both nuclear membranes [16], although the data of Cook & Braze11 [21] suggest that constraints important for supercoiling that are internal to the erythrocyte nucleus are also disrupted by Triton treatment. It would be of interest in this respect to correlate the changes in volume observed here with

69

electron microscope observations of the type performed by Rowlatt & Smith [22] on mouse salivary gland nuclei digested with micrococcal nuclease. Since DNase I does not have the same effect on nuclear volume as micrococcal nuclease, it appears that the initial cuts in the chromatin made by the respective enzymes differ significantly in their impact on higher order packing. This may be related to the marked preferential digestion by DNase I of globin gene sequences in the erythrocyte [23, 241, whereas micrococcal nuclease shows no such specificity [25]. A detailed study of changes in the distribution of soluble chromatin fragment sizes as a function of digestion in the two cases might shed more light on this possibility. Finally, the fact that over-harsh treatments of nuclei or erythrocytes lead to a loss of the expansion effect suggests that another criterion for the integrity of higher order chromatin structures has been established, at least for one type of nucleus. I wish to thank Dr Chris Skidmore for helpful discussions and Drs Skidmore and Mike Antoniou for allowing me extensive use of their facilities.

REFERENCES 1. McGhee, J D & Felsenfeld, G, Ann rev biochem 49 (1980) 1115. 2. Benyajati, C & Worcel, A, Cell 9 (1976) 393. 3. Igo-Kemenes, T & Zachau, H G, Cold Spring Harbor symp quant bio142 (1977) 109. 4. Wu, C, Wong, Y C & Elgin, S C R, Cell 16 (1979) 807. 5. Staider, J, Larsen. A, Engel, J D, Dolan, M, Groudine,.M & Weintraub, H, Cell 20 (1980) 451. 6. Straetlina. W H. Mueller, LJ & Zentgraf, H, Exp cell res l-l7 (1978) 301. I. Butt, T R, Jump, D B & Smulson, M E, Proc natl acad sci US 76 (1979) 1628. 8. Gurdon, J B & Woodland, H R, Biol rev 43 (1968) 233. 9. Barry, J M & Merriam, R W, Exp cell res 71(1972) 90. 10. Kraemer, R J & Coffey, D S, Biochim biophys acta 224 (1970) 568. 11. Leake, R E, Trench, M E & Barry, J M, Exp cell res 71 (1972) 17. Exp Cell Res 140 (1982)

70

J. E. Hyde

12. Mirsky, A E, Proc natl acad sci US 68 (1971) 2945. 13. Nell, M, Thomas, J 0 & Kornberg, R D, Science 187 (1975) 1203. 14. Hyde, J E, Igo-Kemenes, T & Zachau, H G, Nucleic acids res 7 (1979) 31. 15. Ringertz, N R & Bolund, L, The cell nucleus (ed H Busch) vol. 3, pp. 417-446. Academic Press, New York (1974). 16. Aaronson, R P & Blobel, G, J cell biol 62 (1974) 746. 17. Johnson, T S, Swartzendruber, DE & Martin, J C, Exp cell res 134 (1981) 201. 18. Cook, P R & Brazell, I A, Eur j biochem 84 (1978) 465. 19. Nakane, M, Ide, T, Anzai, K, Ohara, S & Andoh, T, J biochem 84 (1978) 145.

&p

Cell

Res

140 (1982)

20. Comings, D E, The cell nucleus (ed H Busch) vol. 4, pp. 345-371. Academic Press, New York (1978). 21. Cook, P R h Brazell, I A, J cell sci 22 (1976) 287. 22. Rowlatt, C & Smith, G J, J cell sci 48 (1981) 171. 23. Mathis, D, Oudet, P & Chambon, P, Progress in nucleic acids research and molecular biology (ed W E Cohn) vol. 24, pp. l-55. Academic Press, New York (1980). 24. Zasloff, M & Camerini-Otero, R D, Proc natl acad sci US 77 (1980) 1907. 25. Bellard, M, Cannon, F & Chambon, P, Cold Spring Harbor symp quant bio142 (1977) 779. Received October 15, 1981 Accepted January 18, 1982

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