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Columnar structure of human telomeric chromatin

Abstract

Telomeres, the ends of eukaryotic chromosomes, play pivotal parts in ageing and cancer and are targets of DNA damage and the DNA damage response1,2,3,4,5. Little is known about the structure of telomeric chromatin at the molecular level. Here we used negative stain electron microscopy and single-molecule magnetic tweezers to characterize 3-kbp-long telomeric chromatin fibres. We also obtained the cryogenic electron microscopy structure of the condensed telomeric tetranucleosome and its dinucleosome unit. The structure displayed close stacking of nucleosomes with a columnar arrangement, and an unusually short nucleosome repeat  length that comprised about 132 bp DNA wound in a continuous superhelix around histone octamers. This columnar structure is primarily stabilized by the H2A carboxy-terminal and histone amino-terminal tails in a synergistic manner. The columnar conformation results in exposure of the DNA helix, which may make it susceptible to both DNA damage and the DNA damage response. The conformation also exists in an alternative open state, in which one nucleosome is unstacked and flipped out, which exposes the acidic patch of the histone surface. The structural features revealed in this work suggest mechanisms by which protein factors involved in telomere maintenance can access telomeric chromatin in its compact form.

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Fig. 1: Heterogeneous nucleosome occupancy of telomeric chromatin.
Fig. 2: Structure of the telomeric tetranucleosome.
Fig. 3: Histone-mediated interactions stabilize the columnar structure of telomeric chromatin.
Fig. 4: Features of the telomeric chromatin.

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Code availability

The code used for MMT analysis is available at https://doi.org/10.5281/zenodo.6810919.

Data availability

Cryo-EM density maps for mono-NCP (EMD-31806), Di-NCP (EMD-31810 (3.9 Å), EMD-31908 (5 Å), EMD-31907 (4.6 Å)), open Di-NCP (EMD-31815 (4.5 Å), EMD-31909 (6.6 Å)), Tri-NCP (EMD-31816), open Tri-NCP (EMD-31826), open Tetra-NCP (EMD-31832) and Telo-tetra (EMD-31823) have been deposited in the Electron Microscopy Data Bank. Fitted models for mono-NCP (7V90), Di-NCP (7V96), open Di-NCP (7V9C), Tri-NCP (7V9J), open Tri-NCP (7V9S) and open Tetra-NCP (7VA4) and Telo-tetra (7V9K) have been deposited in the Protein Data Bank. Raw uncropped gels are provided in Supplementary Fig. 1Source data are provided with this paper.

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Acknowledgements

We thank staff at the NTU Institute of Structural Biology (NISB), Nanyang Technological University, for providing access to facilities; staff at the Facility for Analysis, Characterisation, Testing and Simulation, Nanyang Technological University, for use of their electron microscope and A. Wong and E. Smith for assistance with data collection; staff at the Cryo-Electron Microscopy Facility at the Center for Bioimaging Science, Department of Biological Science, National University of Singapore, and J. Shi for scientific and technical assistance; W. Shum for assistance with the preparations of histone and DNA template constructs; and C. Davey and S. Sandin for their valuable discussions and suggestions. This work has been supported by the Singapore Ministry of Education (MOE) Academic Research Fund (AcRF) Tier 2 (MOE2018-T2-1-112) and Tier 3 (MOE2012-T3-1-001) grants.

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Contributions

A.S., S.Y.W., D.R. and L.N. conceptualized and designed the study. A.S. prepared grids, collected and processed the cryo-EM data, carried out model building and refinement, prepared constructs and biochemical assays. S.Y.W. prepared constructs and carried out negative-stain EM and AUC-SV experiments. S.Y.W., W.S. and S.L. designed and performed cloning and construct preparations. S.L. conceptualized and designed the MCS. S.Y.W., W.S. and Q.C. prepared samples for force spectroscopy. V.K.V. prepared grids and collected cryo-EM data. N.V.B. and J.v.N. built the magnetic tweezer and performed measurements. N.K. and J.v.N. performed and analysed force spectroscopy measurements. A.S., S.Y.W., D.R. and L.N. wrote the paper with the help and input of all authors. All authors contributed to the interpretation of the data.

Corresponding authors

Correspondence to Daniela Rhodes or Lars Nordenskiöld.

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The authors declare no competing interests.

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Extended data figures and tables

Extended Data Fig. 1 Construction design of the pTelo constructs.

a, Plasmid map of the 2844-bp pTelo-1 construct. The pTelo-1 vector consists of a 151-bp human telomeric DNA insert flanked by two MCS (5′-MCS and 3′-MCS). The various restriction sites embedded inside the MCSs enable different manipulations and modifications of the telomeric insert. Digestion with EcoRI and HindIII releases the telomeric insert bearing overhangs that can be ligated to asymmetric handles, such as those used for MMT measurements in this study. The two EcoRV sites enable releasing of the telomeric insert with blunt ends for nucleosome reconstitution and the production of longer arrays. BbsI and BtgZI are Type IIS restriction enzymes that cleave the construct within the 5′- and 3′-AvaI sites. They let inserting additional array elements unilaterally or releasing telomeric arrays bearing AvaI overhangs suitable for multimerisation of telomeric arrays. b, The elementary unit of the telomeric DNA template used in this study consists of a 151-bp human telomeric fragment (green) flanked by AvaI restriction sites (red) at each extremity. Once multimerised, the resulting arrays consist of integral multiples of 157-bp units. The 157-bp telomeric units are released from the pTelo-1 construct upon AvaI digestion and subsequently multimerised (1) into an array by ligation. The non-palindromic nature of the AvaI overhangs ensures control over strand orientation and prevents the G-rich and C-rich strands from mingling. The resulting multimers consist of integral multiples of 157-bp units, and the required multimer size is obtained by colony screening coupled with restriction digest analysis. (2) The entire process can be accelerated by ligating multimers together (e.g. 3×157-bp repeat fragments into 6×, 9× or 12× repeats). The release of 157-bp telomeric arrays bearing AvaI overhangs at each extremity is achieved by cutting the plasmid with BbsI and BtgZI. The resulting insert is subsequently ligated into integral multimers of the parental array. c, Alternatively, a pre-existing array can be extended unilaterally by selectively opening the vector either at the upstream or downstream AvaI site and inserting an extra telomeric array. This approach enables the array to be extended indefinitely in a controlled and stepwise process. Similarly, hybrid arrays bearing non-telomeric terminal sequences (e.g. 601 Widom or alpha-satellite DNA sequences) can be obtained using the same strategy. The array extension procedure is outlined in the Materials and Methods section and herein is illustrated with pTelo-4 plasmid. (1) Firstly, pTelo-4 is treated with BbsI and alkaline phosphatase to generate a linearised and dephosphorylated recipient vector with AvaI overhangs. (2) Separately, a Telo-4 array is excised from pTelo-4 by EcoRV digestion. (3) The resulting purified Telo-4 DNA fragment is digested with BtgZI to create a distal AvaI overhang and (4) ligated to the BbsI-linearized pTelo-4 from step 1. (5) The ligation product is subsequently deprotected with BbsI yielding a proximal AvaI overhang which enables (6) the circularisation of the pTelo-8 plasmid. d, Large-scale preparation of long telomeric DNA array template. (1) A 10×157-bp telomeric DNA array template (Telo-10) is excised from the pTelo-10 vector by EcoRV digestion. The resulting purified DNA fragment is digested with BtgZI (2) or BbsI (3) to generate complementary AvaI overhangs at the downstream and upstream extremities. Subsequently, the resulting reaction products are ligated to yield a 20×157-bp telomeric DNA array template (Telo-20).

Extended Data Fig. 2 Preparation of the telomeric DNA and 601-20 DNA for in-vitro nucleosome array reconstitution.

a, Schematic diagram of various 20-mer 157-bp DNA array templates employed in this study. Telo-20 DNA consists of twenty repeats of 157-bp Telo-1 fragment. #Telo-18# DNA denotes a chimeric array template consisting of eighteen repeats of 157-bp Telo-1 unit flanked by one unit of 157-bp Widom 601 sequence (blue) at each terminus. 601-20 DNA represents an array template made of twenty repeats of the 157-bp Widom 601 sequence. b, pTelo-10 was digested with EcoRV and AvaI for quality analysis. The resulting fragments were fractionated on 1.0% TBE-agarose gel. The quality of the amplified Telo-10 was assessed with EcoRV digestion of the plasmid to reveal any possible recombined telomeric DNA (lane 2). Additional partial digestion with AvaI (lane 3) showed the number of telomeric repeats the plasmid contains. c, Equimolar mixture of the BtgZI- and BbsI-digested Telo-10 fragments were ligated overnight and analysed on 0.7% TBE-agarose gel. Densitometry analysis showed that the ligated mixture contained 70–75% of the Telo-20 DNA. d, Equimolar of BtgZI-digested #Telo-8 and BbsI-digested Telo-10# fragments were ligated overnight and analysed on 0.7% TBE-agarose gel. Densitometry analysis showed that the ligated mixture contained 70–75% of the #Telo-18# DNA. e, Purified 601-20 DNA partial digested with AvaI to reveal exactly 20 repeats analysed on 0.7% TBE-agarose gel. f, Telo-4 DNA template was released from the vector by EcoRV digestion followed by PEG size fractionation and ion-exchange purification (lane 2). This template was used to generate a DNA template containing multiple Telo-4 DNA by self-ligation mediated by T4 ligase and assisted by T4 polynucleotide kinase (lane 3). In g, h, i, j, l, m and n, the black triangle denotes the ratio of histone octamer (HO)/DNA producing the optimal saturation. g and h, Telo-20 (g) and #Telo-18# (g) reconstituted with recombinant human histone octamer titration analysed on 0.7% Tris-borate agarose gel and post-stained with SYBR® Gold Nucleic Acid Gel Stain. i, 601-20 reconstituted with recombinant human histone octamer titration, with 147 bp pUC57 backbone fragment as competitor DNA. Reconstituted arrays were analysed on 0.7% Tris-borate agarose gel and post-stained with SYBR® Gold Nucleic Acid Gel Stain. j, Telo-tetra was reconstituted with recombinant human histone octamer in the presence of 145 bp telomeric DNA as competitor DNA to determine optimal saturation. The saturation of the reconstituted arrays was assessed by its migration on a 1.2% 0.25x TBE-agarose gel. k, Reconstitution of self-ligated Telo-4 template to give arrays containing multiples of the Telo-tetra units. l, The 601-tetra nucleosome was reconstituted as a control for comparison with Telo-tetra. m, Reconstitution of the native sequence telomeric DNA devoid of AvaI restriction sites. n, The Telo-10 DNA was reconstituted with recombinant human histone octamer in the presence of 145 bp telomeric DNA as competitor DNA to determine optimal saturation o, The reconstitution of Telo-tetra with C-terminal truncated H2A (1-122) (H2AΔC) histone octamer (lane 4). The telo-tetra with ΔH2A histone octamer migrates to approximately the same distance as the Telo-tetra on a 1.2% 0.25xTBE-agarose gel. p, SDS gel analysis of the human histone octamer used in the study. Human histone octamers were reconstituted with either wild-type (WT) H2A (lane 1) or C-terminal truncated H2A (1-122) H2AΔC, lane 2]. For b–p, all experiments were repeated at least thrice independently with similar results.

Extended Data Fig. 3 Analytical ultracentrifugation-sedimentation velocity (AUC-SV), negative stain EM and single-molecule magnetic tweezer analysis of telomeric chromatin fibres.

a, AUC-SV data, normalised to the maximum s-value of the sedimentation coefficient distribution, (c(s)). The distribution for Telo-20 (green) has two broad peaks, indicating the presence of the long Telo-20 (39.4 S) and the shorter and unligated Telo-10 arrays (29.6 S). The full width at half maximum (FWHM) value of the main peak at 39.4 S is 4.3 S. The broad main peak of Telo-20 reflects the heterogeneity in nucleosome number of the arrays from ensemble-averaged measurements of fibres in solution. #Telo-18# (red) shows three overlapping peaks that reflect the presence of #Telo-18# (39.2 S), unligated Telo-10# (32.5 S) and #Telo-8 arrays (27.8 S). The FWHM value of the main peak at 39.2S is 2.5 S. The sharper main peak for #Telo-18# indicates higher homogeneity compared to Telo-20. The 601-20 data (blue) shows a single peak at 40.2 S, with an FWMH value of 2.7 S that indicates homogeneity of the array. b, c, and d, Representative negative stain electron-microscopy micrographs of Telo-20 in 0.6 mM Mg2+ (b), #Telo-18# in 0.6 mM Mg2+ (c), and 601-20 in 0.8 mM Mg2+ (d). Boxes show an enlarged view of selected arrays to illustrate the stacked features of the telomeric arrays or the ladder arrangement of the 601-20 array. For each array in b–d, at least 3 independent EM experiments were performed with similar results. e, Cryo-EM images of reconstituted 601-157 tetranucleosome arrays showing zig-zag compaction under identical conditions (1 mg/ml nucleosome array, 20 mM potassium cacodylate (pH 6.0), 0.08 mM MgCl2, 1 mM DTT) where Telo-tetra in f shows columnar packaging. The zig-zag arrangement is highlighted with a red arrow, and a close-up view is shown on the right. The 601-157 tetranucleosome retained zig-zag conformation in more than three independent experiments spanning varying Mg2+ concentrations, with > 3 observations from each Mg2+ concentration. As the data agreed with previous observation zig-zag conformation, biological replicates were not carried out for e. f, Representative cryo-EM micrograph of Telo-tetra. The orientation of the selected particles is depicted with blue (top views) and black (side views) boxes. Each dataset was obtained from separate batches of histone octamer and DNA and contained >4500 micrographs with similar conformations. g and h, Representative data of stretch-relief force spectroscopy measurements of #Telo-18# (g, red triangles and lines) and 601-20 (h, blue squares and lines). Points are experimental data; lines are fitting fibre-stretching data to the statistical mechanics model; dashed lines show stretching of the bare DNA calculated by the WLC model. In each panel, parameters determined by the model are displayed (Ntotal, Nfolded, k, ∆G1, and ∆G2). For the #Telo18# and 601-20 arrays. i, Fitted fibre stiffness, characterised by the stretching modulus (k); #Telo18# – red, mean 0.49, s.d. 0.18, n = 167, range 0.21-1.03; 601-20 – blue, mean 0.53, s.d. 0.19, n = 319, range 0.20-1.12. The fibre stiffness difference between the #Telo18# and 601-20 arrays is marginally statistically significant (p = 0.034). j, Fitted free energies ∆G1 and ∆G2. ∆G1: #Telo18# – mean 12.6, s.d. 4.0, n = 167, range 5.2-23.4; 601-20 – mean 13.6, s.d. 3.4, n = 319, range 6.3-22.8. ∆G1 values differ significantly for the #Telo18# and 601-20 arrays (panel j left; p = 0.004). ∆G2: #Telo18# – mean 4.5, s.d. 1.8, n = 167, range 0.8-8.3; 601-20 – mean 4.6, s.d. 1.7, n = 319, range 0.7-8.8. ∆G2 values are similar (panel j, right; p = 0.65). In (i) and (j), data calculated for each trace are shown as small symbols; mean values are indicated in the graphs, boxes show mean ± s.d.; whiskers indicate the 10-90% data range. A two-sample t-test with equal variance was carried out for data in i and j. k and l, Telomeric nucleosomes unwrap at lower forces than 601 nucleosomes. Heat maps showing the dependence of rupture force events, Frupt, on the rate of applied force and the values of the d (the distance between the bound state and the activation barrier peak) and koff (the rate constant for bond disruption under zero external force) were determined as described in the Methods section. The data for the #Telo18# (k) and 601-20 (l) arrays. Magenta (#Telo-18#) and blue (601-20) lines are linear fitting of the Frupt in the 10 pN < Frupt < 30 pN range. For the #Telo-18# array, we obtained d = 0.880 ± 0.036 nm and koff = 0.0128 ± 0.0016 1/s (2102 of total 3120 points were used); for the 601-20 array, d = 0.919 ± 0.024 nm and koff = 0.0077 ± 0.0007 1/s (4303 of total 6256 points were included).

Source data

Extended Data Fig. 4 Cryo-EM screening of native sequence telomeric arrays, H2A(ΔC) Telo-tetra and Telo-10 fibres. Analysis of the repeating structural unit in telomeric fibres.

a, Representative micrograph of a native sequence telomeric array reconstituted from an approximately 600-bp repetitive TTAGGG DNA template devoid of intermittent AvaI sites. The arrays display the columnar conformation (green circle); some particles are in the open-state conformation with about 90° plane-plane angle between the nucleosomes (red circle). Close-up views of selected particles are shown to the right (columnar) and underneath (open-state). b, Representative micrograph of Telo-tetra arrays reconstituted from a histone octamer comprising a truncated H2A lacking the seven last amino acids of the C-terminal tail [H2A(ΔC)]. The arrays display decreased compaction compared to the wild-type H2A arrays at identical conditions (1 mg/ml arrays, 20 mM potassium cacodylate (pH 6.0), 0.08 mM MgCl2). The arrays reconstituted with the H2A(ΔC) isoform form less compact columnar-like structures than the wild-type construct (enlarged views underneath the micrograph). c, Representative micrograph of a Telo-10 array showing coexistence of compact columns (black arrow) and open-state conformations at both ends (green arrows) and in the middle (red arrows) of the fibre. For panels ac, observed conformations were verified in more than three independent experiments spanning varying Mg2+ concentrations, with about 5 or more observations for each Mg2+ concentration. As the data agreed with previous observation columnar conformation, biological replicates were not carried out for ac. d, Refined map of the Tri-NCP with side and top view of the EM map and fitting of the modelled structure (middle). The right panel shows the analysis of the stacking parameters in this modelled Tri-NCP structure, which suggests that the Di-NCP is the repeating structural unit. Relative orientations of the NCP-1 versus NCP-2 compared to NCP-2 versus NCP-3 are similar: NCP-NCP distances are 57.4 and 56.4 Å; dyad-dyad angles are 135o and 131o (red triangles in Fig. 2g of the main text); plane-plane angles are 18o and 17o. Nucleosomes are approximated as planes with normal vectors; dyad axes are shown as arrows in the planes.

Extended Data Fig. 5 Flowchart of data processing of the Telo-tetra Dataset 1 and merging of Datasets 1 and 2.

The flowchart illustrates the data processing employed for Dataset 1 (Top) and the merging of Datasets 1 and 2 (bottom). Processing of Dataset 2 was done in the same way as for dataset 1, except that the particle picking was carried out using a 3D template of the Di-NCP from Dataset 1. The individual Telo-tetra EM maps obtained from both Datasets showed missing angular views. Hence, Datasets 1 and 2 were merged (bottom), followed by RELION auto-picking. Merging of datasets made it possible to get a homogenous set of particles (26,580) showing improved angular views and better resolution (8.1 Å) (Extended Data Fig. 7a, b) compared to individual datasets.

Extended Data Fig. 6 Flowchart of data processing of Dataset 3 and the merging of Datasets 1, 2 and 3 for the telomeric Di-NCP.

The left side shows the Di-NCP, and the right side shows the extraction of the Mono-NCP and open Di-NCP maps. The bottom left box depicts the merging of the three Di-NCP datasets, Dataset 1 (4.9 Å), Dataset 2 (4.5 Å) and Dataset 3 (3.9 Å), to generate a combined map at 4.5-Å resolution is illustrated in the bottom left box.

Extended Data Fig. 7 Resolution and angular distribution of the Telo-tetra, Tri-NCP and Di-NCP.

Panels a, c, e, g, i and k show gold-standard FSC curves for the corresponding substructures of Telo-tetra with resolution estimated at an FSC threshold of 0.143. The Corrected, Unmasked, Masked and Phase Randomised curves are coloured black, green, blue and red, respectively. a, The Telo-tetra map was refined to a resolution of 8.1 Å (Datasets 1 and 2). b, Angular distribution of particles used for refinement shows missing view and orientation preference (Dataset 1 and 2). c, The Tri-NCP density map was determined to a resolution of 8 Å (Dataset 1). d, Angular distribution of particles used to determine the Tri-NCP structure (Dataset 1). e, The Di-NCP (Dataset 2) was refined to a resolution of 4.5 Å. f, Angular distribution of views of particles used for the Di-NCP refinement (Dataset 2). g, The Di-NCP from Dataset 3 gave a resolution of 3.9 Å for the Di-NCP density map. h, Angular distribution of particles used for building the Di-NCP map with a 3.9 Å resolution (Dataset 3). i, The Mono-NCP (Dataset 3) was refined to a resolution of 3.5 Å. j, Angular distribution of the Mono-NCP particles (Dataset 3). k, The open Di-NCP structure was determined to a resolution of 4.5 Å (Dataset 3). l, Angular distribution of the open Di-NCP (Dataset 3)

Source data

Extended Data Fig. 8 Contribution of histone tails to stabilisation of the columnar structure of the telomeric chromatin fibre.

a, AUC-SV data for the Telo-tetra arrays with intact (WT) histone tails (right, Telo-tetra) and with the C-terminal truncated H2A (left, Telo-tetra H2A(ΔC)) in the unfolded (0 mM Mg2+) or compacted (in the presence of 1 mM Mg2+) forms. In the absence of Mg2+ (which gives an extended beads-on-a-string conformation), the s-value distribution of both Telo-tetra fibres shows two peaks. We identified the peak at the larger and lower s-values as tetramers and under-saturated fibres (with three or two nucleosomes). In the absence of Mg2+, the peak positions of these two forms of the WT arrays are at approximately 21 S and 17 S. For the H2A(ΔC) isoform, these s-values are 21 S and 18 S. Mg2+-induced compaction of the arrays shifts these peaks, respectively to 25 S and 19 S for the WT fibres14; and to 23 S and 19 S for the H2A(ΔC) isoform. The less pronounced increase in the s-value for the main peak of the H2A(ΔC) arrays (from 21S to 23 S) compared to the WT (from 21 S to 25 S) is proposed to be due to the reduced compaction of the H2A(ΔC) array caused by the truncation of the H2A C-termini. Buffer conditions for the AUC-SV analysis: 10 mM NaCl, 10 mM Tris (pH 7.5), 0.1 mM EDTA and containing 1 mM MgCl2 where indicated. b, The merged telomeric Di-NCP electron density map showing tube-like electron densities tentatively assigned to the histone tails (H3 blue, H4 green, H2A yellow, and H2B red). The H2B N-terminal tails mainly occupy locations between the two DNA superhelices of a single NCP. The red arrows denote the H2B αC-helix located between the DNA from two stacked NCPs. The minor bridging density between the two DNAs of the αH2B C-helices is suggested to screen DNA-DNA repulsion as observed in nucleosome crystals27. c, Surface model (left) and electrostatic surface potential (right) representation of the histone octamer interface in Telo-tetra corresponding to the orientation in b, right side. The histone octamer interface in this orientation is mediated by the interaction of the C-terminal tail (Pro117- Lys129) and loop 2 (Arg71-Lys74) of H2A from adjacent NCPs. The acidic patch formed by H2A-H2B is not involved in mediating stacking and is buried in the interior between the two stacked octamers (dotted magenta box). d, Each stacked histone octamer interface is mediated by four sets of histone-histone interactions: two pairs of H2A C-terminal tail (Pro117- Lys129) and loop 2 (Arg71-Lys74) interactions and two pairs of interactions between H3 and H4. The protein-protein stacking interactions between the histone octamers form a helical intertwined network of protein-protein electrostatic interaction along the axis of the Telo-tetra columnar structure. A global sphere model representation view of this helical network is shown with the histones H2A (yellow), H3 (blue) and H4 (green) shown as spheres. H2B is not participating in histone interactions and is not depicted

Source data

Extended Data Fig. 9 Digestions of Telo-tetra and structural analysis of the NRL in telomeric tetranucleosome.

a, AvaI, MNase and DNase digestion of the Telo-tetra. Lane M is a DNA marker; two lanes marked “Ar” show Telo-tetra. The left “Ar” (lane 2) shows Telo-tetra in low salt buffer, and the right lane (lane 3) shows Telo-tetra in DNase buffer. Lanes “AvaI”, “MN”, and “DN” are respectively digestion of the Telo-tetra by AvaI endonuclease, Micrococcal nuclease (MNase), and DNase I nuclease. The bands marked by a black asterisk in the “AvaI” lane indicate 1×157, 2×157, 3×157, and 4×157 products of the AvaI digestion. Both MNase and DNase digest the array to DNA nucleosome core length (145-157 bp) as indicated by the yellow asterisks in the MN” and “DN” lanes. MNase and DNase I can also digest further into the core of the nucleosome, as seen by the smear in the “MN” lane and marked by a red asterisk in the “DN” lane. Under standard nuclease buffer conditions, the presence of divalent ions causes array precipitation and reduces digestion, as shown with the aggregation of the reactants at the top of the gel. b, DNase I digestion of telomeric and 601 NCPs. Lane M denotes DNA marker; “601D” and “TD” lanes show respectively the digestion of the 601-and Telo-mono NCPs. The 601 NCP displays a prominent DNase I-resistant band peak at 145 bp; the telomeric mononucleosome is digested to shorter DNA fragments with a first DNase I-resistant band at about 130 bp. For panels a and b, similar observations were made across more than 3 independent experiments; though differences in rate of digestion were observed across independent experiments, the general distribution pattern of band sizes was similar. c and d, The columnar structure of the Telo-tetra is stabilised by the histone tails, H2A-C (yellow) and H3 (blue). The H2A C-terminal domain protects the DNA at the boundary of the histone octamers separated by ~157 bp of DNA (shown in cyan), suggesting that this protection may be the cause of the observed nuclease-resistant band at 157 bp. The extended DNase treatment results in the digestion of the adjacent DNA positioned between the H2A C-terminal domain and the H3 helix (magenta), leading to the appearance of the ~130 bp nuclease-resistant band.

Extended Data Fig. 10 Open-state of the telomeric Di-NCP, Tri-NCP and Telo-Tetra- and comparison with archaeal chromatin.

In panels d, e and f (right), the histones H3, H4, H2A and H2B are coloured blue, green, yellow and red, respectively. a, Electron density map with heat map depicting the local resolution of the telomeric Di-NCP in the open conformation. b, Superposition of the open state of the telomeric Di-NCP (blue) and the archaeal chromatin (green)32. The two structures show almost identical global features with minor deviation in the opening angle. c, Left, electron density map of the open state in the telomeric Tri-NCP. The map was reconstructed from 11,086 particles from Dataset 2. The particles used for the telomeric open Tri-NCP structure come from the 130,951 particles discarded from the Telo-tetra data processing. The particles underwent multiple rounds of 3D classification to select the open Tri-NCP particles. Right, the modelled atomic structure of the open conformation of the telomeric Tri-NCP fitted to the electron density map. d, Left, electron density map and modelled structure (right) of the open conformation in the Telo-tetra. The map was reconstructed from 5,305 particles selected from the 3D classification of the 130,951 picks (Dataset 2). e, The zig-zag structure of the 6×187 601 array (6hkt)12. f. Left, the representative structure of the higher-order packing in the crystal of the histone-based chromatin in archaea (pdb code 5T5K33). The DNA and protein are shown as respectively white and blue spheres. Right, for comparison, the columnar structure of the Telo-tetra is shown.

Extended Data Table 1 Data collection and electron density map refinement of Telo-tetra and substructures
Extended Data Table 2 Structure refinement statistics of Telo-tetra and substructures

Supplementary information

Supplementary Fig. 1

Uncropped images used in the Extended Data. ao, Uncropped gel images of Extended Data Fig. 2b–p. p, Uncropped image of Extended Data Fig. 3f. q,r, Uncropped gel images of Extended Data Fig. 9a,b.

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Supplementary Video 1

Structure of Telo-tetra. The video shows in 3D, the rotation of the electron density map and refined modelled structure of the telomeric tetranucleosome corresponding to Fig. 2c and illustrates the columnar folding.

Supplementary Video 2

Structure of Telo Di-NCP. The video shows in 3D, the rotation of the dinucleosome unit electron density map within the Telo-tetra, obtained from dataset 3 at 3.9 Å. The refined built structure illustrates the close stacking between nucleosomes and histone contacts.

Supplementary Video 3

Structure of open Telo Di-NCP. The video illustrates the open dinucleosome conformation. The built refined open structure shows show the acidic patch is exposed and accessible to protein factors.

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Soman, A., Wong, S.Y., Korolev, N. et al. Columnar structure of human telomeric chromatin. Nature 609, 1048–1055 (2022). https://doi.org/10.1038/s41586-022-05236-5

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