DNA-mediated association of two histone-bound complexes of yeast Chromatin Assembly Factor-1 (CAF-1) drives tetrasome assembly in the wake of DNA replication
- Howard Hughes Medical Institute, University of Colorado Boulder, United States
- Colorado State University, United States
- Weill Cornell Graduate School of Medical Sciences, United States
- Memorial Sloan Kettering Cancer Center, United States
- University of Colorado Boulder, United States
Figures
Figure 1 with 1 supplement
tCAF-1 is competent for nucleosome assembly in absence of other factors.
(a) Products from the nucleosome assembly assay on 207 bp DNA, with FL CAF-1 or tCAF-1. DNA is 200 nM, (H3–H4)2 200 nM and H2A-H2B-AttoN647 400 nM; 100-200-400-800 nM CAF-1 is titrated. Assemblies …
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Figure 1—source data 1
FL and tCAF-1 assemble nucleosomes in vitro.
- https://doi.org/10.7554/eLife.22799.003
Figure 1—figure supplement 1
Control experiments for Figure 1.
(a) FL CAF-1 binds DNA that is not assembled into nucleosomes. DNA and salt-assembled nucleosomes (Nuc) are 50 nM; CAF-1 was titrated from 50-100-200-400 nM. (b) DNA protection at 126–160 bp from …
Figure 2 with 2 supplements
CAF-1 has a single binding site for a H3-H4 dimer.
(a) van Holde-Weischet analysis of SV-AUC runs performed with tCAF-1 and WTH3-H4, titrated in a 1:1 or 1:2 molar ratio of CAF-1 to H3-H4 dimer (orange and purple). A constitutively dimeric form of …
Figure 2—figure supplement 1
Control experiments for Figure 2.
(a) DMH3-H4 is primarily a dimer, even under conditions that favor the tetrameric state of WTH3-H4. Gel filtration of WTH3-H4 and DMH3-H4 in refolding buffer (2M NaCl). (b) tCAF-1 binds equally well …
Figure 2—figure supplement 2
HX-MS data on CAF-1•H3-H4 complexes.
(a) HX-MS heatmap of the differences in deuterium uptake per H3 (top) or H4 (bottom) peptide when binding to tCAF-1 (60 min). The difference was calculated as percent uptake in free H3-H4 (unbound …
Figure 3 with 1 supplement
H3-H4 binding activates DNA binding by the CAF-1 complex.
(a) Schematic of the constructs and complexes used in this and subsequent figures. Cac1 domains that are combined with full length Cac2 and/or Cac3 as indicated, are depicted. PIP stands for PCNA …
Figure 3—figure supplement 1
Control experiments for Figure 3.
(a) Gel filtration profiles (S200 10/300 GL column) for the complexes described in Figure 3a. SDS PAGE analysis of the purified samples. The HBM sample is also shown on SDS PAGE in panel b) lane 11. …
Figure 4 with 1 supplement
Two CAF-1•H3-H4 complexes associate on a short DNA fragment.
(a–b) A ternary tCAF-1•histone•18DNA complex can be formed with DMH3-H4(a) or with XL(H3-H4)2 (b). EMSA was performed with 100 nM 18 bp DNA (18DNA). tCAF-1 is titrated 0.37–0.75-1.5–3 µM, and is …
Figure 4—figure supplement 1
Control experiments for Figure 4.
(a) In solution cross-linking experiments with 2 µM of either FL CAF-1 alone, FL CAF-1•H3-H4 complex, or FL CAF_WHD**•H3-H4 complex, and variable amounts of DNA (0-1-2-4 µM). DSS was added at 1 mM …
Figure 5 with 1 supplement
DNA of sufficient length sequesters (H3–H4)2 from CAF-1.
(a) EMSA performed with 100 nM 33mer DNA. tCAF-1 or tCAF-1_WHD** were titrated 0.37–0.75-1.5–3 µM, and were added with WTH3-H4 to the DNA. (b) EMSA performed with 100 nM 33 bp DNA (33DNA). tCAF-1 …
Figure 5—figure supplement 1
Control experiments for Figure 5.
(a) EMSA performed with 100 nM 33mer DNA. tCAF-1_∆WHDa or tCAF-1__∆WHDb were titrated 0.37–0.75-1.5–3 µM, and were added with WTH3-H4 to the DNA. (b) SEC-MALS experiment titrating 33DNA into a …
Figure 6
Model of the molecular mechanism of CAF-1 mediated tetrasome assembly.
(a) The nucleosome assembly mechanism of CAF-1 is activated by H3-H4 binding, which releases the WHD domain from an intramolecular interaction with the acidic region on Cac1. (b) DNA binding …
Figure 7 with 1 supplement
DNA binding by the Cac1 WHD is required for nucleosome assembly in vitro and in vivo.
(a) Quantification of the nucleosome bands (126–160 bp) from the NAQ assays. Mean ± SD is shown from two or more independent measurements. For simplicity, we only show results from experiments with …
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Figure 7—source data 1
Mutation of the Cac1 WHD inhibits nucleosome assembly in tCAF-1.
- https://doi.org/10.7554/eLife.22799.016
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Figure 7—source data 2
Mutation of the Cac1 WHD in tCAF-1 promotes the formation of sub-nucleosomal species.
- https://doi.org/10.7554/eLife.22799.017
Figure 7—figure supplement 1
Control experiments to Figure 7.
(a) Cac1 WHD mutation or deletion result in the deposition of a H3-H4 dimer onto DNA. Tetrasome assembly assay with tCAF-1 WHD mutants on 79 (top) or 147 bp DNA (bottom). (b) Cac1 mutants used in Fig…
Author response image 1
In solution cross-linking studies of the complexes formed by FL CAF-1/H3-H4 in presence of 1 18 bp DNA (18DNA) or PCNA.
CAF-1/H3-H4 was kept at 2 µM, 18DNA and PCNA were added at 4 and 8 µM, respectively. The whole gel is shown on the left, the close-up is shown on the right.
Additional files
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Supplementary file 1
HX-MS data and uptake plots for tCAF-1 alone and in complex with WT or DMH3-H4.
- https://doi.org/10.7554/eLife.22799.019
