Nick translation of HeLa cell nuclei as a probe for locating DNase I-sensitive nucleosomes.

The technique of nick translation of nuclei (Levitt, A., Axel, R., and Cedar, H. (1979) Dev. Biol. 69, 496-505) has been used in HeLa cells to label DNase I-sensitive regions. Micrococcal nuclease digestion of the nick translated nuclei was followed by a low ionic strength gel electrophoresis system which separates different types of mononucleosomes. The major label was observed in the vicinity of high mobility group protein containing mononucleosomes. However, further analysis revealed that the particle does not sediment in the position of mononucleosomes on a sucrose gradient. Two alternative explanations are discussed as the possible source of this particle. It is either a high mobility group protein containing nucleosome in some unfolded conformation or the labeled particle originates from discrete DNA fragments, wrapped around some nonhistone proteins, located in a highly DNase I-sensitive region, which is resistant to micrococcal nuclease digestion.

A., Axel, R., and Cedar, H. (1979) Dev. Biol. 69, 496-505) has been used in HeLa cells to label DNase Isensitive regions. Micrococcal nuclease digestion of the nick translated nuclei was followed by a low ionic strength gel electrophoresis system which separates different types of mononucleosomes. The major label was observed in the vicinity of high mobility group protein containing mononucleosomes. However, further analysis revealed that the particle does not sediment in the position of mononucleosomes on a sucrose gradient. Two alternative explanations are discussed as the possible source of this particle. It is either a high mobility group protein containing nucleosome in some unfolded conformation or the labeled particle originates from discrete DNA fragments, wrapped around some nonhistone proteins, located in a highly DNase Isensitive region, which is resistant to micrococcal nuclease digestion.
Understanding the chromatin organization of active and inactive genes has been a major goal of many laboratories. After the discovery of the nucleosomal structure of chromatin, attempts have been made to elucidate differences existing between active and inactive chromatin. Nucleases, as probes of chromatin structures, have proven to be a powerful tool in such an analysis (1,2). Several years ago, Weintraub and Groudine (3) reported that globin genes in tissues active in globin transcription are extremely sensitive to DNase I, whereas tissues not synthesizing this protein lack such a sensitivity. A logical consequence of this observation was to search for the factors involved in this phenomenon. Nonhistone proteins HMG' 14 and HMG 17, which were first isolated by Goodwin and Johns (4), are apparently important for conferring DNase I sensitivity upon globin genes. When these proteins are removed from erythrocyte chromatin using 0.35 M NaCI, DNase I sensitivity is lost. Reconstitution of HMG proteins with HMG-depleted chromatin restored DNase I sensitivity ( 5 , 6). Based on DNase I digestion of *This research was generously supported in part by Grant GM 17533 from the United States Public Health Service, Grant P-577 from the American Cancer Society, and Grant EP-78-S-02-4962.AOOO from the Department of Energy. This is Publication 1468 from the Graduate Department of Biochemistry, Brandeis University, Waltham, MA 02254. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisernent" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
j To whom correspondence should be addressed. Rosenfield Professor of Biochemistry.
' The abbreviations used are: HMG, high mobility group proteins; SDS, sodium dodecyl sulfate; EGTA, ethylene glycol his(@-aminoethyl ether)-N,N,N',N'-tetraacetic acid; bp, base pair, active chromatin, a method was developed for labeling DNase I-sensitive chromatin (7). It essentially consists of nicking chromatin with DNase I followed by DNA polymerase to incorporate labeled nucleotides into active chromosomal loci. In the present study, we have used this technique in HeLa cell nuclei. Micrococcal nuclease digestion of labeled nuclei followed by low ionic strength polyacrylamide gel electrophoresis, which separated different mononucleosomes (8), has enabled us to locate the specific types of mononucleosomes which have been nick translated. Furthermore, subjecting these mononucleosomes to a second dimension polyacrylamide electrophoresis allowed us to identify different proteins associated with each type of mononucleosome. The major site of incorporation of label in the DNA has been shown to be in a particle sedimenting more slowly than a mononucleosome.

MATERIALS AND METHODS
HeLa S3 cells were prepared as described before (9) and stored at -70 "C in PBS buffer (150 mM NaCl, 3 mM KCI, 8 mM NaH2P04, 1 mM CaCI,, 0.5 mM MgCL, pH 7.4) containing 10% glycerol (the cells were prepared at the Massachusetts Institute of Technology Cell Culture Center). AIl operations were carried out at 4 "C. After several washings and centrifuging in PBS, the pellet was suspended in RSB (10 mM NaCI, 10 mM Tris, 3 mM MgClz, pH 7.6) containing 0.5% Nonidet (British Drug House), and 1 mM phenylmethylsulfonyl fluoride (Sigma, stock solution of 0.1 M in dry dioxane). Using a loosely fitted Dounce homogenizer (Type B pestle), the cells were lysed by about 10 strokes. After centrifugation at 2000 X g, the nuclei were washed two to three times in RSB with 1 mM phenylmethylsulfonyl fluoride until the supernatant was clear. Pasteur pipettes were used during all the steps for dispersing cells and nuclei. Nuclei were used immediately or suspended in RSB with 10% glycerol and stored at -70 "C.
Nick Translation and Micrococcal Nuclease Digestion-The procedure used was similar to that of Gazit and Cedar (IO) and Gazit et al. (11); however, for the nick translation reaction of HeLa nuclei, it is very important to add deoxynucleotides immediately prior to the polymerase reaction. Presumably, the endogenous ATPase hydrolyzes pCi of ["HIdTTP was used). The nuclei (1 ml) were then digested with micrococcal nuclese (80 units, 10 min a t 15 "C). After lysis with EGTA/EDTA, the sample was centrifuged and the supernatant was loaded on a 5-20% sucrose gradient. After centrifugation, 0.55-ml fractions were collected. Absorption measurements and counting of trichloroacetic acid-precipitated samples were carried out (19). M and D donote mononucleosomes and dinucleosomes, respectively.
given in the figure legends.
Sucrose Gradients-Samples containing soluble nucleosomes were analyzed by sedimentation on 5-20% linear sucrose gradients in 10 mM Tris, 1 mM EDTA, pH 7.6. Centrifugation was carried out for 18 h at 32,000 rpm using 13-ml tubes in a Beckman SW 40 rotor at 4 "C.
D N A and Protein Electrophoresis-Low ionic strength electrophoresis for separating mononucleosomes was a combination of published procedures with some modifications (8,13). Gels containing 4% polyacrylamide (30:1, acrylamide/bisacrylamide) in 6.4 mM Tris base, 3.2 mM sodium acetate, 1 mM ECTA, 0.5 mM EDTA, pH 8.0, were used. Slab size was 25 X 17 X 0.3 cm. Pre-electrophoresis was carried out a t 150 V for 1.5 h. After introducing the sample, the electrophoresis was run a t 150 V (-25 mA) in the cold room for 16 h. The buffer (the same as in the gel) was circulated between the upper and lower reservoirs. For protein analysis, a strip of polyacrylamide gel (2-cm width) from the first dimension was equilibrated for 2 h in buffer 0 of O'Farrell (14) (10% glycerol, 5% 2-mercaptoethanol, 2.3% SDS, 62.5 mM Tris HCI, pH 6.8) and placed at the top of 0.1% SDSpolyacrylamide (15% acrylamide) slab according to Laemmli (15) as modified by Weintraub et al. (16). 1% agarose in O'Farrell (14) solution was used for sealing the strip. Slab size was the same as before and electrophoresis was carried out a t 100 V. Protein staining was accomplished using 0.5% Coomassie blue in 10% acetic acid overnight. Destaining was performed in 10% acetic acid, 10% methanol.
DNA sizing was done by employing 4-8% polyacrylamide gel electrophoresis according to Maniatis et al. (17) using 0.09 M TBE buffer (Tris/boric acid/EDTA). Extraction of DNA was as described by Egan and Levy-Wilson (18). Autoradiography was performed according to the procedure described (19).

RESULTS
In order to obtain some information about the distribution of the radioactive label incorporated by the nick translation-L. nuclease digestion procedure, the supernatant from lysed nuclei was examined on sucrose gradients. The results are presented in Fig. 1. The absorption profile shows mono-and dinucleosome peaks, whereas the radioactivity diagram reveals incorporation mainly in mononucleosomes. The major peak of label is near the top of the gradient. Our analysis of the DNA size in this region showed that almost all of the DNA has 4 0 bp (data not shown). It is likely that internucleosomal and subnucleosomal fragments contribute to the radioactivity in this region. Similar results were reported for calf thymus by other investigators (20). Low ionic strength gel electrophoresis was employed to obtain a more precise picture of the type of mononucleosomes being labeled. Gel staining and autoradiography results are shown in Fig. 2 allowed them to distinguish three mononucleosome regions in HeLa nuclei digested by micrococcal nuclease. In Fig. 2 A , the lowest band ( K ) consists of mononucleosome core tailed by an A24-containing core particle. Region (15) is HMG-containing mononucleosomes, and finally (M) is H1-containing mononucleosomes. We will verify some of these assignments later. A corresponding autoradiograph (Fig. 2B) reveals a major band in the vicinity of HMG-containing mononucleosomes in addition to some label in the background.
In order to study the relationship between nick translation and the intensity of the above labeled band, the effect of different DNase I concentrations was studied. Results of such an experiment are shown in Fig. 3 -d). At 0.8 pg/ml of DNase I (lane e ) , overdigestion by DNase I has become a major factor and is a reasonable explanation for the observed reduction of label intensity. In addition, in Fig. 3 there are two faster migrating bands. The upper one of these two is very likely the core particle and the lowest band probably originates from subnucleosomal and internucleosomal regions (21,22). We attribute the relative obscurity of the latter bands in Fig. 2 to the longer electrophoresis time of the gel in Fig. 2 and to a shorter exposure time on the film. In experiments herein, freshly prepared nuclei yield less of the labeled subnucleosomal band than stored nuclei when they are nick translated.
Following digestion of the nuclei with micrococcal nuclease, the sample was centrifuged and the diffused particles in the supernatant (SI) were analyzed. The pellet was then lysed and the nucleoprotein of the supernatant (S2) was obtained.
Alternately, the nuclei can be lysed after digestion and the combined S, and S, fractions obtained by centrifugation. In all of the experiments discussed so far, the total digest was used; there was no separation into SI and S p fractions. However, analysis of the label distribution between SI and S, showed that most of the labeled band in the HMG mononucleosome region was associated with SI (data not shown).
Consequently, the rest of the data presented is concerned with SI (except Fig. 4B which is related to SJ. where the nucleosomes were subjected to low ionic strength electrophoresis. Label is associated with the slower migrating band (Fig. 4 A , strip a). The SDS-polyacrylamide gel (Fig. 4A,  strip c ) shows a number of familiar proteins in the lower region of the gel (histones), as well as a large number of other proteins in the higher molecular weight domain, giving rise to a complex pattern. There is very little histone H1 present in the SI nucleosomes (Fig. 4A, strip c). A similar general picture was obtained for oviduct chromatin (12). The fastest migrating particle in the first dimension (Fig. 4A, strip 6) is the core which lacks the protein A24. AZ4-containing core trails behind this and has been implicated in active chromatin (23,24). We should point out that the width of the strip applied for protein analysis was approximately 2 cm. Consequently, some of the protein bands (i.e. HMG 17) are very diffuse. Based on scanning of the proteins in the HMG- containing region (Fig. 44, strip c), we estimate one molecule of HMG 17/two nucleosomes in this region. The ratio is lower for HMG 14 since this protein probably undergoes proteolysis during isolation from nuclei (25). These ratios represent a lower limit since DNase I digestion of nuclei has been shown to solubilize HMG proteins (26). In general, the ratio of HMG proteins to nucleosomes always showed an increase when nuclei were digested with micrococcal nuclease without being nick translated first. A similar protein analysis for the Sr fraction is shown in Fig. 4B. The HMG 17 protein band is very faint. Histone H1 is present and is associated with the corresponding mono-and higher oligosomes. If one assumes that the labeled band is indeed HMG-containing mononucleosomes, the situation resembles that of the ovalbumin genes as studied by Bloom and Anderson (27), where micrococcal nuclease digestion of nuclei released the ovalbumin gene nucleosomes into the supernatant.
A later study by Goodwin et al. (12) demonstrated that these diffused nucleosomes were enriched with HMG 14 and HMG 17 proteins.
The result of the sizing of the labeled DNA was surprising. Following gel electrophoresis and ethidium bromide staining of SI (similar to Fig. 4A, strip b) upon visualizing on a UVilluminated box, the HMG mononucleosomes region was excised from the gel. After elution and dialysis (19), the DNA was extracted and run on a polyacrylamide sizing gel. The results of the autoradiography are shown in Fig. 5. Besides the nucleosome size DNA band (180-230 bp), there are DNA pieces running in the 100-bp range and lower.

Nick Translation of HeLa Cell Nuclei
This result prompted us to investigate the matter in more detail. A nick-translated SI fraction was subjected to a sucrose gradient fractionation (Fig. 6A), and individual fractions were run on the low ionic strength gel system (Fig. 6B) (note the difference in position of mononucleosomes in Fig. 1 and Fig.  6A). The data of Fig. 6B clearly indicate that the major labeled fragment is in fractions 16 and 17 (indicated by 2). The mononucleosome label is in fraction 14 corresponding to the position of the mononucleosome peak in Fig. 6A. A number of conclusions can be drawn from these results. First, the major labeled band is not in the mononucleosome peak but is associated with the fractions closer to the top of the gradient with minimum UV absorption. If one assumes a value of 11 S for the sedimentation coefficient of the mononucleosome, the calculated value for the observed band is 9 S. Second, the mononucleosomes apper to be mainly labeled in the core particle (Fig. 6B, fraction 14). This very likely reflects the high concentration of the core particle relative to HMGcontaining mononucleosomes giving rise to a stronger label in the core region. Third, the band observed with lower migration in fractions 14 and 15 (designated by +) is not due to an oligonucleosome since it appears in the monosome fraction. The lowest portion of the gel in fractions 15 and higher reflects the presence of subnucleosome and internucleosome fragments.
The main conclusion is that the observed band which migrates in the HMG mononucleosome region on low ionic strength gel electrophoresis has a lower sedimentation coefficient than mononucleosomes. There is a very small amount present based on the UV absorption measurements, but it is highly labeled. DNA and protein analysis of mononucleosomes and the labeled particle obtained from sucrose gradient fractions (Figs. 6, A and B ) are shown in Fig. 7 . It is clear that the DNA size of the particle is smaller (Fig. 7A, lane b) than the mononucleosomes (Fig. 7A, lane a). Based on the length of the DNA markers, the DNA of the labeled material is estimated to be 50-130 bp with subnucleosomal DNA pieces contributing to the lower level of this range. It is interesting to note that the band designated by + in Fig. 6B which migrates with mononucleosomes (fraction 14) on a sucrose gradient must have a DNA length of mononucleosome size since no DNA of larger size is observed in Fig. 7A, lane a. It is reasonable to assume that the slower mobility of this particle on the gel compared with mononucleosomes is likely due to its association with some nonhistone proteins. SDS electrophoresis of the fractions is presented in Fig. 7B. There is a small amount of core histones and HMG proteins in the labeled particle fraction (lane b). Farther up on the gel there are a number of nonhistone proteins, some of which are cadidates for interaction with the labeled DNA fragment. The protein composition of mononucleosomes (Fig. 7B, lane c ) shows a full complement of histones and smaller amounts of nonhistone proteins.

DISCUSSION
Our aim in undertaking this investigation was to find out whether, upon digesting nick translated chromatin with micrococcal nuclease and subsequent low ionic strength gel electrophoresis, any radioactive enrichment of HMG-containing mononucleosomes could be detected. Nick translation of HeLa cell nuclei results in a highly labeled nucleoprotein particle with a sedimentation coefficient of approximately 9 S. The corresponding band on gel electrophoresis is sharp and does not change its position as a result of further micrococcal nuclease digestion. However, our results show that the size of DNA extracted from such a complex is dependent on the level of enzyme concentration. Increased digestion by micrococcal nuclease gives rise to smaller DNA fragments upon extraction of DNA from the nucleoprotein samples (data not shown).
We can suggest a number of possible sources responsible for the formation of the observed particle. It could originate from nonspecific (or specific) association of internucleosomal DNA fragments with some nonhistone proteins. One then has to assume that the DNA fragments are of a special type since they represent a very small population.
A second possibility is to assume that the labeled particle is indeed a HMG mononucleosome in an unfolded configuration causing the particle to move more slowly on a sucrose gradient? The open structure may account for its susceptibility to internal cleavage by micrococcal nuclease. Such a particle is expected to remain intact on the low ionic strength gel and would indicate cleavage when DNA is extracted. If this interpretation is correct, we have to conclude that only a small population of HMG mononucleosomes exists in this open configuration.
An alternative is to attribute the labeled DNA to a nuclease- sensitive region free of nucleosomes. One may consider the presence of histones and HMGs (Fig. 7B, lane b) as a contamination from the mononucleosome region and instead focus attention on the nonhistone proteins present. Such a DNAprotein complex must lie in a DNase I-sensitive region. Since the DNase I concentration is sufficiently low to introduce only nicks (7), translation by DNA polymerase results in a labeled particle. Later digestion by micrococcal nuclease generates nucleoprotein particles which are to some extent protected against digestion. The DNA-protein complex could be assembled by wrapping DNA around a nonhistone protein; such a structure is well documented in the case of DNA gyrase (28). Evidence of nucleosome-free regions being sensitive to DNase I has been reported in a number of transcribing genes (29-35). The upstream region of the 5' end is apparently the major site. A recent paper contains information which resembles our data; Pauli et al. (36), during an investigation of nucleosomal organization of ribosomal genes of Physarum polycephalum, using micrococcal nuclease, reported that a repeat length of 30-40-bp fragments was generated which was superimposed on the usual nucleosomal pattern. Hybridization analysis showed that the small DNA fragments originate from upstream of the initiation site of rRNA transcription. It was pointed out earlier under "Results" that the endogenous nuclease appears to introduce significant nicks into DNA. This is not surprising in view of the other reports demonstrating the role of endogenous nuclease in cleaving DNase I-sensitive regions (30, 31).
In conclusion, nick translation of HeLa cell nuclei followed by micrococcal nuclease digestion results in a distinct labeled nucleoprotein. Whether this particle is an unfolded HMG mononucleosome or a DNA protein (nonhistone) complex with the possibility of being located in a sensitive DNase I region will await further experiments.

Nick
Translation of HeLa Cell Nuclei