Eur. J. Biochem. 96, 257-265 (1979)
DNA-Protein Interactions in Nucleosomes and in Chromatin Structural Studies of Chromatin Stabilized by Ultraviolet-Light-Induced Crosslinking Richard MANDEL, Calina KOLOMIJTSEVA, and J. Georges BRAHMS Institut de Recherche en Biologie Moleculaire du Centre National de la Recherche Scientifique, UniversitC Paris VII (Received June 2/December 8, 1978)
Crosslinking induced by ultraviolet light irradiation at 254 nm has been utilized to investigate the structure of chromatin and isolated nucleosomes. The results presented here imply that the four core histones, as well as histone H1, have reactive groups within a bond length of the DNA bases. In nucleosomes depleted of H1, all of the core histones react similarly with the DNA and form crosslinks. In chromatin, the rate of crosslinking of all histones to DNA is essentially similar. Comparison of mononucleosomes, dinucleosomes and whole chromatin shows that the rate of crosslinking increases significantly with increasing number of connected nucleosomes. These differences in the rate of crosslinking are interpreted in terms of interactions between neighbouring nucleosomes on the chromatin fiber, which are absent in an isolated mononucleosome.
Considerable progress has been achieved in the understanding of chromatin structure as a result of the discovery of the structural subunit in chromatin [l - 101. These particles, known as nucleosomes, and generated by nuclease digestion, consist of a DNA segment approximately 200-base-pairs long and two each of the four histones H2A, H2B, H3 and H4 [7]. Structural studies of nucleosome core particles by neutron scattering and X-ray diffraction [8- 101 provide a model of flattened disc-shaped particles with a diameter of 11 nm and a height of 5.7 nm in which the core of the particle is occupied by histones with the DNA wrapped around the outside. Much indirect evidence suggests that the four histones comprising the core interact in a specific way forming dimers, tetramers and an octamer [ l l - 131. Chromatin fibers have been visualized by electron microscopy and been shown to consist of beads of nucleosomes on a string [6] or, in a more compacted state, consisting of a thin 10-nm nucleofilament [14,15]. This thin nucleofilament has been shown to undergo further condensation to a thick 20-30-nm fiber in the presence of small amounts of divalent cations or 0.14 M NaCl [14-161. Despite this progress, the fine structure of the subunits and the overall structures of the chromatin or of the nucleofilament remain unclear. Crosslinking of protein to DNA in eukaryotic cells has been shown to occur upon exposure to ultraviolet light (for a review, see [17,18]). It has been '
demonstrated in previous studies that irradiation produces covalent linkages between different amino acids and nucleotides, and that adducts between thymine (a pyrimidine) and the basic amino acids lysine, arginine and also cysteine are highly favored photoproducts [17,19]. It has been shown that the formation of a covalent linkage of DNA to protein can only occur when the interacting molecules are close and when .their reactive groups are within one bond length apart. Thus a variety of proteins have been covalently attached to nucleic acids. These include bovine serum albumin [20], DNA polymerase [21], RNA polymerase [27], lac repressor, aminoacyl tRNA synthetase [23] and ribosomal proteins of Escherichia coli [24]. These covalent complexes of DNA (or RNA) with proteins were formed in solution by irradiation at 254 nm under conditions of salt and pH which favor binding of the protein to the nucleic acid. The crosslinking of protein to DNA has been shown to occur in both prokaryotic and eukaryotic cells in v i m upon exposure to ultraviolet light [12,18]. Evidence that light of 254 nm produces crosslinking of protein to DNA in chromatin is presented in this paper using four independent methods : filter binding, chloroform extraction assay, gel filtration chromatography and polyacrylamide electrophoresis. Up to 85 :d of the histones are shown to be capable of crosslinking to DNA under the conditions employed. Further, evidence for protein to DNA adducts in isolated nucleo-
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somes are presented, and rates and extent of crosslinking between chromatin and nucleosomes are compared.
MATERIALS AND METHODS
Preparation of Chromatin Chromatin from calf thymus was prepared by three methods. These methods utilize either low ionic strength (I) or precipitation in 0.15 M salt (I1 and 111). In addition methods I and I11 try to avoid shearing whereas method I1 uses homogenization to solubilize the chromatin fiber. Method I. Chromatin was prepared according to the method of Reeder [25] modified by de Pomerai et al. [26], a method designed to minimize disruption of the native state. In brief, homogenizations were all carried out using a hand-held Potter glass homogenizer in the presence of sucrose and 5 mM MgC12. The nuclei were lysed in the presence of EDTA and glycerol. At all stages of preparation 0.1 mM phenylmethylsulfonyl fluoride and only low-ionic-strength solution were used. Method 11. This method utilizes 0.14 M NaCl without either divalent cations or EDTA. In brief, calf thymus was suspended in 0.14 M NaCl (pH 7.5), homogenized in a Potter homogenizer and filtered through four layers of Miracloth. The homogenate was centrifuged for 10 min at 2500 x g. The pellet was resuspended in 0.14 M NaCl, homogenized for 60 s and purified by further centrifugation and resuspension four times in 0.14 M NaCl. The final pellet was resuspended in a low-ionic-strength solution at pH 8.0 by stirring slowly overnight [27]. Method I l l . Chromatin was prepared by the same procedure as method 11, but all possibility of shearing was severely avoided. No treatment more severe than the gentle stirring by magnetic stirrer and a hand-held Potter homogenizer was used.
Preparation of Nucleosomes Nucleosomes were prepared from calf thymus nuclei isolated in 5 mM Tris-cacodylate pH 7.3, 0.1 mM CaCh and 0.04 M sucrose. Digestion was carried out with staphylococcal nuclease to 6 % acid solubility. The resulting nucleosomes were separated on Bio-Gel A-5m [28]. Several distinct peaks were present which were subsequently analyzed on 4 % polyacrylamide gels. Clean preparation of mononucleosomes and dinucleosomes was effected in this manner.
Irradiation Irradiation was carried out using an unfiltered, germicidal, low-pressure mercury lamp (15 W). The
DNA-Protein Interactions in Nucleosomes and in Chromatin
incident intensity of the 253.7-nm light was measured with a Latarjet dosimeter. Samples of chromatin and of nucleosomes were irradiated on ice in quartz cuvettes of ultraviolet optical grade. All irradiation was performed under anaerobic conditions at 0 "C. Oxygen was displaced from the chromatin solutions by gassing with helium. Irradiation at 280 nm was carried out using a Rayonet RPR 208 source and by filtering wavelengths shorter than 280 nm with Corex glass tubes [19].
Filter Binding Assay A filter binding assay similar to many others (see for example [29,30]) was carried out with HAWP 0.45-pM-pore, 25-mm-diameter Millipore filters. The chromatin was dissociated in 3.0 M NaC104, filtered through filters presoaked with 3.0 M NaC104 and washed with an equal volume of solvent. The amount of DNA passed by the filter was measured by the absorbance of the filtrate at 260 nm.
Chlorojbrm E.xtraction Assay Irradiated samples of chromatin were adjusted to 1 '%,sodium dodecylsulphate, 1 .O M NaCl and vortexed for 15 s with twice the volume of chloroform/iso-amyl alcohol (24/1). The two phases were separated by centrifugation at 6000 rev./min for 15 min and the absorbance at 260nm of the aqueous phase was measured.
Column Chromutography Irradiated samples of chromatin were adjusted to 3.0 M NaC104 for complete dissociation of the proteins [31]. The samples were incubated 1-2 h at 4 "C and then applied to a column of Bio-Gel A-5m previously equilibrated with 3.0 M NaC104. Fractions were collected and the absorbances at 260 nm and 230 nm were measured.
Gel Electrophoresis Polyacrylamide gel electrophoresis was carried out using both 15 % dodecylsulphate gels and 4 % trisborate nondenaturing gels to separate intact mononucleosomes and dinucleosomes. 15 % dodecylsulphate/polyacrylamide gels were prepared using the procedure of Laemmli, but with only 0.4 % bisacrylamide and without a stacking gel 1321. Samples of chromatin containing 10-25 pg DNA (previously irradiated) were lyophilized to dryness and redissolved in electrophoresis buffer as described by Laemmli [32]. 4 % polyacrylamide gels with an acrylamide: bisacrylamide ratio of 40:l were utilized to assay and separate nucleosomes. Nucleosomes containing 1030 pg of protein were applied and electrophoresed.
R. Mandel, G. Kolomijtseva, and J. G. Brahms
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Fig. 1. Filter. binding and chlorof~rme.rtraction. (A) Fraction of DNA passing the Millipore filter (0 0)and fraction of DNA removed from the aqueous phase with CHC13 ( x x ) with irradiation performed at low Salt (1 mM NaCI): fraction of D N A extracted with CHC13 ( 0 -- - --0)with irradiation performed at 2.0 M NaCI, all as a function of irradiation time. (B) Fraction of DNA removed from the aqueous phase by chloroform extraction. Chomatin (O---O), mononucleosomes (A----A) and dinucleosomes (0. . - . . 0)
Under these conditions, the mononucleosome has an RF of approximately 0.50 and the dinucleosome has an RF of approximately 0.25. Gels were stained for 24 h at 37 ”C with a 0.1 ”/, solution of Coomassie brilliant blue R-250 and subsequently destained at 37°C as described by Maize1 [33]. Destained gels were scanned at 510 nm using an Isco gel scanner model 659 with Isco model U-5 absorbance monitor. The Coomassie blue absorption coefficient is linear over the concentration range examined [34].
RESULTS
Filter Binding and Chloroform Extraction Assays of’ Crosslinked DNA-Protein Complex The results of the filter binding assay performed on chromatin are shown in Fig.lA, where the fraction of the DNA retained on the filter as a function of irradiation time is plotted. The background retention of the filter was approximately 40% of the DNA under a variety of dissociating conditions (5 M NaCl or 3 M NaC104 + 2 % heparin) for unirradiated chromatin. Thus the fraction of DNA crosslinked to protein as a function or irradiation time is calculated as :
where A260 is the absorbance of the filtrate passing the filter. The fraction crosslinked reaches a maximum of f = 0.80 after 20 min of irradiation and then remains constant. This phenomena has been observed by others in the case of complexes of lac repressor
with DNA [29] and is probably due to less than 100 2) retention efficiency of the filter for complex. The chloroform extraction assay was found to be rapid and reproducible without any measurable background. The results, as seen in Fig.lA, show that after 5 min half the DNA is extracted by the chloroform and after 30 min nearly 100 is extracted from the aqueous phase. In contrast, not more than 5 % of the DNA is extractable when irradiation is carried out under dissociating conditions of 2.0 M NaCl, as seen in Fig. 1A. Both assays show the same dependence of the fraction of DNA crosslinked as a function of time. The filter binding assay apparently is more sensitive to small quantities of undissociable proteins, probably non-histone proteins which are known to be tightly bound to the DNA under strongly dissociating conditions. These results are in good agreement with a study of ultraviolet crosslinking of chromatin from Chinese hamster cells which found 70 ”/, of the DNA remaining on the filter at a dose of 12 kJ/m2 (at 15 min irradiation time our dose is comparable : 13 kJ/mZ); we also find 70 % retention on the filter (Fig. 1A) [35]. A comparison of the crosslinking of chromatin, mononucleosomes and dinucleosomes is shown in Fig. 1 B as a function of irradiation time. It can be seen that the DNA in chromatin is crosslinked most rapidly with nearly all the DNA extractable from the aqueous phase after 30 min. The kinetics of nucleosome crosslinking are much slower and there is a small but consistent difference between mononucleosomes and dinucleosomes, with the dinucleosome crosslinking of DNA to protein more rapid than for mononucleosomes. By contrast, not more than 5 % of the DNA is
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DNA-Protein Interactions in Nucleosomes and in Chromatin
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Fig. 2. G K /,/i//ru/ioti of' cliromutin. Absorbance at 260 nm (-~~--) and at 230 nm ( ~)versus fraction number for (A) 0, (B) 30 and (C) 120 min of irradiation
extracted from the aqueous phase when irradiation is carried out under dissociating conditions of 2.0 M NaCl for either chromatin or nucleosomes. Gel Chromatography
and there is a decrease in A230 of the protein peak. This value of A 4 A 2 6 0 corresponds to 29% of the protein crosslinked to the DNA. After a 120-min irradiation (Fig. 2C), the A230/A260 ratio is found to be 0.756, about the same value as that the original chromatin with no second peak corresponding to free protein. Thus there is a progressive increase in the fraction of protein covalently crosslinked to DNA as a function of irradiation time.
In order to determine which proteins are crosslinked to DNA and their percentage crosslinked as a function of time, dodecylsulphate/polyacrylamidegel electrophoresis was performed on irradiated chromatin to resolve the histone proteins dissociated with 2$4 sodium dodecylsulphate in the presence of 5 % 2-mercaptoethanol. All proteins crosslinked to DNA will remain at the gel origin of migration, since the DNA is too large to penetrate the 15% polyacrylamide gels. Specific interprotein crosslinks will give rise to new bands migrating more slowly than the constituent protein monomers. It is shown in this paper that only two different histone dimers in small quantity are formed and these migrate at approximately the same rate ( R F )as histone H1. Higher protein aggregates are not seen and probably do not constitute a major photoproduct. Thus, the progressive disappearance of stain from the histone bands can be used to measure their crosslinking. Fig.3 is a photograph of a typical chromatin electrophoresis with irradiations times between zero and 120 min. It can be seen that there is a progressive disappearance of the major bands consisting of the histone proteins. The disappearance of the histones is plotted in Fig.4 with the fraction crosslinked Cr, versus time ( t ) where:
Chromatin, irradiated and subsequently dis,X l i (1) , f ( t ) = 1 -. !---~ sociated in 3.0 M NaC104, was analyzed using column 7l i (0) chromatography to separate the dissociable protein from the covalently attached DNA-protein complex. in which Z iis the staining intensity of band i; the sumThe high-molecular-weight DNA and all crosslinked mation is carried out over the appropriate bands. proteins elute out at the void volume of Bio-Gel Fig.4 shows the change in the stained intensity of the sum of the histone protein bands for three different A-5 m whereas the dissociable proteins, having much lower molecular weights, are retarded on the column. preparations of chromatin and of mononucleosomes, all normalized to t = 0. These three preparations The relative amount of DNA and protein is detershow similar rates of crosslinking. The two methods mined by measuring the absorbance of the fractions using 0.14 M NaCl (I1 and 111) yield a product which at 260 nm and 230 nm. The ratio of absorbance for DNA at 230 nm and 160 nm is 0.43. crosslinks at the same rate. Method I, using divalent cation and EDTA and low ionic strength only, shows a The control, with no irradiation, displays two peaks, as seen in Fig. 2A; the first peak appearing at slightly slower rate of crosslinking than the other the column void volume with a ratio of A z ~ o / A ~ ~preparations. o Mononucleosomes, on the other hand, = 0.43, corresponds to DNA completely dissociated show a significantly slower rate of crosslinking than from any protein; the second peak, largely retarded all three preparations of chromatin. These results on the column, has absorbance only at 230 nm and are in general agreement with those obtained by corresponds to protein. After a 30-min irradiation chloroform extraction, indicating the difference in (Fig.2B), the DNA peak has an A230/A260 = 0.654 the rate of crosslinking between mononucleosomes,
R. Mandel. G. Kolomijtseva, and J. G. Brahms
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Fig. 4. ~ o t / i ~ c . ~ ~ . s u / ~ ~ ~ i t r l c ~ / ~ 7 o / ~ ge/ ~ r 1 ce/c~i~lr.ophoi.r.c.;,s . r . ~ ~ / r i n 1 ~ c / cof~ dwo. i?zo.somiiIprofi’ins. Fraction of histones crosslinked as a function of irradiation time. Chromatins: method I ( x x ) . method I 1 (0.-- ~ O )method , 111 (A - -- A) and mononucleosornes (0. . . 0) ~
dinucleosomes and chromatin (see Fig. 1 B). Quantitative comparison of the relative kinetics of crosslinking by the two methods is difficult because the chloroform extraction detects crosslinking on the part of DNA to protein, whereas protein acrylamide gel electrophoresis measures crosslinking on the part of proteins to DNA and the other proteins. It can be seen, however, that the chloroform extraction assay is 5 - 10 times more ‘sensitive’ to the presence of DNAprotein crosslinks than is the gel electrophoresis, at low doses of irradiation. The detailed pattern of the protein gels for chromatin (prepared by method 111) at irradiation times between 0 and 60 min and for mononucleosomes between 0 and 90 min, is shown and analyzed in order to compare both DNA-protein and protein-protein crosslinks in chromatin and mononucleosomes. Fig. 5 A shows the trace of the Coomassie-bluestained gels for chromatin at 0, 30 and 60 min of irradiation time. It can be seen at t = 0 irradiation time that there are three bands on the left (bottom of the gel), that is H4, H2A H2B and H3, and two
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bands of H1 in the middle of the gel. After 30 min of irradiation, there is a decrease in the amplitudes of all histone bands with the appearance of one and possibly two new bands which comigrate with the two band of H 1. The more pronounced one can be clearly seen between the two HI bands and the second can be seen as a shoulder on the left-band side of the H1 bands (migrating more rapidly). That there are two new bands will be clearly shown on the gel patterns of the mononucleosomes and in chromatin prepared by method I deplected of HI histone. In addition, a difference curve of chromatin at 0 and 15 min irradiation time clearly indicate the presence of two new bands (not shown). Finally, there is a buildup of colour intensity near the top of the gel which increases with irradiation time. After 2 h of irradiation time approximately 80 ‘%, of the histones are crosslinked and excluded from the gel. Mononu Llrosomrs
A similar analysis was carried out for mononucleosomes. Fig.6A shows the results of the densitometer tracing of a typical experiment performed at 0, 30, 60 and 90 min of irradiation. There is a progressive disappearance of the three major bands at the left-hand side (bottom) of the gel, representing H4, H2A H2B and H 3 histones due to their crosslinking. In the middle of the gel there are two new bands (DI and D2), which are probably histone dimers and which are not present initially but appear at all later times of irradiation. Since the mononucleosomes prepared in this study do not show any H I present, the new bands can be clearly seen. These two bands correspond well in their rates of migration and relative intensities to the two bands observed near H I in irradiated native chromatin. The electrophoresis of
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DNA-Protein Interactions in Nucleosomes and in Chromatin
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Fig. 5 . ( A ) Dodecylsulphate/polyacrylarnidegel electrophoresis of chromosomal proteins, from chromatin prepared by method I I at irradiation times between 0 and 60 rnin; ( B ) fraction of stain in the histone bands HI + D , H2A + H2B, H 3 and H4. NHP = non-histone protein
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Fig. 6 . ( A ) Dodecylsulphate/polyacrylarnidegel electrophoresis of chromosomal proteins, from mononucleosomes stained with Coomassie blue and scanned at 510 nm at irradiation times between 0 and 90 min; ( B )fraction of stain in the histone bands H2A -f H2B, H 3 , H4 and DI and D 2 for mononucleosomes
irradiated H 1-depleted chromatin is in agreement with these results and shows the same two dimer bands in the same relative proportions (figure not shown). A more detailed analysis of the crosslinking pattern was carried out by decomposing the gel patterns into their constituent bands. Fig. 5 B shows the fraction of stain in each band H4, H2A + H2B, H3 and H l + D as a function of time between 0 and 60 min. Histones H2A H2B and H4 remain constant in their relative stained intensity, H3 decreases in relative importance in a fairly monotonic way and H1 D increases between 0 and 15 min and then remains fairly constant.
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The increase in relative intensity in H1 + D probably results from the appearance of the dimer bands which thus represent approximately 6 % of the total absorbance by the stained proteins. There are several features which concern the crosslinking of non-histone proteins to DNA and these can be observed in Fig.5A. The photograph shows between four and six non-histone protein bands situated between the histone H1 and H3 bands which are crosslinked and disappear. Due to their small quantity, they can no longer be seen at times of 60 min or more. Near the origin of the gels there are at least
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R. Mandel, G. Kolornijtseva, and J. G. Brahrns
two other non-histone bands. These bands appear to remain constant in their intensity up to irradiation times of 2.5 h and apparently are not crosslinked to DNA. In order to assure that the histone proteins were not simply being degrated by the ultraviolet light, chromatin under dissociating conditions of 2.0 M NaCl, was irradiated and the proteins run on the same gel system. Irradiation times of 30 and 60 min show no extensive broadening of the bands and no new bands with low molecular weights. Finally, all the gels show an increase in absorbance near the origin with increasing irradiation time. This corresponds to products of protein and DNA crosslinked together. Fig. 6 B shows the relative fraction of stain in each band as a function of 254-nm-irradiation time for mononucleosomes. Histones H2A + H2B and H4 remain a relative constant fraction of the stained protein bands whereas H3 decreases, especially at times greater than 15 min. This figure also shows the growth of the two new bands and indicates a relative ratio of around 2: 1 in their stained intensity, with the slower-migrating band (high-molecular-weight band D2) with greater intensity. The relative fraction of the two dimers reaches a maximum value at around 45 min of irradiation and this represents approximately 15 % of the total stain. However, since the histones do not all stain equally or in a simple stoichiometric fashion, this does not necessarily represent the same percentage of histone. At times between 45 and 90 min, the quantitity of dimer increases slightly to 13% of the stain. Thus more dimer is being continually formed but at the same time is being depleted by further crosslinking. It should be added that nucleosomes remain intact after irradiation up to 60 min as tested by their migration on 4 % polyacrylamide gels under nondissociating conditions.
DISCUSSION Evidence f o r Intrinsic Protein-DNA Covalent Binding
We have presented evidence for ultraviolet-lightinduced covalent protein-DNA products using four different methods: (a) binding of the DNA on membrane filters under dissociating conditions; (b) extraction of DNA by chloroform from the aqueous solution under dissociating conditions ; (c) elution of protein coincident with DNA at the void volume of Bio-Gel A-5 m columns and the disappearance of protein from later-elution fractions under dissociating conditions; (d) progressive disappearance of protein bands on polyacrylamide/dodecylsulphate gel electrophoresis. These results, obtained by four independent methods, when considered together imply the formation of a protein-DNA complex. Some of the results (methods a, b and c) yield information about crosslinking
with protein on the part of DNA. The other results (methods c and d) indicate crosslinking with the DNA on the part of the protein. In addition, electrophoresis (method d) indicates the formation of a limited number of specific protein-crosslinked products. The evidence for protein-DNA crosslinks is supported by the formation of a non-dissociable nucleoprotein complex sedimenting in a neutral CsCl equilibrium density gradient at a density between 1.5 and 1.7 g/cm3. This product yields free protein and small oligonucleotides when extensively digested with DNAase and phosphodiesterase (Mandel and Minsky, unpublished data). These findings are in agreement with the data of Strniste and Rall [35] who showed that the treatment of irradiated chromatin with DNAase eliminated high-molecular-weight ''C-labeled-protein-containing aggregates as observed by agarose column chromatography under dissociating conditions. Finally, under dissociating conditions, i.e. 2 M NaCl and 3 M NaC104, the irradiation is ineffective and no covalent bonds between protein and DNA are detected. It is evident from the data presented, that the chloroform extraction (Fig. 1) is more sensitive to small amounts of crosslinking than the methods of gel filtration (Fig. 2) or dodecylsulphate/polyacrylamide gel electrophoresis (Fig. 4). For example, at 30 min of irradiation, all the DNA (98 %) is extractable from the aqueous phase by chloroform, whereas this same irradiation time corresponds to only 26 % of the histones crosslinked as assayed by electrophoresis, in good agreement with 32.5% of the protein crosslinked as measured by gel chromatography (average of two experiments). This result supports the notion that a ratio, r, of 0.1 - 0.2 mg protein/mg DNA crosslinked is insufficient to render the DNA chloroformextractable. This corresponds to one covalent bond between a histone and DNA every 100 base pairs for r = 0.2. Proteins Bound in Chromatin
The present results indicate that the four core histones, H2A, H2B, H3 and H4, as well as H1 are bound to DNA. Fig.5 and 6 show the progressive disappearance, as a function of time, of the histones. Since the histones crosslink at approximately the same rate and formation of protein dimers and higher oligomers represent only a small fraction of the irradiation product, we can conclude that all five histone fractions form crosslinks with DNA and have reactive amino acid side chains in intimate contact with the DNA bases. This is supported by the fact that, on the basis of relative absorption coefficients of DNA and proteins in chromatin, more than 96% of the light at 254nm is absorbed by the DNA; therefore the major activated photoproducts involve the nucleotide bases.
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The results of the electrophoresis show that both histone and non-histone chromosomal proteins can be crosslinked to the DNA but also indicate that there are non-histone proteins which d o not crosslink to DNA. This class of non-histone chromosomal proteins is notably observed in electrophoresis by two distinct protein bands near the origin of the gels which remain of constant intensity at all times of irradiation. The molecular weight marker of bovine serum albumin comigrated with the heavier non-histone protein band. Thus its molecular weight is approximately 65 000. These proteins may either not be directly interacting with the DNA or they may be result from the method of chromatin preparation. The major products of irradiation at 254 nm appear to involve DNA-histone crosslinks, although the gel patterns of irradiated chromatin reveal the existence of two protein dimers. There is no evidence for a significant proportion of higher oligomers of histones (such as trimers) on the gels, whereas the origin of the gels show a buildup of staining intensity corresponding to aggregates, that is, histones crosslinked to segments of DNA of heterogeneous but high molecular weight. Thus protein-DNA adducts rather than protein-protein adducts largely account for the presence of the high-molecular-weight aggregates which d o not enter the gel. The two observed dimers appear at all times of irradiation beginning with the shortest measured time, 15 min. These dimers have the same relative rate of migration as the bands associated with H1 histone. Between t = 0 and t = 15 min, the stained intensity of the protein bands at H1 increases by about 6 % with respect to the total stained intensity, and at later' times remains constant. This initial increase and subsequent levelling off probably represents the attainment of a steady state between loss of free protein dimer due to crosslinking with DNA and gain due to formation of new protein dimers from uncrosslinked histones. This implies that formation of DNAprotein adducts occurs concurrently with proteinprotein crosslinks but that the latter never represent a large proportion of the. 254-nm-irradiation products. The data presented cannot assign unambiguously which histones are contributing to the dimers. However, one of these protein dimers has been convincingly identified by Martinson et al. [36] as that of H2AH2B, both in whole chromatin and in reconstitution experiments using H2A and H2B only. Although they found evidence for this dimer after irradiation of chromatin at 254 nm, irradiation at 280 nm (i.e. in the spectral region where the aromatic amino acids, mainly tyrosine and tryptophan, have their absorption), is most favorable for the activation and production of protein-protein crosslinked products. In fact, the absorption band of the histones in this region of the ultraviolet shows a very small peak at 278 nm
DNA-Protein Interactions in Nucleosomes and in Chromatin
with an average absorption coefficient of 50 mol residue-' cm-'. At 254 nm, the wavelength used in our study, the aromatic amino acids contained in the histones are at a relative absorption minimum, with an absorption coefficient of approximately a quarter of this peak value (approximately 12.5). The absorption coefficient of DNA at 254 nm is about 1000 times greater. Consequently, it is understandable that photoactivation is mostly due to the bases of DNA with the subsequent formation of adducts with reactive amino acid side chains and with other bases of DNA. In order to help identify the dimers, we have irradiated the chromatin at 280 nm and electrophoresed the product on our acrylamide gel system. We find, in agreement with Martinson et al. [37], that there is largely one dimer formed at this wavelength and that the DNA-protein crosslinking is diminished. The dimer product of 280-nm irradiation, identified by them as H2A-H2B dimer, migrates however at a rate intermediate between the two products at 254-nm irradiation, though closer to the more rapidly migrating band. The assignment of the other dimer band as H3-H3 is consistent with the more rapid disappearance of H3 on the gels. Such a product could form by a photo-induced crosslink at cysteine. In fact, it is known that cysteine is the most photoreactive amino acid [38] and directly absorbs radiation at 254 nm with consequent photoactivation. Previously, another study of ultraviolet crosslinking appeared [39], which claims that there is protein breakage by irradiation at rather high doses, at wavelengths of less than 295 nm, and that this breakage interferes with the crosslinking. Our results do not support this conclusion, using irradiation at 254 nm. It was possible to crosslink up to 80 7"of the histones to DNA with the remaining uncrosslinked proteins migrating on polyacrylamide gels at the same RF as unirradiated control. In addition, these protein bands show no appreciable broadening (Fig. 3), with no smaller protein fragments appearing on the gels. This conclusion has been confirmed in a paper by Martinson et al. [37] in which they present evidence for ultraviolet crosslinking of chromatin in good agreement with the present study. Finally, electrophoresis of chromatin irradiated in the presence of 2 M NaCl under dissociating conditions show the same histone and non-histone protein bands as unirradiated control without noticeable broadening or the appearance of new low-molecular-weight bands.
Nucleosomrs The comparison of chloroform-extractable DNA as a function of irradiation time between mononucleosomes, dinucleosomes and chromatin shows that the DNA is crosslinked more rapidly to protein when
R. Mandel. G. Kolomijtseva, and J. G. Brahms
there are more subunits together. This implies that there are important interactions on the chromatin chain between the nucleosomes and specifically between the protein and DNA which are absent in isolated nucleosomes. This conclusion is supported when the results of the gel electrophoresis are compared between mononucleosomes and chromatin. Particularly at low irradiation doses (i.e. at times under 30 min of irradiation) the extent of crosslinking is about 2 - 3 times more rapid in the case of whole chromatin than in mononucleosomes. The electrophoretic pattern of the mononucleosomes (lacking H I ) shows more clearly the presence of the two distinct dimers produced by irradiation at 254nm. These two dimers represent a greater proportion of the total stained intensity on the mononucleosome gels, reaching a peak of approximately 15 ?;), at 45 min of irradiation. This is 2.5 times greater than for the irradiated chromatin. This, taken with the evidence that the histone bands disappear more slowly in the mononucleosome gels, implies that protein-DNA interactions are stronger in chromatin than in nucleosomes. This may be due either to stabilization of the individual nucleosome by adjacent nucleosomes or to quaternary interactions between the histone proteins on one nucleosome with the DNA on nearestneighbor nucleosomes, or that additional proteinDNA interactions arise from the assembly of the nucleosomes along the chromatin filament. Finally, since the protein dimers form in both mononucleosomes and in chromatin, they are therefore of intranucleosomal origin and probably reflect interactions in the histone octamer core. Note. After this article was submitted for publication, another article appeared on a similar subject [40]. The authors have performed ultraviolet crosslinking of nucleosomes with results in substantial agreement with the present study. However, the authors used irradiation conditions substantially different from this present study. Their study utilized a 5 ”/, acetone solution as a sensitizer for ultraviolet radiation with the wavelength greater than 290 nm, conditions which lead to the activation of guanine and cytosine, possibly explaining the preferential crosslinking of H2A and H2B. observed by them. By contrast, a t 254 nm absorption of light by all four bases occurs, giving rise to crosslinking by all four histones to D N A at similar rates. The comparison of mononucleosomes, dinucleosomes and chromatin indicates that the rate ofcrosslinking increases with the number ofconnected nucleosomes.
REFERENCES I . Hewish, D. R. & Burgoyne, L. A. (1973) Biochem. Bioplzys. Rrs. Commun. 52. 504- 510. 2. Sahasrabuddhe, C. G. & Van Holde, K. E. (1974) J . B i d . Clirm. 249, 152 - 156. 3. Noll. M. (1974) Nature (Lond.) 251, 249-251. 4. Kornberg, R. D. (1974) Science (Wash. D.C.) 184, 868-871. 5. Van Holde, K. E., Sahasrabuddhe, C. G. & Shaw, B. R. (1974) Nucleic. Acids Res. 1. 1579 - 1586. 6. Olins. A . L. & Olins, D. E. (1974) Sciencc (Wash. D.C.) 183, 330- 332.
265 7. Oudet. P., Gross-Bellard, M. & Chambon. R . (1975) Cvll, 4. 281 - 300. 8. Pardon. J . F., Worcester. D. L., Wooley, J . C., Tatchell, K., Van Holde, K. E. & Richards, B. M. (1975) Nuclric,. Ac,ici.s Res. 2, 2163-~2176. 9. Baldwin, J . P.. Boseley. P. G., Bradbury. E. M . Sr lbel, K. P. (1975) Ntrture (Lond.) 253, 245 - 249. 10. Finch, J. T., Lutter, L. C., Rhodes, D., Brown, R. S., Rushton, B., Levitt, M. s( Klug, A. (1977) Nature (Lond.) 269, 29-36. 1 1 . D’Anna, J . A. & Isenberg, I. (1974) Biochcw7i.stry, 13, 4992-4997. 12. Kornberg. R. D. Sr Thomas. J. 0.(1974) Si,irncv (W(rsh.D.C.) 184. 865 -~ 868. 13. Thomas, J . 0. & Kornberg, R. D. (1975) Proc,. h’crtl AUK/. Sci. U . S . A . 72,2626-2630. 14. Finch, J. T. 91 Klug, A. (1976) P w c . Nut1 A w d . Sci. U.S.A. 73, 1897 - 1901. 1%. Ris, H. & Chandler, B. (1963) Cold Spring Harbor Sjwp. Quunt. Biol. 28, 1 --8. 1%. Ris, H . (1974) Ciha Found. Sivp. 28, 7--23. 16. Worcel, A. Sr Benyajati, C. (1977) (’c.11, 12, 83-100. 17a. Smith, K. C. (1975) Pliotoc.lienii.str). and Photohio10,qj~o / N i r clc~ic.Acids (Wang, S. Y . , ed.) vol. 2, pp. 187-218, Academic Press, New York. 17b. Varghese, A. J . (1 976) in A ~ i r i gand Ccrrcinogcnc.sis and Ra(fiation Biology (Smith, K. C., ed.) pp.207-223, Plenum Press, New York. 18. Weintraub, H. (1973) Cold Spring Harbor Sjwp. Qucint. Biol. 38,247 - 256. 19. Shetler, M. D., Schott, H. M., Martinson, H. G . & Lin, E. T. (1975) BiochcJrn. Biophys. Res. Commun. 66, 83-93. 20. Braun, A . & Merrick, B. (1975) Pliotoclzem. Pl7otohio1. 21, 243 - 247. 21. Markovitz, A . (1972) Biochim. Biophj..S. Acta. 281, 522- 534. 22. Strniste, G. F. & Smith, D. A. (1974) Bioc/7r~mistr),13, 4 8 5 S 488. 23. Schoemaker, H. J . P. & Schimmel, P. R. (1974) J . Mol. Biol. 84,503 - 51 3. 24. Ehresman, B.. Reinbott, J . & Ebel, J. P. (1975) FEBS Lett. 58. 106 -- 11 1 . 25. Reeder, R. H. (1973) J . Mol. Biol. 80. 229 -- 241. 26. De Pomerai. D. I., Chesterton, C . J. & Butterworth, P. H. W. (1974) Eur. J . Biochrm. 46, 461 -471. 27. Kadykov, V. A., Kolomijtseva, G. Ya., Manamsh‘yan, T. A., Senchenkov, E. P. & Chepyzheva, M. A. (1973) Mol. Biol. 7, 192 - 200. 28. Shaw, B. R., Corden, J. L., Sahasrabuddhe, C . E. & Van Holde, K. E. (1974) Bioc,hrm. Biopliys. Res. Comniun.61, 1193- 1198. 29. Riggs, A . D., Suzuki, H. & Bourgeois. S . (1970) J . Mol. Biol. 48, 67 -~ 83. 30. Jones, 0. W. Sr Berg, P. (1 966) J . Mol. Biol. 22, 199 - 209. 31. Ohlenbusch, H. N., Olivera, B. M., Tuan, D. & Davidson, N. (1961) J . Mol. Biol. 25. 299-315. 32. Laemmli, U . K. (1970) Nature (Lond.) 227, 680-685. 33. Maizel, J. V., J r (1971) Methods Virol. 5, 180-247. 34. Elgin, S. C. R. (1975) Method.s Enzymol. 40, 144- 160. 35. Strniste, G. F. & Rall, S. C . (1976) Biochemistrj,, I S , 17121719. 36. Martinson, H. G. & McCarthy, B. J. (1976) Bioc,lienii.stry, 13. 4126--4130. 37. Martinson. H. G., Shetlar, M. D. & McCarthy, B. J. (1976) Biochemistry. I S , 2002 - 2007. 38. Shettar, M. D.. Schott, H. M., Martinson, H. G. & Lin, E. T. (1973) Biochem. Biophys. Rcs. Commun. 66, 83- 93. 39. Celis, J. R., Fink, M. & Kattof (1976) Nucleic Acids Res. 3. 1065- 1069. 40. Sperling, J. & Sperling, R. (1978) Nucleic Acids Res. 5, 27552773.
R. Mandel, G. Kolomijtseva, and J . G. Brahms, Institut de Recherche en Biologie Moleculaire du C.N.R.S., Universite Paris VII, Tour 43, 2 Place Jussieu, F-75221 Paris-Cedex-05, France
DNA-Protein Interactions in Nucleosomes and in Chromatin Structural Studies of Chromatin Stabilized by Ultraviolet-Light-Induced Crosslinking Richard MANDEL, Calina KOLOMIJTSEVA, and J. Georges BRAHMS Institut de Recherche en Biologie Moleculaire du Centre National de la Recherche Scientifique, UniversitC Paris VII (Received June 2/December 8, 1978)
Crosslinking induced by ultraviolet light irradiation at 254 nm has been utilized to investigate the structure of chromatin and isolated nucleosomes. The results presented here imply that the four core histones, as well as histone H1, have reactive groups within a bond length of the DNA bases. In nucleosomes depleted of H1, all of the core histones react similarly with the DNA and form crosslinks. In chromatin, the rate of crosslinking of all histones to DNA is essentially similar. Comparison of mononucleosomes, dinucleosomes and whole chromatin shows that the rate of crosslinking increases significantly with increasing number of connected nucleosomes. These differences in the rate of crosslinking are interpreted in terms of interactions between neighbouring nucleosomes on the chromatin fiber, which are absent in an isolated mononucleosome.
Considerable progress has been achieved in the understanding of chromatin structure as a result of the discovery of the structural subunit in chromatin [l - 101. These particles, known as nucleosomes, and generated by nuclease digestion, consist of a DNA segment approximately 200-base-pairs long and two each of the four histones H2A, H2B, H3 and H4 [7]. Structural studies of nucleosome core particles by neutron scattering and X-ray diffraction [8- 101 provide a model of flattened disc-shaped particles with a diameter of 11 nm and a height of 5.7 nm in which the core of the particle is occupied by histones with the DNA wrapped around the outside. Much indirect evidence suggests that the four histones comprising the core interact in a specific way forming dimers, tetramers and an octamer [ l l - 131. Chromatin fibers have been visualized by electron microscopy and been shown to consist of beads of nucleosomes on a string [6] or, in a more compacted state, consisting of a thin 10-nm nucleofilament [14,15]. This thin nucleofilament has been shown to undergo further condensation to a thick 20-30-nm fiber in the presence of small amounts of divalent cations or 0.14 M NaCl [14-161. Despite this progress, the fine structure of the subunits and the overall structures of the chromatin or of the nucleofilament remain unclear. Crosslinking of protein to DNA in eukaryotic cells has been shown to occur upon exposure to ultraviolet light (for a review, see [17,18]). It has been '
demonstrated in previous studies that irradiation produces covalent linkages between different amino acids and nucleotides, and that adducts between thymine (a pyrimidine) and the basic amino acids lysine, arginine and also cysteine are highly favored photoproducts [17,19]. It has been shown that the formation of a covalent linkage of DNA to protein can only occur when the interacting molecules are close and when .their reactive groups are within one bond length apart. Thus a variety of proteins have been covalently attached to nucleic acids. These include bovine serum albumin [20], DNA polymerase [21], RNA polymerase [27], lac repressor, aminoacyl tRNA synthetase [23] and ribosomal proteins of Escherichia coli [24]. These covalent complexes of DNA (or RNA) with proteins were formed in solution by irradiation at 254 nm under conditions of salt and pH which favor binding of the protein to the nucleic acid. The crosslinking of protein to DNA has been shown to occur in both prokaryotic and eukaryotic cells in v i m upon exposure to ultraviolet light [12,18]. Evidence that light of 254 nm produces crosslinking of protein to DNA in chromatin is presented in this paper using four independent methods : filter binding, chloroform extraction assay, gel filtration chromatography and polyacrylamide electrophoresis. Up to 85 :d of the histones are shown to be capable of crosslinking to DNA under the conditions employed. Further, evidence for protein to DNA adducts in isolated nucleo-
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somes are presented, and rates and extent of crosslinking between chromatin and nucleosomes are compared.
MATERIALS AND METHODS
Preparation of Chromatin Chromatin from calf thymus was prepared by three methods. These methods utilize either low ionic strength (I) or precipitation in 0.15 M salt (I1 and 111). In addition methods I and I11 try to avoid shearing whereas method I1 uses homogenization to solubilize the chromatin fiber. Method I. Chromatin was prepared according to the method of Reeder [25] modified by de Pomerai et al. [26], a method designed to minimize disruption of the native state. In brief, homogenizations were all carried out using a hand-held Potter glass homogenizer in the presence of sucrose and 5 mM MgC12. The nuclei were lysed in the presence of EDTA and glycerol. At all stages of preparation 0.1 mM phenylmethylsulfonyl fluoride and only low-ionic-strength solution were used. Method 11. This method utilizes 0.14 M NaCl without either divalent cations or EDTA. In brief, calf thymus was suspended in 0.14 M NaCl (pH 7.5), homogenized in a Potter homogenizer and filtered through four layers of Miracloth. The homogenate was centrifuged for 10 min at 2500 x g. The pellet was resuspended in 0.14 M NaCl, homogenized for 60 s and purified by further centrifugation and resuspension four times in 0.14 M NaCl. The final pellet was resuspended in a low-ionic-strength solution at pH 8.0 by stirring slowly overnight [27]. Method I l l . Chromatin was prepared by the same procedure as method 11, but all possibility of shearing was severely avoided. No treatment more severe than the gentle stirring by magnetic stirrer and a hand-held Potter homogenizer was used.
Preparation of Nucleosomes Nucleosomes were prepared from calf thymus nuclei isolated in 5 mM Tris-cacodylate pH 7.3, 0.1 mM CaCh and 0.04 M sucrose. Digestion was carried out with staphylococcal nuclease to 6 % acid solubility. The resulting nucleosomes were separated on Bio-Gel A-5m [28]. Several distinct peaks were present which were subsequently analyzed on 4 % polyacrylamide gels. Clean preparation of mononucleosomes and dinucleosomes was effected in this manner.
Irradiation Irradiation was carried out using an unfiltered, germicidal, low-pressure mercury lamp (15 W). The
DNA-Protein Interactions in Nucleosomes and in Chromatin
incident intensity of the 253.7-nm light was measured with a Latarjet dosimeter. Samples of chromatin and of nucleosomes were irradiated on ice in quartz cuvettes of ultraviolet optical grade. All irradiation was performed under anaerobic conditions at 0 "C. Oxygen was displaced from the chromatin solutions by gassing with helium. Irradiation at 280 nm was carried out using a Rayonet RPR 208 source and by filtering wavelengths shorter than 280 nm with Corex glass tubes [19].
Filter Binding Assay A filter binding assay similar to many others (see for example [29,30]) was carried out with HAWP 0.45-pM-pore, 25-mm-diameter Millipore filters. The chromatin was dissociated in 3.0 M NaC104, filtered through filters presoaked with 3.0 M NaC104 and washed with an equal volume of solvent. The amount of DNA passed by the filter was measured by the absorbance of the filtrate at 260 nm.
Chlorojbrm E.xtraction Assay Irradiated samples of chromatin were adjusted to 1 '%,sodium dodecylsulphate, 1 .O M NaCl and vortexed for 15 s with twice the volume of chloroform/iso-amyl alcohol (24/1). The two phases were separated by centrifugation at 6000 rev./min for 15 min and the absorbance at 260nm of the aqueous phase was measured.
Column Chromutography Irradiated samples of chromatin were adjusted to 3.0 M NaC104 for complete dissociation of the proteins [31]. The samples were incubated 1-2 h at 4 "C and then applied to a column of Bio-Gel A-5m previously equilibrated with 3.0 M NaC104. Fractions were collected and the absorbances at 260 nm and 230 nm were measured.
Gel Electrophoresis Polyacrylamide gel electrophoresis was carried out using both 15 % dodecylsulphate gels and 4 % trisborate nondenaturing gels to separate intact mononucleosomes and dinucleosomes. 15 % dodecylsulphate/polyacrylamide gels were prepared using the procedure of Laemmli, but with only 0.4 % bisacrylamide and without a stacking gel 1321. Samples of chromatin containing 10-25 pg DNA (previously irradiated) were lyophilized to dryness and redissolved in electrophoresis buffer as described by Laemmli [32]. 4 % polyacrylamide gels with an acrylamide: bisacrylamide ratio of 40:l were utilized to assay and separate nucleosomes. Nucleosomes containing 1030 pg of protein were applied and electrophoresed.
R. Mandel, G. Kolomijtseva, and J. G. Brahms
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.o
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Fig. 1. Filter. binding and chlorof~rme.rtraction. (A) Fraction of DNA passing the Millipore filter (0 0)and fraction of DNA removed from the aqueous phase with CHC13 ( x x ) with irradiation performed at low Salt (1 mM NaCI): fraction of D N A extracted with CHC13 ( 0 -- - --0)with irradiation performed at 2.0 M NaCI, all as a function of irradiation time. (B) Fraction of DNA removed from the aqueous phase by chloroform extraction. Chomatin (O---O), mononucleosomes (A----A) and dinucleosomes (0. . - . . 0)
Under these conditions, the mononucleosome has an RF of approximately 0.50 and the dinucleosome has an RF of approximately 0.25. Gels were stained for 24 h at 37 ”C with a 0.1 ”/, solution of Coomassie brilliant blue R-250 and subsequently destained at 37°C as described by Maize1 [33]. Destained gels were scanned at 510 nm using an Isco gel scanner model 659 with Isco model U-5 absorbance monitor. The Coomassie blue absorption coefficient is linear over the concentration range examined [34].
RESULTS
Filter Binding and Chloroform Extraction Assays of’ Crosslinked DNA-Protein Complex The results of the filter binding assay performed on chromatin are shown in Fig.lA, where the fraction of the DNA retained on the filter as a function of irradiation time is plotted. The background retention of the filter was approximately 40% of the DNA under a variety of dissociating conditions (5 M NaCl or 3 M NaC104 + 2 % heparin) for unirradiated chromatin. Thus the fraction of DNA crosslinked to protein as a function or irradiation time is calculated as :
where A260 is the absorbance of the filtrate passing the filter. The fraction crosslinked reaches a maximum of f = 0.80 after 20 min of irradiation and then remains constant. This phenomena has been observed by others in the case of complexes of lac repressor
with DNA [29] and is probably due to less than 100 2) retention efficiency of the filter for complex. The chloroform extraction assay was found to be rapid and reproducible without any measurable background. The results, as seen in Fig.lA, show that after 5 min half the DNA is extracted by the chloroform and after 30 min nearly 100 is extracted from the aqueous phase. In contrast, not more than 5 % of the DNA is extractable when irradiation is carried out under dissociating conditions of 2.0 M NaCl, as seen in Fig. 1A. Both assays show the same dependence of the fraction of DNA crosslinked as a function of time. The filter binding assay apparently is more sensitive to small quantities of undissociable proteins, probably non-histone proteins which are known to be tightly bound to the DNA under strongly dissociating conditions. These results are in good agreement with a study of ultraviolet crosslinking of chromatin from Chinese hamster cells which found 70 ”/, of the DNA remaining on the filter at a dose of 12 kJ/m2 (at 15 min irradiation time our dose is comparable : 13 kJ/mZ); we also find 70 % retention on the filter (Fig. 1A) [35]. A comparison of the crosslinking of chromatin, mononucleosomes and dinucleosomes is shown in Fig. 1 B as a function of irradiation time. It can be seen that the DNA in chromatin is crosslinked most rapidly with nearly all the DNA extractable from the aqueous phase after 30 min. The kinetics of nucleosome crosslinking are much slower and there is a small but consistent difference between mononucleosomes and dinucleosomes, with the dinucleosome crosslinking of DNA to protein more rapid than for mononucleosomes. By contrast, not more than 5 % of the DNA is
260
DNA-Protein Interactions in Nucleosomes and in Chromatin
1 .c
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Fig. 2. G K /,/i//ru/ioti of' cliromutin. Absorbance at 260 nm (-~~--) and at 230 nm ( ~)versus fraction number for (A) 0, (B) 30 and (C) 120 min of irradiation
extracted from the aqueous phase when irradiation is carried out under dissociating conditions of 2.0 M NaCl for either chromatin or nucleosomes. Gel Chromatography
and there is a decrease in A230 of the protein peak. This value of A 4 A 2 6 0 corresponds to 29% of the protein crosslinked to the DNA. After a 120-min irradiation (Fig. 2C), the A230/A260 ratio is found to be 0.756, about the same value as that the original chromatin with no second peak corresponding to free protein. Thus there is a progressive increase in the fraction of protein covalently crosslinked to DNA as a function of irradiation time.
In order to determine which proteins are crosslinked to DNA and their percentage crosslinked as a function of time, dodecylsulphate/polyacrylamidegel electrophoresis was performed on irradiated chromatin to resolve the histone proteins dissociated with 2$4 sodium dodecylsulphate in the presence of 5 % 2-mercaptoethanol. All proteins crosslinked to DNA will remain at the gel origin of migration, since the DNA is too large to penetrate the 15% polyacrylamide gels. Specific interprotein crosslinks will give rise to new bands migrating more slowly than the constituent protein monomers. It is shown in this paper that only two different histone dimers in small quantity are formed and these migrate at approximately the same rate ( R F )as histone H1. Higher protein aggregates are not seen and probably do not constitute a major photoproduct. Thus, the progressive disappearance of stain from the histone bands can be used to measure their crosslinking. Fig.3 is a photograph of a typical chromatin electrophoresis with irradiations times between zero and 120 min. It can be seen that there is a progressive disappearance of the major bands consisting of the histone proteins. The disappearance of the histones is plotted in Fig.4 with the fraction crosslinked Cr, versus time ( t ) where:
Chromatin, irradiated and subsequently dis,X l i (1) , f ( t ) = 1 -. !---~ sociated in 3.0 M NaC104, was analyzed using column 7l i (0) chromatography to separate the dissociable protein from the covalently attached DNA-protein complex. in which Z iis the staining intensity of band i; the sumThe high-molecular-weight DNA and all crosslinked mation is carried out over the appropriate bands. proteins elute out at the void volume of Bio-Gel Fig.4 shows the change in the stained intensity of the sum of the histone protein bands for three different A-5 m whereas the dissociable proteins, having much lower molecular weights, are retarded on the column. preparations of chromatin and of mononucleosomes, all normalized to t = 0. These three preparations The relative amount of DNA and protein is detershow similar rates of crosslinking. The two methods mined by measuring the absorbance of the fractions using 0.14 M NaCl (I1 and 111) yield a product which at 260 nm and 230 nm. The ratio of absorbance for DNA at 230 nm and 160 nm is 0.43. crosslinks at the same rate. Method I, using divalent cation and EDTA and low ionic strength only, shows a The control, with no irradiation, displays two peaks, as seen in Fig. 2A; the first peak appearing at slightly slower rate of crosslinking than the other the column void volume with a ratio of A z ~ o / A ~ ~preparations. o Mononucleosomes, on the other hand, = 0.43, corresponds to DNA completely dissociated show a significantly slower rate of crosslinking than from any protein; the second peak, largely retarded all three preparations of chromatin. These results on the column, has absorbance only at 230 nm and are in general agreement with those obtained by corresponds to protein. After a 30-min irradiation chloroform extraction, indicating the difference in (Fig.2B), the DNA peak has an A230/A260 = 0.654 the rate of crosslinking between mononucleosomes,
R. Mandel. G. Kolomijtseva, and J. G. Brahms
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0 0
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Fig. 4. ~ o t / i ~ c . ~ ~ . s u / ~ ~ ~ i t r l c ~ / ~ 7 o / ~ ge/ ~ r 1 ce/c~i~lr.ophoi.r.c.;,s . r . ~ ~ / r i n 1 ~ c / cof~ dwo. i?zo.somiiIprofi’ins. Fraction of histones crosslinked as a function of irradiation time. Chromatins: method I ( x x ) . method I 1 (0.-- ~ O )method , 111 (A - -- A) and mononucleosornes (0. . . 0) ~
dinucleosomes and chromatin (see Fig. 1 B). Quantitative comparison of the relative kinetics of crosslinking by the two methods is difficult because the chloroform extraction detects crosslinking on the part of DNA to protein, whereas protein acrylamide gel electrophoresis measures crosslinking on the part of proteins to DNA and the other proteins. It can be seen, however, that the chloroform extraction assay is 5 - 10 times more ‘sensitive’ to the presence of DNAprotein crosslinks than is the gel electrophoresis, at low doses of irradiation. The detailed pattern of the protein gels for chromatin (prepared by method 111) at irradiation times between 0 and 60 min and for mononucleosomes between 0 and 90 min, is shown and analyzed in order to compare both DNA-protein and protein-protein crosslinks in chromatin and mononucleosomes. Fig. 5 A shows the trace of the Coomassie-bluestained gels for chromatin at 0, 30 and 60 min of irradiation time. It can be seen at t = 0 irradiation time that there are three bands on the left (bottom of the gel), that is H4, H2A H2B and H3, and two
+
bands of H1 in the middle of the gel. After 30 min of irradiation, there is a decrease in the amplitudes of all histone bands with the appearance of one and possibly two new bands which comigrate with the two band of H 1. The more pronounced one can be clearly seen between the two HI bands and the second can be seen as a shoulder on the left-band side of the H1 bands (migrating more rapidly). That there are two new bands will be clearly shown on the gel patterns of the mononucleosomes and in chromatin prepared by method I deplected of HI histone. In addition, a difference curve of chromatin at 0 and 15 min irradiation time clearly indicate the presence of two new bands (not shown). Finally, there is a buildup of colour intensity near the top of the gel which increases with irradiation time. After 2 h of irradiation time approximately 80 ‘%, of the histones are crosslinked and excluded from the gel. Mononu Llrosomrs
A similar analysis was carried out for mononucleosomes. Fig.6A shows the results of the densitometer tracing of a typical experiment performed at 0, 30, 60 and 90 min of irradiation. There is a progressive disappearance of the three major bands at the left-hand side (bottom) of the gel, representing H4, H2A H2B and H 3 histones due to their crosslinking. In the middle of the gel there are two new bands (DI and D2), which are probably histone dimers and which are not present initially but appear at all later times of irradiation. Since the mononucleosomes prepared in this study do not show any H I present, the new bands can be clearly seen. These two bands correspond well in their rates of migration and relative intensities to the two bands observed near H I in irradiated native chromatin. The electrophoresis of
+
262
DNA-Protein Interactions in Nucleosomes and in Chromatin
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Fig. 5 . ( A ) Dodecylsulphate/polyacrylarnidegel electrophoresis of chromosomal proteins, from chromatin prepared by method I I at irradiation times between 0 and 60 rnin; ( B ) fraction of stain in the histone bands HI + D , H2A + H2B, H 3 and H4. NHP = non-histone protein
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Fig. 6 . ( A ) Dodecylsulphate/polyacrylarnidegel electrophoresis of chromosomal proteins, from mononucleosomes stained with Coomassie blue and scanned at 510 nm at irradiation times between 0 and 90 min; ( B )fraction of stain in the histone bands H2A -f H2B, H 3 , H4 and DI and D 2 for mononucleosomes
irradiated H 1-depleted chromatin is in agreement with these results and shows the same two dimer bands in the same relative proportions (figure not shown). A more detailed analysis of the crosslinking pattern was carried out by decomposing the gel patterns into their constituent bands. Fig. 5 B shows the fraction of stain in each band H4, H2A + H2B, H3 and H l + D as a function of time between 0 and 60 min. Histones H2A H2B and H4 remain constant in their relative stained intensity, H3 decreases in relative importance in a fairly monotonic way and H1 D increases between 0 and 15 min and then remains fairly constant.
+
+
The increase in relative intensity in H1 + D probably results from the appearance of the dimer bands which thus represent approximately 6 % of the total absorbance by the stained proteins. There are several features which concern the crosslinking of non-histone proteins to DNA and these can be observed in Fig.5A. The photograph shows between four and six non-histone protein bands situated between the histone H1 and H3 bands which are crosslinked and disappear. Due to their small quantity, they can no longer be seen at times of 60 min or more. Near the origin of the gels there are at least
263
R. Mandel, G. Kolornijtseva, and J. G. Brahrns
two other non-histone bands. These bands appear to remain constant in their intensity up to irradiation times of 2.5 h and apparently are not crosslinked to DNA. In order to assure that the histone proteins were not simply being degrated by the ultraviolet light, chromatin under dissociating conditions of 2.0 M NaCl, was irradiated and the proteins run on the same gel system. Irradiation times of 30 and 60 min show no extensive broadening of the bands and no new bands with low molecular weights. Finally, all the gels show an increase in absorbance near the origin with increasing irradiation time. This corresponds to products of protein and DNA crosslinked together. Fig. 6 B shows the relative fraction of stain in each band as a function of 254-nm-irradiation time for mononucleosomes. Histones H2A + H2B and H4 remain a relative constant fraction of the stained protein bands whereas H3 decreases, especially at times greater than 15 min. This figure also shows the growth of the two new bands and indicates a relative ratio of around 2: 1 in their stained intensity, with the slower-migrating band (high-molecular-weight band D2) with greater intensity. The relative fraction of the two dimers reaches a maximum value at around 45 min of irradiation and this represents approximately 15 % of the total stain. However, since the histones do not all stain equally or in a simple stoichiometric fashion, this does not necessarily represent the same percentage of histone. At times between 45 and 90 min, the quantitity of dimer increases slightly to 13% of the stain. Thus more dimer is being continually formed but at the same time is being depleted by further crosslinking. It should be added that nucleosomes remain intact after irradiation up to 60 min as tested by their migration on 4 % polyacrylamide gels under nondissociating conditions.
DISCUSSION Evidence f o r Intrinsic Protein-DNA Covalent Binding
We have presented evidence for ultraviolet-lightinduced covalent protein-DNA products using four different methods: (a) binding of the DNA on membrane filters under dissociating conditions; (b) extraction of DNA by chloroform from the aqueous solution under dissociating conditions ; (c) elution of protein coincident with DNA at the void volume of Bio-Gel A-5 m columns and the disappearance of protein from later-elution fractions under dissociating conditions; (d) progressive disappearance of protein bands on polyacrylamide/dodecylsulphate gel electrophoresis. These results, obtained by four independent methods, when considered together imply the formation of a protein-DNA complex. Some of the results (methods a, b and c) yield information about crosslinking
with protein on the part of DNA. The other results (methods c and d) indicate crosslinking with the DNA on the part of the protein. In addition, electrophoresis (method d) indicates the formation of a limited number of specific protein-crosslinked products. The evidence for protein-DNA crosslinks is supported by the formation of a non-dissociable nucleoprotein complex sedimenting in a neutral CsCl equilibrium density gradient at a density between 1.5 and 1.7 g/cm3. This product yields free protein and small oligonucleotides when extensively digested with DNAase and phosphodiesterase (Mandel and Minsky, unpublished data). These findings are in agreement with the data of Strniste and Rall [35] who showed that the treatment of irradiated chromatin with DNAase eliminated high-molecular-weight ''C-labeled-protein-containing aggregates as observed by agarose column chromatography under dissociating conditions. Finally, under dissociating conditions, i.e. 2 M NaCl and 3 M NaC104, the irradiation is ineffective and no covalent bonds between protein and DNA are detected. It is evident from the data presented, that the chloroform extraction (Fig. 1) is more sensitive to small amounts of crosslinking than the methods of gel filtration (Fig. 2) or dodecylsulphate/polyacrylamide gel electrophoresis (Fig. 4). For example, at 30 min of irradiation, all the DNA (98 %) is extractable from the aqueous phase by chloroform, whereas this same irradiation time corresponds to only 26 % of the histones crosslinked as assayed by electrophoresis, in good agreement with 32.5% of the protein crosslinked as measured by gel chromatography (average of two experiments). This result supports the notion that a ratio, r, of 0.1 - 0.2 mg protein/mg DNA crosslinked is insufficient to render the DNA chloroformextractable. This corresponds to one covalent bond between a histone and DNA every 100 base pairs for r = 0.2. Proteins Bound in Chromatin
The present results indicate that the four core histones, H2A, H2B, H3 and H4, as well as H1 are bound to DNA. Fig.5 and 6 show the progressive disappearance, as a function of time, of the histones. Since the histones crosslink at approximately the same rate and formation of protein dimers and higher oligomers represent only a small fraction of the irradiation product, we can conclude that all five histone fractions form crosslinks with DNA and have reactive amino acid side chains in intimate contact with the DNA bases. This is supported by the fact that, on the basis of relative absorption coefficients of DNA and proteins in chromatin, more than 96% of the light at 254nm is absorbed by the DNA; therefore the major activated photoproducts involve the nucleotide bases.
264
The results of the electrophoresis show that both histone and non-histone chromosomal proteins can be crosslinked to the DNA but also indicate that there are non-histone proteins which d o not crosslink to DNA. This class of non-histone chromosomal proteins is notably observed in electrophoresis by two distinct protein bands near the origin of the gels which remain of constant intensity at all times of irradiation. The molecular weight marker of bovine serum albumin comigrated with the heavier non-histone protein band. Thus its molecular weight is approximately 65 000. These proteins may either not be directly interacting with the DNA or they may be result from the method of chromatin preparation. The major products of irradiation at 254 nm appear to involve DNA-histone crosslinks, although the gel patterns of irradiated chromatin reveal the existence of two protein dimers. There is no evidence for a significant proportion of higher oligomers of histones (such as trimers) on the gels, whereas the origin of the gels show a buildup of staining intensity corresponding to aggregates, that is, histones crosslinked to segments of DNA of heterogeneous but high molecular weight. Thus protein-DNA adducts rather than protein-protein adducts largely account for the presence of the high-molecular-weight aggregates which d o not enter the gel. The two observed dimers appear at all times of irradiation beginning with the shortest measured time, 15 min. These dimers have the same relative rate of migration as the bands associated with H1 histone. Between t = 0 and t = 15 min, the stained intensity of the protein bands at H1 increases by about 6 % with respect to the total stained intensity, and at later' times remains constant. This initial increase and subsequent levelling off probably represents the attainment of a steady state between loss of free protein dimer due to crosslinking with DNA and gain due to formation of new protein dimers from uncrosslinked histones. This implies that formation of DNAprotein adducts occurs concurrently with proteinprotein crosslinks but that the latter never represent a large proportion of the. 254-nm-irradiation products. The data presented cannot assign unambiguously which histones are contributing to the dimers. However, one of these protein dimers has been convincingly identified by Martinson et al. [36] as that of H2AH2B, both in whole chromatin and in reconstitution experiments using H2A and H2B only. Although they found evidence for this dimer after irradiation of chromatin at 254 nm, irradiation at 280 nm (i.e. in the spectral region where the aromatic amino acids, mainly tyrosine and tryptophan, have their absorption), is most favorable for the activation and production of protein-protein crosslinked products. In fact, the absorption band of the histones in this region of the ultraviolet shows a very small peak at 278 nm
DNA-Protein Interactions in Nucleosomes and in Chromatin
with an average absorption coefficient of 50 mol residue-' cm-'. At 254 nm, the wavelength used in our study, the aromatic amino acids contained in the histones are at a relative absorption minimum, with an absorption coefficient of approximately a quarter of this peak value (approximately 12.5). The absorption coefficient of DNA at 254 nm is about 1000 times greater. Consequently, it is understandable that photoactivation is mostly due to the bases of DNA with the subsequent formation of adducts with reactive amino acid side chains and with other bases of DNA. In order to help identify the dimers, we have irradiated the chromatin at 280 nm and electrophoresed the product on our acrylamide gel system. We find, in agreement with Martinson et al. [37], that there is largely one dimer formed at this wavelength and that the DNA-protein crosslinking is diminished. The dimer product of 280-nm irradiation, identified by them as H2A-H2B dimer, migrates however at a rate intermediate between the two products at 254-nm irradiation, though closer to the more rapidly migrating band. The assignment of the other dimer band as H3-H3 is consistent with the more rapid disappearance of H3 on the gels. Such a product could form by a photo-induced crosslink at cysteine. In fact, it is known that cysteine is the most photoreactive amino acid [38] and directly absorbs radiation at 254 nm with consequent photoactivation. Previously, another study of ultraviolet crosslinking appeared [39], which claims that there is protein breakage by irradiation at rather high doses, at wavelengths of less than 295 nm, and that this breakage interferes with the crosslinking. Our results do not support this conclusion, using irradiation at 254 nm. It was possible to crosslink up to 80 7"of the histones to DNA with the remaining uncrosslinked proteins migrating on polyacrylamide gels at the same RF as unirradiated control. In addition, these protein bands show no appreciable broadening (Fig. 3), with no smaller protein fragments appearing on the gels. This conclusion has been confirmed in a paper by Martinson et al. [37] in which they present evidence for ultraviolet crosslinking of chromatin in good agreement with the present study. Finally, electrophoresis of chromatin irradiated in the presence of 2 M NaCl under dissociating conditions show the same histone and non-histone protein bands as unirradiated control without noticeable broadening or the appearance of new low-molecular-weight bands.
Nucleosomrs The comparison of chloroform-extractable DNA as a function of irradiation time between mononucleosomes, dinucleosomes and chromatin shows that the DNA is crosslinked more rapidly to protein when
R. Mandel. G. Kolomijtseva, and J. G. Brahms
there are more subunits together. This implies that there are important interactions on the chromatin chain between the nucleosomes and specifically between the protein and DNA which are absent in isolated nucleosomes. This conclusion is supported when the results of the gel electrophoresis are compared between mononucleosomes and chromatin. Particularly at low irradiation doses (i.e. at times under 30 min of irradiation) the extent of crosslinking is about 2 - 3 times more rapid in the case of whole chromatin than in mononucleosomes. The electrophoretic pattern of the mononucleosomes (lacking H I ) shows more clearly the presence of the two distinct dimers produced by irradiation at 254nm. These two dimers represent a greater proportion of the total stained intensity on the mononucleosome gels, reaching a peak of approximately 15 ?;), at 45 min of irradiation. This is 2.5 times greater than for the irradiated chromatin. This, taken with the evidence that the histone bands disappear more slowly in the mononucleosome gels, implies that protein-DNA interactions are stronger in chromatin than in nucleosomes. This may be due either to stabilization of the individual nucleosome by adjacent nucleosomes or to quaternary interactions between the histone proteins on one nucleosome with the DNA on nearestneighbor nucleosomes, or that additional proteinDNA interactions arise from the assembly of the nucleosomes along the chromatin filament. Finally, since the protein dimers form in both mononucleosomes and in chromatin, they are therefore of intranucleosomal origin and probably reflect interactions in the histone octamer core. Note. After this article was submitted for publication, another article appeared on a similar subject [40]. The authors have performed ultraviolet crosslinking of nucleosomes with results in substantial agreement with the present study. However, the authors used irradiation conditions substantially different from this present study. Their study utilized a 5 ”/, acetone solution as a sensitizer for ultraviolet radiation with the wavelength greater than 290 nm, conditions which lead to the activation of guanine and cytosine, possibly explaining the preferential crosslinking of H2A and H2B. observed by them. By contrast, a t 254 nm absorption of light by all four bases occurs, giving rise to crosslinking by all four histones to D N A at similar rates. The comparison of mononucleosomes, dinucleosomes and chromatin indicates that the rate ofcrosslinking increases with the number ofconnected nucleosomes.
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R. Mandel, G. Kolomijtseva, and J . G. Brahms, Institut de Recherche en Biologie Moleculaire du C.N.R.S., Universite Paris VII, Tour 43, 2 Place Jussieu, F-75221 Paris-Cedex-05, France
