Robinson et al. 10.1073/pnas.0601212103. |
Supporting Figure 5
Supporting Figure 6
Supporting Materials and Methods
Fig. 5. Reconstitution and compaction of nucleosome arrays containing both histone octamer and linker histone H5. (A) Size determination of long 601 DNA tandem arrays. 0.25 mg of long array constructed with 177-bp (lane 1), 187-bp (lane 2), 197-bp (lane 3), 207-bp (lane 4), 217-bp (lane 5), 227-bp (lane 6), and 237-bp (lane 7) 601 DNA repeats were each analyzed in lanes containing 2- to 80-kbp DNA size markers. 0.8- to 10-kbp DNA size markers (lane M) were included in separate lanes as a reference. (B) Reconstituting histone octamer onto a tandem DNA array containing 22 copies of a 177-bp 601 nucleosome positioning sequence (22 × 177-bp 601 DNA). 22 × 177-bp 601 DNA at a concentration of 9.3 nM was incubated with histone octamer at increasing molar input ratios: 0, 0.50, 0.76, 1.0, 1.27, and 1.52 histone octamer molecules per 177-bp repeat (lanes 1–8). (C) Reconstitution with linker histone H5. The histone octamer saturated array in lane 5 of B was incubated with linker histone H5 at increasing molar input ratios of 0, 0.94, 1.87, 2.34, 2.81, 3.28, and 3.75 H5 molecules per nucleosome core (lanes 1–7). (D) Increased electrophoretic migration resulting from Mg2+-induced chromatin compaction. A long array containing 72 repeats of a 177-bp 601 DNA sequence (lane 1) was reconstituted with both histone octamer (lane 2) and histone octamer plus H5 (lanes 3 and 4). The +H5 array was analyzed after dialysis into folding buffer (1.6 mM Mg2+/20 mM TEA, pH 7.4) both before (lane 3) and after (lane 4) fixation with glutaraldehyde at a final concentration of 0.1% (vol/vol). The positions of the naked 22 × 177-bp 601 DNA (DNA), the octamer (+Oct), and the fully reconstituted array (+H5), as well the glutaraldehyde fixed array (+Fix), are indicated.
Fig. 6. Geometric representation of the maximum spacing between successive nucleosomes with 10 bp of linker DNA (177-bp DNA). (A) Angles and distances relating dyad positions of nucleosome 1 (D1) and nucleosome 2 (D2), shown in the form of a projection down the helix axis. The dyads D1 and D2 lie on an imaginary circle (dashed), which delineates the circumference of an inner hole formed through the radial offset of nucleosomal subunits. The superimposed points O1 and O2 mark the intersection of the helix axis with imaginary lines (dashed) projected along dyad axes of nucleosome 1 and 2, respectively. The xy projection angle between these imaginary lines represents the maximal azimuthal rotation angle (f) for a repeat length of 177 bp, which contains only 10 bp of linker DNA. In a maximally extended form, 10 bp of linker DNA is equivalent to a maximum distance of 3.4 nm, equivalent to 3.25 nm in the xy plane. The maximum separation of successive nucleosomes is achieved when 10 bp of straight linker exits from a point adjacent to the dyad in nucleosome 1 (E1) (same xy plane with 0° nucleosome tilt) and enters at an equivalent entry point on the opposite side of the dyad in nucleosome 2 (E2). The 2.7-nm spacing of points E1 and E2 from their respectively dyad positions corresponds to the spacing of adjacent DNA gyres on the surface of the nucleosome core [Luger, K., Mader, A. W., Richmond, R. K., Sargent, D. F. & Richmond, T. J. (1997) Nature 389, 251–260]. The two main assumptions on which this diagram are based include that (i) successive nucleosomes are following the path of a simple one-start helix, and (ii) the completion of two full superhelical turns on the surface of the nucleosome requires 167 bp of nucleosomal DNA, the ends of which become positioned adjacent to the nucleosome dyad with a spacing of 2.7 nm. The summed angle f was calculated to be ≈83°, equivalent to a minimum of ≈4.3 nucleosomes per turn. (B) Three-dimensional representation of the view in A showing the derivation of the calculated distance between exit (E1) and entry (E2) points in the xy plane. In the calculation of this distance, 1 nm was used as the rise per nucleosome in the helix (Fig. 2B).
Supporting Materials and Methods
Construction of Long 601 DNA Arrays.
The assembly of tandem arrays containing a large number of repeats was done in several steps. Firstly, up to 30 tandem DNA repeats of each nucleosome repeat length were cloned into pUC18 (1) and grown up in DH5a Escherichia coli. Secondly, these clones were digested with three different combinations of restriction enzymes (EcoRI, HindIII, and AvaI) to release fragments that would undergo a three-way ligation into precut pETcoco-1 vector with a resulting amplification of repeat number. Thirdly, the number of 601 DNA inserts in pETcoco-1 was increased by using a partial digestion procedure: long tandem repeat inserts flanked by ends complementary to (i) a site in the vector (HindIII or NheI) and (ii) the AvaI overhang from another long insert (AvaI) were prepared by complete digestion of the flanking site (HindIII or NheI) followed by partial AvaI digestion of the linearized construct. The largest fragments in the resulting ladder were gel-purified and added to a three-way ligation reaction with a complementary long insert and pETcoco-1 predigested with HindIII and NheI. To produce the long inserts on a large scale, the pETcoco-1 plasmid was amplified by induction with 0.01% L-arabinose. The 601 DNA arrays were excised from both pUC18 and pETcoco-1 as blunt-ended fragments by using flanking EcoRV sites and purified by differential PEG precipitation.EM.
The fixation of folded chromatin arrays was performed at a final chromatin concentration of ≈40 mg/ml in 0.1% glutaraldehyde (vol/vol) on ice for 30 min. For negative stain images, a 4-ml drop of the fixed material was applied to a continuous carbon layer covering a 200-mesh copper/palladium microscopy grid that had been airglow discharged. After 1 min the sample was blotted from the edge of the grid by using Whatman1 filter paper, 40 ml of 2% uranyl acetate was added and immediately blotted off, and the grid was left to air-dry. For electron cryo-microscopy of the folded 72 × 177-bp nucleosome array, 4 ml of fixed chromatin sample (≈40 mg/ml) were applied to an amylamine treated thin continuous carbon layer covering the surface of a standard carbon holey grid. The grid was mounted in tweezers on a gravity-driven plunging device at room temperature within a humidity chamber. After 1 min the sample drop was blotted by using a strip of P81 cation exchange paper (Whatman) before rapid freezing in liquid ethane (2). The shorter chromatin fibers containing 22 nucleosomes were captured in ice by using standard carbon holey grids and blotted as described above by using Whatman1 filter paper. Images of negatively stained samples were recorded by using a Tecnai T12 microscope at an operating voltage of 120 keV at [times]42,000 direct magnification with 1-mm defocus. For electron cryo-microscopy, frozen grids were transferred at liquid nitrogen temperature to either a Tecnai T12 or F20 microscope by using a Gatan model 626-DH cryo-holder, and images were recorded at an operating voltage of either 120 keV (T12) or 200 keV (F20) at [times]50,000-52,000 direct magnification with 2- to 4-mm defocus by using a low-dose operating mode.1. Huynh, V. A., Robinson, P. J. & Rhodes, D. (2005) J. Mol. Biol. 345, 957–968.
2. Dubochet, J., Adrian, M., Chang, J. J., Homo, J. C., Lepault, J., McDowall, A. W. & Schultz, P. (1988) Q. Rev. Biophys. 21, 129–228.
3. Luger, K., Mader, A. W., Richmond, R. K., Sargent, D. F. & Richmond, T. J. (1997) Nature 389, 251–260.