1. Structural Biology and Molecular Biophysics
  2. Chromosomes and Gene Expression
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The Cac1 subunit of histone chaperone CAF-1 organizes CAF-1-H3/H4 architecture and tetramerizes histones

  1. Wallace H Liu
  2. Sarah C Roemer
  3. Yeyun Zhou
  4. Zih-Jie Shen
  5. Briana K Dennehey
  6. Jeremy L Balsbaugh
  7. Jennifer C Liddle
  8. Travis Nemkov
  9. Natalie G Ahn
  10. Kirk C Hansen
  11. Jessica K Tyler  Is a corresponding author
  12. Mair EA Churchill  Is a corresponding author
  1. University of Colorado School of Medicine, United States
  2. Weill Cornell Medicine, United States
  3. MD Anderson Cancer Center, United States
  4. University of Colorado, Boulder, United States
Research Article
Cite this article as: eLife 2016;5:e18023 doi: 10.7554/eLife.18023
7 figures, 4 tables and 1 additional file

Figures

Figure 1 with 1 supplement
The Cac1 subunit is sufficient for (H3/H4)2 tetramerization.

(A) Schematic of domains in the individual CAF-1 subunits. (B) H3/H4(Py) binding to individual CAF-1 subunits. Fluorescence anisotropy of 25 nM pyrene-labeled H3/H4 was monitored with titration of individually purified Cac1, Cac2, or Cac3 in Histone Buffer (H.B.: 20 mM Tris, 150 mM KCl, 2 mM MgCl2, 0.5 mM TCEP, 1% Glycerol, 0.05% BRIJ-35.) The CAF-1 complex was titrated into 5 nM H3/H4(Py). The curves were fitted using Equation 3. (C) A representative EMSA separating histone:DNA species as disomes or tetrasomes. 1.6 µM of the indicated histone chaperone or CAF-1 subunit was incubated with 0.2 µM H3/H4(FM) dimer, prior to addition of 0.4 µM 80 bp DNA. The bar graph shows the mean and standard deviation of fraction of tetrasomes formed, calculated by Equation 5, from at least three independent experiments. (D) FRET of mixed labeled H3/H4(CPM/FM). Spectra were obtained for 10 nM of labeled histones incubated with 0.2 µM CAF-1 or DNA, or 1 µM CAF-1 subunit. The FRET Effect was calculated using Equation 4 from at least three independent experiments.

https://doi.org/10.7554/eLife.18023.003
Figure 1—figure supplement 1
Purified proteins used in this study.

Coomassie-stained SDS PAGE of the (A) individual, purified CAF-1 subunits, Cac1 truncations, and (B) CAF-1 and CAF-1-H3/H4 complexes cross-linked by DSS or EDC.

https://doi.org/10.7554/eLife.18023.004
Figure 2 with 1 supplement
Hydrogen/deuterium exchange of CAF-1, and CAF-1-H3/H4 complexes.

(A) The sequences of the three individual CAF-1 subunits are shown. Each bar represents an individual identical peptide observed in the protein between the compared samples, plotted as the difference in deuteron uptake between the CAF-1 and CAF-1-H3/H4 samples (i.e., difference = CAF-1-H3/H4 – CAF-1 only). The differences in deuteron uptake at 60’ are colored according to the legend. The 'cooler' colors (green, blue, and purple) represent an increase in apparent protection for the peptide in CAF-1-H3/H4 compared to the CAF-1 sample, whereas the 'warmer' colors (orange, yellow, and red) represent decreased apparent protection. Peptide coverage was approximately 60%, 80%, and 80% for Cac1, Cac2 and Cac3, respectively. (B) Differences in HX at 60’ were mapped on PHYRE2 models of Cac2 and Cac3. The coloring scheme is the same as for A, but amino acids with no coverage are colored dark gray to distinguish these residues from those that have coverage but did not exchange significantly. (C) The top panel shows five fold serial dilution analysis of strain CFY53 (cac1) with the vector pCac1 introduced that was either empty, expressed wild type Cac1 or Cac1 with the indicated amino acid changes. The bottom shows five fold serial dilution analysis of strain CFY54 (cac2) with the vector pCac2 introduced that was either empty, expressed wild type Cac2 or Cac2 with the indicated amino acid changes.

https://doi.org/10.7554/eLife.18023.006
Figure 2—figure supplement 1
Peptide coverage in HX-MS.

Coverage maps of Cac1, Cac2, and Cac3, from side-to-side comparisons between CAF-1 and CAF-1-H3/H4 samples in the HX study. All identical peptides between multiple samples were calculated for differences in deuteron uptake at 60’ and colored accordingly, using the same scheme as in Figure 2B.

https://doi.org/10.7554/eLife.18023.007
Figure 3 with 1 supplement
Chemical cross-linking of CAF-1 and CAF-1-H3/H4 complexes.

(A) CAF-1 or (B) CAF-1-H3/H4 complexes were covalently cross-linked with DSS or EDC, then digested and run on an LTQ-Orbitrap. Cross-linked peptides were analyzed using Protein Prospector. The primary sequences are depicted in gray bars, with each gray circle marking 50 amino acid segments. DSS cross-links are shown in purple and EDC cross-links are in red. DSS leaves a 11.4 Å spacer arm between covalently-linked amine groups. EDC treatment results in a zero length cross-link between amine and carboxyl groups. The inter-subunit cross-links are represented as solid lines and cross-links to H3 and H4 are shown as dotted lines. (C) Analysis of Cac3 mutants in yeast. Cac3 mutants were subjected to zeocin-induced DNA damage response in vivo. The panel shows five fold serial dilution analysis of strain CFY58 (cac3) with the vector pCac3 introduced that was either empty (EV), expressed wild type Cac1, or Cac1 with the indicated amino acid changes.

https://doi.org/10.7554/eLife.18023.010
Figure 3—figure supplement 1
Intra-Cac1 cross-links.

(A) DSS (purple) and EDC (red) cross-links detected within the Cac1 protein in the CAF-1 complex and (B) CAF-1-H3/H4 complex.

https://doi.org/10.7554/eLife.18023.011
Figure 4 with 1 supplement
The C-terminus of Cac1 binds and tetramerizes H3/H4.

(A) EMSA evaluating tetrasome formation by Cac1 N-terminal truncations Cac1386, Cac1421 and Cac1457 in H.B. The graph shows the mean and standard deviation from at least three independent experiments. Arrows point to complexes of DNA bound to H3/H4 dimers (D) or tetramers (T), respectively. (B) Change in FRET Effect of H3/H4(CPM/FM) induced by 2 µM Cac1386, Cac1421 or Cac1457. The Cac1 spectrum is included from Figure 2B for reference. (C) Fluorescence anisotropy of Cac1386(Py) or Cac1454(Py) titrated with H3/H4 in H.B. The schematic indicates two labeled residues on Cac1386 (cysteines 440 and 454), and one on Cac1454.

https://doi.org/10.7554/eLife.18023.012
Figure 4—figure supplement 1
Histone deposition assay of Cac1 truncations in Minimal Buffer (M.B.).

1.6 µM of each Cac1 truncation was incubated with 0.2 µM H3/H4FM, then allowed to interact with 0.4 µM 80 bp DNA. The EMSA (upper panel) is representative of at least four independent experiments that were used for comparisons in the bar graph (lower panel).

https://doi.org/10.7554/eLife.18023.013
Figure 5 with 2 supplements
The Cac1 C-terminal winged helix (WH) domain can form a homodimer.

(A) Crystal structure of amino acids 520–600 at a resolution of 2.9 Å (PDB ID 5JBM), shown as two crystallographically related monomers colored separately (light gray and dark gray). The inset shows major interacting residues buried in half of the homodimer interface, which is arranged in a head-to-tail symmetry with identical interactions on both halves. (B) Homo-dimerization of the Cac1 C-terminus quantified by titrating unlabeled Cac1386 or Cac1454 into 10 nM of labeled Cac1386(Py) or Cac1454(Py), respectively. The pyrene anisotropy of Cac1386(Py) or Cac1454(Py) increases in Minimal Buffer (M.B.: 20 mM HEPES, 150 mM NaCl, 1 mM DTT, pH 7.5), but homo-dimerization does not occur in H.B. (C) Binding affinity of the Cac1386(Py)-Cac2 interaction. Pyrene fluorescence anisotropy of 10 nM Cac1386(Py) titrated with increasing concentration of Cac2 in Histone Buffer (H.B.). The KD was determined to be 1.3 µM (Table 1). (D) Pyrene fluorescence spectra of Cac1386(Py) alone, Cac1454(Py) alone, and Cac1386(Py) bound to 2 µM H3/H4 or 13 µM Cac2. The excimer band that peaks at 465 nm is indicated.

https://doi.org/10.7554/eLife.18023.014
Figure 5—figure supplement 1
Purification of full-length Cac1 and Cac1457 from baculovirus-infected Sf9 cells.

(A) Cac1 elutes from a 120 mL Sephadex 200 column in 3 peaks. (B) Western blotting for the Strep II epitope present on the Cac1 C-terminus. Peak 1 is full-length Cac1, whereas Peak 3 is truncated from the N-terminus. (C) MALDI identified the C-terminal regions as residues 457–606 (expected mass 18285.3; observed mass 18276.4).

https://doi.org/10.7554/eLife.18023.015
Figure 5—figure supplement 2
Structural analysis of the Cac1457 WH domain.

(A) The Cac1 WH domain monomer and one of the symmetry mates are depicted in both a ribbon and surface representation, in two orientations. The Cac1 monomers are colored light gray and dark gray, respectively, with the electrostatic potential shown mapped onto the surface, colored from red to blue, indicating negatively charged to positively charged regions. (B) The Cac1 WH domain monomer is depicted in both a ribbon and surface representation. The putative dimerization interface faces to the right. HX changes are colored in orange to represent an increase in HX with H3/H4 bound to CAF-1 (Figure 3). Amino acids that cross-link to H3/H4 are labeled and colored in red.

https://doi.org/10.7554/eLife.18023.016
Architectural model of the CAF-1-H3/H4 complex.

Cac2 and Cac3 are presented using PHYRE2 models. Cac1 is presented with respect to the domains observed in this study: the 'Middle Domain,' which consists of amino acids 118–334 and includes the KER region; the 'ED-rich Region' that includes the ED domain and the adjacent amino acids; and the 'C-terminus' that includes the WH domain. The nucleosomal (H3/H4)2 tetramer is shown (1ID3.pdb) with H3 (blue) and H4 (bright green) colored to distinguish the histones. CAF-1 proteins are colored according to the 60’ HX data and coloring scheme in Figure 2B. The cross-linking data is incorporated using the same coloring and line schemes as in Figure 3B.

https://doi.org/10.7554/eLife.18023.018
Potential equilibrium for CAF-1 association with H3/H4.

D and T indicate dimers or tetramers of H3/H4, respectively.

https://doi.org/10.7554/eLife.18023.019

Tables

Table 1

KD values from pyrene fluorescence anisotropy of CAF-1 subunits and H3/H4.

https://doi.org/10.7554/eLife.18023.005
Pyrene-labeled proteinBinding partnerKD or KDapp (M)
H3/H4CAF-15.3 ± 0.9 × 10−9
H3/H4Cac19.7 ± 1.8 × 10−8
H3/H4Cac2n.c.
H3/H4Cac3n.c.
Cac1386Cac13862.6 ± 0.2 × 10−8
Cac1454Cac14542.5 ± 0.2 × 10−8
Cac1386H3/H42.1 ± 0.5 × 10−7
Cac1454H3/H4n.c.
Cac1386Cac21.3 ± 0.4 × 10−6
  1. n.c. not calculated

Table 2

Yeast strains and plasmids.

https://doi.org/10.7554/eLife.18023.008
StrainMutationGenotypeReference
w1588-4aWTMat alpha; leu2-3,112; ade2-1; can1-100; his3-11,15; ura3-1; trp1-1; RAD5Gift from R. Rothstein
CFY53cac1ΔMat alpha; leu2-3,112; ade2-1; can1-100; his3-11,15; ura3-1; trp1-1; RAD5 cac1Δ::NATThis study
CFY54cac2ΔMat alpha; leu2-3,112; ade2-1; can1-100; his3-11,15; ura3-1; trp1-1; RAD5 cac2Δ::NATThis study
CFY58cac3ΔMat alpha; leu2-3,112; ade2-1; can1-100; his3-11,15; ura3-1; trp1-1; RAD5 cac3Δ::NATThis study
JKT004rad52ΔMAT a rad52::TRP1; trp1-1; ura3-1; can1-100; ADE; bar1::LEU2; his3-11; GALRamey et al. (2004)
PlasmidCharacteristicsReference
pRS315 (EV)CEN6 ARSH4 LEU2Sikorski and Hieter (1989)
pCac1pRS315-Cac1This study
pCac2pRS315-Cac3This study
pCac3pRS315-Cac3This study
pCac1Δ233-237pRS315-Cac1 aa 233-237 deletedThis study
pCac1Δ280-284pRS315-Cac1 aa 280 to 284 deletedThis study
pCac1Δ304-322pRS315-Cac1 aa 304 to 322 deletedThis study
pCac1Δ340-360pRS315-Cac1 aa 340-360 deletedThis study
pCac1Δ428-432pRS315-Cac1 aa 428-432 deletedThis study
pCac1K442E/R443E/K444EpRS315-Cac1 with the mutation K442E/R443E/K444EThis study
pCac1Δ463-473pRS315-Cac1 aa 463 to 473 deletedThis study
pCac1Δ497-501pRS315-Cac1 aa 497 to 501 deletedThis study
pCac1Δ574-584pRS315-Cac1 aa 574-584 deletedThis study
pCac1Δ578-580pRS315-Cac1 aa 578 to 580 deletedThis study
pCac1Δ576-606pRS315-Cac1 aa 576-606 deletedThis study
pCac1Δ578-580pRS315-Cac1 aa 578 to 580 deletedThis study
pCac2Δ1-15pRS315-Cac2 aa 1 to 15 deletedThis study
pCac2E70KpRS315-Cac2 with the mutation E70KThis study
pCac2D91K/D92KpRS315-Cac2 with the mutation D91K/D92KThis study
pCac2S206A/A207GpRS315-Cac2 with the mutation S206A/A207GThis study
pCac2V273A/P275A/S276A/G277ApRS315-Cac2 with the mutation V273A/P275A/S276A/G277AThis study
pCac2I274A/S276ApRS315-Cac2 with the mutation I274A/S276AThis study
pCac2D248K/E285KpRS315-Cac2 with the mutation D248K/E285KThis study
pCac2R295EpRS315-Cac2 with the mutation R295EThis study
pCac2K306A/N307A/R308ApRS315-Cac2 with the mutation K306A/N307A/R308AThis study
pCac2L316A/K318ApRS315-Cac2 with the mutation L316A/K318AThis study
pCac2L316E/K318EpRS315-Cac2 with the mutation L316E/K318EThis study
pCac2Δ371-373pRS315-Cac2 aa 371 to 373 deletedThis study
pCac2M417A/H418A/E420ApRS315-Cac2 with the mutation M417A/H418A/E420AThis study
pCac2Δ425-468pRS315-Cac2 aa 425-468 deletedThis study
pCac2Δ445-468pRS315-Cac2 aa 445-468 deletedThis study
pCac2K447E/K448EpRS315-Cac2 with the mutation K447E/K448EThis study
pCac3K284A/K285A/E286ApRS315-Cac3 with the mutation K284A/K285A/E286AThis study
pCac3Δ306-309pRS315-Cac3 deleted aa 306 to 309This study
pCac3Δ287-290pRS315-Cac3 deleted aa 287 to 290This study
Table 3

Yeast mutants and phenotypes observed.

https://doi.org/10.7554/eLife.18023.009
MutantRationale for mutantZeocin resistanceProtein expression
Cac1Δ233-237Cross-link to Cac3 (Figure 3B)SensitiveNo
Cac1Δ280-284Cross-link to Cac3 (Figure 3A,B)SensitiveNo
Cac1Δ304-322HX change with H3/H4 and cross-link to Cac2 (Figure 2A,3B)Very sensitiveYes
Cac1Δ340-360HX change with H3/H4 (Figure 2A)SensitiveNo
Cac1Δ428-432In ED-rich Region (Figure 1A)Not sensitiveYes
Cac1K442E/K443E/K444ECross-link to H3 (Figure 3B)Not sensitiveYes
Cac1Δ463-473HX change and cross-link to H3/H4 (Figure 2A, 3B)SensitiveYes
Cac1Δ497-501Cross-link to Cac2 (Figure 3B)Little sensitiveYes
Cac1Δ574-584HX change and cross-link to H3/H4 (Figure 2A, 3B)Little sensitiveYes
Cac1Δ578-580HX change and cross-link to H3/H4 (Figure 2A, 3B)SensitiveYes
Cac1Δ575-606HX change and cross-link to H3/H4 (Figure 2A, 3B)Very sensitiveNo
Cac2Δ1-15HX change with H3/H4 (Figure 2A)Very sensitiveYes
Cac2E70KLoop next to Cac2 N-terminal loop (Figure 2A)Not sensitiveYes
Cac2D91K/D92KCross-link to Cac1 (Figure 3A)SensitiveYes
Cac2S206A/A207GHX change with H3/H4 (Figure 2A)SensitiveYes
Cac2V273A/P275A/S276A/G277AHX change with H3/H4 (Figure 2A)SensitiveYes
Cac2I274A/S276AHX change with H3/H4 (Figure 2A)Not sensitiveYes
Cac2D284K/E285KCross-link to Cac1 (Figure 3A)Not sensitiveYes
Cac2R295ELoop between Cac2 blades 5 and 6 (Figure 2A)SensitiveNo
Cac2K306A/N307A/R308AHX change with H3/H4 (Figure 2A)Not sensitiveYes
Cac2L316A/K318AHX change with H3/H4 (Figure 2A)SensitiveYes
Cac2L316E/K318EHX change with H3/H4 (Figure 2A)SensitiveYes
Cac2Δ371-373Loop next to Cac2 N-term loop and blade 6 (Figure 2A)SensitiveYes
Cac2M417/H418A/E420AC-terminal loop in Cac2Not sensitiveYes
Cac2Δ425-468HX change with H3/H4 (Figure 2A)Not sensitiveYes
Cac2Δ445-468C-terminal loop in Cac2Not sensitiveYes
Cac3K284A/K285A/E286ACross-link to Cac1 (Figure 3B)Not sensitiveYes
Cac3Δ287-290Cross-link to Cac1 (Figure 3B)SensitiveYes
Cac3Δ306-309Cross-link to Cac1 (Figure 3A)SensitiveYes
Table 4

Data collection and refinement statistics.

https://doi.org/10.7554/eLife.18023.017
Wavelength1.0 Å
Resolution range – data collection29.43–2.91 (3.01–2.91)
Space groupP 41 2 2
Unit cell (Å)
(deg)
58.850 58.830 97.929
90 90 90
Total reflections26,419 (5001)
Unique reflections4117 (393)
Multiplicity6.42 (6.63)
Completeness (%)99.3 (99.7)
Mean I/sigma(I)12.9 (1.7)
Wilson B-factor92.56
R-meas0.099 (0.557)
Resolution range - refinement29.43–3.00 (3.107–3.00)
Reflections used in refinement3761 (365)
Reflections used for R-free360 (42)
R-work0.233 (0.408)
R-free0.275 (0.324)
Number of non-hydrogen atoms654
 Macromolecules653
 Protein residues81
RMS(bonds)0.007 Å
RMS(angles)0.93 deg
Ramachandran favored (%)88
Ramachandran allowed (%)12
Ramachandran outliers (%)0
Rotamer outliers (%)4.3
Clashscore6.85
Average B-factor48.8
Number of TLS groups3
  1. Statistics for the highest-resolution shell are shown in parentheses.

  2. Friedel mates were averaged when calculating data collection statistics.

Additional files

Supplementary file 1

Supplementary tables.

(A) Peptides identified in HX studies. (B) Primers used in the studies in yeast. (C) Chemically cross-linked peptides identified by XL-MS. (D) Cac1C putative dimer contacts.

https://doi.org/10.7554/eLife.18023.020

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