XB-ART-46611Nucleic Acids Res. November 1, 2012; 40 (20): 10139-49.
Yeast CAF-1 assembles histone (H3-H4)2 tetramers prior to DNA deposition.
Following acetylation, newly synthesized H3-H4 is directly transferred from the histone chaperone anti-silencing factor 1 (Asf1) to chromatin assembly factor 1 (CAF-1), another histone chaperone that is critical for the deposition of H3-H4 onto replicating DNA. However, it is unknown how CAF-1 binds and delivers H3-H4 to the DNA. Here, we show that CAF-1 binds recombinant H3-H4 with 10- to 20-fold higher affinity than H2A-H2B in vitro, and H3K56Ac increases the binding affinity of CAF-1 toward H3-H4 2-fold. These results provide a quantitative thermodynamic explanation for the specific H3-H4 histone chaperone activity of CAF-1. Surprisingly, H3-H4 exists as a dimer rather than as a canonical tetramer at mid-to-low nanomolar concentrations. A single CAF-1 molecule binds a cross-linked (H3-H4)2 tetramer, or two H3-H4 dimers that contain mutations at the (H3-H4)2 tetramerization interface. These results suggest that CAF-1 binds to two H3-H4 dimers in a manner that promotes formation of a (H3-H4)2 tetramer. Consistent with this idea, we confirm that CAF-1 synchronously binds two H3-H4 dimers derived from two different histone genes in vivo. Together, the data illustrate a clear mechanism for CAF-1-associated H3-H4 chaperone activity in the context of de novo nucleosome (re)assembly following DNA replication.
PubMed ID: 22941638
PMC ID: PMC3488248
Article link: Nucleic Acids Res.
Grant support: F32GM096531 NIGMS NIH HHS , GM- 067777 NIGMS NIH HHS , GM72719 NIGMS NIH HHS , Howard Hughes Medical Institute , R01 GM072719 NIGMS NIH HHS , P50 CA108961 NCI NIH HHS , F32GM096531 NIGMS NIH HHS , R01 GM072719 NIGMS NIH HHS , GM72719 NIGMS NIH HHS , Howard Hughes Medical Institute , GM- 067777 NIGMS NIH HHS
Genes referenced: acvrl1 asf1a cnot7 hist2h2ab hist2h2be ncr3
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|Figure 1. CAF-1 binds H3-H4 with ∼20-fold higher affinity than H2A-H2B. Titration of CAF-1 into fluorescently labeled histone complexes (H3-H4 and H2A-H2B) results in a change in fluorescent signal, indicating direct binding events. The normalized fluorescent change was plotted against increasing CAF-1 concentration (log [CAF-1]) to produce a binding curve where the CAF-1 concentration at 50% fluorescent change equals the apparent dissociation constant (Kdapp) for the CAF-1/histone complex. CAF-1 binds H3-H4 in the low nanomolar range with the averaged data points shown as black circles. The data points were fit with a non-linear regression curve to establish the apparent dissociation constant and Hill coefficient. CAF-1 binds H2A-H2B with significantly lower affinity, which is seen by a rightward shifted binding curve (gray squares). The error bars represent the standard error within individual data points. The total data points for a single experiment are 48.|
|Figure 2. Acetylation of H3K56 increases CAF-1 affinity for H3-H4, whereas removal of the terminal histone tails has no measurable effect. (A) The binding curves for H3K56Ac-H4 (gray squares) and wild-type H3-H4 (dashed line) with increased concentration of CAF-1. CAF-1 binds the acetylated form of H3-H4 with ∼2.5-fold higher affinity than that of the wild-type H3-H4 complex as shown through increased binding (more fluorescence change) at lower CAF-1 concentrations. (B) The effect of the histone tails on CAF-1 binding. H3-H4 complex lacking amino-terminal tails (gray squares) (H3 res. 27-135 and H4 res. 20-102) are titrated with CAF-1 and produce a markedly similar binding curve as the wild-type H3-H4 (dashed line). The error bars represent the standard error within individual data points.The total data points for a single experiment are 48.|
|Figure 3. The oligomeric state of the histone H3-H4 complex is dynamic across the nanomolar range. The size-exclusion elution profile for wild-type H3-H4 gradually shifts toward the right upon dilution from 1000 nM to 50 nM. At 1000 nM, the H3-H4 complex elutes at a volume (14 ml) consistent with a tetrameric conformation. At 50 nM, the H3-H4 peak elutes at 16 ml, consistent with a dimeric state. Elution profiles for cross-linked (H3-H4)2 tetramers remain at 14 ml at both 1000 and 50 nM (top right panel). The H3(L126R, I130R)-H4 double mutation prohibits H3:H3′ four-helix bundle formation and thus precludes (H3-H4)2 tetramer formation. This mutant histone complex elutes at 16 ml for both high and low concentrations (bottom right panel).|
|Figure 4. CAF-1 binds (H3-H4)2 tetramers and H3-H4 dimers with similar affinities, yet 2-fold different stoichiometries. (A) An overlay of the binding curves for CAF-1 titrated into a H3(L126R, I130R)-H4 double mutant that effectively prevents tetramer formation (gray squares) and a cross-linked (H3-H4)2 tetramer (triangle) with the wild-type H3-H4 curve from Figure 1 (dashed line). The curves are very similar to wild-type and produce dissociation constants of 5.5 nM for the double mutant and 9.0 nM for the cross-linked complex (Table 1). (B) Determination of the stoichiometry of the CAF-1•WT H3-H4 complex by titration of CAF-1 into a constant concentration of labeled H3-H4. Fluorescence change occurs until wild-type H3-H4 is saturated with CAF-1 and the ratio at this inflection point is equal to the number of CAF-1 molecules bound to a single H3-H4 complex. (C) Determination of the stoichiometry of CAF-1/H3(L126R, I130R)-H4 complex. The fluorescence change plateaus similar to that of wild-type (H3-H4)2 tetramer and corresponds to 0.5 CAF-1 molecules per 1 H3(L126R, I130R)-H4 dimer or a 1:2 stoichiometry. (D) The cross-linked (H3-H4)2 tetramer is bound by a single CAF-1 molecule, as shown by the near 1:1 ratio obtained by the titration.The error bars represent the standard error within individual data points.The total data points for a single experiment are 48.|
|Figure 5. Determination of the apparent molecular weight of CAF-1/H3-H4 complex using SEC-MALS. (A) The elution profiles for CAF-1 (gray) and H3-H4 bound CAF-1 (black). H3-H4 binding by CAF-1 results in earlier elution volumes (10 ml) than CAF-1 alone (12 ml), suggesting a larger molecular weight for the complex. Light scattering confirms a consistently larger molecular weight across the earlier eluting peak (Panel A, right axis and Table 2). (B) The same size exclusion peak profile as (A), but with the measured root mean square (RMS) radius for each peak highlighted on the right axis. The CAF-1/H3-H4 complex has only a slightly larger radius in solution than CAF-1 alone (Table 2).|
|Figure 6. Two H3-H4 molecules are co-purified with CAF-1 in yeast cells. (A–B) CAF-1 binds both H3 and H3-HA. The sequential IP procedure is outlined in panel A. Cac2-TAP is first purified from a yeast strain with either HHT1 or HHT2 tagged with the HA epitope using IgG sepharose.The eluted proteins are either immunoprecipitated with antibodies against the HA epitope (Panel B, lanes 1–3) or precipitated with TCA (lanes 4–6). After precipitation, H3 and H3-HA were detected by western blot using antibodies against H3K56Ac and both the HA-tagged and untagged H3 were found in complex with CAF-1.|