Biochemistry 1981, 20, 2127-2132
2127
Micrococcal Nuclease Cleavage of Chromatin Displays Nonrandom Propertiest Richard E. LaFond,* Jon Goguen,! Leo Einck,s and C. L. F. Woodcock
ABSTRACT: A statistical analysis of the products of digestion of chicken erythrocyte chromatin by micrococcal nuclease was used to test for randomness of the cutting process. DNA fragment size classes corresponding to mononucleosome, dinucleosome, trinucleosome, tetranucleosome, and all fragments larger than tetranucleosome were evaluated. In every case, fragments in the mononucleosome and greater-than-tetranucleosome classes were produced in excess of the level expected on the basis of random cleavage while those in the dinucleosome-tetranucleosome classes exhibited a shortage. The
pattern of nonrandomness appears to depend on substrate size: the magnitude of deviations from randomness was large when substrates of genomic size are compared with polynucleosomal segments whereas the direction of deviation is identical. Nonrandomness was independent of ionic conditions known to affect the state of chromatin condensation and also appeared to be unaffected by depletion of histones H1 and H5. The possible universality of nonrandom cleavage was suggested when other data from the literature were analyzed. Some possible mechanisms to account for this property are discussed.
%e digestion of chromatin with endonucleases has been an important tool in the development of the nucleosome model of chromatin structure (Hewish & Burgoyne, 1973; Noll, 1974; Kornberg, 1974) and in subsequent studies of chromatin structure and function (Felsenfeld, 1978). It has generally been assumed that micrococcal (staphylococcal) nuclease cuts internucleosomal linker DNA at random, and, indeed, in cases where this aspect has been examined, random cutting has been observed (Burgoyne & Mobbs, 1975; Lohr et al., 1977a). Tests for random nuclease attack have compared the observed fragmentation of chromatin with the model system of random cleavage of an infinitely long linear polymer discussed by Tanford (1961). This model gives the weight fraction (W) of an oligomer of size n as
chromatin is a nonrandom process regardless of the ionic strength (and hence the degree of chromatin condensation) in which the experiments are performed. A similar nonrandom digestion is exhibited by multimeric chromatin segments. The pattern of nonrandom cleavage is consistent: mononucleosomes are produced in excess, while the classes of small oligonucleosomes (n = 2-4) are under-represented. Possible explanations for this phenomenon are considered. Materials and Methods
W,, = np"'(1 - P ) ~ (1) where p is the fraction of the linker regions intact at any given time. A plot of weight fractions for mononucleosomes through tetranucleosomes, and the class of digestion products greater in size than tetranucleosomes, as a function of percent linkers remaining intact on the basis of the random model is given in Figure 1. Using this relationship, Lohr et al. (1977a) found that the digestion of yeast chromatin by micrococcal nuclease showed no significant deviation from randomness, and Burgoyne & Mobbs (1975) reached the same conclusion regarding the digestion of rat liver chromatin by endogenous endonuclease. However, in examining the products of micrococcal nuclease digestion of chicken erythrocyte chromatin, it appeared to us that the ratio of mononuclemomes to dinucleosomes was much higher than expected on the basis of random cleavage. We have, therefore, reinvestigated the question of randomness and developed a more rigorous test for comparing the observed products to that expected for random cleavage of an infinitely long linear polymer at regularly spaced sites (Tanford, 1961). The studies reported here offer evidence that micrococcal nuclease cleavage of linker DNA in whole chicken erythrocyte
Isolation of Cells and Nuclei. Chicken erythrocytes were collected and washed in buffer consisting of 10 mM Pipes' and 150 mM KCl, pH 8.0, and the cells were disrupted by mixing at room temperature for 15 min in the presence of 0.4% Nonidet P-40. Nuclei were recovered after at least two washings in the same buffer by sedimentation at 375g. The amount of DNA was estimated from the absorbance at 260 nm in 1.0 M NaOH by using the relation 1.0 A260= 50 clg/mL. Digestion with Micrococcal Nuclease. Nuclease digestion buffer consisted of 10 mM Pipes and 1.0 mM CaC12,pH 8.0, which was, for some experiments, supplemented with salts to give final concentrations of 150 mM KC1, 1.O mM MgC12, or 5.0 mM MgC12. In some experiments, NaCl and Tris buffer were used without altering the results. Nuclei were washed in digestion buffer and resuspended, digestion was initiated by adding 1 unit of micrococcal nuclease (Worthington Biochemical Corp.) per absorbance unit of chromatin DNA, and the reaction was allowed to proceed for various times at 37 "C. Enzyme activity was halted by adding Na2EDTA to 10 mM and cooling to 0 OC. Treatment of Digests. Phenol extraction: DNA was isolated by making the digest samples 1.0 M in NaCl and extracting according to Sollner-Webb et al. (1976) with an equal volume of chloroform-isoamyl alcohol (24:l v/v). The phases were separated by centrifugation at lOOOg for 10 min, and the aqueous phase was reextracted with an equal volume of
From the Department of Zoology, University of Massachusetts, Amherst, Massachusetts 01003. Receiued September 30, 1980. Supported by National Institutes of Health Grant GM-25305. Present address: Department of Microbiology, University of Alabama at Birmingham, Birmingham, AL 35294. 8 Present address: National Cancer Institute, Bethesda, MD 20014.
Abbreviations used: Pipes, piperazine-N,N'-bis(2-ethanesulfonic acid); Tris, tris(hydroxymethy1)aminomethane; EDTA, ethylenediaminetetraacetic acid; NaDodS04, sodium dodecyl sulfate; HaeIII, Haemophilus aegyprius restriction endonuclease; CHO, Chinese hamster ovary.
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%Linkers Intact ( p ) FIGURE 1: Weight fraction ( W) of mononucleosomes (I), dinucleosomes (II), trinucleosomes (III), tetranucleosomes (IV), and all products greater in size than IV (IVC) expected on the basis of random cleavage as a function of percent linkers remaining intact. This profile is derived from eq 1.
phenol-chloroform-isoamyl alcohol (24:24:1 v/v). DNA was precipitated from the aqueous phase by adding 2 volumes of cold absolute ethanol and allowing to stand at -20 O C overnight. The DNA was then collected by sedimentation at 15000g for 15 min. NaDodSO, method: Whole digest samples were precipitated by making the suspension 5.0 mM in MgC12,adding 2 volumes of cold absolute ethanol, and allowing to stand at -20 O C overnight. The precipitate was collected by centrifuging at 2000g for 15 min, and the samples were prepared for NaDodS0, gel electrophoresis by the method of Varshavsky et al. (1978). Polyacrylamide Gel Electrophoresis. Electrophoresis was in 3.5% polyacrylamide slab gels [ 19: 1 acrylamide/bis(acrylamide)] utilizing the buffer system of Loening (1 967) and, in the case of NaDodS04-treated samples, incorporating 1.0% NaDodS04 according to Varshavsky et al. (1978). NaDodS04-treated samples were readissolved in running buffer consisting of 10% glycerol, 0.16% bromophenol blue, and 1.Wo NaDodSO,, heated at 55 "C for 15 min, and electrophoresed at 100 V and 75 mA for approximately 1.5 h or until the bromophenol blue tracking dye reached the end of the 12.5-cm gel. The gel was then washed in three successive changes of 1.O m M Na2EDTA, pH 7.5, stained with ethidium bromide ( 2 pg/mL in water) for 30 min, and destained overnight in water. In cases where phenol extraction was employed, DNA was redissolved and electrophoresed in the absence of NaDodSO,. No difference in results was detected between the two extraction techniques. Gels were calibrated with 4 X174 HaeIII restriction fragments (BRL, Inc.). Isolation of Multimeric Nucleosomes and Sucrose-Gradient Sedimentation. Multimeric nucleosomes were prepared by digesting nuclei in our buffer at 0 OC for either 30 min or 1 h as suggested by Stratling (1979). The digests were sedimented at 18000g for 10 min and the supernatants applied to linear 15-45% sucrose gradients containing either 150 mM or 500 mM NaCl and 0.2 mM Na2EDTA, pH 8.0. Centrifugation in an S W 27 rotor (Beckman) was at lOOOOOg for 16 h at 4 O C . Gradients were fractionated from the top, passed through a UV absorbance monitor (Isco, Inc.), and scanned at 254 nm. Multimer fractions were collected and dialyzed overnight at 4 "C against 150 mM or 500 mM NaCl in the presence of 0.2 mM Na,EDTA, pH 8.0, or, in one experiment, against 0.2 mM Na2EDTA, pH 8.0, alone. Fractions were repurified by centrifugation in an identical sucrose gradient in an SW 41 (Beckman) rotor at 200000g for 4 h at 4 OC. Purified multimeric nucleosome fractions were redigested after dialyzing overnight against the appropriate salt concentrations
together with 0.2 mM Na2EDTA, pH 8.0, at 4 O C . Photography and Scanning. Stained gels were displayed on a shortwave UV transilluminator (Ultraviolet Products) and photographed with Polaroid Type P/N 55 film, employing a Vivitar (02) orange filter. The negatives were scanned at 560 nm in a Gilford 250 spectrophotometer equipped with a linear transport scanner. Areas corresponding to the various DNA fragments were estimated by tracing the area bounded by each peak and lines drawn vertically from the lowest points in the adjacent troughs to the bottom of the scan. Areas were then determined by weighing each silhouette and correcting for the background density of the negative. Analysis. The weight of the silhouettes for DNA fragments in the size classes monomer-tetramer and the weight corresponding to the total ethidium-staining material on each gel were then applied to a computerized test of agreement with the theory of random scission defined by eq 1 for an infinitely long linear polymer with regularly spaced, equally accessible sites for nuclease cleavage (linkers). This test consisted of two steps. First, the weight of the silhouette corresponding to each DNA fraction was normalized to one with respect to the total, and the proportion of total DNA not included in the monomer-tetramer classes was assigned to the greater-than-tetramer class (IV
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