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Chromatin Studies by DNA–Protein Cross-Linking
METHODS: A Companion to Methods in Enzymology 12, 36–47 (1997) Article No. ME970445
Chromatin Studies by DNA–Protein Cross-Linking Dmitry Pruss*,1 and Sergey G. Bavykin†,2 *Laboratory of Molecular Embryology, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892; and †Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow 117984, Russia
Our current level of understanding of chromatin structure was to a large extent achieved with the help of DNA–protein crosslinking. The versatile inventory of cross-linking techniques allows the identification of the contacts between DNA and proteins with a single nucleotide–single amino acid precision, to detect minor components of the complex nucleoprotein systems, to reveal the interactions of the flexible protein domains with DNA, and to assay for conformational changes in the nucleosomes. q 1997 Academic Press
Intermolecular covalent cross-linking is a high-resolution approach with unique potential for structural molecular biology studies. Cross-linking-based techniques can work in situations where the ‘‘heavy guns’’ of structural biology, such as X-ray crystallography or 2-dimensional NMR, fail. Indeed, flexible or labile molecular interfaces present a formidable challenge for the crystallographic methods, whereas cross-linking still works in these sorts of situations. Importantly, minuscule amounts of materials do not present a problem for cross-linking-based techniques. While it may not decipher the structures of macromolecular complexes in their entirety, crosslinking can provide a wealth of information about the contacts between their constituent molecules. Thus, this biochemical approach and the biophysical methods supplement one another: X-ray crystallography tells about the structure of molecular blocks and of the more defined or more stable domains, and cross-linking helps to fit these blocks together and to pinpoint the more flexible, otherwise ‘‘invisible’’ domains. This is particu1 Present address: Myriad Genetics, 390 Wakara Way, Salt Lake City, UT 84108. 2 Corresponding author. Present address: Center for Mechanistic Biology and Biotechnology, Argonne National Laboratory, Building 202-A233, 9700 S. Cass Avenue, Argonne, IL 60439. Fax: (630) 2523387. E-mail: sbavykinqeverest.bim.anl.gov.
larly important in the case of chromatin, where welldefined molecular structures and flexible tails or linkers are both crucial structural elements of the nucleosomes (1–3). Historically, a major role in elucidating nucleosome structure has been played by the Mirzabekov cross-linking reaction. In the early 1970s, Andrei Mirzabekov, then a tRNA researcher, was trying to apply the chemical approaches developed for RNA structure studies to DNA. Dimethyl sulfate (DMS, a purine-modifying reagent) emerged as a particularly useful chemical probe (4). The early experiments showed, unexpectedly, that the histone does not leave a DMS footprint on chromatin DNA (5). Indeed, as we know now, the nucleosome essentially lacks direct DNA base – protein interactions (1). In 1974, Gilbert, Maxam, and Mirzabekov (6) reported the first highresolution DMS footprinting protocol. This discovery has become one of the key events that spawned the era of gene sequencing (7). In parallel, using of high lability of DMS-methylated bases, Mirzabekov and colleagues developed a new method of DNA-protein cross-linking (8). In an attempt to extend this approach to the newly discovered nucleosomes, they came across what initially seemed to be a bizarre side reaction: single-stranded splitting that appeared at the site of protein – DNA cross-linking (9). A meticulous investigation not only clarified the mechanism of this reaction but also allowed the development of applications as a powerful tool for nucleoprotein studies (9, 10). In the classic Mirzabekov protocol, a nucleophilic group of a protein catalyzes a process similar to the process catalyzed by piperidine in the Maxam – Gilbert sequencing reaction (Fig. 1). Each cross-linking event is accompanied by DNA strand scission, and in the aftermath of the reaction, only the 5*-terminal portion of the DNA strand remains attached to the protein. Therefore, to pinpoint a DNA contact made by a protein the size of the DNA fragment crosslinked to this protein must be measured exactly.
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Two-dimensional electrophoretic procedures developed in Mirzabekov’s laboratory make it possible to examine numerous DNA fragments cross-linked to several proteins in the course of a single experiment. This technique, sometimes referred to as ‘‘nucleoprotein sequencing,’’ was instrumental in the elucidation of the structural organization of the nucleosome core (11, 12), linker histone-containing nucleosomes (13), and internucleosomal DNA within the higher-order chromatin structure (14), and it has been recently extended to the nucleosomes formed on specific DNA sequences (15, 16). The cross-linking approach was also used to map DNA contacts made by l cro (17), Lac repressor (18), and prokaryotic RNA polymerase (19).
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Another important application of DNA – protein cross-linking methodologies is to estimate the relative abundance of a protein on different DNA fragments in chromatin preparations or in vivo. Within the scope of such an approach, the high precision of the canonic Mirzabekov reaction is dispensable. Therefore, it is possible to use the 2-dimensional electrophoresis technique in conjunction with different means of cross-linking, e.g., UV light-induced cross-linking or an alternative Mirzabekov protocol (modified so as to trade the DNA strand scission for a lower chemical selectivity and higher yield of the reaction) (20 – 22). Finally, it is possible to determine which functional group of a protein gave rise to a cross-linking event.
FIG. 1. The chemistry of Mirzabekov cross-linking reaction.
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One may use the covalently attached nucleic acid fragment as a vehicle to plant 32P label on a protein and then use the inventory of protein biochemistry to identify the labeled site within the protein molecule. The sites of cross-linking within the molecules of histones H4 (23), H5 (24), H1 (25), H2A, and H2B (26) have been thus identified with a single amino acid precision.
DNA–PROTEIN CROSS-LINKING TO PARTLY DEPURINATED DNA Methylation-induced protein–DNA cross-linking is initiated by spontaneous hydrolysis of the N-glycosidic bond of the methylated purine nucleotide. The spontaneous apurination proceeds very slowly at neutral pH at room temperature, and it can be drastically accelerated at slightly elevated temperatures. An unmasked glycosilic center of the resulting abasic site exists in tautomeric equilibrium between a semiacetal form (I) (Fig. 1) and an aldehyde form (II) (the latter accounts for merely 1% of the tautomeric mixture (28)). An aldehyde moiety may react with a nearby nucleophilic functional group of a protein (such as aminoterminal a-amino groups, e-amino groups of lysines, and, possibly imidazole rings of histidines) and thus becomes reversibly covalently bound to the protein. The resulting imines (III) are prone to fast hydrolysis. They may, however, be converted into stable adducts in the presence of a reducing agent, e.g., sodium borohydride (IV) (9). The imine-specific reducers, such as sodium cyanoborohydride or pyridine borane complex, are particularly useful, since they may be continually present during the reaction to selectively remove the imines (III) from the equilibrium, while the parental aldehyde (II) remains unchanged. In the absence of such a selective mild reducing agent, the imine formation is only a prelude for a chain on conversions, which begins with the breakdown of the 3*-phosphodiester bond. The DNA strand cleavage proceeds by the mechanism of b-elimination and results, via the a,b-unsaturated imine intermediate (V), in the formation of an a,b-unsaturated aldehyde (VI) (9, 28–30). Under more harsh reaction conditions, the 5*-phosphate may eventually undergo d-elimination (VIII) (30–32). The 3*-phosphodiester bond scission is a trademark feature of the canonical Mirzabekov cross-linking reaction. It should be noted, however, that neither the a,bunsaturated imines (V) nor the corresponding reduced amines (VII) have been isolated from the reaction products. Several findings suggest that the major product of Mirzabekov cross-linking may not be an a,b-unsatu-
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rated imine. For one thing, the cross-links cannot be quantitatively hydrolyzed, even at acidic pH (9). More importantly, the majority of the postelimination crosslinks are formed by the side chains of histidines (20, 23, 24), rather than by primary amino groups, whereas only the latter may efficiently catalyze b-elimination under physiological conditions (34). The alternative mechanism of adduct formation is electrophilic alkylation of proteins by the b-elimination products (Michael addition), resulting in protein attachment via the 3* position (IX). It has been shown to account for adduct formation between apurine sites and 3-aminocarbazole (35), phenylhydrazine (36), thiols (37, 38), and water (31), and it is responsible for inhibition of excision– repair enzymes by 9-aminoellipticine (40). Michael addition is expected not only to attach the apurine sites to His and Cys residues of proteins following b-elimination but also to prevent subsequent d-elimination. Technical Notes DNA may be methylated either directly within nucleoprotein complexes or prior to their assembly in the presence of 2–5 mM DMS on a chilling bath for 18 h (resulting in incorporation of a single methyl residue per approximately 100–1000 base pairs of DNA). Depurination of the methylated bases and subsequent cross-linking occur at a slightly elevated temperature (377C for 24 h; 427C for 12 h; or 457C for 8 h). The crosslinks and the unbound aldehydes are then reduced by the addition of 20 mM sodium borohydride. The crosslinking reaction mixture may be supplemented with 5 mM pyridine–borane complex to selectively trap protein–DNA adducts prior to b-elimination. The yield of methylated purine loss routinely does not exceed 20%, and no more than 1–5% of the protein becomes cross-linked. In the case of prolonged storage or high-temperature treatment of the cross-linked complexes, some residual methylated nucleotides continue to decay and the resulting aldehyde moieties might continue to bind the proteins. As a routine precaution, we recommend using primary amines to compete out the protein amino groups from the nascent aldehydes.
LOCALIZATION OF PROTEIN CONTACT SITES ON A DNA MOLECULE The DNA–protein adducts may be resolved on a denaturing gel in the presence of urea and SDS. Since Mirzabekov cross-linking is accompanied by DNA strand scission, so that only the 5*-terminal fragments of DNA remain attached to the proteins, each site of cross-linking gives rise to a band of unique mobility,
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roughly defined by the sum of sizes of the protein and the nucleic acid portions of the cross-linking product. To separate the increments attributable to the protein portion and the DNA portion of the adduct, one can selectively hydrolyze either the protein or the DNA component and measure the size of the remaining part. This is routinely achieved by running a second dimension DNA or protein gel (Figs. 2 and 3). The size of the destroyed component may then be inferred. An interesting variation of this protocol employs immunoprecipitation to fractionate the nucleoproteins prior to 2D electrophoresis. It has been used to study interaction of HMG 14/17 proteins with nucleosomes (41). DNP-DNA 2D Gel Electrophoresis The denaturing gel of the first dimension is followed by the gel separation of free DNA components of the parental cross-linked adducts, released by exhaustive enzymatic digestion of the proteins directly in the second dimension gel (Fig. 2). Since both of the dimensions use the same SDS – urea gel formulation, the uncross-linked DNA fragments migrate the same distance in both directions and form a diagonal (not
FIG. 2. Two-dimensional DNP-DNA gel electrophoresis schematic. A reconstituted 5S rRNA gene nucleosome was used for this particular experiment. The positions of 32P-labeled DNA fragments crosslinked to different histones are revealed by autoradiography (each horizontal line corresponds to a particular DNA fragment size, i.e., to a particular distance between the cross-linking/strand scission site and the 5* terminus of DNA), with the hot spots corresponding to different sizes of proteins that were covalently attached to a protein during the first dimension of electrophoresis. Note that the spots derived from each protein are found on a diagonal projection, with its position depending on the respective size of the protein. The rightmost diagonal (not shown) corresponds to zero protein size, i.e., it contains residual unbound DNA fragments.
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shown). Covalently bound proteins would slow DNA chains in the first-dimension gel: the bulkier the protein molecule, the larger is the resulting deflection from the free DNA diagonal. As a result, each covalently bound protein is manifested by a separate diagonal array of DNA spots: the larger the protein mass, the steeper is the cross-linked DNA diagonal. To quantitatively digest proteins in the gel, one may load either pronase or proteinase K, suspended in SDScontaining loading buffer, directly on the second-dimension gel (10 mg/cm2 of the gel cross section). Although SDS does not inhibit the nonspecific proteases, urea does. Therefore, one has to wash out urea from the first-dimension gel strip and to embed it into a urea-less gel section on top of the second-dimension SDS–urea gel. Upon the entry of the protease into the underlying gel, the protein digestion becomes complete within 1 h. DNP-Protein 2D Gel Electrophoresis The denaturing gel of the first dimension is followed by the gel separation of free protein components of the
FIG. 3. Two-dimensional DNP-protein gel electrophoresis schematic. The positions of 125I-labeled histones cross-linked to different DNA sites within random-sequence nucleosomes are revealed by autoradiography (each horizontal line corresponds to a particular protein type, with the hot spots corresponding to different sizes of DNA that was covalently attached to a protein during the first dimension of electrophoresis). The extreme right group of hot spots contains uncross-linked proteins (and, possibly, proteins bound to very short DNA fragments).
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cross-linked adducts (Fig.3). The proteins are liberated in the course of chemical hydrolysis of DNA directly in the first-dimension gel strip (70% HCOOH, 2% diphenylamine, 707C for 20 min). The uncross-linked proteins migrate identically in the first and in the second dimensions; therefore, their spots form a diagonal. Covalently bound DNA fragments slow the attached proteins in the first dimension gel; the longer the DNA molecule, the larger is the resulting deflection from the free protein spot. As a result, each covalently bound protein is separated into a horizontal array of spots. The Choice of the Two-Dimensional Electrophoretic System Since either the DNA or the protein component of the cross-linked complexes must be destroyed in the process of 2D gel separation, the choice of which system to use depends on the subsequent steps of analysis. The DNP-DNA gels generally offer a higher resolution and are less laborious. They are the only choice if the cross-linked DNA is needed for subsequent blotting. The DNP-protein gels may be indispensable if a further analysis (e.g., peptide mapping) of the cross-linked proteins has to be performed.
‘‘PROTEIN SHADOWS’’ OF TRANSCRIPTIONALLY ACTIVE CHROMATIN: LOCALIZATION OF DNA-BINDING PROTEINS AT SPECIFIC DNA SEQUENCES On a DNP-DNA gel, the specific DNA sequences within each diagonal can be detected by hybridization, while the overall amount of DNA in the diagonals can be estimated by ethidium bromide staining. Therefore, the assay detects proteins bound to each DNA sequence of interest and allows the quantitation of the relative abundance of the proteins on DNA fragments. This approach was dubbed ‘‘protein image,’’ or, perhaps more aptly, ‘‘protein shadow’’ hybridization (since the crosslinked proteins are already gone by the time their presence on DNA is detected). Protein image hybridization
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analysis of highly transcribed hsp70 (20) and ribosomal (21) genes as well as of constitutive DHFR genes (22) showed that histones were absent from the promoter regions. Transcription also resulted in decreased DNA cross-linking to the globular (histone fold) domains of the core histones (particularly H2A and H2B) within coding regions (20, 21) and in some essential parts of the 5*- and 3*-flanks (21, 22). However, cross-linking via histone ‘‘tails’’ remained unaffected by the level of transcription (20). This raises the possibility that upon passage of the transcription machinery, nucleosomal DNA is transiently released from the surface of the histone octamer but retains the contacts with the histone tails, in a manner reminiscent of the process suggested by Miller et al. (42). Belikov et al. (43) have dramatically enhanced the resolution power of protein image mapping by treating the products of UV light-induced cross-linking by restriction endonucleases and exonuclease III. This results in DNA strands with one end matching a restriction site and another end covalently attached to a protein, and it thus allows the DNP-DNA gel mapping procedure to achieve the same high resolution as does the canonical Mirzabekov protocol. Belikov et al. (43) found that histone H1 and two newly identified nuclosome-binding proteins, rABP50 and rABP70, are precisely positioned at Alu repeat DNA and account for the formation of unusual 240-bp nucleosomal particles at Alu repeats.
ANALYSIS OF STOICHIOMETRY OF NEWLY REPLICATED CHROMATIN Newly replicated DNA may be easily detected upon BrdUrd labeling by an anti-BrdUrd antiserum. This technique was used to probe cross-linked DNA resolved by two-dimensional DNP-DNA electrophoresis (44) to give rise to protein shadows of newly replicated Ehrlich ascites carcinoma chromatin. We found that histone H1 is deposited on nascent DNA simultaneously with or very soon after the core histones. Within 3 min fol-
FIG. 4. Cross-linked peptide mapping. (A) Preparation and analysis of 32P-labeled nucleotide-tagged peptides. (B) Two-dimensional gel electrophoresis is a powerful way to resolve multiple cross-linked peptides. Cross-linking of a purified globule domain of histone H5 with DNA (left) proceeds almost exclusively via His25 (arrows) and His62 (arrowheads). In contrast, cross-linking of the full-length histone H5 (right) gives rise to numerous additional 32P-labeled nucleotide-tagged peptides. (C) Pseudo-2D protein electrophoresis followed by crosslinked peptide analysis (24). A heterogenous population of histone H5-containing nucleosomes (170–200 bp in length) was cross-linked, and an aliquot was 125I-labeled and resolved on a 2-dimensional DNP-protein gel (both the first-dimension lane and the 2D gel are shown). The bulk of the products of cross-linking proteins was resolved in a parallel lane of the first-dimension gel, and the proteins were liberated by DNA acid hydrolysis and eluted from three gel sections, which correspond to the positions marked on the 2D gel (with approximate lengths of the linker histone bound DNA chains of ca. 170–200, 150–170, and 130–150 nt, correspondingly). After 5* labeling and tryptic digestion, the nucleotide-tagged peptides were resolved by acid electrophoresis (inset). Notably, cross-linking via His25 (arrow) occurs proximally to and possibly within the core particle boundary.
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lowing replication, the stoichiometry of the newly replicated chromatin was the same as that of the bulk chromatin. The interaction of histones with the newly replicated DNA was weaker than in the bulk chromatin, although the efficiency of cross-linking via the histone fold domains seems to be the same in both types of chromatin.
DNA-BINDING DOMAIN/PEPTIDE IDENTIFICATION The task of identifying DNA attachment sites on a protein is complicated by the fact that cross-linking often involves multiple amino acid residues of a protein. The strategy is to trim the DNA chain of a crosslinked complex so as to leave only a small nucleotide tag covalently linked to protein (45). This is achieved by acidic hydrolysis of DNA in the presence of diphenylamine and/or by extensive exonucleolytic digestion (e.g., by MNase at pH 9.0). Upon polynucleotide kinase labeling of the cross-linking site, the proteins are purified by gel electrophoresis (Fig. 4A). The technique is essentially very close to the technique described above for DNP-protein 2D electrophoresis. Indeed, the nucleotide-tagged proteins may be successfully eluted and analyzed from DNP-protein 2D gels (46). A more convenient ‘‘pseudo-two-dimensional’’ technique for separating nucleotide-tagged proteins de-
rived from different DNA segments (24) supplements chemical digestion of DNA directly in the gel by elution, dephosphorylation, and 32P labeling prior to separation in a new SDS gel, which effectively plays the role of second dimension of a canonical 2D electrophoresis (Fig. 4C). The nucleotide-tagged peptides are excised by a specific protease (e.g., trypsin) and separated by gel electrophoresis (multiple cross-linked peptides, such as the ones derived from linker histone tails, may require a two-dimensional peptide electrophoresis (Fig. 4B)). An indirect strategy for peptide identification, based on consecutive cleavages and/or chemical modifications of the peptides, is schematically illustrated in Fig. 4A; e.g., a cross-linked peptide map depicted at the bottom of Fig. 4A would imply that the second slowest peptide comes from a particular proteolytic fragment (since it is present in lane 2) and contains a particular amino acid residue (since it shifts upon its chemical modification, lane 3). In such a manner, a combination of limited trypsinolysis (Fig. 4B), Asp- and Met- specific protein cleavage, and Lys- and Tyr-specific chemical modification allowed us to identify the major DNA cross-linking sites within linker histones H5 (24) and H1 (25). In H5-containing mononucleosomes, the linker histone cross-linking proceeds via the His25 residue of its globular domain. Additional DNA contacts by His62 and the a-amino group of Thr1 emerge in chromatin.
TABLE 1 Histone–DNA Contacts: X-ray Crystallography vs Cross-Linking (Modified from (48)) DNA positiona (turns from the dyad) 031 032 131 132 231 232 331 332 431 432 531, 532 631 632, 731
H3 H4 H4 H3 H3 H4 H2B H2B H2A H2A H2B H2A
Histone contacts as implied from the X-ray crystallography data (1–3, 48)
Histone cross-links (23, 27, 47)b
Loop from C-terminus of long a-helix Loop from N-terminus of long a-helix N-terminus a-helix Penultimate N-terminus a-helix Loop from N-terminus of long a-helix Loop from C-terminus of long a-helix Loop from C-terminus of long a-helix Loop from C-terminus of long a-helix N-terminus a-helix N-terminus a-helix Loop from N-terminus of long a-helix Loop from C-terminus of long a-helix (Not resolved)
H3, H2A C-terminus H4 N terminus H3, H4 N-terminus H3, H4 H3 H4 H2B H2B H2B, H2A H2B H2A, H2B H3 H2A (histone fold), H3
a The DNA site designations are adopted from (48); i.e., the n32 positions are on the sugar–phosphate chain with the distal portion being a 3* terminus, and n31 positions are on the opposite strand (with its distal portion being a 5* terminus). Only the contacts on one-half of the nucleosome are shown; the contacts on the remaining half are assumed to be identical. b In random sequence nucleosome cores.
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In the sea urchin sperm histone H1, one of the two principal cross-linking sites is homologous to His25 of histone H5, while the other site is found within its putative DNA-binding a-helix. The DNA cross-linking site of histone H4 , His18 of its N-terminal ‘‘tail,’’ was identified by synthesis from purified components and a combination of HPLC and mass spectroscopy (23).
NUCLEOSOME STRUCTURE AS PROBED BY DNA–PROTEIN CROSS-LINKING The first zero-length cross-linking studies of the Mirzabekov laboratory (11, 12) provided high-resolution data on the sequential arrangement of histones along the nucleosomal DNA. Subsequent cross-linking studies proved that the fine structure of the nucleosome core is invariable throughout the three eukaryotic kingdoms (animals, plants, and fungi) (47). Contacts of histones H3 and H4 with DNA were
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found to be concentrated in more central DNA positions, while histone H2A and H2B cross-linking occurred mostly at the periphery of nucleosomal DNA, with the bulk of the cross-linking occurring at one side of the DNA helix (Fig. 6D, Table 1). These results played a crucial role in shaping our current view of the nucleosome as that of a tripartite histone complex with DNA wrapped around it. Nucleosome X-ray diffraction studies have only recently achieved a level of resolution sufficient to reach (but not yet to surpass) the resolution of cross-linking – 2D gel mapping (48) (Table 1). Cross-linking analysis has been the most demanding assay used thus far to prove that reconstitution of the core particles from purified histones generates particles indistinguishable from the nucleosome cores excised from the bulk chromatin (49). This finding opened the way for in vitro studies of the role of the nucleosomes within specific gene contexts. The reconstitution/cross-linking approach applied to the 5S rRNA gene nucleosomes (15) provided a single nucleotide resolution of the histone –
FIG. 5. (A) A DNP-DNA 2D gel reveals core histone tail contacts with the key TFIIIA binding site of the Xenopus 5S rRNA gene (15). (B) Acetylation of the lysine residues of the tails allows TFIIIA to bind: a model.
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DNA contacts (Fig. 2B) and resolved an array of additional core histone – DNA contacts on the outer surface of the nucleosome DNA superhelix, as well as in the linker DNA.
INTERACTION OF THE HISTONE ‘‘TAILS’’ WITH DNA The extended flexible ‘‘tail’’ domains of the core histones play an extremely important role in gene regulation, as evidenced by a large body of data on tail posttranslational modification, mutagenesis, and proteolysis (reviewed in 50–52), yet virtually nothing is known about their place within chromatin structure (2, 48). Cross-linking remains essentially the only method capable of identifying the transient and/or flexible contacts of the tails with DNA.
FIG. 6. Linker DNA and the conformational transitions within the nucleosomal core. (A–C) Schematic drawings of a condensed polynucleosomal ‘‘solenoid’’ (A), an extended polynucleosomal fiber (note that changed shape of the nucleosomes) (B), and isolated mononucleosomes (C) (53). (D) The transition as manifested by a shift in the pattern of histone–DNA cross-linking. Dashed arrows represent the altered contacts that disappear during chromatin unfolding and reappear after core particle isolation (53).
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The first DNA cross-linking site identified within the core histone tails was His18 of histone H4 (23), which may be cross-linked to three adjacent DNA sites at approximately 1.5 helical turns from the dyad axis (see Table 1) where DNA is sharply bent (1) (Fig. 6D). This result suggests that a highly basic amino acid cluster Lys16-Arg-His-Arg-Lys-Val-Leu-Arg23 of the N-terminal tail of histone H4 is involved in the sharp bending of nucleosomal DNA. A different approach used limited proteolysis of the core particle in addition to the standard cross-linking– 2D gel procedure to pinpoint DNA localization of the C-terminal tail of histone H2A (27). We have mapped protein–DNA contacts in nucleosomes either with both N- and C-termini or with only a C-terminus of histone H2A removed by selective proteolysis (by trypsin or clostripain, respectively). We found that a peptide comprising the 10 most C-terminal amino acid residues contacts the DNA at the nucleosome dyad axis (an earlier cross-linked peptide study has identified His122 of this peptide as a site of cross-linking (26)). This contact appears only in the absence of linker DNA. Nuclease digestion study indicated that e-terminal domain of histone H2A may interact with linker DNA (52a). The rearrangement of the CH2A tail from the linker to the nucleosome dyad may play a biological role during chromatin unfolding and in the retention of the H2A–H2B dimer (or the whole octamer) during the passing of polymerases through the nucleosome. Cross-linking of the 5S rRNA gene nucleosomes has been mapped with a high resolution sufficient to determine the rotational orientation of histone–DNA contacts (15). The contacts at the outer surface of the nucleosomal DNA are presumably made by the peptides adjacent to the histone tails. In addition to the contacts of histones H4 and H2A tails mentioned above, histone H3 cross-linking a half-helical turn away from the nucleosome dyad axis and several histone H3, H2A, and H2B contacts between 4 and 7 DNA turns from the dyad were localized. These core histone linker DNA interactions are likely to be responsible for deacetylation-dependent inhibition of TFIIIA binding by the core histone tails (54) (Fig. 5).
CROSS-LINKING STUDIES OF THE LINKER HISTONES AND HIGHER-ORDER CHROMATIN STRUCTURE Chromatin unfolding alters core particle structure. Recent cross-linking study of the conformation of the nucleosome cores within polynucleosomal arrays (53), demonstrated a reversible conformational change caused by stretching of the linker DNA during chroma-
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tin unfolding (Fig. 6). The so-called ‘‘stretched nucleosome’’ conformation is characterized by altered strength of the H4 and H2A/H2B-DNA contacts with the regions of sharply bent nucleosomal DNA in ca. {1, 4, and 5 helical turns from the nucleosome twofold symmetry axis, respectively (Fig. 6D). These conformational changes may affect binding of sequence-specific factors to nucleosomal DNA. The path of internucleosomal linker DNA in chromatin is a major matter of controversy. The existing concepts of chromatin fiber structure are based on the opposite concepts of linker DNA organization. All multihelical models of 30-nm fibers (55–59) and the ‘‘accordion’’ model (60) suggest that the DNA between the contiguous nucleosomes is extended. The nucleosomes are therefore arranged in a ‘‘zigzag’’ structure. Other models of 30-nm chromatin fibers are based on the idea that the linker DNA forms a superhelical coil, much like the nucleosomal core DNA (61–63). Histone cross-linking with the linker DNA may provide the crucial body of data to distinguish between these models. The interaction of the linker histone globule with linker DNA adjacent to the nucleosome core boundaries is well documented by electron microscopy (64) and nuclease digestion experiments (65, 66). Our DNA– protein cross-linking study of the highly condensed chromatin within sea urchin sperm nuclei (14) unexpectedly revealed interactions of the globular part of histone H1 with the central region of internucleosomal linker DNA, whereas the DNA segments immediately adjacent to the edges of the core particle remained essentially free of H1 interactions. We also found unambiguous interactions of the globular part of core histones (H3 and H2B) along the entire length of long (ca. 90 bp) linker DNA (14). These findings would be difficult to explain within a model in which the linker DNA is extended beyond the nucleosome boundaries, e.g., if it links the neighboring particles in a zigzag configuration. We suggest that in the 30-nm chromatin fibers of highly condensed chromatin, both nucleosomal DNA and linker DNA form a continuous superhelix (Fig.7). Chromatin decondensation is accompanied by a rearrangement of histone H1 toward the boundaries of the core particles, providing an appearance of zigzag structure. This transition is also manifested by a dramatic change in the linker histone cross-linking peptide pattern. (24). Histone H5 residue His25 is an exclusive cross-linking site in mononucleosomes and decondensed chromatin arrays, and it binds to DNA in the vicinity of the nucleosome core boundaries (Fig. 4C). In contrast, His62 is most effectively cross-linked to DNA within highly condensed chromatin, and the bulk of the cross-linking occurs at a distance from the nucleosomes. The higher resolution of 2-dimensional gel mapping,
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which has become available with the use of the nucleosomes reconstituted at specific DNA sequences, allowed us for the first time to localize linker histone– DNA cross-linking in an isolated nucleosome with a single nucleotide precision (16). As expected, the crosslinking occurred between the linker histone globule and a terminal segment of the nucleosomal DNA. Importantly, however, the precise position of the GH5– DNA contact found in our experiments appears to exclude any chromatosome model that places the globule between the two ends of the nucleosomal DNA, roughly across the nucleosome dyad axis. Our data suggest a drastically asymmetric chromatosome structure, with the linker histone globule bound at one edge of the nucleosome (67). The position of linker histones within the nuclear chromatin remains to be studied.
FIG. 7. A model for the arrangement of histones H1, H2B, and H3 on superhelical nucleosomal and spacer DNA in sea urchin sperm nuclei (14). The core DNA together with the spacer forms a lefthanded superhelix containing about 80 bp. Long thin arrows mark the sites of additional interactions of the core histones with the spacer DNA on the adjacent superhelical turn. A short solid arrow indicates the center of the spacer DNA. Histones interacting with the core DNA and spacer DNA are designated C and S, respectively. Distances along the nucleosomal DNA are given by numbers of superhelical turns from the dyad axis.
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14. Bavykin, S. G., Usachenko, S. I., Zalensky, A. O., and Mirzabekov, A. D. (1990) J. Mol. Biol. 212, 495–511.
CONCLUDING REMARKS/FUTURE DIRECTIONS
15. Pruss, D., and Wolffe, A. P. (1993) Biochemistry 32, 6810–6814.
DNA–protein cross-linking is uniquely suited for studying a structure as complex and dynamic as chromatin. Extending the traditional Mirzabekov technique to the nucleoprotein complexes formed at specific DNA sequences will not only dramatically enhance the resolution of the method but also address specific structural details that make the nucleosomes work as a part of the regulatory machinery within the cell nucleus. DNA-binding peptide mapping may pinpoint the DNA contacts by flexible regions of chromatin proteins. Twodimensional cross-link identification methodologies may be used to probe the chromatin structure of particular transcriptionally or replicationally relevant sequences.
16. Hayes, J. J., Pruss, D., and Wolffe, A. P. (1994) Proc. Natl. Acad. Sci. USA 91, 7817–7821. 17. Ebralidze, K. K., Volkov, S. K., Kirpichnikov, M. P., Mirzabekov, A. D., and Baev, A. A. (1986) Dokl. Akad. Nauk. SSSR 287, 1013–6. 18. Kamashev, D. E., Esipova, N. G., Ebralidse, K. K., and Mirzabekov, A. D. (1995) FEBS Lett. 375, 27–30. 19. Chenchick, A., Beabealashvilli, R., and Mirzabekov, A. (1981) FEBS Lett. 128, 46–50. 20. Nacheva, G. A., Guschin, D. Y., Preobrazhenskaya, O. V., Karpov, V. L., Ebralidse, K. K., and Mirzabekov, A. D. (1989) Cell 58, 27–36. 21. Belikov, S. V., Dzherbashyajan, A. R., Preobrazhenskaya, O. V., Karpov, V. L., and Mirzabekov, A. D. (1990) FEBS Lett. 273, 205–207. 22. Pemov, A., Bavykin, S., and Hamlin, J. L. (1995) Biochemistry 34, 2381–2392. 23. Ebralidse, K., Grachev, S., and Mirzabekov, A. (1988) Nature 331, 365–367.
ACKNOWLEDGMENTS
24. Mirzabekov, A. D., Pruss, D. V., and Ebralidse, K. K. (1990) J. Mol. Biol. 211, 479–491.
We thank Drs. S. A. Grachev, A. Mazumder, Y. Pommier, and A. P. Wolffe for stimulating discussions.
25. Pruss, D. V. (1989) Identification of Contact Sites between Histones H1/H5 and DNA at Different Levels of Chromatin Organization. Diss. Cand. Sci. Chim., Moscow. 26. Gushchin, D. Y., Ebralidze, K. K., and Mirzabekov, A. D. (1991) Mol. Biol. 25, 1400–1411.
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55. Worcel, A., Strogartz, S., and Riley, D. (1981). Proc. Natl. Acad. Sci. USA 78, 1461–1465. 56. Woodcock, C. L. F., Frado, L.-L. Y., and Rattner, J. B. (1984) J. Cell. Biol. 99, 41–52. 57. Makarov, V., Dimitrov, S., Smirnov, V., and Pashev J. (1985) FEBS Lett. 181, 357–361. 58. Williams, S. P., Athey, B. D., Muglia, L. J., Schappe, R. S., Gough, A. H., and Langmore, J. P. (1986). Biophys. J. 49, 233– 248. 59. Borda, J., Perez-Grau, L., Koch, M. H. J., Vega, M. C., and Nave, C. (1986) Eur. Biophys. J. 13, 175–185. 60. Subirana, J. A., Munoz-Guerra, S., Aymami, J., Radermacher, M., and Frank, J. (1985) Chromosoma 91, 377–390. 61. Finch, J. T., and Klug, A. (1976) Proc. Natl. Acad. Sci. USA 73, 1897–1901. 62. Worcel, A., and Benyajati, C. (1977) Cell 12, 83–100. 63. McGhee, J. D., Nickol, J. M., Felsenfeld, G., and Rau, D. C. (1983) Cell 33, 831–841. 64. Thoma, F., Koller, T., and Klug, A. (1979) J. Cell. Biol. 83, 403– 427. 65. Allan, J., Hartman, P. G., Crane-Robinson, C., and Aviles, F. X. (1980) Nature 288, 675–679. 66. Allan, J., Mitchell, T., Harborne, N., Bohm, L., and Crane-Robinson, C. (1986) J. Mol. Biol. 187, 591–601. 67. Pruss, D., Hayes, J. J., and Wolffe, A. P. (1995) BioEssays 17, 161–170.
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Chromatin Studies by DNA–Protein Cross-Linking Dmitry Pruss*,1 and Sergey G. Bavykin†,2 *Laboratory of Molecular Embryology, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892; and †Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow 117984, Russia
Our current level of understanding of chromatin structure was to a large extent achieved with the help of DNA–protein crosslinking. The versatile inventory of cross-linking techniques allows the identification of the contacts between DNA and proteins with a single nucleotide–single amino acid precision, to detect minor components of the complex nucleoprotein systems, to reveal the interactions of the flexible protein domains with DNA, and to assay for conformational changes in the nucleosomes. q 1997 Academic Press
Intermolecular covalent cross-linking is a high-resolution approach with unique potential for structural molecular biology studies. Cross-linking-based techniques can work in situations where the ‘‘heavy guns’’ of structural biology, such as X-ray crystallography or 2-dimensional NMR, fail. Indeed, flexible or labile molecular interfaces present a formidable challenge for the crystallographic methods, whereas cross-linking still works in these sorts of situations. Importantly, minuscule amounts of materials do not present a problem for cross-linking-based techniques. While it may not decipher the structures of macromolecular complexes in their entirety, crosslinking can provide a wealth of information about the contacts between their constituent molecules. Thus, this biochemical approach and the biophysical methods supplement one another: X-ray crystallography tells about the structure of molecular blocks and of the more defined or more stable domains, and cross-linking helps to fit these blocks together and to pinpoint the more flexible, otherwise ‘‘invisible’’ domains. This is particu1 Present address: Myriad Genetics, 390 Wakara Way, Salt Lake City, UT 84108. 2 Corresponding author. Present address: Center for Mechanistic Biology and Biotechnology, Argonne National Laboratory, Building 202-A233, 9700 S. Cass Avenue, Argonne, IL 60439. Fax: (630) 2523387. E-mail: sbavykinqeverest.bim.anl.gov.
larly important in the case of chromatin, where welldefined molecular structures and flexible tails or linkers are both crucial structural elements of the nucleosomes (1–3). Historically, a major role in elucidating nucleosome structure has been played by the Mirzabekov cross-linking reaction. In the early 1970s, Andrei Mirzabekov, then a tRNA researcher, was trying to apply the chemical approaches developed for RNA structure studies to DNA. Dimethyl sulfate (DMS, a purine-modifying reagent) emerged as a particularly useful chemical probe (4). The early experiments showed, unexpectedly, that the histone does not leave a DMS footprint on chromatin DNA (5). Indeed, as we know now, the nucleosome essentially lacks direct DNA base – protein interactions (1). In 1974, Gilbert, Maxam, and Mirzabekov (6) reported the first highresolution DMS footprinting protocol. This discovery has become one of the key events that spawned the era of gene sequencing (7). In parallel, using of high lability of DMS-methylated bases, Mirzabekov and colleagues developed a new method of DNA-protein cross-linking (8). In an attempt to extend this approach to the newly discovered nucleosomes, they came across what initially seemed to be a bizarre side reaction: single-stranded splitting that appeared at the site of protein – DNA cross-linking (9). A meticulous investigation not only clarified the mechanism of this reaction but also allowed the development of applications as a powerful tool for nucleoprotein studies (9, 10). In the classic Mirzabekov protocol, a nucleophilic group of a protein catalyzes a process similar to the process catalyzed by piperidine in the Maxam – Gilbert sequencing reaction (Fig. 1). Each cross-linking event is accompanied by DNA strand scission, and in the aftermath of the reaction, only the 5*-terminal portion of the DNA strand remains attached to the protein. Therefore, to pinpoint a DNA contact made by a protein the size of the DNA fragment crosslinked to this protein must be measured exactly.
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Two-dimensional electrophoretic procedures developed in Mirzabekov’s laboratory make it possible to examine numerous DNA fragments cross-linked to several proteins in the course of a single experiment. This technique, sometimes referred to as ‘‘nucleoprotein sequencing,’’ was instrumental in the elucidation of the structural organization of the nucleosome core (11, 12), linker histone-containing nucleosomes (13), and internucleosomal DNA within the higher-order chromatin structure (14), and it has been recently extended to the nucleosomes formed on specific DNA sequences (15, 16). The cross-linking approach was also used to map DNA contacts made by l cro (17), Lac repressor (18), and prokaryotic RNA polymerase (19).
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Another important application of DNA – protein cross-linking methodologies is to estimate the relative abundance of a protein on different DNA fragments in chromatin preparations or in vivo. Within the scope of such an approach, the high precision of the canonic Mirzabekov reaction is dispensable. Therefore, it is possible to use the 2-dimensional electrophoresis technique in conjunction with different means of cross-linking, e.g., UV light-induced cross-linking or an alternative Mirzabekov protocol (modified so as to trade the DNA strand scission for a lower chemical selectivity and higher yield of the reaction) (20 – 22). Finally, it is possible to determine which functional group of a protein gave rise to a cross-linking event.
FIG. 1. The chemistry of Mirzabekov cross-linking reaction.
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One may use the covalently attached nucleic acid fragment as a vehicle to plant 32P label on a protein and then use the inventory of protein biochemistry to identify the labeled site within the protein molecule. The sites of cross-linking within the molecules of histones H4 (23), H5 (24), H1 (25), H2A, and H2B (26) have been thus identified with a single amino acid precision.
DNA–PROTEIN CROSS-LINKING TO PARTLY DEPURINATED DNA Methylation-induced protein–DNA cross-linking is initiated by spontaneous hydrolysis of the N-glycosidic bond of the methylated purine nucleotide. The spontaneous apurination proceeds very slowly at neutral pH at room temperature, and it can be drastically accelerated at slightly elevated temperatures. An unmasked glycosilic center of the resulting abasic site exists in tautomeric equilibrium between a semiacetal form (I) (Fig. 1) and an aldehyde form (II) (the latter accounts for merely 1% of the tautomeric mixture (28)). An aldehyde moiety may react with a nearby nucleophilic functional group of a protein (such as aminoterminal a-amino groups, e-amino groups of lysines, and, possibly imidazole rings of histidines) and thus becomes reversibly covalently bound to the protein. The resulting imines (III) are prone to fast hydrolysis. They may, however, be converted into stable adducts in the presence of a reducing agent, e.g., sodium borohydride (IV) (9). The imine-specific reducers, such as sodium cyanoborohydride or pyridine borane complex, are particularly useful, since they may be continually present during the reaction to selectively remove the imines (III) from the equilibrium, while the parental aldehyde (II) remains unchanged. In the absence of such a selective mild reducing agent, the imine formation is only a prelude for a chain on conversions, which begins with the breakdown of the 3*-phosphodiester bond. The DNA strand cleavage proceeds by the mechanism of b-elimination and results, via the a,b-unsaturated imine intermediate (V), in the formation of an a,b-unsaturated aldehyde (VI) (9, 28–30). Under more harsh reaction conditions, the 5*-phosphate may eventually undergo d-elimination (VIII) (30–32). The 3*-phosphodiester bond scission is a trademark feature of the canonical Mirzabekov cross-linking reaction. It should be noted, however, that neither the a,bunsaturated imines (V) nor the corresponding reduced amines (VII) have been isolated from the reaction products. Several findings suggest that the major product of Mirzabekov cross-linking may not be an a,b-unsatu-
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rated imine. For one thing, the cross-links cannot be quantitatively hydrolyzed, even at acidic pH (9). More importantly, the majority of the postelimination crosslinks are formed by the side chains of histidines (20, 23, 24), rather than by primary amino groups, whereas only the latter may efficiently catalyze b-elimination under physiological conditions (34). The alternative mechanism of adduct formation is electrophilic alkylation of proteins by the b-elimination products (Michael addition), resulting in protein attachment via the 3* position (IX). It has been shown to account for adduct formation between apurine sites and 3-aminocarbazole (35), phenylhydrazine (36), thiols (37, 38), and water (31), and it is responsible for inhibition of excision– repair enzymes by 9-aminoellipticine (40). Michael addition is expected not only to attach the apurine sites to His and Cys residues of proteins following b-elimination but also to prevent subsequent d-elimination. Technical Notes DNA may be methylated either directly within nucleoprotein complexes or prior to their assembly in the presence of 2–5 mM DMS on a chilling bath for 18 h (resulting in incorporation of a single methyl residue per approximately 100–1000 base pairs of DNA). Depurination of the methylated bases and subsequent cross-linking occur at a slightly elevated temperature (377C for 24 h; 427C for 12 h; or 457C for 8 h). The crosslinks and the unbound aldehydes are then reduced by the addition of 20 mM sodium borohydride. The crosslinking reaction mixture may be supplemented with 5 mM pyridine–borane complex to selectively trap protein–DNA adducts prior to b-elimination. The yield of methylated purine loss routinely does not exceed 20%, and no more than 1–5% of the protein becomes cross-linked. In the case of prolonged storage or high-temperature treatment of the cross-linked complexes, some residual methylated nucleotides continue to decay and the resulting aldehyde moieties might continue to bind the proteins. As a routine precaution, we recommend using primary amines to compete out the protein amino groups from the nascent aldehydes.
LOCALIZATION OF PROTEIN CONTACT SITES ON A DNA MOLECULE The DNA–protein adducts may be resolved on a denaturing gel in the presence of urea and SDS. Since Mirzabekov cross-linking is accompanied by DNA strand scission, so that only the 5*-terminal fragments of DNA remain attached to the proteins, each site of cross-linking gives rise to a band of unique mobility,
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roughly defined by the sum of sizes of the protein and the nucleic acid portions of the cross-linking product. To separate the increments attributable to the protein portion and the DNA portion of the adduct, one can selectively hydrolyze either the protein or the DNA component and measure the size of the remaining part. This is routinely achieved by running a second dimension DNA or protein gel (Figs. 2 and 3). The size of the destroyed component may then be inferred. An interesting variation of this protocol employs immunoprecipitation to fractionate the nucleoproteins prior to 2D electrophoresis. It has been used to study interaction of HMG 14/17 proteins with nucleosomes (41). DNP-DNA 2D Gel Electrophoresis The denaturing gel of the first dimension is followed by the gel separation of free DNA components of the parental cross-linked adducts, released by exhaustive enzymatic digestion of the proteins directly in the second dimension gel (Fig. 2). Since both of the dimensions use the same SDS – urea gel formulation, the uncross-linked DNA fragments migrate the same distance in both directions and form a diagonal (not
FIG. 2. Two-dimensional DNP-DNA gel electrophoresis schematic. A reconstituted 5S rRNA gene nucleosome was used for this particular experiment. The positions of 32P-labeled DNA fragments crosslinked to different histones are revealed by autoradiography (each horizontal line corresponds to a particular DNA fragment size, i.e., to a particular distance between the cross-linking/strand scission site and the 5* terminus of DNA), with the hot spots corresponding to different sizes of proteins that were covalently attached to a protein during the first dimension of electrophoresis. Note that the spots derived from each protein are found on a diagonal projection, with its position depending on the respective size of the protein. The rightmost diagonal (not shown) corresponds to zero protein size, i.e., it contains residual unbound DNA fragments.
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shown). Covalently bound proteins would slow DNA chains in the first-dimension gel: the bulkier the protein molecule, the larger is the resulting deflection from the free DNA diagonal. As a result, each covalently bound protein is manifested by a separate diagonal array of DNA spots: the larger the protein mass, the steeper is the cross-linked DNA diagonal. To quantitatively digest proteins in the gel, one may load either pronase or proteinase K, suspended in SDScontaining loading buffer, directly on the second-dimension gel (10 mg/cm2 of the gel cross section). Although SDS does not inhibit the nonspecific proteases, urea does. Therefore, one has to wash out urea from the first-dimension gel strip and to embed it into a urea-less gel section on top of the second-dimension SDS–urea gel. Upon the entry of the protease into the underlying gel, the protein digestion becomes complete within 1 h. DNP-Protein 2D Gel Electrophoresis The denaturing gel of the first dimension is followed by the gel separation of free protein components of the
FIG. 3. Two-dimensional DNP-protein gel electrophoresis schematic. The positions of 125I-labeled histones cross-linked to different DNA sites within random-sequence nucleosomes are revealed by autoradiography (each horizontal line corresponds to a particular protein type, with the hot spots corresponding to different sizes of DNA that was covalently attached to a protein during the first dimension of electrophoresis). The extreme right group of hot spots contains uncross-linked proteins (and, possibly, proteins bound to very short DNA fragments).
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cross-linked adducts (Fig.3). The proteins are liberated in the course of chemical hydrolysis of DNA directly in the first-dimension gel strip (70% HCOOH, 2% diphenylamine, 707C for 20 min). The uncross-linked proteins migrate identically in the first and in the second dimensions; therefore, their spots form a diagonal. Covalently bound DNA fragments slow the attached proteins in the first dimension gel; the longer the DNA molecule, the larger is the resulting deflection from the free protein spot. As a result, each covalently bound protein is separated into a horizontal array of spots. The Choice of the Two-Dimensional Electrophoretic System Since either the DNA or the protein component of the cross-linked complexes must be destroyed in the process of 2D gel separation, the choice of which system to use depends on the subsequent steps of analysis. The DNP-DNA gels generally offer a higher resolution and are less laborious. They are the only choice if the cross-linked DNA is needed for subsequent blotting. The DNP-protein gels may be indispensable if a further analysis (e.g., peptide mapping) of the cross-linked proteins has to be performed.
‘‘PROTEIN SHADOWS’’ OF TRANSCRIPTIONALLY ACTIVE CHROMATIN: LOCALIZATION OF DNA-BINDING PROTEINS AT SPECIFIC DNA SEQUENCES On a DNP-DNA gel, the specific DNA sequences within each diagonal can be detected by hybridization, while the overall amount of DNA in the diagonals can be estimated by ethidium bromide staining. Therefore, the assay detects proteins bound to each DNA sequence of interest and allows the quantitation of the relative abundance of the proteins on DNA fragments. This approach was dubbed ‘‘protein image,’’ or, perhaps more aptly, ‘‘protein shadow’’ hybridization (since the crosslinked proteins are already gone by the time their presence on DNA is detected). Protein image hybridization
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analysis of highly transcribed hsp70 (20) and ribosomal (21) genes as well as of constitutive DHFR genes (22) showed that histones were absent from the promoter regions. Transcription also resulted in decreased DNA cross-linking to the globular (histone fold) domains of the core histones (particularly H2A and H2B) within coding regions (20, 21) and in some essential parts of the 5*- and 3*-flanks (21, 22). However, cross-linking via histone ‘‘tails’’ remained unaffected by the level of transcription (20). This raises the possibility that upon passage of the transcription machinery, nucleosomal DNA is transiently released from the surface of the histone octamer but retains the contacts with the histone tails, in a manner reminiscent of the process suggested by Miller et al. (42). Belikov et al. (43) have dramatically enhanced the resolution power of protein image mapping by treating the products of UV light-induced cross-linking by restriction endonucleases and exonuclease III. This results in DNA strands with one end matching a restriction site and another end covalently attached to a protein, and it thus allows the DNP-DNA gel mapping procedure to achieve the same high resolution as does the canonical Mirzabekov protocol. Belikov et al. (43) found that histone H1 and two newly identified nuclosome-binding proteins, rABP50 and rABP70, are precisely positioned at Alu repeat DNA and account for the formation of unusual 240-bp nucleosomal particles at Alu repeats.
ANALYSIS OF STOICHIOMETRY OF NEWLY REPLICATED CHROMATIN Newly replicated DNA may be easily detected upon BrdUrd labeling by an anti-BrdUrd antiserum. This technique was used to probe cross-linked DNA resolved by two-dimensional DNP-DNA electrophoresis (44) to give rise to protein shadows of newly replicated Ehrlich ascites carcinoma chromatin. We found that histone H1 is deposited on nascent DNA simultaneously with or very soon after the core histones. Within 3 min fol-
FIG. 4. Cross-linked peptide mapping. (A) Preparation and analysis of 32P-labeled nucleotide-tagged peptides. (B) Two-dimensional gel electrophoresis is a powerful way to resolve multiple cross-linked peptides. Cross-linking of a purified globule domain of histone H5 with DNA (left) proceeds almost exclusively via His25 (arrows) and His62 (arrowheads). In contrast, cross-linking of the full-length histone H5 (right) gives rise to numerous additional 32P-labeled nucleotide-tagged peptides. (C) Pseudo-2D protein electrophoresis followed by crosslinked peptide analysis (24). A heterogenous population of histone H5-containing nucleosomes (170–200 bp in length) was cross-linked, and an aliquot was 125I-labeled and resolved on a 2-dimensional DNP-protein gel (both the first-dimension lane and the 2D gel are shown). The bulk of the products of cross-linking proteins was resolved in a parallel lane of the first-dimension gel, and the proteins were liberated by DNA acid hydrolysis and eluted from three gel sections, which correspond to the positions marked on the 2D gel (with approximate lengths of the linker histone bound DNA chains of ca. 170–200, 150–170, and 130–150 nt, correspondingly). After 5* labeling and tryptic digestion, the nucleotide-tagged peptides were resolved by acid electrophoresis (inset). Notably, cross-linking via His25 (arrow) occurs proximally to and possibly within the core particle boundary.
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lowing replication, the stoichiometry of the newly replicated chromatin was the same as that of the bulk chromatin. The interaction of histones with the newly replicated DNA was weaker than in the bulk chromatin, although the efficiency of cross-linking via the histone fold domains seems to be the same in both types of chromatin.
DNA-BINDING DOMAIN/PEPTIDE IDENTIFICATION The task of identifying DNA attachment sites on a protein is complicated by the fact that cross-linking often involves multiple amino acid residues of a protein. The strategy is to trim the DNA chain of a crosslinked complex so as to leave only a small nucleotide tag covalently linked to protein (45). This is achieved by acidic hydrolysis of DNA in the presence of diphenylamine and/or by extensive exonucleolytic digestion (e.g., by MNase at pH 9.0). Upon polynucleotide kinase labeling of the cross-linking site, the proteins are purified by gel electrophoresis (Fig. 4A). The technique is essentially very close to the technique described above for DNP-protein 2D electrophoresis. Indeed, the nucleotide-tagged proteins may be successfully eluted and analyzed from DNP-protein 2D gels (46). A more convenient ‘‘pseudo-two-dimensional’’ technique for separating nucleotide-tagged proteins de-
rived from different DNA segments (24) supplements chemical digestion of DNA directly in the gel by elution, dephosphorylation, and 32P labeling prior to separation in a new SDS gel, which effectively plays the role of second dimension of a canonical 2D electrophoresis (Fig. 4C). The nucleotide-tagged peptides are excised by a specific protease (e.g., trypsin) and separated by gel electrophoresis (multiple cross-linked peptides, such as the ones derived from linker histone tails, may require a two-dimensional peptide electrophoresis (Fig. 4B)). An indirect strategy for peptide identification, based on consecutive cleavages and/or chemical modifications of the peptides, is schematically illustrated in Fig. 4A; e.g., a cross-linked peptide map depicted at the bottom of Fig. 4A would imply that the second slowest peptide comes from a particular proteolytic fragment (since it is present in lane 2) and contains a particular amino acid residue (since it shifts upon its chemical modification, lane 3). In such a manner, a combination of limited trypsinolysis (Fig. 4B), Asp- and Met- specific protein cleavage, and Lys- and Tyr-specific chemical modification allowed us to identify the major DNA cross-linking sites within linker histones H5 (24) and H1 (25). In H5-containing mononucleosomes, the linker histone cross-linking proceeds via the His25 residue of its globular domain. Additional DNA contacts by His62 and the a-amino group of Thr1 emerge in chromatin.
TABLE 1 Histone–DNA Contacts: X-ray Crystallography vs Cross-Linking (Modified from (48)) DNA positiona (turns from the dyad) 031 032 131 132 231 232 331 332 431 432 531, 532 631 632, 731
H3 H4 H4 H3 H3 H4 H2B H2B H2A H2A H2B H2A
Histone contacts as implied from the X-ray crystallography data (1–3, 48)
Histone cross-links (23, 27, 47)b
Loop from C-terminus of long a-helix Loop from N-terminus of long a-helix N-terminus a-helix Penultimate N-terminus a-helix Loop from N-terminus of long a-helix Loop from C-terminus of long a-helix Loop from C-terminus of long a-helix Loop from C-terminus of long a-helix N-terminus a-helix N-terminus a-helix Loop from N-terminus of long a-helix Loop from C-terminus of long a-helix (Not resolved)
H3, H2A C-terminus H4 N terminus H3, H4 N-terminus H3, H4 H3 H4 H2B H2B H2B, H2A H2B H2A, H2B H3 H2A (histone fold), H3
a The DNA site designations are adopted from (48); i.e., the n32 positions are on the sugar–phosphate chain with the distal portion being a 3* terminus, and n31 positions are on the opposite strand (with its distal portion being a 5* terminus). Only the contacts on one-half of the nucleosome are shown; the contacts on the remaining half are assumed to be identical. b In random sequence nucleosome cores.
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In the sea urchin sperm histone H1, one of the two principal cross-linking sites is homologous to His25 of histone H5, while the other site is found within its putative DNA-binding a-helix. The DNA cross-linking site of histone H4 , His18 of its N-terminal ‘‘tail,’’ was identified by synthesis from purified components and a combination of HPLC and mass spectroscopy (23).
NUCLEOSOME STRUCTURE AS PROBED BY DNA–PROTEIN CROSS-LINKING The first zero-length cross-linking studies of the Mirzabekov laboratory (11, 12) provided high-resolution data on the sequential arrangement of histones along the nucleosomal DNA. Subsequent cross-linking studies proved that the fine structure of the nucleosome core is invariable throughout the three eukaryotic kingdoms (animals, plants, and fungi) (47). Contacts of histones H3 and H4 with DNA were
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found to be concentrated in more central DNA positions, while histone H2A and H2B cross-linking occurred mostly at the periphery of nucleosomal DNA, with the bulk of the cross-linking occurring at one side of the DNA helix (Fig. 6D, Table 1). These results played a crucial role in shaping our current view of the nucleosome as that of a tripartite histone complex with DNA wrapped around it. Nucleosome X-ray diffraction studies have only recently achieved a level of resolution sufficient to reach (but not yet to surpass) the resolution of cross-linking – 2D gel mapping (48) (Table 1). Cross-linking analysis has been the most demanding assay used thus far to prove that reconstitution of the core particles from purified histones generates particles indistinguishable from the nucleosome cores excised from the bulk chromatin (49). This finding opened the way for in vitro studies of the role of the nucleosomes within specific gene contexts. The reconstitution/cross-linking approach applied to the 5S rRNA gene nucleosomes (15) provided a single nucleotide resolution of the histone –
FIG. 5. (A) A DNP-DNA 2D gel reveals core histone tail contacts with the key TFIIIA binding site of the Xenopus 5S rRNA gene (15). (B) Acetylation of the lysine residues of the tails allows TFIIIA to bind: a model.
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DNA contacts (Fig. 2B) and resolved an array of additional core histone – DNA contacts on the outer surface of the nucleosome DNA superhelix, as well as in the linker DNA.
INTERACTION OF THE HISTONE ‘‘TAILS’’ WITH DNA The extended flexible ‘‘tail’’ domains of the core histones play an extremely important role in gene regulation, as evidenced by a large body of data on tail posttranslational modification, mutagenesis, and proteolysis (reviewed in 50–52), yet virtually nothing is known about their place within chromatin structure (2, 48). Cross-linking remains essentially the only method capable of identifying the transient and/or flexible contacts of the tails with DNA.
FIG. 6. Linker DNA and the conformational transitions within the nucleosomal core. (A–C) Schematic drawings of a condensed polynucleosomal ‘‘solenoid’’ (A), an extended polynucleosomal fiber (note that changed shape of the nucleosomes) (B), and isolated mononucleosomes (C) (53). (D) The transition as manifested by a shift in the pattern of histone–DNA cross-linking. Dashed arrows represent the altered contacts that disappear during chromatin unfolding and reappear after core particle isolation (53).
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The first DNA cross-linking site identified within the core histone tails was His18 of histone H4 (23), which may be cross-linked to three adjacent DNA sites at approximately 1.5 helical turns from the dyad axis (see Table 1) where DNA is sharply bent (1) (Fig. 6D). This result suggests that a highly basic amino acid cluster Lys16-Arg-His-Arg-Lys-Val-Leu-Arg23 of the N-terminal tail of histone H4 is involved in the sharp bending of nucleosomal DNA. A different approach used limited proteolysis of the core particle in addition to the standard cross-linking– 2D gel procedure to pinpoint DNA localization of the C-terminal tail of histone H2A (27). We have mapped protein–DNA contacts in nucleosomes either with both N- and C-termini or with only a C-terminus of histone H2A removed by selective proteolysis (by trypsin or clostripain, respectively). We found that a peptide comprising the 10 most C-terminal amino acid residues contacts the DNA at the nucleosome dyad axis (an earlier cross-linked peptide study has identified His122 of this peptide as a site of cross-linking (26)). This contact appears only in the absence of linker DNA. Nuclease digestion study indicated that e-terminal domain of histone H2A may interact with linker DNA (52a). The rearrangement of the CH2A tail from the linker to the nucleosome dyad may play a biological role during chromatin unfolding and in the retention of the H2A–H2B dimer (or the whole octamer) during the passing of polymerases through the nucleosome. Cross-linking of the 5S rRNA gene nucleosomes has been mapped with a high resolution sufficient to determine the rotational orientation of histone–DNA contacts (15). The contacts at the outer surface of the nucleosomal DNA are presumably made by the peptides adjacent to the histone tails. In addition to the contacts of histones H4 and H2A tails mentioned above, histone H3 cross-linking a half-helical turn away from the nucleosome dyad axis and several histone H3, H2A, and H2B contacts between 4 and 7 DNA turns from the dyad were localized. These core histone linker DNA interactions are likely to be responsible for deacetylation-dependent inhibition of TFIIIA binding by the core histone tails (54) (Fig. 5).
CROSS-LINKING STUDIES OF THE LINKER HISTONES AND HIGHER-ORDER CHROMATIN STRUCTURE Chromatin unfolding alters core particle structure. Recent cross-linking study of the conformation of the nucleosome cores within polynucleosomal arrays (53), demonstrated a reversible conformational change caused by stretching of the linker DNA during chroma-
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tin unfolding (Fig. 6). The so-called ‘‘stretched nucleosome’’ conformation is characterized by altered strength of the H4 and H2A/H2B-DNA contacts with the regions of sharply bent nucleosomal DNA in ca. {1, 4, and 5 helical turns from the nucleosome twofold symmetry axis, respectively (Fig. 6D). These conformational changes may affect binding of sequence-specific factors to nucleosomal DNA. The path of internucleosomal linker DNA in chromatin is a major matter of controversy. The existing concepts of chromatin fiber structure are based on the opposite concepts of linker DNA organization. All multihelical models of 30-nm fibers (55–59) and the ‘‘accordion’’ model (60) suggest that the DNA between the contiguous nucleosomes is extended. The nucleosomes are therefore arranged in a ‘‘zigzag’’ structure. Other models of 30-nm chromatin fibers are based on the idea that the linker DNA forms a superhelical coil, much like the nucleosomal core DNA (61–63). Histone cross-linking with the linker DNA may provide the crucial body of data to distinguish between these models. The interaction of the linker histone globule with linker DNA adjacent to the nucleosome core boundaries is well documented by electron microscopy (64) and nuclease digestion experiments (65, 66). Our DNA– protein cross-linking study of the highly condensed chromatin within sea urchin sperm nuclei (14) unexpectedly revealed interactions of the globular part of histone H1 with the central region of internucleosomal linker DNA, whereas the DNA segments immediately adjacent to the edges of the core particle remained essentially free of H1 interactions. We also found unambiguous interactions of the globular part of core histones (H3 and H2B) along the entire length of long (ca. 90 bp) linker DNA (14). These findings would be difficult to explain within a model in which the linker DNA is extended beyond the nucleosome boundaries, e.g., if it links the neighboring particles in a zigzag configuration. We suggest that in the 30-nm chromatin fibers of highly condensed chromatin, both nucleosomal DNA and linker DNA form a continuous superhelix (Fig.7). Chromatin decondensation is accompanied by a rearrangement of histone H1 toward the boundaries of the core particles, providing an appearance of zigzag structure. This transition is also manifested by a dramatic change in the linker histone cross-linking peptide pattern. (24). Histone H5 residue His25 is an exclusive cross-linking site in mononucleosomes and decondensed chromatin arrays, and it binds to DNA in the vicinity of the nucleosome core boundaries (Fig. 4C). In contrast, His62 is most effectively cross-linked to DNA within highly condensed chromatin, and the bulk of the cross-linking occurs at a distance from the nucleosomes. The higher resolution of 2-dimensional gel mapping,
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which has become available with the use of the nucleosomes reconstituted at specific DNA sequences, allowed us for the first time to localize linker histone– DNA cross-linking in an isolated nucleosome with a single nucleotide precision (16). As expected, the crosslinking occurred between the linker histone globule and a terminal segment of the nucleosomal DNA. Importantly, however, the precise position of the GH5– DNA contact found in our experiments appears to exclude any chromatosome model that places the globule between the two ends of the nucleosomal DNA, roughly across the nucleosome dyad axis. Our data suggest a drastically asymmetric chromatosome structure, with the linker histone globule bound at one edge of the nucleosome (67). The position of linker histones within the nuclear chromatin remains to be studied.
FIG. 7. A model for the arrangement of histones H1, H2B, and H3 on superhelical nucleosomal and spacer DNA in sea urchin sperm nuclei (14). The core DNA together with the spacer forms a lefthanded superhelix containing about 80 bp. Long thin arrows mark the sites of additional interactions of the core histones with the spacer DNA on the adjacent superhelical turn. A short solid arrow indicates the center of the spacer DNA. Histones interacting with the core DNA and spacer DNA are designated C and S, respectively. Distances along the nucleosomal DNA are given by numbers of superhelical turns from the dyad axis.
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14. Bavykin, S. G., Usachenko, S. I., Zalensky, A. O., and Mirzabekov, A. D. (1990) J. Mol. Biol. 212, 495–511.
CONCLUDING REMARKS/FUTURE DIRECTIONS
15. Pruss, D., and Wolffe, A. P. (1993) Biochemistry 32, 6810–6814.
DNA–protein cross-linking is uniquely suited for studying a structure as complex and dynamic as chromatin. Extending the traditional Mirzabekov technique to the nucleoprotein complexes formed at specific DNA sequences will not only dramatically enhance the resolution of the method but also address specific structural details that make the nucleosomes work as a part of the regulatory machinery within the cell nucleus. DNA-binding peptide mapping may pinpoint the DNA contacts by flexible regions of chromatin proteins. Twodimensional cross-link identification methodologies may be used to probe the chromatin structure of particular transcriptionally or replicationally relevant sequences.
16. Hayes, J. J., Pruss, D., and Wolffe, A. P. (1994) Proc. Natl. Acad. Sci. USA 91, 7817–7821. 17. Ebralidze, K. K., Volkov, S. K., Kirpichnikov, M. P., Mirzabekov, A. D., and Baev, A. A. (1986) Dokl. Akad. Nauk. SSSR 287, 1013–6. 18. Kamashev, D. E., Esipova, N. G., Ebralidse, K. K., and Mirzabekov, A. D. (1995) FEBS Lett. 375, 27–30. 19. Chenchick, A., Beabealashvilli, R., and Mirzabekov, A. (1981) FEBS Lett. 128, 46–50. 20. Nacheva, G. A., Guschin, D. Y., Preobrazhenskaya, O. V., Karpov, V. L., Ebralidse, K. K., and Mirzabekov, A. D. (1989) Cell 58, 27–36. 21. Belikov, S. V., Dzherbashyajan, A. R., Preobrazhenskaya, O. V., Karpov, V. L., and Mirzabekov, A. D. (1990) FEBS Lett. 273, 205–207. 22. Pemov, A., Bavykin, S., and Hamlin, J. L. (1995) Biochemistry 34, 2381–2392. 23. Ebralidse, K., Grachev, S., and Mirzabekov, A. (1988) Nature 331, 365–367.
ACKNOWLEDGMENTS
24. Mirzabekov, A. D., Pruss, D. V., and Ebralidse, K. K. (1990) J. Mol. Biol. 211, 479–491.
We thank Drs. S. A. Grachev, A. Mazumder, Y. Pommier, and A. P. Wolffe for stimulating discussions.
25. Pruss, D. V. (1989) Identification of Contact Sites between Histones H1/H5 and DNA at Different Levels of Chromatin Organization. Diss. Cand. Sci. Chim., Moscow. 26. Gushchin, D. Y., Ebralidze, K. K., and Mirzabekov, A. D. (1991) Mol. Biol. 25, 1400–1411.
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