Cross-linking of DNA with trimethylpsoralen is a probe for chromatin structure

Cell, Vol. 11, 631-640,

July 1977,

Copyright

0 1977 by MIT

Cross-Linking of DNA with Trimethylpsoralen a Probe for Chromatin Structure Thomas Cech and Mary Lou Pardue Department of Biology Massachusetts Institute of Technology Cambridge, Massachusetts 02139

Summary Me,-psoralen (4,5’,8-trimethylpsoralen) undergoes a photochemical reaction with DNA, resulting in the formation of covalent monoadducts and interstrand cross-links. DNA was photoreacted with 3H-me,-psoralen inside mouse liver nuclei to the extent of one covalently bound me,-psoralen molecule per 114 or per 246 base pairs (bp) on the average. The photoreacted nuclei were digested with micrococcal nuclease, which is known to degrade the DNA preferentially between nucleosomes (subunits of chromatin containing approximately 200 bp of DNA). The digestion of nuclei with micrococcal nuclease produced DNA fragments of the same molecular weights whether or not the nuclei had been reacted with me,-psoralen. The rate of acid solubilization of covalently bound 3H-me,-psoralen was much greater than that of the bulk DNA when the digestion was performed in nuclei, even though me,-psoralen-containing regions of DNA were digested more slowly in control experiments with deproteinized DNA. At the digestion limit, when 45% of the DNA had been degraded to acid solubility, 92% of the 3Hme,-psoralen had been released from chromatin by micrococcal nuclease. Throughout the nuclease digestion, the me,-psoralen was found to be covalently bound to DNA fragments with the molecular weights previously characterized for micrococcal nuclease digestion products. We conclude that the major site of me,-psoralen reaction in nuclei is with the DNA most susceptible to micrococcal nuclease-that is, with the DNA between nucleosomes. Because mes-psoralen cross-links can be located in DNA by electron microscopy, this probe provides a possible method for determining the in vivo location of chromosomal proteins along high molecular weight DNA, such as a transciption unit. Introduction A great deal of recent work has been directed toward understanding the structure and function of the basic repeating subunit of chromatin, the “nucleosome.” Each nucleosome contains eight histone proteins (probably two each of histones H2A, H2B, H3 and H4) and about 200 bp of DNA (Kornberg, 1974; Van Holde, Sahasrabuddhe and Shaw, 1974a; Weintraub, Palter and Van Lente, 1975).

Is

Digestion of nuclei either with endogenous Ca+‘Mg+‘-dependent nuclease (Hewish and Burgoyne, 1973) or with micrococcal nuclease (Nell, 1974) results in the production of a set of nucleoprotein particles containing DNA fragments whose molecular weights are multiples of the monomeric 200 bp unit. Both this arithmetic series of DNA fragments produced by nuclease digestion and electron micrographs of chromatin (Olins and Olins, 1974; Oudet, Gross-Bellard and Chambon, 1975; Woodcock, Safer and Stanchfield, 1976) and chromatin fragments (Van Holdeet al., 1974b; Finch, Noll and Kornberg, 1975) provide evidence for tandem arrangement of the nucleosome subunits. Me,-psoralen (4,5’,8-trimethylpsoralen) is a low molecular weight, planar molecule that intercalates in DNA.

CH3

+$y+JJ*

CH3 Upon exposure to long wavelength ultraviolet light (360 nm), me,-psoralen undergoes photochemical reactions with pyrimidines, forming covalent monoadducts and interstrand cross-links. (The photoreactive double bonds are in the 3-4 and 4’-5’ positions.) The advantages of me,-psoralen for cross-linking DNA include the high efficiency of the reaction, the high stability of the interstrand bonds, the lack of DNA degradation and the absence of a detectable reaction with proteins. Of special interest is the ability of me,-psoralen to cross-link DNA inside living cells (see Pathak and Kramer, 1969; Cole and Zusman, 1970; Cole, 1970, 1971; Musajo and Rodighiero, 1972; Cech and Pardue, 1976.) Hanson, Shen and Hearst (1976) first investigated the sites in chromatin that were available for the me,-psoralen photoreaction. They used electron microscopy under totally denaturing conditions to locate me,-psoralen cross-links in DNA that had been reacted with the drug in Drosophila embryo nuclei. Even after extensive photoreaction, about 60% of the DNA appeared to be protected from cross-linking except at intervals of 160-200 bp and intervals of twice that length. This periodicity was consistent with me,-psoralen reacting mainly with the DNA between nucleosomes. In the present study, we investigate further the specificity of the reaction of me,-psoralen with DNA in nuclei. We are particularly interested in using me,-psoralen as a probe for chromatin structure because it

Cell 632

can be used in vivo, eliminating the uncertainties about whether isolated nuclei or isolated chromatin have retained their native arrangement of chromosomal proteins. Furthermore, if me,-psoralen reacts only at certain accessible regions of chromatin to produce cross-links, the location of the cross-links in purified, high molecular weight DNA gives a map of the location of chromosomal proteins in vivo.

The Effect of Me,-Psoralen on Micrococcal Nuclease Digestion of Purified DNA We wished to use micrococcal nuclease digestion to investigate further the sites of me,-psoralen photoreaction in DNA in nuclei. To interpret such a study, it was necessary to know the extent to which psoralen affects the action of the nuclease. We therefore compared the nuclease digestion of uniformly 32P-labeled DNA in the absence of me,-psoralen, DNA in the presence of free me,-psoralen (not photoreacted with the DNA) and DNA containing covalently bound me,-psoralen (monoadducts and cross-links). The DNA was labeled with 32P to allow monitoring the solubilization of all four nucleotides; a radioactive label present only in thymidine might be preferentially solubilized by micrococcal nuclease (Roberts et al., 1962). Tritium-labeled me,-psoralen was used to allow a comparison of

the solubilization of me,-psoralen-reacted nucleotides with the solubilization of the bulk DNA. 32P-labeled mouse deproteinized DNA was photoreacted with 3H-me,-psoralen using different times of irradiation to control the extent of reaction. The amount of covalently bound me,-psoralen was determined from the tritium radioactivity after removal of free merpsoralen and its photolysis products (see Experimental Procedures). Table 1 gives the amount of covalently bound 3H-mes psoralen in each sample. Mixtures of me,-psoralen and purified DNA either photoreacted as described above or else kept in the dark were treated for various amounts of time with micrococcal nuclease. The presence of free me,-psoralen did not detectably alter the rate or final extent of solubilization of the DNA (data not shown). Photoreaction of me,-psoralen with the DNA, however, did inhibit the nuclease to some extent. As shown in Figure 1 and Table 1, both the rate and the final extent of hydrolysis decreased with increasing amounts of psoralen photoreaction. Even the most highly cross-linked sample, however, was degraded to 97% ethanol solubility and 99.5% acid solubility, as summarized in Table 1. [When these two methods of assaying nuclease digestion were compared on the same DNA, the acid precipitation method always gave a faster rate of digestion and a more complete final level of digestion. We attribute this difference to oligonu-

Table

with

Results

1. Micrococcal

Nuclease

Digestion

of Purified

32P-DNA

Reacted

% Not Digested

Irradiation (Min)a Ob

% of bp Reactedd

Calculated Average Distance between Me,-Psoralen (bp)

(0.03)e

3H-me,-Psoralen by 40 Min

=P-DNA

3H-me3-Psoralen

Acid*

Ethanolg

0.2

1 .2

0.5

1 .2

Acid’

initial Digestion Rate’ (Relative to Sample 0)

EthanoP

1 .o

1.6 10

0.29

340

0.1

1 .l

4.6

17

1.23

+ 0.22 (2)

60

0.92

109

0.3

1.6

ND”

16

0.73

f 0.25 (4)

0.69

k 0.20 (3)

1.7

20

2.4 120c

1.85

54

0.5

2.6 2.9

11.5

13 14

a 6 pg/ml 3H-merpsoralen. b DNA not irradiated, but mixed with 3H-me,-psoralen that had been irradiated for 80 min. c A second 6 pg/ml 3H-me9psoralen were added at 60 min. d Determined from the 3H/32P ratio after removal of unreacted 3H-me,-psoralen. e The background level of BH-me,-psoralen co-purified with the DNA. Sucrose gradient sedimentation the DNA. f Undigested DNA determined by HCIO, precipitation onto filters. g Undigested DNA determined by aqueous counting after ethanol precipitation. ’ Not determined. r Numbers in parentheses = number of experiments from which initial rate was calculated; (2) range

showed

of values.

that

it was not reacted

with

Psoralen 633

Is a Probe

for Chromatin

Structure

cleotide digestion products ~20 bp in size, which are largely perchloric acid-soluble but still ethanolprecipitable (Cleaver and Boyer, 1972).] As shown in Figure 1, covalently bound me3psoralen was solubilized less completely than the bulk DNA, a result observed consistently (Table 1). The effect appears to be accentuated after longer photoreaction in Figure 1, but this has not been extensively studied. The initial rates of 3H-me,psoralen and of 32P-DNA solubilization were very similar, as in Figure 1. Later in the reaction, however, the rate of me,-psoralen solubilization decreased relative to that of the 32P-DNA. The conclusion from these studies is that at levels of me,-psoralen photoreaction up to 1 meRpsoralen per 54 bp, covalently bound me,-psoralen only slightly reduces the rate and the final extent of DNA digestion by micrococcal nuclease. In addition, the 3H-me,-psoralen is solublized less completely than the rest of the DNA, presumably because regions of the DNA that are extensively reacted with the drug are resistant to micrococcal nuclease digestion. The Effect of Me,-Psoralen Photoreaction on Micrococcal Nuclease Digestion of Chromatin in Nuclei Mouse liver nuclei were isolated as described in Experimental Procedures and divided into two samples. Me,-psoralen was added to one sample and both were irradiated for 30 min. After addition of Ca+‘, each sample was further divided into 0.5 ml aliquots which were subjected to different

amounts of micrococcal nuclease digestion. DNA was extracted from each sample of nuclei and analyzed by 6% acrylamide gel electrophoresis. As shown in Figure 2, micrococcal nuclease cleaves both me,-psoralen-reacted and unreacted DNA in nuclei into the same series of discrete fragments. At early times of digestion, the DNA fragments form bands with molecular weights that are integral multiples of 196 bp as first described by Hewish and Burgoyne (1973) and Noll (1974). The 196 bp is calculated from the dimer and tr,imer molecular weights. The monomer fragments have presumably been degraded further to give the broad band centered at 155 bp (Noll, 1974, 1976). After prolonged digestion, both samples show the same “limit digest” series of low molecular weight fragments, with a gap in the series at about 70 bp. The limit digest pattern appears to be a property of the internal structure of native nucleosomes (Axe1 et al., 1974; Axel, 1975; Sollner-Webb and Felsenfeld, 1975; Camerini-Otero, Sollner-Webb and Felsenfeld , 1976).

0

B A I 0’ 4 20 60 0 0+ 4 20 60

-590 bp -390

-155

0

5

IO

15 TIME

Figure 1. Micrococcal Nuclease alen Cross-Linked DNA

20 (MIN) Digestion

Figure 2. DNA Fragments Digestion of Mouse Nuclei of Purified,

MeQ-Psor-

Purified, 32P-labeled mouse DNA was irradiated for various times in the presence of 3H-me3-psoralen (see Table 1 and Experimental Procedures). Unbound merpsoralen was removed, and the DNA was digested at a concentration of 160 pg/ml with 2 pg/ml micrococcal nuclease. (Sample 40+, 7 pg/ml nuclease for 40 min.) Digestion was assayed by the acid precipitation method. IO min irradiation: (-•-) 3ZP-DNA (3100 cpm per point at t = 0); (- -0-) 3H-me,-psoralen covalently bound to DNA (2650 cpm). 120 min irradiation: (-A-) 32P-DNA (2850 cpm); (--A-) 3Hme,-psoralen covalently bound to DNA (15,300 cpm).

Produced

by Micrococcal

Nuclease

(A) Nuclei irradiated in the presence of mea-psoralen such that 0.95% of the base pairs reacted with the drug. Ca+’ was added to 1 mM, and the nuclei were treated with micrococcal nuclease or incubated without nuclease. DNA was then extracted and analyzed by 6% polyacrylamide gel electrophoresis. (0) no nuclease, nuclei kept on ice; (Of) no nuclease, nuclei incubated at 37°C for 20 min; (4) nuclease added at 6 pg/ml, 4 min digestion; (20) 20 min digestion: (80) 60 min digestion. (B) Nuclei irradiated as in (A), but without mea-psoralen. Nuclei were then incubated with (or without) micrococcal nuclease exactly as in (A). The molecular weights were determined from a different gel which contained h Hin fragments, similar to Figure 5.

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Although the nuclei used in the Figure 2 experiment were isolated according to the method of Axe1 (1975), the observation that me,-psoralen crosslinked DNA in nuclei is cleaved into the same products as uncross-linked DNA in nuclei also held true for mouse nuclei isolated according to a modification of the Hewish and Burgoyne (1973) procedure, as described in Experimental Procedures. Wiesehahn and Hearst (1976) have previously concluded that me,-psoralen cross-linking does not alter the micrococcal nuclease digestion fragment pattern of Drosophila embryo nuclei. The rates at which me,-psoralen-reacted and unreacted chromatin were digested appear to be similar from the gel in Figure 2. The kinetics of nuclease digestion of the two types of nuclei were further studied by perchloric acid precipitation (Figure 3). The me,-psoralen reaction has little effect on initial digestion rate, but does decrease the final level of hydrolysis to some extent. Because micrococcal nuclease cleaves me,psoralen-reacted chromatin into the same molecular weight products as unreacted chromatin in nuclei and with similar kinetics, we can use the nuclease to probe the location of the me,-psoralen in chromatin. Furthermore, to the extent that the micrococcal nuclease digestion pattern is indicative of native chromatin structure (Camerini-Otero et al., 1976), the photoreaction of megpsoralen with DNA in nuclei does not appear to perturb the chromatin structure. The Removal of Covalently Bount 3H-me3Psoralen from DNA in Nuclei by Micrococcal Nuclease The use of 3H-labeled me,-psoralen allowed the nuclease digestion of both bulk DNA and me,-psoralen-reacted portions of the DNA to be monitored in the same experiment. Figure 4 shows the kinetics of nuclease digestion of DNA in nuclei at two levels of mes-psoralen photoreaction: one covalently bound meapsoralen per 246 bp and one per 114 bp. In both cases, the me3-psoralen-containing DNA was degraded to 92% acid solubility when the DNA approached the digestion limit, in this case about 45% acid solubility. If the 8% of the 3H-me3psoralen that remained acid-precipitable after extensive nuclease digestion were protected from digestion by chromosomal proteins, we would expect the DNA to become almost completely nuclease-sensitive after deproteinization. When the zero time DNA sample in Figure 4B was deproteinized and subjected to multiple micrococcal nuclease treatments, however, 7-15% of the 3H-me3psoralen remained acid-precipitable. This finding suggests that a minor fraction of the DNA in nuclei was so heavily cross-linked that micrococcal nu-

0

I

I

5

IO

I 15 TIME

I 20 (MIN)

J+d

Figure 3. Micrococcal Nuclease Digestion of Mouse with and without Me,-Psoralen Photoreaction

Liver

Nuclei

Nuclei were isolated by a modification of the procedure of Hewish and Burgoyne (1973), as described in Experimental Procedures. After merpsoralen treatment, micrococcal nuclease was added at 12 pg/ml, and nuclei were incubated at 37°C. (40+) 30 ag/ml nuclease for 40 min. (A) (--O-) nuclei (160 pg DNA/ml) plus 6 pg/ml merpsoralen were irradiated for 36 min at 4°C (1 mes-psoralen bound per 246 bp). (-- 0--) the same nuclei-me,-psoralen mixture was kept in the dark for 36 min at 4°C. (------) the same nuclei were incubated at 37% without added nuclease. The digestion was monitored by absorbance after perchloric acid precipitation, as described in Experimental Procedures. (B) (d-) nuclei (130 pg DNA/ml) plus 4 pg/ml merpsoralen were irradiated for 15 min at 4°C; me,-psoralen addition and irradiation were repeated 2 more times (1 mes-psoralen bound per 114 bp). (- - 0--) same nuclei with 95% ethanol added instead of me,-psoralen dissolved in 95% ethanol. Irradiation as above. Each point represents a sample of nuclei that was independently treated with micrococcal nuclease, followed by DNA purification as described in Experimental Procedures. The amount of digestion was calculated from the amount of DNA (A& recovered in each sample compared to the amount recovered in two undigested controls. A previous study had shown that the DNA purification gave 100 * 5% recovery (after making a small correction for any volume losses during extraction).

clease could not degrade it to acid solubility. In any case, the data do not allow us to draw conclusions about the site of reaction of the 8% of the me3psoralen that is nuclease-resistant. The fraction of 3H-me3-psoralen not converted to ethanol solubility is higher than that not converted to acid solubility (Figure 4B), suggesting that a significant

Psoralen 635

Is a Probe

for Chromatin

Structure

amount of the final digestion product had length ~20 bp, making it ethanol-precipitable but largely acid-soluble (Cleaver and Boyer, 1972). The initial rate of the release of merpsoralen from DNA in nuclei is much greater than the initial rate of solubilization of the bulk nuclear DNA. The early time points in Figure 4 allow the determination of the initial rate of DNA solubilization, but provide only a lower limit for the initial rate of me3psoralen solubilization. Even so, after 1 min of nuclease treatment, 5.6 and 7.0 times as much 3Hme,-psoralen as bulk DNA was converted to acid solubility in the two studies in Figure 4. At 0.5 min of digestion in Figure 4B, the ratio of me,-psoralen to bulk DNA solubilization was about 12. When me,-psoralen-reacted protein-free DNA was treated with the nuclease under the same conditions (See Figure l), this ratio was about 1.0. We therefore conclude that the major part of the reaction of me3psoralen with DNA in chromatin occurs with a subset of the chromatin DNA that is particularly sensitive to nuclease. The products of the micrococcal nuclease digestion of me,-psoralen-reacted nuclei were analyzed on polyacrylamide gels as in Figures 5A and 58. The gels showed that there was no nucleolytic cleavage within nucleosomes (no low molecular weight fragments were produced) after 2 min of digestion in the experiment of Figure 4A (see gel in Figure 5A) or after 3 min in Figure 48 (gel not shown). At this point in the digestion, the solubilization of 3H-me,psoralen was 61% (56/92 x 100) and 73% (67/92 x 100) complete in the two experiments, respectively. Approximately 55 t 10% of the chromatin had been cleaved to monomer-size fragments up to this point. At later times, while the remaining me,-psoralen was being solubilized, most of the remaining chromatin DNA was being cleaved to fragments ~200 bp, and digestion within nucleosomes also occurred. The data therefore demonstrate that the majority of the me,-psoralen reacted with the accessible regions of DNA between nucleosomes and are consistent with all the me,--psoralen having reacted with these regions. The Molecular Weight of the Micrococcal Nuclease Digestion Products That Contain 3Hme,-Psoralen The DNA fragments produced by micrococcal nuclease digestion of nuclei (Figure 4A) were analyzed by polyacrylamide gel electrophoresis (Figure 5A). Because the DNA was unlabeled except for the covalently bound 3H-me9psoralen, tritium fluorography could be used to locate the me,-psoralen in the gel. Preexposed film was used for the fluorography, so the areas under the densitometer scans should be proportional to the amount of radioactivity (Laskey and Mills, 1975). The decrease in peak

t

B

=

IOO-

Figure 4. The from Chromatin coccal Nuclease

Removal of Covalently in Nuclei by Digestion

Bound 3H-me3-Psoralen of the Nuclei with Micro-

(A) 1 me,-psoralen per 246 bp. (8) 1 me,-psoralen per 114 bp. (- -A- -) release of covalently bound 3H-me,-psoralen from high molecular weight DNA by micrococcal nuclease digestion, determined by perchloric acid precipitation onto filters as described in Experimental Procedures. 100% acid insolubility was 2700 cpm per point in (A), 4100 in (B). (-XI-) release of 3H-me3psoralen determined by ethanol precipitation and aqueous counting (see Experimental Procedures). 100% insolubility was 4900 cpm per point in (B); assay not performed in (A). (-) digestion of the DNA in the same me,-psoralen-reacted nuclei, from Figure

area as a function of digestion time in Figure 5D was proportional to the decrease in acid-precipitable 3H-me,-psoralen from Figure 4. This confirms the conclusion that >90% of the 3H-me,-psoralen was released from chromatin during the micrococcal nuclease digestion. Comparison of the gel in Figure 5A with the fluorogram in Figure 5C or comparison of the densitometer scans in Figures 5B and 5D shows that the 3H-me,-psoralen co-migrated with the nucleasecleaved DNA throughout most of the digestion. [Late in the reaction, when limit digest fragments of 20-140 bp can be seen (Figure 5B), the most nuclease-resistant components of the covalently bound me,-psoralen appear to be located predominantly in the 90-140 bp fragments (Figure 5D). This observation, which has been made several times, may reflect a slight nonlinearity of the fluorography at low 3H doses; it has not been further investigated .]

Cell 636

Figure

5. The Location

of Covalently

Bound

3H-me,-Psoralen

in Nuclease

Digestion

Products

(A) DNA was isolated from portions of the same me,-psoralen-reacted nuclei that were assayed in Figures 3A and 4A, and 2.0 fig of each sample were analyzed on a 6% acrylamide slab gel, stained with ethidium bromide. (0) No nuclease, nuclei kept on ice; (0+) no nuclease, nuclei incubated for 40 min at 37°C; (A) phage A DNA cleaved by Hind II + Ill restriction endonucleases; white arrows point to the 248, 216, 155 and 122 bp fragments: (1) nuclei digested with 12 pg/ml nuclease for 1 min; (2) 2 min; (7) 7 min; (15) 15 min; (40) 40 min; (40+) 40 min with 30 *g/ml nuclease; (hv-) unirradiated nuclei (therefore unreacted with me,-psoralen) digested under the same conditions as sample (40+). (6) Densitometer scan of the negative of the photograph shown in (A). (T) top of gel; (D) position of bromophenol bluedye markerat end of electrophoresis; (B) bottom of gel. Molecular weights (bp) were calculated from the mobilities of the A Hin fragments. (C) The same digested mouse nuclei DNA samples showin in (A) were run on a second 6% polyacrylamide gel, 70 +g of DNA per slot. Two slots contained A Hin fragments from which molecular weights were calculated. The gel was stained with ethidium bromide to allow location of the DNA and was then prepared for fluorography. This x-ray film was exposed for 24 days. Samples designated as in (A). (D) Densitometer scan of the x-ray film shown in (C). (bg) background exposure of film.

In the experiment of Figure 4B, where me,-psoralen was reacted with mouse nuclei to the greater extent of 1 me,-psoralen per 114 bp, the molecular weights of the DNA fragments containing 3H-psoralen were also determined by gel electrophoresis and fluorography (not shown). Once again, the covalently bound mehpsoralen was found associated with the bulk DNA throughout most of the digestion (but again appeared to be depleted in the low molecular weight fragments of the limit digest).

Discussion Micrococcal Nuclease Studies We have used micrococcal nuclease, a probe for chromatin structure, to characterize further the sites in chromatin that are available to the reaction of me,-psoralen. The experimental rationale was as follows. If me,-psoralen reacted randomly with the DNA in nuclei, as it does with purified DNA (our unpublished results), micrococcal nuclease would

Psoralen 637

Is a Probe

for Chromatin

Structure

be expected to degrade me,-psoralen-containing regions of the chromatin at about the same rate as regions not reacted with the drug. In fact, the me,psoralen-containing regions should be attacked more slowly late in the reaction (see the first section of Results). At the limit of digestion, when approximately one half of the DNA had been degraded, no more than one half of the me,-psoralen would be released from the chromatin. If, on the other hand, the me,-psoralen photoreaction occurred only with some most accessible subfraction of the DNA in nuclei, as suggested by the electron microscopic data, micrococcal nuclease might preferentially degrade the me3-psoralen-containing regions of the chromatin. The almost complete release of mes-psoralen from chromatin by micrococcal nuclease (Figure 4) does not support a hypothesis of random reaction. Most (92%) of the mes-psoralen reacts with the half of the chromatin that is accessible to micrococcal nuclease (Clark and Felsenfeld, 1971, 1974). The kinetics of the release of the drug suggest that the primary sites of mes-psoralen photoreaction are the same sites (or a subset of the same sites) that are preferentially degraded by micrococcal nuclease-the DNA between nucleosomes (Noll, 1974; Van Holde et al., 1974a, 197413; Finch et al., 1975). Although these data are consistent with all the nuclease-sensitive mes-psoralen having reacted between nucleosomes, we cannot eliminate the possibility that a small amount of the nucleasesensitive drug reacted with nuclease-sensitive sites within the nucleosomes. In addition, 8% of the me,-psoralen in chromatin was not degraded by micrococcal nuclease and did not become nuclease-sensitive even after DNA purification. We therefore draw no conclusions about the site of reaction of this small fraction of the me,-psoralen. An alternate interpretation of these data is that most of the me,-psoralen reacts with regions of protein-free DNA in nuclei. A small amount of such regions might always exist in chromosomes (reviewed by Simpson, 1973; Varshavsky, llyin and Georgiev, 1974; Oudet et al., 1975) or might be produced by me,-psoralen intercalation. Any such regions would be expected to be highly nucleasesensitive, so nuclease digestion would quickly and completely release the mespsoralen from the chromatin. This alternate interpretation is not consistent with the fluorography in Figure 5, which shows that the me,-psoralen is bound to nucleosome DNA digestion products. Extensive cross-linking of protein-free regions of chromatin also seems improbable when one considers the results of electron microscopic mapping of me,-psoralen cross-links. When Drosophila embryo nuclei (Hanson et al., 1976) or mouse tissue

culture cells (Cech and Pardue, 1976) are reacted with mes-psoralen, the spacing of the cross-links is not random but shows a 160-200 bp periodicity. DNA extracted from undigested portions of the me,-psoralen-reacted nuclei described in Figures 3A and 3B was also spread for electron microscopy under totally denaturing conditions. In both DNA samples, the distances between adjacent crosslinks occurred in discrete size classes which were integral multiples of a 190 bp “monomer” molecular weight (T. Cech, D. Potter and M. L. Pardue, manuscript in preparation). This is exactly the periodicity expected if me,-psoralen reacted mainly with the DNA between nucleosomes. Neither the electron microscopy nor the gel fluorography experiments eliminate the possibility that a minority fraction of the me,-psoralen might react with protein-free regions. Although the levels of mespsoralen reaction studied in this paper are high, in the range of 1-2 molecules per nucleosome, they are subsaturation levels. G. Wiesehahn, J. Hyde and J. E. Hearst (manuscript submitted) determined the saturation of Drosophila nuclei with me,-psoralen. Because of rapid photolysis of psoralen compounds, reaching saturation required numerous cycles of drug addition and irradiation. The levels of me,-psoralen reaction we used correspond to about 16% and 35% of the level at which me,-psoralen saturates the Drosophila chromatin.

The Restricted Availability of Chromatin to Me,Psoralen The reaction of mea-psoralen with DNA occurs in two steps: binding of the drug to DNA by intercalation and photoreaction of the drug with pyrimidines upon exposure of the system to 360 nm light. The DNA involved in nucleosome structure could be protected from me,-psoralen reaction because of restricted intercalation or restricted photoreaction or both. Studies with other DNA intercalating molecules-actinomycin D, ethidium bromide, quinacrine and acridine orange-show that chromatin has fewer sites for intercalation relative to purified DNA (Kleiman and Huang, 1971; Lurquin and Seligy, 1972; Brodie, Giron and Latt, 1975). We therefore expect that at least part of the specificity of the reaction of me,-psoralen with chromatin results from restricted intercalation. The four major histones are probably the main components of chromatin that restrict the reaction of regions of DNA with me3-psoralen, but may not be the only components. We are interested particularly in how the reaction of me,-psoralen with DNA might be affected by histone Hi, RNA polymerase and nonhistone chromosomal proteins.

Cell 636

Comparison of Me,-Psoralen Chromatin Structure

to Other Probes for

The titration of chromatin with chemical probes such as Mn+2, polylysine, various dyes and histones has given information about the accessibility of DNA in chromatin relative to that of pure DNA (reviewed by Huberman, 1973; Simpson, 1973; Clark and Felsenfeld, 1974). To determine the relationship of the accessible sites to each other or their location in the bulk chromatin, however, the probe must be able to label its site of interaction permanently. Micrococcal nuclease “labels” its sites of interaction in chromatin by degrading the accessible DNA. As mentioned earlier, use of this nuclease has helped reveal the subunit structure of chromatin. Interpretations of nuclease digestion data, however, must always consider the perturbations of chromatin structure caused by such a destructive probe (for example, ltzhaki, 1974; Axel et al., 1974). Two other enzymes have been successfully used to label accessible sites in chromatin. Using DNA methylase to attach a radioactive label to DNA bases, Bloch and Cedar (1976) found that the saturation level of methylation of chromatin was 44% that of DNA. Eco RI restriction endonuclease cleaves a specific base sequence that occurs at 1400 bp intervals in calf satellite I DNA (Botchan, 1974). Lipchitz and Axel (1976) found that 57% of these Eco RI sites were accessible in calf nuclei. Even at saturation, both enzymes “label” purified DNA of an average of about once per 1000 bp, which limits their applicability. Me,-psoralen has several important advantages when compared to the other molecules that have been used as probes for chromatin structure. The drug reacts randomly with pyrimidines in DNA, so high levels of reaction can be attained. It can be used to probe chromatin structure in vivo as well as in isolated nuclei or chromatin preparations. It can be used over a wide range of temperature and ionic strength, and in the presence or absence of divalent cations. Possible perturbations of chromatin structure caused by nucleolytic cleavage of the DNA are eliminated. Finally, because covalent cross-links can be located on DNA using denaturation electron microscopy, me,-psoralen has the potential of allowing the position of chromosomal proteins to be mapped along transcription units. Experimental

Procedures

Mouse DNA Cross-Linked with 3H-me3-Psoralen SVT2 mouse tissue culture cells were grown as described previously (Cech, Rosenfeld and Hearst, 1973). 32P-phosphate (New England Nuclear: carrier-free) was added to the medium at a final concentration of 0.5 &i/ml. After 36 hr of labeling, the cells were harvested by trypsinization. DNA was isolated by the pH 10.5 procedure described previously (Cech et al., 1973). The initial

specific activity was 1900 cpm/wg when counted in Triton X-100 fluor (1 part Triton X-100/2 parts toluene containing 4 g PPO and 50 mg POPOP per liter). The spillover from the 32P channel of the scintillation counter into the $H channel was 0.5% under these counting conditions. 32P-labeled DNA at 60 ELg/ml in buffer A (the nuclei isolation buffer described below) was made 6 pg/ml in 3H-me3-psoralen (72,000 cpm/pg; a gift from S. T. Isaacs, C. V. Hanson and J. E. Hearst). The DNA in 25 cm* plastic tissue culture flasks (Falcon) was irradiated with long wave-length ultraviolet light asdescribed by Cech and Pardue (1976), except that the irradiation was performed at 4°C. Quantitation of the Photoreaction The amount of 3H-me9psoralen covalently bonded with the DNA was determined from the 3H-me3-psoralen/32P-DNA ratio after unbound me,-psoralen and its photolysis products had been removed. The photolysis products are particularly troublesome because they co-precipitate with DNA in ethanol and are retained on filters after acid precipitation. They can be largely eliminated by chloroform extractions (C.-K. J. Shen and J. E. Hearst, personal communication). The irradiated DNA solution was made 0.15 M in NaCI, extracted 3 or 4 times with chloroform containing 4% isoamyl alcohol and precipitated with 2 vol of cold 95% ethanol. The precipitate was pelleted by centrifugation (Sorvall HB-4 rotor, 30 min at 10,000 rpm, O”C), washed with cold 70% ethanol, centrifuged again for 10 min and dissolved in a low salt buffer. The DNA was then counted in Triton X-100 fluor as above. The background of unbound 3H-mes-psoralen that remained with the DNA during the extraction and precipitation steps was determined by irradiating 3H-merpsoralen without DNA for 60 min, adding 3ZP-DNA in the dark, repurifying the DNA as above and determining the SH/32P ratio. This background was about 7 3H cpmlpg 32P-DNA, corresponding to 3 x 10m4 me,-psoralen molecules per DNA base pair. Sucrose gradient sedimentation analysis of this sample showed that the small residual amount of 3H-me,-psoralen photoproducts did not co-sediment with the DNA, but pelleted in the centrifuge tube. Sedimentation analysis of the DNA irradiated in the presence of merpsoralen and then extracted, on the other hand, showed the 3H-mea-psoralen co-sedimenting with the 32P-DNA, as expected for covalently bound merpsoralen. Mouse Liver Nuclei Balb/c and 129/S, mice were provided by M. Kamarck and S. Epstein. Their livers were dissected and weighed, 1 g of wet liver yielding about 1.2 mg of purified DNA. All subsequent steps were carried out in an ice bath or a 4°C cold room. The livers were minced in buffer A containing 1.3 mM EDTA and 0.3 mM EGTA. Our buffer A was similar to that of Hewish and Burgoyne (1973), but was made two thirds as concentrated to avoid the extremely shrunken nuclei we observed with full-strength buffer. The buffer A used here contained 0.22 M sucrose, IO mM Tris buffer, 10 mM NaCI, 45 mM KCI, 0.67 mM dithiothreitol, 0.33 mM spermidine, 0.10 mM spermine, and was adjusted to pH 7.4 with HCI. The minced livers were gently homogenized (Dounce homogenizer, loose “A” pestle), filtered through 4 layers of cheesecloth and centrifuged in siliconized tubes for 10 min at 4000 rpm in the Sorvall HB-4 rotor at 0°C. Gentle homogenization followed by centrifugation was repeated twice in buffer A containing 0.1 mM EDTA, 0.1 mM EGTA and 0.17% Triton X-100, and then once in buffer A. After the last centrifugation, the nuclei were resuspended in buffer A and transferred to siliconized glass petri dishes. After addition of 3H-me3-psoralen in 95% ethanol (or 95% ethanol for control samples), the nuclei remained in the dark for IO min and were then irradiated as described above for purified DNA. (The final concentration of ethanol was I%.) In early experiments, nuclei were isolated according to the protocol of Axel (1975). Our mouse nuclei were unstable at the very low ionic strength involved in this method: the volume of the

Psoralen 639

Is a Probe

for Chromatin

Structure

pelleted “nuclei” increased several fold during late stages of the isolation, and microscopic examination showed only empty nuclear ghosts. In our hands, “nuclei” prepared by this method are essentially isolated chromatin. Thus except in the one experiment in Figure 2, we used the Buffer A nuclear isolation method de scribed above. Micrococcal Nuclease Treatment Purified DNA or nuclei in buffer A containing 1 .O mM CaCI, were divided into 0.5 or 1.0 ml portions in glass tubes. Micrococcal nuclease (Worthington: 29,000 U/mg) was dissolved at 150 pg/ml in 0.25 M sucrose, 0.1 mM CaCI,, 1 mM Tris buffer (pH 7.9), and used within 1 week. Each incubation mixture was prewarmed for 2 min at 37°C before nuclease was added. Digestion proceeded at 37°C and was stopped by adding EDTA to 5 mM and cooling the tube in ice. Nuclease Digestion Assay-Purified DNA Solubilization of covalently bound 3H-merpsoralen could be assayed only after unbound 3H-me,-psoralen had been removed. This removal was accomplished by addition of carrier tRNA (60 yglml), followed by chloroform extraction and ethanol precipitation as described above. Percentage of digestion was then determined for both 32P-DNA and 3H-meapsoralen by scintillation counting according to two methods-ethanol precipitation and acid precipitation. Using the first method, ethanol-precipitated DNA was dissolved in a low salt buffer and counted after solubilization in Triton X-100 fluor, described above. With the second method, the DNA was precipitated by adding an equal volume of cold 2 M HC10r2 M NaCl for 30-60 min on ice. The precipitate was collected on a 0.45 wrn millipore filter and washed under suction with 20 ml of 0.5 M HC104-0.5 M NaCl followed by 10 ml of 70% ethanol. When dry, the filter was counted in PPO-POPOPtoluene. Nuclease Digestion Assay-Nuclei The kinetics of nuclease digestion of nuclei were assayed by perchloric acid precipitation (Axel, 1975). An equal volume of cold 2 M HC104-2 M NaCl was added to the tube of digested nuclei, they were kept on ice for 30-60 min and the precipitate was pelleted by centrifugation (Sorvall HB-4 rotor, IO min at 4000 rpm, 0°C). The supernatant was warmed to room temperature, and its absorbance was measured at 350, 315, 280, 260 and 240 nm to determine a rough spectrum. The absorbance of the supernatant from undigested nuclei that had been precipitated in the same manner was subtracted from each value, and the AZsO was corrected for any scattering. Percentage of digestion was calculated As AZsO x 100/(A~$@eS’ed x 1.67), where AZBO is the absorbance of the acid supernatant of the digested sample and A&;psested is the absorbance of an equal portion of the same nuclei that was lysed without being digested or precipitated, and 1.67 corrects for the hyperchromicity of nucleotides in acid (Sollner-Webb, CameriniOtero and Felsenfeld, 1976). AZeO Undigestedwas normally about 3. Identical results were obtained by purifying DNA from the nucleasetreated nuclei and calculating percentage of digestion as [l .O of these methods indiV&d&m undipested)] x 100. (The equivalence cated that low molecular weight acid-precipitable DNA fragments were extracted with the same efficiency as high molecular weight DNA, using the DNA isolation technique described below.) The amount of 3H-merpsoralen covalently bound to DNA in nuclei was determined after complete purification of the DNA. Both the ethanol and acid precipitation methods (described above) were used. The JH-merpsoralen could not be determined by acid precipitation of a nuclear lysate because merpsoralen photolysis products precipitated along with the me,-psoralen bound to DNA. This resulted in an intolerably high background. Isolation of DNA from Nuclei After micrococcal nuclease treatment, digested 0.4 M in NaCl and 0.4% in Na dodecylsulfate.

nuclei were made The mixture was

shaken at 37°C until clear. Proteinase K [E. Merck; 1 mg/ml in IO mM Tris buffer, 10 mM NaCI, 10 mM EDTA, 0.2% Na dodecylsulfate (pH 7.9)] was added to a final concentration of 0.3 mg/ml. After 4 hr at 37”C, the solution was extracted once with phenolchloroform-isoamyl alcohol (25:24:1) and once with choloroformisoamyl alcohol, and then ethanol-precipitated as described above. Gel Electrophoresis and Fluorography 6% polyacrylamide gels were prepared and run in Tris-borateEDTA buffer (Peacock and Dingman, 1968) exactly as described by Maniatis, Jeffrey and van de Sande (1975), except that the gels (12 x 13 x 0.16 cm) were electrophoresed for 2 hr at 25 mA. Gels werestained with 1 pg/ml ethidium bromide in running buffer and photographed on Polaroid 55 P/N or 57 film through a red filter. The molecular weight of a DNA fragment was calculated from its electrophoretic mobility by comparison with the mobilities of Hind Ii + III fragments of A DNA run on the same gel. The molecular weights of the A Hin fragments weredetermined by Maniatis et al. (1975) by comparison with +X174 Hin fragments. We have adjusted these A Hin molecular weights in accordance with the recently available exact values for the +X174 Hin fragment molecular weights (Sanger et al., 1977). This adjustment increases the values of Maniatis et al. (1975), but never by more than 11%. The location of 3H-merpsoralen in the gel was determined by fluorography (Bonner and Laskey, 1974) using Kodak X-Omat RP x-ray film that had been preexposed (Laskey and Mills, 1975) to Awe = 0.20 above the background for unexposed film. Film exposure was quantitated using a Joyce-Loebl microdensitometer. Acknowledgments T. C. is grateful to J. E. Hearst for the suggestion that psoralen could be used as a probe for chromatin structure. We thank G. Wiesehahn, J. Hydeand J. E. Hearstforapreprint oftheirwork; B. Meyer for A Hin fragments; M. Kamarck and S. Epstein for mice; and S. T. lsaacs and C. V. Hanson for 3H-me3-psoralen. This work was supported by a National Cancer Institute Research Fellowship to T. C. and an NSF grant to M. L. P. Received

March

8, 1977

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