Structural plasticity of histones H3-H4 facilitates their allosteric exchange between RbAp48 and ASF1

Abstract

The mechanisms by which histones are disassembled and reassembled into nucleosomes and chromatin structure during DNA replication, repair and transcription are poorly understood. A better understanding of the processes involved is, however, crucial if we are to understand whether and how histone variants and post-translationally modified histones are inherited in an epigenetic manner. To this end we have studied the interaction of the histone H3-H4 complex with the human retinoblastoma-associated protein RbAp48 and their exchange with a second histone chaperone, anti-silencing function protein 1 (ASF1). Exchange of histones H3-H4 between these two histone chaperones has a central role in the assembly of new nucleosomes, and we show here that the H3-H4 complex has an unexpected structural plasticity, which is important for this exchange.

Figure 1

Stoichiometry of the RbAp48 interaction with the histone H3–H4 complex. a, Refolded recombinant histones gH3 and gH4 were analyzed on a Superdex 200 size exclusion column, either alone (red trace), or in the presence of RbAp48 in a 1:1 ratio (blue trace). Eluted complexes were analyzed using SDS-PAGE gels (right). b, Non-denaturing nano-ESI mass spectrum of the RbAp48–gH3–gH4 complex. The m/z region 3500 to 5500 is expanded (below) to show the ion series for RbAp48–gH3–gH4 (~50 % of total intensity), RbAp48–gH3 (~20 %) and RbAp48–gH4 (~30 %), represented by purple, green and orange dots, respectively. c, The RbAp48–gH3–gH4 species was selected for collision-induced dissociation leading to release of either gH3 (~40 %) or gH4 (~60 %). (The remaining RbAp48–gH4 and RbAp48–gH3 complexes are above m/z 4500, and are not shown.) A comparison of the experimental vs theoretical m/z values is provided in the Supplementary Table.

Figure 2

RbAp48 binds to histone H3–H4 dimers. a, Background corrected PELDOR data for the H3Q125C spin-labeled (H3–H4)₂ complex alone (black), and following the addition of two equivalents of RbAp48 (blue) (left panel). Tikhonov derived distance distributions show a clear peak at ~30 Å for the H3Q125-labelled H3–H4 tetramer, but none for the RbAp48–H3–H4 complex (right panel). b, Background corrected PELDOR data for the H3–H4 complex, where both H3Q125C and H4T71C were spin-labeled, before (black) and after (blue) the addition of two equivalents of RbAp48 (left panel). Tikhonov derived distance distributions show peaks at ~20, 37 and 56 Å for the H3–H4 tetramer and several overlapping peaks for the RbAp48 complex (right panel). In both a and b, the most appropriate time trace simulation (obtained using Tikhonov regularization) is shown in red. A C110A mutation in H3 was used such that only the unique cysteine at Gln-125 was labeled with MTSL.

Figure 3

The H3–H4 complex undergoes substantial structural rearrangement upon binding of RbAp48. a, Model of two possible conformations for H3–H4 in solution. The model was generated using PyMol (ref. 42). Helix-1 from H4 may unfold to interact with the RbAp46 and RbAp48 binding pocket as observed in the RbAp46–H4 crystal structure. b, Background corrected PELDOR data for an H3–H4 complex, where H3L65C and H4N25C were spin-labeled, before and after the addition of an equimolar amount of RbAp48 are shown in black and blue, respectively (left panel). Distance distribution peaks are observed for the H3–H4 dimer (~30 Å), and for the RbAp48 complex (~55 Å) (right panel). c, Background corrected PELDOR data for an H3–H4 complex, where H3M90C and H4N25C were spin-labeled, before and after the addition of an equimolar amount of RbAp48 (black and blue, respectively, see left panel). The distance distribution shows a predominant peak for the H3–H4 dimer (~23 Å) and a major peak (~61 Å) for the RbAp48 complex (see right panel). In b and c, the most appropriate time trace simulation (obtained using Tikhonov regularization) is shown in red. A C110E mutation in H3 was used to reduce H3–H4 tetramerization and create an obligate H3-H4 dimer (ref. 33).

Figure 4

Both RbAp48 and ASF1 can simultaneously bind the histone H3–H4 complex. a, gH3 and gH4 were refolded and mixed with RbAp48 in a 1:1 molar ratio together with excess ASF1^1-159 prior to analysis on a Superdex 200 size exclusion column. The eluted proteins were analyzed by SDS-PAGE (right panel). b, Non-denaturing nano-ESI mass spectrum of the RbAp48–gH3–gH4–ASF1^1-159 complex. The ion series from the RbAp48–gH3–gH4–ASF1^1-159, RbAp48, and ASF1^1-159 species are shown by magenta, brown, and light green diamonds, respectively. A comparison of the experimental vs theoretical m/z values is provided in the Supplementary Table. (The peak series marked with asterisks corresponds to a 1:2:2:2 RbAp48–H3–H4–ASF1^1-159 complex, which perhaps forms due to oxidation and dimerization of ASF1^1-159 in the sample.)

Figure 5

Comparison of the affinities of ASF1 for the histone H3–H4 and RbAp48–H3–H4 complexes. a, and b, r_obs, the measured fluorescence anisotropy, is plotted against increasing concentrations of either ASF1^1-159 or RbAp48 to demonstrate binding to H3-H4. H3 was labeled with the Cy3 fluorophore at either Gln-125 or Leu-65. c, Plot of r_obs against increasing concentrations of ASF1^1-159 for binding to the RbAp48–H3–H4 complex, labeled with Cy3 at Gln-125 on H3. In a, and b, a ligand-depletion binding isotherm was fitted to the data using the Levenberg-Marquardt algorithm. In c, the data were fitted with either a ligand-depletion binding isotherm using the Levenberg-Marquardt algorithm (dashed line), or with an anisotropy ligand-depletion binding isotherm (with drift) using a Monte Carlo algorithm (solid line). (Note that in b, the concentration of Cy3-labeled H3–H4 was lower than that in a and c, giving the curves a different appearance) All experiments were carried out in triplicate – data points are the mean of three independent experiments and the error bars represent ± 1 standard deviation. A C110A mutation in H3 was used such that only the unique cysteine was labeled with the fluorophore.