Tài liệu Báo cáo khoa học: The sequentiallity of nucleosomes in the 30 nm chromatin fibre pptx

The sequentiallity of nucleosomes in the 30 nm chromatin fibre Dontcho Z. Staynov 1 and Yana G. Proykova 2 1 Imperial College London, National Heart and Lung Institute, UK 2 School of Earth and Environmental Sciences, University of Portsmouth, UK The DNA is packed on several levels as chromatin in the eukaryotic nucleus. The first level of packing, the highly conserved nucleosome, allows transcrip- tion, after remodelling and ⁄ or histone modifications ⁄ replacements. The nucleosome core particles have been reconstituted and crystallized and their structure solved in detail at 1.9 A ˚ resolution [1–3]. The second level of packing is the transcriptionally dormant 30 nm chro- matin fibre. Understanding its structure, as well as the processes that determine its folding and unfolding, is a prerequisite for studying the epigenetic mechanism, which leads to poised-for-transcription or dormant chromatin [4]. The fibre consists of the entire chroma- tin of the nucleated avian erythrocytes and comprises approximately 85% of the chromatin in other cell types [5]. The structure of chicken erythrocyte chromatin is the most widely studied in the whole nucleus, as well as in solution. Using small angle X-ray and neutron scattering, it has been shown that all the high mole- cular weight material that diffuses out of the nuclei after micrococcal nuclease (MNase) digestion is in the 30 nm fibre conformation. It consists of a regular helix with a diameter of approximately 33 nm and a variable mass per unit length, which approaches 0.6 nucleo- somesÆnm )1 with an 11 nm pitch at 80 mm salt concen- trations. This implies that there are seven nucleosomes per helical turn with their flat surfaces almost parallel to the fibre axis [6–11]. The unusually small cross- sectional radius of gyration (9.5 nm at 80 mm salt) suggests a very compact structure with close nucleo- some–nucleosome contacts. There are several basic models for the structure of the fibre that were proposed in the late 1970s and early 1980s, and some variants have been published subse- quently [4,5,12]. They all comprise regular helices of more or less seven nucleosomes per turn and thus approximately satisfy the results obtained by small angle X-ray and neutron scattering and low resolution electron microscopy with respect to the packing of Keywords 30 nm fibre; chromatin structure; nucleosome Correspondence D. Z. Staynov, Imperial College London, National Heart and Lung Institute, Guy Scadding Building, Dovehouse Street, London SW3 6LY, UK Tel: +44 207 6223644 E-mail: d.staynov@imperial.ac.uk (Received 29 March 2008, revised 20 May 2008, accepted 23 May 2008) doi:10.1111/j.1742-4658.2008.06522.x The folding of eukaryotic DNA into the 30 nm fibre comprises the first level of transcriptionally dormant chromatin. Understanding its structure and the processes of its folding and unfolding is a prerequisite for under- standing the epigenetic regulation in cell differentiation. Although the shape of the fibre and its dimensions and mass per unit length have been described, the path of the internucleosomal linker DNA and the sequential- lity of the nucleosomes in the fibre are poorly understood. In the present study, we have chemically crosslinked adjacent nucleosomes along the helix of chicken erythrocyte oligonucleosome fibres, digested the inter- nucleosomal linker DNA and then examined the digestion products by sucrose gradient sedimentation. We found that the digestion products con- tain considerable amounts of mononucleosomes but less dinucleosomes, which suggests that there are end-discontinuities in the fibres. This can be explained by a nonsequential arrangement of the nucleosomes along the fibre helix. Abbreviations as, acid soluble; DSP, dithiobis-(succinimidyl propionate); EDC, 1-ethyl-3(3-dimethylaminopropyl)-carbodiimide; MNase, micrococcal nuclease. FEBS Journal 275 (2008) 3761–3771 ª 2008 The Authors Journal compilation ª 2008 FEBS 3761 nucleosomes in the fibre. However, they were proposed before the crystal structure of the nucleosome was solved and do not take into account the topological constraints imposed on the relationship with respect to nucleosome orientation and tilt versus chromatin repeat length. Thus, they differ with respect to the ori- entation of the nucleosomes and the path of the linker DNA within the fibre. High definition structures have not been achieved because the native fibres comprise a mixture of differ- ent repeat lengths and could not be crystallized. To avoid this problem, several studies recently reconsti- tuted oligonucleosome arrays on a nucleosome by positioning DNA sequence-repeats differing by multi- ples of 10 or 11 bp [13–16]. Dorigo et al. [14] used cys- teine substituted recombinant core histones with or without linker histone. The electron micrographs of their reconstitutes show flat ribbons with approxi- mately five instead of seven nucleosomes per 11 nm, which do not fold into helical fibres, although they refer to them as two-start helices. To study nucleosome sequentiallity in their reconstitutes, Dorigo et al. [14] crosslinked the samples and digested the linker DNA with a restriction enzyme. The resultant oligonucleo- somes migrate in a ‘native’ gel as a ladder with dimers up to half the size of the original material and support a two-start helix. In a subsequent study by Schalch et al. [16], a reconstituted tetranucleosome with a 20 bp linker DNA was crystallized. It was speculated that this construct allows a two-stranded helix with close nucleosome contacts in which the flat surfaces of the nucleosomes are perpendicular instead of parallel to the fibre axis. Very different results were obtained by Robinson et al. [15] using native chicken erythrocyte core histones and H5 linker histone. They observed two helical arrays: one for repeat lengths below 210 bp with a diameter of 33 nm and another for repeat lengths above 210 bp with a diameter of 45 nm and with the flat surfaces of the nucleosomes close to parallel to the fibre axis. The overall shapes of their reconstitutes are very similar to the fibres observed in ‘native’ chromatin. Most striking are the two very different structures presented by the two groups for the 177 bp as well as the 207 and 208 bp repeat lengths, which differ by the presence ⁄ absence of the linker histone. Apparently, the reconstitutes of the two groups cannot represent the same structure and additional evidence is needed. Both groups have discussed their results with respect to discriminating between single-start (sequentially arranged nucleosomes) and two-start nonsequential helices. Other possible nonsequential helices were ignored. Neither group considered the very informa- tive results obtained by DNase I digestion of native chromatin, which produces ‘dinucleosome repeat’ pat- terns. Such patterns in which the even multiples are strong can be produced only if the adjacent nucleo- somes are digested at alternating sites and, thus, the odd multiple fragments are attenuated and the even multiple fragments dominate the pattern. These results unambiguously show that there is a common structure of the fibre in which the consecutive nucleosomes in samples of several different repeat lengths have alter- nating orientations, as extensively discussed elsewhere [5,12,17,18]. The question of the sequentiallity of the nucleo- somes in the fibre is essential. Because a variety of higher order structures might be capable of reconstitu- tion with a repeat-sequence DNA, a key question is how do the reconstitutes of the two groups compare with the fibres obtained from natural chromatin? In the present study, we examined the sequentiallity of the nucleosomes in 30 nm fibres, which diffuse out of chicken erythrocyte nuclei after a mild MNase diges- tion without further manipulations. We used the ratio- nale of Dorigo et al. [14], which involved crosslinking and nuclease digestion. To demarcate the adjacent nucleosomes along the fibre, we used nonspecific protein–protein crosslinkers with two different spans: (a) dithiobis-(succinimidyl propionate) (DSP) (also known as Lomant’s reagent), a cleavable bifunctional ester with 1.2 nm span and a noncleavable, contact-site crosslinker and (b) 1-ethyl-3(3-dimethylaminopropyl)- carbodiimide (EDC). Subsequently, the samples were redigested with MNase and fractionated by sedimenta- tion on sucrose gradients. Instead of obtaining half the size of the original material, we observed only a slight decrease of its size and a considerable number of mononucleosomes. These results do not support the two-start helix arrangement, but a higher order nonse- quential arrangement of the nucleosomes in the fibre with end-defects as described below. Experimental rationale It has been shown that, at moderate ionic strength dif- ferent, crosslinkers can covalently crosslink linker- and core-histones beyond a single nucleosome and thus are able to demarcate adjacent nucleosomes along the helix of the 30 nm fibre [5]. In the present study, we used internucleosome histone crosslinking and subse- quent nuclease digestion to distinguish between differ- ent arrangements of the nucleosomes in the fibre. The rationale is illustrated in Fig. 1. Three topologically different arrangements of the nucleosomes along the fibre have been suggested [5]. Nucleosome sequentiallity in the 30 nm fibre D. Z. Staynov and Y. G. Proykova 3762 FEBS Journal 275 (2008) 3761–3771 ª 2008 The Authors Journal compilation ª 2008 FEBS A sequential arrangement The order of nucleosomes along the fibre helix follows their order along the DNA. If a 9-mer fragment of continuous helix of nucleosomes (Fig. 1Aa) is exten- sively crosslinked, the adjacent nucleosomes will be crosslinked and the continuity of the linker DNA will not be required to keep them together. Figure 1Ab shows the sedimentation profile of an oligonucleosome sample comprising 7- to 9-mers and half the quantity of 6- to 10-mers. After 100% crosslinking and nuclease digestion to mononucleosome size DNA, the sedimen- tation profile of the sample remains the same. The 80% crosslinked sample will show a decrease of the average size of the original oligonucleosome sample and smaller size oligomers will appear (Fig. 1Ac). If the nucleosomes are interdigitated, as shown by Rob- inson et al. [15], there will be more internucleosomal crosslinks, which will stabilize the fibre structure, and the sedimentation profiles of digestion products will be intermediate between those shown in Fig. 1Ab,Ac. A multi-start helix Nucleosomes are arranged in a multistrand sequence and are not consecutive. Figure 1Ba illustrates a rib- bon, which can fold into a two-start helix fibre, with linkers either parallel or perpendicular to its axis. Fig. 1. Schematic presentation of different nucleosome arrangements in the 30 nm fibre and the expected sucrose gradient sedimentation profiles after 100%, 90% or 80% chemical crosslinking of nucleosomes and digestion of the DNA to mononucleosome size of samples con- sisting of a mixture of hepta-, octa- and nonanucleosomes and half the amount of hexa- and decanucleosomes (6- to 10-mer sample). (A) (a) Nonamer of a sequential single helix. (b, c) Sedimentation profiles after crosslinking and subsequent digestion of all DNA linkers of the 6- to 10-mer sample: (b) 100% crosslinked and (c) 80% crosslinked. (B) (a) Nonamer in a two start nonsequential (ribbon) arrangement. Numbers indicate the number of consecutive nucleosomes along the DNA chain. (b–d) Sedimentation profiles of the oligonucleosome sample before digestion (b) and after digestion of 100% crosslinked (c) and 90% crosslinked sample (d). (C) (a) Hexamer in a single helix nonsequential arrangement. (b–d) Sedimentation profiles of the 6- to 10-mer sample: (b) original, (c) after digestion of 100% crosslinked and (d) 90% cross- linked sample. The thick red line demarcates adjacent nucleosomes, expected to be crosslinked. Numbers under the horizontal axes of the sedimentation profiles denote mono- di-, etc. nucleosomes. D. Z. Staynov and Y. G. Proykova Nucleosome sequentiallity in the 30 nm fibre FEBS Journal 275 (2008) 3761–3771 ª 2008 The Authors Journal compilation ª 2008 FEBS 3763 Digestion of the same mixture of 6- to 10-mers (Fig. 1Bb) of a 100% crosslinked sample will produce half the size of the original sample (Fig. 1Bc). If the crosslinking is not complete, smaller size oligomers will appear (Fig. 1Bd) but the maximum of the main peak will be approximately half the size of the original sample. Single-stranded helices with nonsequentially arranged nucleosomes Due to nucleosomes’ nonsequentiallity, the fibres have end-defects (not envisaged in either of the structures shown in Fig. 1A,B); namely, one or two missing end- nucleosomes, as well as one or two end-nucleosomes separated from the rest by longer distances. Thus, these end-nucleosomes are probably non-interacting with the continuous helix and may not be crosslinked to the rest. One such arrangement, the (–3,5) arrange- ment, is shown in Fig. 1Ca [18]. Thus, nuclease diges- tion will shorten the size of the original sample on average by three nucleosomes and will produce a frac- tion of 3 ⁄ n mononucleosomes, where n is the average number of nucleosomes per fragment. The expected sedimentation profiles of the same 6- to 10-mer sam- ple, before and after nuclease digestion, are shown in Fig. 1Cb–d. Because the closely interacting nucleo- somes are always an even number (Fig. 2), digestion will produce a mononucleosome fraction and a mix- ture of even multiples. If some of the end-nucleosomes are crosslinked to the rest via linker–linker or linker– core histone crosslinks, the mononucleosome fraction will be less than 3⁄ n and crosslinking will produce some odd number oligonucleosomes in the digest. Thus, the sedimentation profile might not be as clear- cut as shown in Fig. 1Cc,d, but there would be a mononucleosome fraction and enriched even-multiples of oligonucleosomes in the main fraction. Incomplete (90%) crosslinking will also produce some odd number oligomers. As shown in Fig. 1, the differences among the expected sedimentation profiles of the oligonucleosome samples after crosslinking and MNase digestion are expected to be considerable and some incomplete crosslinking, or cross-chain crosslinking, will not change their characters. Figure 2 shows that, in the nonsequential (–3,5) arrangement, the number of nucleosomes making close contacts is always even [18]. Thus, there are no close contacts in the di- and trinucleosomes, whereas only two nucleosomes are close to each other in tetra- and pentanucleosomes. In hexanucleosomes, there are four nucleosomes in close proximity. Results Chicken erythrocyte nuclei were digested with MNase and the material that diffused out of the nuclei was fractionated by sedimentation through sucrose gradi- ents. All chromatin samples from dinucleosomes to high molecular weight material contained indistin- guishable ratios of linker to core histones (see supple- mentary Fig. S1). Chromatin crosslinked with DSP DSP has been used previously for histone crosslinking to establish the proximity of different histones inside the nucleosome or of nucleosomes in the fibre [5,19]. It is a cleavable crosslinker with two succinimidyl groups, which react with lysines independently. The maximum span of crosslinking is 1.2 nm. A sample of oligonucleosomes, consisting mainly of tetra-, penta-, hexamers, minor tri- and heptamer com- ponents with an average number of nucleosomes in the main peak of 4.9, was extensively crosslinked. It was digested with MNase for different lengths of time and agarose gel electrophoresis of the DNA exhibited the characteristic nucleosome repeat (not shown). After removal of free crosslinker by dialysis, it was fraction- ated on sucrose gradients. Figure 3A,B shows that the di tri tetra penta hexa 1 1 1 1 1 2 2 2 2 2 3 3 3 3 4 4 4 5 5 6 Fig. 2. Schematic presentation of di- to hexanucleosomes in the (–3,5) arrangement. The thick red lines illustrate closely spaced nucleosomes. Numbers indicate consecutive nucleosomes along the DNA chain. Nucleosome sequentiallity in the 30 nm fibre D. Z. Staynov and Y. G. Proykova 3764 FEBS Journal 275 (2008) 3761–3771 ª 2008 The Authors Journal compilation ª 2008 FEBS crosslinking resulted in a partial loss of resolution and a drop in the sedimentation velocities. Digestion with MNase produced 10% mononucleosomes and as little as 3.5% dinucleosomes (Fig. 3C). The average number of nucleosomes per chain in the main peak decreased to 4.1. One third of the optical density sedimented slower than the mononucleosome fraction. It com- prised an acid soluble (as) oligo-nucleotide fraction (14%) at the top of the gradient, and a well-defined band, S, of mononucleosome-size naked DNA (17%). Further MNase digestion (Fig. 3D,E) led to an increase of band S by up to 50%, but did not change the overall result; the proportion of mononucleosomes increased to 15% and dinucleosomes to 7%. The main peak was centered at tetranucleosomes (approximately 40%) with the even numbers (2-, 4- and 6-mers) slightly more pronounced than the odd numbers (3- and 5-mers). At longer times of digestion, more than 50% of the sample was converted into the frac- tion S. Breaking the disulfate bond in the middle of the crosslinker produced only mononucleosomes and the band S. DNA gel electrophoresis of the fractions from Fig. 3D,E showed that approximately 90% of the DNA in the main peak as well as the mononucleo- somes and band S are all in the 140–160 bp size interval (not shown). The high percentage of mono- nucleosomes obtained after MNase digestion with only a small amount of dinucleosomes, as well as the grad- ual decrease of the number of nucleosomes in the main peak, indicates that crosslinking is almost complete in the middle of the fibre, but some of the end-nucleo- somes are not crosslinked to the rest and therefore must originate from end-defects in the fibre. In differ- ent experiments, the mononucleosome fraction was always in the range 0.9–1.6 per chain (and often higher than 1.0). A sample of eight to 12 nucleosomes was cross- linked, digested with MNase, and further digested with trypsin for different lengths of time. The sedimentation profiles are shown in Fig. 4. It is seen that MNase (Fig. 4B) produces a similar profile as in Fig. 3B, with prominent fractions as, S and mononucleosomes, but that di- to penta-nucleosomes are of negligible amounts due to the larger size of the starting material. Absorbance at 254 nm F E D C B A S123456as Distance from top of the gradient Fig. 3. UV absorbance profiles of sucrose gradients of an oligonu- cleosome sample mainly comprising tetra- penta- and hexamers and minor tri- and heptamer components. (A) Control (no crosslink- ing). (B–E) Extensively crosslinked with DSP and digested with 20 unitsÆmL )1 MNase for 0, 8, 16 and 32 min respectively. (F) Showing the sample used in (E) but reduced to break the crosslin- ker. Numerals 1, 2, etc., denote mono-, di-, etc., nucleosomes. Fig. 4. UV absorbance profiles of sucrose gradients of a sample of eight to 12 nucleosomes. (A) Control (no crosslinking). (B–F) Exten- sively crosslinked with DSP, digested with 20 unitsÆmL )1 MNase for 20 min and subsequently digested with trypsin for 0, 1.5, 5, 15 and 45 min. Numerals 1, 2, etc., denote mono-, di-, etc., nucleo- somes. D. Z. Staynov and Y. G. Proykova Nucleosome sequentiallity in the 30 nm fibre FEBS Journal 275 (2008) 3761–3771 ª 2008 The Authors Journal compilation ª 2008 FEBS 3765 Short trypsin digestion times caused an increase of the mononucleosome fraction without appearance of di- to penta-nucleosomes (Fig. 4C,D,E) and all the oligonu- cleosome fractions appeared only after a long digestion (Fig. 4F). Histone gel electrophoresis of the samples cross- linked with DSP showed that crosslinking of core particles produced a histone octamer, whereas cross- linking of oligonucleosomes produced higher multiples corresponding to their size, as reported previously [5]. It is not clear why treatment with DSP slows down the sedimentation of all oligonucleosomes including the mononucleosome fraction. The final product of MNase digestion of all the fractions, including S, is mononu- cleosome size DNA and the ten nucleotide periodicity is preserved in the DNase I digests (not shown). Tryp- sin digestion leads to digest products of the same limit as that observed in the untreated nucleosomes (see supplementary Fig. S2). This suggests that, in all prob- ababilty, DSP changes the buoyant density of the samples without changing the structure of the nucleo- somes. Bearing in mind that the two-succinimidyl groups react independently, some lysines that normally interact with DNA are bound by one of these groups. Therefore, several of their positive charges are neutral- ized and the histone-DNA interactions are weakened, with or without the establishment of covalent bonds with other lysines. Apart from the as oligonucleotides at the top of the gradients, the remainder of the DNA, including the naked DNA in fraction S, comes from digested nucleosomes. Thus, after crosslinking, the nucleosomes must have been intact. However, these nucleosomes become less stable and some of them do not survive the subsequent dialysis. Chromatin crosslinked with EDC The water-soluble carbodiimide EDC has been used to crosslink H1-histone to itself and to core histones. Although it is noncleavable and does not allow easy identification of the crosslinked products, it offers some important advantages over the cleavable crosslin- kers. First, it is a contact-site (zero length) crosslinker and thus it excludes long-range bridges between non- interacting amino acids. Second, it binds to an acidic aminogroup first, and only subsequently makes a pep- tide bond with an adjacent lysine [20]. Thus, it does not interfere with the majority of the lysines that inter- act with DNA and the chromatin structure is less likely to be damaged. In a repetition of the experiments shown in Figs 3 and 4, a sample comprising 6–18 nucleosomes per chain was crosslinked with EDC and sedimented in a sucrose gradient (Fig. 5A). Extensive digestion with MNase, which broke more than 90% of the DNA linkers according to DNA electrophoresis, produced 13.5% mononucleosomes (approximately two per chain), 0.5% dinucleosomes and a negligible amount of tri- and tetranucleosomes (Fig. 5B). The average size of the oligonucleosome fraction decreased from approximately 14 to 12 nucleosomes per chain. This result is principally as that obtained by DSP cross- linking, although crosslinking does not change the sedimentation velocity of the samples and does not produce the free DNA fraction S. It is interesting that MNase digestion produces less than three mononucleo- somes per chain, even after crosslinking with this zero length crosslinker. Most probably, some of the end- nucleosomes are crosslinked to the rest via linker– linker or linker–core histone links. Digestion of this material with trypsin initially caused a gradual decrease of the number of nucleosomes per chain in the main peak, with a corresponding increase in the Fig. 5. UV absorbance profiles of sucrose gradients of an oligonu- cleosome sample of six to 18 nucleosomes per chain crosslinked with EDC. (A) Crosslinked but undigested. The profile of the un- crosslinked sample is identical to (A) (not shown). (B–D) Digested with 20 units ÆmL)1 MNase at 37 °C for 40 min and with trypsin for (B) 0 min; (C) 0.5 lgÆmL )1 trypsin for 6 min; and (D) 2 lgÆmL )1 tryp- sin for 30 min. Nucleosome sequentiallity in the 30 nm fibre D. Z. Staynov and Y. G. Proykova 3766 FEBS Journal 275 (2008) 3761–3771 ª 2008 The Authors Journal compilation ª 2008 FEBS percentage of mononucleosomes by up to 25%; approximately 2.7 nucleosomes per chain (Fig. 5C). The oligonucleosome fraction has a maximum at ten nucleosomes and two shoulders around six and eight nucleosome sizes. There are other shoulders beyond ten nucleosomes but their sizes cannot be estimated accurately (Fig. 5C). As with the samples that were crosslinked with DSP, after extensive digestion with trypsin, almost all of the material was converted into mononucleosomes (Fig. 5D). This experiment was repeated several times with preparations consisting of a different number of nucleosomes per chain and crosslinked for different lengths of time (from 30 min up to 5 h) in a 30–80 mm Na + ion concentration. The proportion of mononucleosomes in the sucrose gradi- ents after MNase digestion was always more than two per chain. Most probably, the histones were cross- linked mainly via their N- and C-terminal tails and extensive digestion with trypsin separated them from each other (Fig. 5D). Because the EDC crosslink is not cleavable, how far the histones are digested by trypsin in the oligo- nucleosome fraction cannot be determined but trypsin digestion of crosslinked mononucleosomes shows very similar digest limits to the noncrosslinked mono- nucleosomes (see supplementary Fig. S3). Crosslinking with EDC does not change the sedimentation veloci- ties, nor does it cause sliding of the nucleosomes along the DNA. The repeat length is preserved and the background is low (see supplementary Fig. S4A). It only slows down the digestion rate of the MNase to approximately one quarter of the original rate (see supplementary Fig. S4B). Prior trypsin digestion of this material increases the rate of digestion by MNase several fold without causing nucleosome sliding (see supplementary Fig. S4C). There is no indication that DNA is crosslinked to histones and thereby protected from nuclease digestion. Some crosslinked samples were dialyzed against distilled water and dried. Poly- acrylamide gels of this material were stained first for DNA and subsequently for proteins. It was seen that the DNA and histones moved independently. Further- more, when this material was first dissolved in 0.25 m HCl and the supernatant was precipitated with 20% trichloroacetic acid, no DNA was observed in the gels (results not shown). Thus, the histones are not cross- linked to DNA and the reduction in digestion rate and its recovery after partial trypsin treatment suggests that the histone–histone crosslinking has introduced some steric hindrance to nuclease around the DNA linkers (i.e. the linkers are buried inside the fibre). Discussion When compared with the expected results from the three topologically different arrangements in the Experimental rationale, our results are incompatible with the single-helix sequential and the two-start helix arrangements (Fig. 1A,B) because neither would yield end-of-fibre discontinuities. The results are consistent, however, with the (–3,5) nonsequential arrangement shown in Fig. 1C [18] or some other unenvisaged non- sequential nucleosome arrangement. The sedimentation profiles of the chromatin fragments digested with MNase after crosslinking with two very different cross- linkers show remarkable similarities and must reflect the actual proximities of the nucleosomes in the fibre. There is a considerable amount of mononucleosomes and much less di- ⁄ trinucleosomes in the products. The mononucleosomes evidently come from the ends of the fibre because of the corresponding decrease of the average number of nucleosomes per chain in the main peak. Digestion of the EDC crosslinked samples with MNase produced less than the three mononucleo- somes per chain expected for the (–3,5) arrangement, but partial trypsin digestion, which cuts the linker his- tone tails first, increased their number to approxi- mately three per chain, with a negligible increase of di- and trinucleosomes. In other similar experiments with EDC, there were between two and three mono- nucleosomes per chain. Thus, some of the end-nucleo- somes are crosslinked to the rest, even by the zero-length crosslinker, perhaps via linker histones H1 and H5. Indeed, there is evidence that the tails of these histones follow the path of the linker DNA [5]. They are crosslinked first to each other first and subse- quently to the core histones [19]. The even number nucleosome fragments (six, eight and ten) is more prominent in the main peak, as expected from Fig. 2. The rest of the nucleosomes must interact either directly or via histone tails and are also crosslinked by the zero-length crosslinker. Early in trypsin digestion, when the tails of all H1 and H5 and some H3 histones were digested, only end-nucleosomes become separated (Fig. 5C), whereas, after breaking the core histone tails, all oligonucleosome sizes appeared and, when the trypsin limit digest was approached, the whole sample was converted to mononucleosomes (Fig. 5D). Two alternative explanations of these results were consid- ered but both appear very unlikely. In the first explanation, oligonucleosomes smaller than 6-mers have different hydrodynamic behaviour compared to longer oligomers and such short oligo- mers might not fold into a fibre [21]. Such oligomers D. Z. Staynov and Y. G. Proykova Nucleosome sequentiallity in the 30 nm fibre FEBS Journal 275 (2008) 3761–3771 ª 2008 The Authors Journal compilation ª 2008 FEBS 3767 would not be crosslinked and, after MNase digestion, they will produce only mononucleosomes, whereas, in the longer oligomers, adjacent nucleosomes will be crosslinked to each other. If this were the case, the sucrose gradient profiles would be very different from those observed. Because the mononucleosomes would be produced from the shorter oligomers, the average number of nucleosomes in the main peak would increase, and not decrease as actually observed. More- over, the gradients shown in Fig. 3C–E show that con- siderable amounts of nucleosomes are crosslinked, even in a mixture of tetra- to hexanucleosomes, and suggest that there are closely interacting nucleosomes in such short oligonucleosomes. For the second explanation, it was reported that oligomers tend to lose some H1 and H5 histones and the loss is approximately inversely proportional to the size of the fragments [22]. It can be speculated that, in a sequentially folded fibre, the lost H1 and H5 histones come from the ends of the fibre and the end-nucleo- somes therefore no longer interact with the remainder. This explanation is highly unlikely for the two reasons. First, early during trypsin digestion when it is mainly the linker histones that are cut, more end-nucleosomes are converted into mononucleosomes. These nucleo- somes must therefore have been crosslinked to the rest via the linker histones (i.e. linker histones were present at the ends of the fibre). Second, the loss of H1 and H5 depends on the procedure of chromatin extraction. We used the same protocol of mild digestion with MNase to footprint H1 ⁄ H5 histones on the chromato- some and found that only the mononucleosome frac- tion loses some of the linker histones. The ratio of H5 to H4 histones (which have similar abundances and can be assessed quantitatively) in the dinucleosome sample is indistinguishable from that of high-molecular weight chromatin. Furthermore, in the DNase I foot- print of the dinucleosome, the band resulting from a cut on the dyad axis (Band S0 at 70 bp) becomes undetectable as a result of protection by linker histone [23]. How then do the present results compare with those from the reconstituted fibres of Dorigo et al. [14] and Robinson et al. [15]? Both groups show real regular structures, as indicated by electon micrographs. They both considered two cases: a continuous helix and a two-strand fibre and they neglected any possible non- sequential arrangement of the nucleosomes (Fig. 1C). Nor did they consider the topological constraints imposed by the length of the linker DNA on the tilts of the nucleosomes, as discussed elsewhere [24]. Dorigo et al. [14] studied the sequentiallity of the nucleosomes in the fibre using crosslinking and digestion rationale but with cysteine-substituted recom- binant histone mutants. They obtained unambiguous proof that their reconstitutes are two-start ribbons. The most probable explanation for the discrepancy between the results obtained by Dorigo et al. [14] and those of the present study is that we examined differ- ent structures and the reconstitutes used do not repre- sent the native 30 nm fibre of higher eukaryotes. In the selected micrographs of Dorigo et al. [14] (see Figs 4 and S2 therein), the samples appear less con- densed and look very different from previously pub- lished micrographs of 30 nm fibres [25], as well as their Fig. 1. The reconstituted decamers, which are large enough to be in the fibre conformation [21], are flat ribbons with approximately five instead of seven nucleosomes per 11 nm. The reconstituted oligonucleosomes of Dorigo et al. [14] can be crosslinked in the absence of H1 histone, suggesting that H1 is not required for the close nucleo- some–nucleosome contacts. However, H1 histone is essential for the fibre stability of chicken erythrocyte chromatin [5,26]. The loss of linker histone causes exposure of the linker DNA to DNase I [27] and leads to shortening of the chromatin repeat length in the mouse [28]. It can be speculated that the particular repeat lengths used by Dorigo et al. [14] bring the nucleosomes into contact. Other causes for the differ- ences between reconstituted and native fibres might be the type of the crosslinking used, which, in the study by Dorigo et al. [14], comprised selective crosslinking using cysteine modified core histones that may facili- tate direct nucleosome interactions. The crystallized tetranucleosome [16] from the same laboratory is out- side the repeat length interval of higher eukaryotic chromatin and might have relevance to viral, telomeric or yeast chromatin in which the presence of linker his- tone is questionable. Furthermore, the presence of all oligomers, with dimers up to the half the size of the original samples after digestion of the linkers, was sug- gested by Dorigo et al. [14] to indicate incomplete crosslinking. However, incomplete but random cross- linking should produce a Poisson distribution of all sizes with a single maximum. In their gels, there are two maxima. As shown in Fig. 3A,B of Dorigo et al. [14], both the even multiples (tetra- and hexamers) are more abundant than the pentamers. This suggests that either the samples comprised a mixture of different structures, the crosslinking is not random, or the nucleosomes are nonsequentially arranged (not dis- cussed in their study). The two structures reported by Robinson et al. [15] are regular helices and conform to the parameters of the natural fibres with corresponding repeat length Nucleosome sequentiallity in the 30 nm fibre D. Z. Staynov and Y. G. Proykova 3768 FEBS Journal 275 (2008) 3761–3771 ª 2008 The Authors Journal compilation ª 2008 FEBS intervals. Robinson et al. [15] did not examine the sequentiallity of the nucleosomes, nor did they identify the path of the linker DNA or the location of the linker histones. They have assumed that the nucleo- somes follow the fibre helix and that the linker histones define the different paths of the linker DNA for different repeat lengths. It is unclear, however, if this would be the case because they used the same linker histone for all repeat lengths [24]. Accordingly, their results do not contradict the results provided in the present study and, thus, further experiments are required to check for compatibility. Nevertheless, the reconstitutes of Robinson et al. [15] are a good start for further high definition structural studies. Conclusions The present study demonstrates that, after crosslinking of oligonucleosomes from native nuclei with two different crosslinkers followed by nuclease digestion, there is a gradual decrease of the size of the main frac- tion, and mainly mononucleosomes are liberated. These mononucleosomes evidently come from end- discontinuities in the fibre, which can be explained only by nonsequential arrangements of the nucleo- somes along the fibre helix. The shoulders in the digests that represent even-numbered nucleosome frag- ments (Fig. 5), as well as the stronger tetra- and hexa- nucleosome bands shown in Fig. 3A,B in the study by Dorigo et al. [14], suggest a nonsequential arrangement of the nucleosomes; whether this is the (–3,5) arrange- ment, or some other as yet unenvisaged structure with end-defects, remains to be seen. Experimental procedures Preparation of chromatin samples To avoid irreversible damage of the fibre and to minimize the redistribution of linker histones, all crosslinking and sucrose gradient fractionation experiments were carried out at Na + ion concentrations in the range 25–60 mm. Chicken erythrocyte nuclei, freshly prepared or frozen at )70 °Cin 40% glycerol, 10 mm Tris–HCl (pH 7.6), 6 mm MgCl 2 , 25 mm KCl, 35 mm NaCl, 0.2 mm phenylmethanesulfonyl fluoride, were washed and suspended at 6 mgÆmL )1 DNA in digestion buffer [0.25 m sucrose, 1 mm CaCl 2 ,5mm Tris–HCl (pH 7.6), 60 mm NaCl]. They were digested with 33 unitsÆmL )1 MNase at 37 °C for 10 min, terminated with 5mm EDTA at final concentration and pelleted by centri- fugation at 2300 g for 1 min in a microcentrifuge. The supernatant contained approximately 10–15% of the DNA and consisted mainly of acid-soluble material and mononucleosomes with a lower amount of H1 and H5 hi- stones. The pellet was resuspended in 1 mm EDTA, 5 mm Tris–HCl (pH 7.6) and 25 mm NaCl, dialysed against the same buffer overnight and pelleted again. The supernatant usually contained 60–70% of the total DNA and all the histones. It was fractionated according to size in 6–40% isokinetic sucrose gradients in the same buffer on an SW27 Beckman rotor (Beckman Coulter, Fullerton, CA, USA) at 5 °C, 29 500 g. for different times. The material consisted of a mixture of mono- to 30–40 nucleosomes size frag- ments, with the most abundant comprising 10–20 nucleo- somes. Dialysis of the fractionated samples against higher salt concentration buffers (15–40 mm NaH 2 PO 4 , pH 8.0) was performed when the sucrose was dialysed out. In this way, no apparent aggregation was observed. We have pre- viously shown that, when using this protocol for the isola- tion of chromatin, only mononucleosomes lost some of the linker histones [23]. All samples that were used contained equal ratios of linker to core histones. Histone gel electrophoresis of samples used in Figs 3–5 are shown in the supplementary Fig. S1. Although the mean sizes of the three samples are approximately 5, 10 and 14 nucleosomes per chain, respec- tively, the ratio of intensities of the bands of H5 to H4 remains the same. In some experiments, the nuclei were digested with 60 unitsÆmL )1 MNase and oligonucleosomes were extracted directly with 60 mm NaCl, 5 mm Tris–HCl (pH 7.6) and 5mm EDTA. The supernatant contained 40–50% of the total DNA up to 15–20 nucleosomes long. This material did not show any difference in histone content compared to the samples shown in Fig. S1. Crosslinking with DSP Crosslinking with DSP (Pierce, Rockford, IL, USA) was carried out in 15–40 mm NaH 2 PO 4 (pH 8), 1 mm EDTA (31–59 mm Na + ions) with 3 mgÆmL )1 DSP at room temperature for different times and terminated with 15 mm glycine and the crosslinker dialysed out. We started with 5 h of crosslinking, but subsequently found out that 10 min was sufficient. Crosslinking with EDC Crosslinking with EDC (Pierce) was carried out in 22–40 mm NaH 2 PO 4 (pH 6.8), 1 mm EDTA (31–62 mm Na + ions) with 5 mgÆmL )1 EDC at room temperature for 30 min to 12 h and terminated with 1% 2-mercaptoethanol. No difference was found for the results obtained using different times for crosslinking. Redigestion of crosslinked oligonucleosome samples with MNase was carried out in 30 mm NaH 2 PO 4 (pH 7.6), 1.2 mm CaCl 2 , either at 37 °Corat6°C and terminated D. Z. Staynov and Y. G. Proykova Nucleosome sequentiallity in the 30 nm fibre FEBS Journal 275 (2008) 3761–3771 ª 2008 The Authors Journal compilation ª 2008 FEBS 3769 with 5 mm EDTA. Digestion with trypsin (type II; Sigma, St Louis, MO, USA) was carried out at room temperature and terminated with soybean trypsin inhibitor (Sigma). Isokinetic sucrose gradients (6–40%) of crosslinked and (or) redigested material were run in 30 mm NaH 2 PO 4 (pH 6.8), 5 mm EDTA (46 mm Na + ions) in a SW41 rotor (Beckman) at 5 °C (200 000 g for 12–18 h). Agarose gel electrophoresis of DNA was carried out in 2% gels in 30 mm NaH 2 PO 4 (pH 6.8), 0.5 mgÆL )1 ethidium bromide. Additional results on linker histone abundance and MNase and trypsin digestions are presented in the supple- mentary Figs S1–S4. Acknowledgements We are grateful to Drs Daniela Rhodes and Venki Ramakrishnan for useful discussions. Funding by Wellcome Trust grant no. 037008 to D. Z. S. is grate- fully acknowledged. 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Fan Y, Nikitina T, Morin-Kensicki EM, Zhao J, Magnuson TR, Woodcock CL & Skoultchi AI (2003) H1 linker histones are essential for mouse development and affect nucleosome spacing in vivo Mol Cell Biol 23, 4559–4572 Nucleosome sequentiallity in the 30 nm fibre Fig S1 Linker to core histones ratios Fig S2 Trypsin digestion of samples crosslinked with DSP Fig S3 Trypsin digestion of samples crosslinked with... digests of samples crosslinked with EDC This material is available as part of the online article from http://www.blackwell-synergy.com Please note: Blackwell Publishing are not responsible for the content or functionality of any supplementary materials supplied by the authors Any queries (other than missing material) should be directed to the corresponding author for the article Supplementary material The. .. supplied by the authors Any queries (other than missing material) should be directed to the corresponding author for the article Supplementary material The following supplementary material is available online: FEBS Journal 275 (2008) 3761–3771 ª 2008 The Authors Journal compilation ª 2008 FEBS 3771 . decrease of the number of nucleosomes in the main peak, indicates that crosslinking is almost complete in the middle of the fibre, but some of the end-nucleo- somes. do the reconstitutes of the two groups compare with the fibres obtained from natural chromatin? In the present study, we examined the sequentiallity of the