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Short nucleosome repeats impose rotational modulations on chromatin fibre folding

2012; Springer Nature; Volume: 31; Issue: 10 Linguagem: Inglês

10.1038/emboj.2012.80

1460-2075

Sarah Correll, Michael Schubert, Sergei A. Grigoryev,

RNA Research and Splicing

Article30 March 2012free access Short nucleosome repeats impose rotational modulations on chromatin fibre folding Sarah J Correll Sarah J Correll Department of Biochemistry and Molecular Biology, Hershey Medical Center, Pennsylvania State University, College of Medicine, Hershey, PA, USAPresent address: Division of Human Genetics, The Children's Hospital of Philadelphia, Philadelphia, PA 19104, USA. Search for more papers by this author Michaela H Schubert Michaela H Schubert Department of Biochemistry and Molecular Biology, Hershey Medical Center, Pennsylvania State University, College of Medicine, Hershey, PA, USA Search for more papers by this author Sergei A Grigoryev Corresponding Author Sergei A Grigoryev Department of Biochemistry and Molecular Biology, Hershey Medical Center, Pennsylvania State University, College of Medicine, Hershey, PA, USA Search for more papers by this author Sarah J Correll Sarah J Correll Department of Biochemistry and Molecular Biology, Hershey Medical Center, Pennsylvania State University, College of Medicine, Hershey, PA, USAPresent address: Division of Human Genetics, The Children's Hospital of Philadelphia, Philadelphia, PA 19104, USA. Search for more papers by this author Michaela H Schubert Michaela H Schubert Department of Biochemistry and Molecular Biology, Hershey Medical Center, Pennsylvania State University, College of Medicine, Hershey, PA, USA Search for more papers by this author Sergei A Grigoryev Corresponding Author Sergei A Grigoryev Department of Biochemistry and Molecular Biology, Hershey Medical Center, Pennsylvania State University, College of Medicine, Hershey, PA, USA Search for more papers by this author Author Information Sarah J Correll1, Michaela H Schubert1 and Sergei A Grigoryev 1 1Department of Biochemistry and Molecular Biology, Hershey Medical Center, Pennsylvania State University, College of Medicine, Hershey, PA, USA *Corresponding author. Department of Biochemistry and Molecular Biology, Hershey Medical Center, Pennsylvania State University, College of Medicine, 500 University Drive, H171, P.O. Box 850, Hershey, PA 17033-0850, USA. Tel.:+1 717 531 8588; Fax: +1 717 531 7072; E-mail: [email protected] The EMBO Journal (2012)31:2416-2426https://doi.org/10.1038/emboj.2012.80 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info In eukaryotic cells, DNA is organized into arrays of repeated nucleosomes where the shorter nucleosome repeat length (NRL) types are associated with transcriptionally active chromatin. Here, we tested a hypothesis that systematic variations in the NRL influence nucleosome array folding into higher-order structures. For NRLs with fixed rotational settings, we observed a negative correlation between NRL and chromatin folding. Rotational variations within a range of longer NRLs (188 bp and above) typical of repressed chromatin in differentiated cells did not reveal any changes in chromatin folding. In sharp contrast, for the shorter NRL range of 165–177 bp, we observed a strong periodic dependence of chromatin folding upon the changes in linker DNA lengths, with the 172 bp repeat found in highly transcribed yeast chromatin imposing an unfolded state of the chromatin fibre that could be reversed by linker histone. Our results suggest that the NRL may direct chromatin higher-order structure into either a nucleosome position-dependent folding for short NRLs typical of transcribed genes or an architectural factor-dependent folding typical of longer NRLs prevailing in eukaryotic heterochromatin. Introduction In eukaryotic cells, the DNA is organized into chromatin structures. At the basic level of compaction, this involves wrapping ∼147 bp of DNA around an octamer of core histones to form the nucleosome core particle (Richmond and Davey, 2003). Nucleosome cores are connected by 10–70 bp of linker DNA forming nucleosome arrays (beads-on-a-string) that are further compacted with the aid of linker histone into higher-order chromosomal structures (Tremethick, 2007; Fussner et al, 2011; Li and Reinberg, 2011; Schlick et al, 2012). One of the key determinants of chromatin structure is the nucleosome repeat length (NRL). The NRL includes the constant length of DNA wrapped around the histone octamer (∼147 bp) and the variable length of linker DNA connecting adjacent nucleosome cores. Linker DNA varies in length between different tissues and in different organisms, producing a range of NRLs from the shortest 155 bp NRL found in fission yeast (Lantermann et al, 2010) to the longest measured NRL occurring in echinoderm sperm (∼240 bp) (Athey et al, 1990). Linker histone levels in chromatin are correlated with the NRL (Pearson et al, 1984; Woodcock et al, 2006). There is also an inverse correlation between linker DNA length and gene activity, where actively transcribed genomes (e.g., yeast, embryonic stem cells, and tumour cells) tend to have shorter NRLs (∼160–189 bp) and mature, transcriptionally inactive genomes (e.g. echinoderm sperm and chicken erythrocytes) have longer NRLs (∼190–240 bp) (Thomas and Furber, 1976; Weintraub, 1978; Athey et al, 1990; Perisic et al, 2010). Biochemical analysis showed that the average NRL in yeast is quantized following the rule of 10n+5 bp per linker (Lohr, 1981); this feature was also notable in the longer-NRL rat liver and chicken erythrocyte chromatin (Strauss and Prunell, 1983; Lohr, 1986). More recently, genome-wide studies of NRLs in yeast and human cells confirmed the quantized 10n+5 bp spacing between yeast nucleosomes (Wang et al, 2008) and showed that, in human chromatin, active genes have a notably shorter repeat (178 bp) than repressed or heterochromatic non-coding sequences (206 bp) (Valouev et al, 2011). While changes in the NRL were initially expected to alter the chromatin fibre diameter proportionally to NRL (Athey et al, 1990), more recent electron microscopic studies of linker histone-dependent chromatin compaction by Rhodes and her colleagues revealed two types of chromatin fibre distinguished by a stepwise increase in chromatin diameter from ∼33 nm for chromatin with 177–207 bp linkers to ∼42 nm for 217–227 bp linkers (Robinson et al, 2006). A third type of structure was observed for very short NRLs typical of yeast and neuronal cells (167 bp). Electron microscopic studies showed that, in contrast to the longer arrays, those with a short 167 bp NRL could fold into compact fibres without linker histone (Routh et al, 2008) in agreement with earlier biochemical observations of linker histone-independent compact folding of 167–177 bp NRL arrays (Dorigo et al, 2003; Shogren-Knaak et al, 2006). The folded 167 bp NRL arrays also had a distinct fibre diameter about 21–23 nm with a clear zigzag morphology (Routh et al, 2008) consistent with nucleosome stacking in the tetranucleosome X-ray crystal structure (Schalch et al, 2005). Due to the helical nature of DNA, smaller alterations to the NRL in the range of several base pairs were predicted to have a strong effect on chromatin fibre folding (Widom, 1992; Woodcock et al, 1993; Leuba et al, 1994; Stehr et al, 2010). Here, we examine whether and how chromatin higher-order folding depends on the internucleosomal rotational setting. This parameter refers to linker DNA length variability under 10.5bp (one turn of the DNA double helix) that is expected to change internucleosomal rotation angle by 36° per 1 bp of DNA length. It is distinct from the more conventional definition of rotational setting as the local orientation of the DNA helix on the histone surface (Jiang and Pugh, 2009), instead referring to the rotational settings relative to adjacent nucleosomes, which may be altered by DNA orientation at the surface and local variations in the linker length. We show that for rotationally similar nucleosomes with different NRLs, there is a general negative correlation of chromatin compaction with DNA linker length. For long-linker arrays, rotational alterations do not affect chromatin folding. In a striking contrast, for shorter NRLs (165–177 bp), we observed a periodic dependence of chromatin fibre folding upon local rotational variations. Our data thus demonstrate that internucleosomal rotational settings are crucial in determining nucleosome packing of arrays with short linker DNA, while long-linker DNAs accommodate rotational variability without a substantial effect on chromatin folding. Our results suggest that internucleosomal rotational settings play a crucial role in the higher-order structure of chromatin with short NRLs such as those found in the yeast genome (Lohr, 1981; Wang et al, 2008) and in transcriptionally active chromatin of human cells (Valouev et al, 2011). Results Nucleosome array folding reflects the intrinsic properties of the underlying NRL Reconstituted nucleosome arrays with various NRLs were assembled using the clone 601 sequence and histone octamers isolated from chicken erythrocyte nuclei. Since precise histone stoichiometry is crucial for proper folding, it was monitored through several independent techniques. First, to ensure that our arrays were not underloaded, the reconstituted arrays were digested with restriction nucleases into mononucleosomes and assayed by agarose DNP electrophoresis in TAE buffer optimized for resolving mononucleosomes and free DNA. On these gels, the presence of free DNA monomer bands was indicative of underloaded material (arrow on Supplementary Figure S1A). Next, the reconstitutes were analysed using agarose DNP gels in HEPES buffer optimized for oligonucleosome separation. On these gels, smearing of carrier DNA indicated that high-affinity (clone 601) templates had been saturated and excess histones had been bound to the low-affinity carrier DNA (Lane 2, Supplementary Figure S1B). These same gels also allowed us to visualize instances where nucleosome arrays were overloaded with histone octamers through the appearance of additional bands above the main array band (brackets on Supplementary Figure S1B). Based on the preliminary experiments, we reconstituted arrays at a larger scale with a typical yield of 100 μg DNA. Agarose DNP gels in parallel with analytical ultracentrifugation provide an efficient way to monitor histone loading in multiple arrays and prepare homogeneous samples for solution and imaging studies (Figure 1). In these experiments, higher mobility of the main electrophoretic band (black arrow, Figure 1A) and sedimentation coefficients S20,w below 28 S (Figure 1B, top panel) indicated underloading and were discarded. Overloaded samples, which showed additional dimer bands above the main array band (white arrows in Figure 1A), also had additional peaks during ultracentrifugation (white arrows in Figure 1B, middle panel) and were discarded. The optimally loaded arrays (grey arrows, Figure 1A and B) were examined by transmission electron microscopy to confirm full saturation of the nucleosome arrays with the majority of the arrays containing 12 nucleosomes by EM (Figure 1C and D). Figure 1.Oligonucleosome reconstitution and characterization. (A) DNP type IV agarose gel in HE buffer, showing reconstituted nucleosome arrays, 169×12 arrays with different histone loadings. Lanes 1–4: 1—DNA molecular weight markers, 2—underloaded (black arrow), 3—overloaded (white arrows), and 4—properly loaded (grey arrow) nucleosome arrays. (B) Distributions of sedimentation coefficients, c(S), for 169×12 arrays with different histone loadings: underloaded (top panel), overloaded (middle panel), and properly loaded (bottom panel) at 0.6 mM MgCl2. (C) Electron micrograph (uranyl acetate staining, dark-field imaging) of 207×12 core arrays (top panels) and 167×12 core arrays (bottom panels) fixed at 5 mM NaCl. (D) Histograms showing distribution of nucleosome arrays containing a certain number of nucleosomes per array calculated from several EM fields of 207×12 arrays (top panel) and 167×12 arrays (bottom panel). Download figure Download PowerPoint The homogeneity and extent of nucleosome array folding for arrays with different NRLs were assayed by sedimentation velocity analysis using the continuous c(S) distribution model (Schuck, 2000) as well as by the boundary analysis of van Holde and Weischet, 1975. For highly homogeneous samples of 167×12 and 207×12 arrays, we observed that the main boundary positions in van Holde–Weischet plots are fully consistent with the main peaks in the c(S) distribution (cf. Figure 2A and B to Supplementary Figure S2A) showing a dramatic difference in sedimentation (36 versus 52 S) at 1 mM MgCl2. We also observed that c(S) analysis was very efficient in resolving sedimentation distribution in heterogeneous systems (cf. Figure 2C to Supplementary Figure S2B). Therefore, we applied the c(S) method for sedimentation analysis of all heterogeneous samples including those partially self-associated due to overloading or excessive ionic strength as well as intentionally heterogeneous samples throughout this work. Figure 2.Nucleosome array folding depends upon the intrinsic properties of the underlying NRL. (A–C) Distribution of sedimentation coefficients, c(S), for 167×12 (A), 207×12 (B), and the 167/207×12 coreconstitute (C) at 1 mM MgCl2. (D–F) Electron micrographs of 167×12 (D), 207×12 (E), and the 167/207×12 coreconstitute (F) at 1 mM MgCl2 showing different degrees of compaction. Download figure Download PowerPoint To independently verify that the observed differences in folding, such as those between arrays with NRLs of 167 and 207 (Figure 2A and B), were not due to variations in histone loading, chromatin arrays were reconstituted with an equimolar mixture of each of 167×12 and 207×12 DNA templates and core histones in the same dialysis bag. These coreconstitutes were then assayed for folding in sedimentation velocity experiments on the analytical ultracentrifuge. The coreconstitutes formed two distinct peaks, indicating two types of particles with different folding extents (Figure 2C), with each one of these double peaks corresponding to the single peak of the uniform array in Figure 2A and B. EM imaging of the 167×12/207×12 coreconstitute further confirms the presence of two distinct states of chromatin folding for 167×12 and 207×12 arrays (Figure 2D and E), as both compact and open arrays are seen in the coreconstituted sample (Figure 2F). NRL with fixed rotational setting is negatively correlated with chromatin folding We constructed and characterized a series of nucleosome core arrays in which the NRL was altered so that due to the 10.5 bp periodicity of DNA, the arrays were expected to maintain the same rotational settings. As such, reconstituted 12-mer chromatin arrays were assembled with NRLs of 167, 177, 188, or 209 bp. While the sedimentation peaks observed at 5 mM NaCl had very similar distributions of sedimentation coefficients around 30 S as expected for unfolded nucleosome arrays (Figure 3A), upon the induction of chromatin folding by 60–150 mM NaCl or 0.6–2 mM MgCl2, we observed a notable increase in sedimentation velocity for all samples (Figure 3B and C and Supplementary Table S3). Remarkably, the salt-dependent increase in S values was much stronger for shorter NRLs—about 20 S for 167×12 compared to a moderate increase of about 5 S for 209×12 at 1 mM MgCl2. This result is consistent with the strong salt-dependent folding previously reported for 167 versus 197 (Routh et al, 2008) and 167 versus 207 (Grigoryev et al, 2009) arrays at the same ionic strength. Now we observed a clear negative correlation between the NRL and the degree of nucleosome array compaction for a range of monovalent and divalent cation concentration known to support compact folding of the nucleosome arrays. Figure 3.Dependence of chromatin compaction upon NRL for nucleosome arrays with constant internucleosomal rotations. Distribution of sedimentation coefficients, c(S), for 12-mer oligonucleosome core arrays with NRL of 167, 177, 188, and 209 bp at 5 mM NaCl (A), 150 mM NaCl (B), and 1 mM MgCl2 (C). Download figure Download PowerPoint Rotational variations do not alter folding for chromatin arrays with long NRL While native chromatin species have NRLs differing ±2 to ±4 from the average repeat lengths (Strauss and Prunell, 1982; Widom, 1992), most experiments with reconstituted nucleosomal arrays employed nucleosomal DNA templates with uniform NRLs. To explore the importance of local variations in the internucleosomal rotations mimicking native chromatin for chromatin folding, we constructed and characterized 12-mer arrays (205–207–209)×4 and (205–209)×6, in which the NRL varied in a defined manner by either ±2 bp or ±4 bp, respectively. These arrays were expected to show structural variations reflecting local changes in the internucleosomal rotation angle (Woodcock et al, 1993). However, for neither of these reconstitutes did we observe any significant difference in chromatin folding between the uniform (207×12) and variable (±2 or±4 bp) arrays at any of the ionic conditions tested (Table I, Supplementary Figure S3A). Table 1. Linker length variations do not affect chromatin folding for uniform and variable 12-mer arrays with long NRLs 5 mM NaCl 60 mM NaCl 150 mM NaCl 0.6 mM MgCl2 1 mM MgCl2 Uniform −LH 29 36 36 35 36 ±2 bp −LH 30 37 38 33 36 ±4 bp −LH 30 36 39 35 36 Uniform +LH 38 53 54 57 60+ ±2 bp +LH 36 55 56 60 60+ ±4 bp +LH 37 52 55 56 58+ Sedimentation coefficient main peak positions (average of three independent experiments) were determined from distributions of sedimentation coefficients, c(S), for 12-mer oligonucleosome core arrays (−LH) and linker arrays (+LH) with uniform (207 bp) and variable (207±2 and 207±4 bp) NRLs at various salt conditions as indicated. Next, the folding of uniform and variable arrays was assayed in the presence of linker histone H5, which is known to be important for the complete folding of nucleosome arrays with 207 bp NRL (Carruthers et al, 1998). As expected, the addition of linker histone increased the sedimentation coefficients to ∼55–60 S in the presence of MgCl2; however, the uniform and variable arrays again had similar sedimentation coefficients both in the presence and absence of linker histone H5 (Table I, Supplementary Figure S3B). To confirm the absence of size dependence of nucleosome folding in our reconstituted arrays, the uniform and ±4-bp arrays were doubled in length to form 24-mer arrays, which were again assayed for folding. There were no significant differences in folding between the uniform and variable 24-mer arrays in either the core arrays or in those containing linker histone H5 (Supplementary Table S2). These results clearly demonstrate that the uniform and variable arrays with net 207 bp NRL are completely folded into the chromatin fibre in the presence of linker histone and that the degree of their folding is independent of internucleosomal rotational settings. Internucleosomal rotational variations affect folding in chromatin arrays with short NRL Next, we asked whether uniform arrays with altered rotational settings would follow a similar pattern of increased NRL leading to decreased S values. To test this, we employed 12-mer core arrays with repeats of 165, 169, 172, 200, 205, and 207 bp, that is, NRL values intermediate between those explored above (167, 177, 188, and 209). Within each array, the rotational setting is constant, but is different relative to the other arrays because the NRL changes are not in increments of DNA helical periodicity (10.5 bp). If the rotational settings did not affect chromatin folding, then we expected the compaction of the nucleosome arrays with intermediate NRLs to fall between the values obtained for arrays with the nearest longer and the nearest shorter NRLs. Indeed, as seen in Figure 4A and B and Supplementary Table S3, we found that core arrays with NRL of 200 bp have similar folding to arrays with NRL of 188 bp (∼38–40 S), and the 205-bp arrays were similar to 207 bp (∼35–36 S). Figure 4.NRL variations affect chromatin folding but not self-association. (A) Graphic plotting of main sedimentation coefficient peaks in c(S) distribution (average of three independent experiments) for 12-mer oligonucleosome core arrays with varying NRL (165,167,169, 172, 177, 188, 200, 205, 207, and 209 bp) at 5 mM NaCl, 60 mM NaCl, or 150 mM NaCl. Student's t-test P-values for significant differences between the data sets are shown over the brackets. (B) Graphic plotting of main sedimentation coefficient peaks in c(S) distribution (average of three independent experiments) for 12-mer oligonucleosome core arrays with varying NRL (165, 167, 169, 172, 177, 188, 200, 205, 207, and 209 bp) at 0.6 mM MgCl2, 1 mM MgCl2, or 2 mM MgCl2. (C) Histograms of the concentration of MgCl2 (average of three independent experiments) that results in 50% precipitation of material for arrays with varying NRL. Download figure Download PowerPoint Surprisingly, in a sharp contrast to the apparently similar folding of the longer-NRL arrays, the shorter arrays displayed strong NRL-dependent structural deviations. Figure 4A and B shows that 172×12 array was dramatically unfolded, with sedimentation coefficient values of 41 S in 150 mM NaCl and 38 S in 1 mM MgCl2, compared to the 167×12 and 177×12 arrays, which have sedimentation coefficients at about 48–54 S under those conditions. The 165×12 and 169×12 arrays showed intermediate folding with S values distributed around 43–47 S in either 150 mM NaCl or 1 mM MgCl2. The NRL-dependent periodic changes of chromatin compaction were also observed for a wider range of Mg2+ concentrations—between 0.6 and 2 mM (Figure 4B). These data show that the unfolded short-NRL arrays (165 and 169) and, especially, the intermediate NRL arrays (188 and 200 bp) undergo additional compaction at 2 mM MgCl2. Sedimentation of neither the most folded or unfolded arrays with short NRLs (167, 172, and 177) nor the unfolded arrays with long NRLs was significantly affected by increased Mg2+, showing that the divalent cation may partially curb the difference between NRLs 165, 167, and 169, but cannot fold the most open arrays (unlike linker histone in experiments shown in Figure 5 below). These results, together with sedimentation analysis of the longer-NRL nucleosome arrays facilitated by linker histone (Table I), show that the internucleosomal rotational settings have a strong periodical effect on chromatin folding for nucleosome core arrays with short NRLs (167–177 bp) but not for the long (>200 bp) NRLs. Our findings are consistent with recent modelling studies predicting a stronger counterion requirement for packing the longer and more negatively charged linkers (Perisic et al, 2010), and the linker DNA geometry being most important for folding of the nucleosome arrays with short NRL (see Discussion). Figure 5.A 5-bp difference in linker DNA length destabilizes chromatin folding for short NRLs. (A–D) Distribution of sedimentation coefficients, c(S), at 1 mM MgCl2 for separately analysed 167×12 and 172×12 core arrays (A), 167/172×12 coreconstitute (B), 167×12 reconstituted with and without linker histone (C), and 172×12 reconstituted with and without linker histone (D). (E–G) Electron micrographs of 172×12 core arrays at 5 mM NaCl (E) and 172×12 (F) and 167×12 (G) at 1 mM MgCl2 showing different degrees of compaction. EM magnification 42K. (H) Histogram showing particle size distribution of 167×12 arrays (white columns) and 172×12 arrays (black columns) condensed in 1 mM MgCl2. The inset shows average particle size measured at the Y-axis and X-axis for the 167×12 arrays and 172×12 arrays. Error bars in the inset represent s.d.'s. (I, J) Electron micrographs of 167×12 and 172×12 core arrays at 1 mM MgCl2 showing different degrees of compaction. The samples were contrasted by platinum shadowing. EM magnification 110K. Download figure Download PowerPoint Nucleosome array self-association does not depend upon the NRL In addition to chromatin folding (secondary chromatin structure), chromatin fibres are compacted by self-association (tertiary chromatin structure) (Woodcock and Dimitrov, 2001; Luger and Hansen, 2005). To examine whether NRL variations affect chromatin tertiary structure, we monitored the extent of self-association for the various constructs differing in NRL by magnesium-dependent self-association assays (Schwarz et al, 1996). We observed that wide changes in NRL, including those that significantly affected chromatin folding, did not notably alter self-association rates, with most chromatin arrays having 50% precipitation between 3.0–3.5 mM MgCl2 (Figure 4C). It thus appears that nucleosome self-association is not affected by the broad range of NRLs, suggesting that neither general NRL nor its local rotational variations can notably affect formation of tertiary chromatin structures. While chromatin precipitation by low-speed centrifugation is typically employed to determine chromatin self-association (Schwarz et al, 1996), we noticed that under certain conditions analytical ultracentrifugation can be successfully used to assay the initial steps of self-association in solution resulting in array dimerization (tertiary structures). The dimers appear as a 'second peak', running at 90 S (or above). This peak is most commonly observed on arrays with NRLs ∼207 bp in the presence of linker histone H5 and 1 mM MgCl2. Using this approach, we also did not observe any significant differences in Mg-dependent formation of the 90 S peak between the 207×12 uniform array and the variable ±2- or ±4-bp arrays (Supplementary Figure S3B, bottom), confirming the results of the precipitation assay. Intrinsic properties of the short-NRL DNA templates determine nucleosome array folding After observing periodic modulations in chromatin folding for the short-NRL arrays, we also wished to independently verify that these differences were not due to variations in histone loading. Therefore, we assembled nucleosome reconstitutes with a mix of 167×12 and 172×12 DNA templates. These coreconstitutes were then assayed for folding by sedimentation velocity experiments on the analytical ultracentrifuge and by transmission EM. The coreconstitutes formed two distinct peaks at 1 mM MgCl2, indicating two samples with different folding extent (Figure 5B), with each one of these double peaks corresponding to the single peak of the 167×12 or 172×12 uniform array (cf. panels A and B in Figure 5). EM imaging (42K magnification) of the 172×12 in the unfolded state (Figure 5E) as well as 172×12 and 167×12 arrays folded at 1 mM MgCl2 (Figure 5F–H) confirms the notably larger diameters of 172×12 arrays at 1 mM MgCl2. This difference is also observed in the EM images of platinum-shadowed samples obtained at 110K magnification (Figure 5, cf. panels I and J). We next asked whether the addition of linker histone, which is required to stabilize compact folding (>50 S) of nucleosome arrays with longer NRLs (Carruthers et al, 1998; Routh et al, 2008), would also promote the folded state of the short-NRL arrays. In this set of experiments, we added linker histone H5 to 167×12 and 172×12 arrays and observed that H5 induced only minor increases in the already folded 167×12 (52–55 S, Figure 5C), consistent with previous findings by Routh et al (2008). Strikingly, adding linker histone caused a dramatic increase in 172×12 sedimentation from 38 to 55 S in 1 mM MgCl2 (Figure 5D). It thus appears that certain short NRLs can be completely folded by linker histone, suggesting that short NRLs following the 10n+5 rule impose an architectural factor-dependent folding on the overlaying chromatin fibre. In contrast, the short NRLs containing an integer number of linker DNA turns (such as 167×12 and 177×12) promote compact folding independently of the architectural proteins. Discussion Arrays of positioned nucleosomes with regular NRLs are widely used in the studies of chromatin higher-order structure. However, nucleosomes are mobile on native DNA (Flaus-Owen-Hughes, 2003) as most native DNA sequences do not bind or position histone octamers as tightly and precisely as they bind to the clone 601 sequence and other strong positioning templates (Lowary and Widom, 1998) utilized in most recent experiments. As such, the NRL is not uniform within native chromatin, but rather contains variations around well-defined average peaks with 10 bp intervals ±2 bp (Widom, 1992). Several models have directly suggested a dependence of chromatin higher-order structure on the internucleosomal rotational variations (Widom, 1992; Woodcock et al, 1993; Leuba et al, 1994; Stehr et al, 2010). This raises the question of whether the regular arrays fold into higher-order structures similar to native chromatin. Our experiments with uniform (207×12) and variable (207±2 and ±4 bp) nucleosome arrays clearly showed that these arrays exhibited similar compaction whether they were in unfolded, partially folded, or completely folded states (Table I and Supplementary Figure S3). Thus, our results imply that nucleosome linkers are flexible enough to be independent from the rotational settings for the NRLs of around 200 bp so that previous biochemical experiments with regular repeats, as well as computative modelling conducted by us and other groups, adequately reproduce native chromatin states