Disordered regions and folded modules in CAF-1 promote histone deposition in Schizosaccharomyces pombe
- Université Paris-Saclay, CEA, CNRS, Institute for Integrative Biology of the Cell (I2BC), Institute Joliot, France
- Institut Curie, PSL Research University, CNRS UMR 3348, INSERM U1278, Université Paris-Saclay, Equipe labellisée Ligue contre le Cancer, France
- Institut Curie, PSL Research University, CNRS, Sorbonne Université,CNRS UMR3664, Nuclear Dynamics Unit, Équipe Labellisée Ligue contre le Cancer, France
- Synchrotron SOLEIL, HelioBio group, l'Orme des Merisiers, France
Figures
Figure 1 with 5 supplements
The large SpCAF-1 subunit includes four Intrinsically Disordered Regions (IDR).
(A) Names for the large, medium and small subunits of CAF-1 in H. sapiens, S. cerevisiae, and S. pombe. (B) Upper panel: The magenta line shows the predicted disorder of Pcf1 (spot disorder …
Figure 1—figure supplement 1
Sequence and constructions of Pcf1, the large subunit of SpCAF-1.
(A) Alignment of the large-subunit of CAF-1 from S. pombe, S. cerevisiae, and human (B) Domain composition of Pcf1 and binding predictions inferred from sequence homology. The delimitation and …
Figure 1—figure supplement 2
Reconstitution and structural analysis of SpCAF-1.
(A–B) SEC analysis of recombinant Pcf1, Pcf2, and Pcf3 proteins purified separately (1-3) mixed by pairs (5-7) or the mix of the three proteins (4). For each chromatograph, the composition of all …
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Figure 1—figure supplement 2—source data 1
Uncropped SDS Page gels for the SEC analysis of recombinant Pcf1, Pcf2, and Pcf3 proteins purified separately presented in Figure 1—figure supplement 2A, B (lower panels).
- https://cdn.elifesciences.org/articles/91461/elife-91461-fig1-figsupp2-data1-v1.pdf
Figure 1—figure supplement 3
Structure prediction of the module Pcf1(403-450)-Pcf2(1-453) from SpCAF-1.
(A) Predicted Alignment Error plot (PAE) calculated by AlphaFold2 for the best model for the Pcf1(403-450)-Pcf2(1-453) complex corresponding the module of SpCAF-1 including the seven WD repeats of …
Figure 1—figure supplement 4
Structure prediction of the module Pcf1(200-335)-Pcf3(1-408) from SpCAF-1.
(A) Predicted Alignment Error plot (PAE) calculated by AlphaFold2 for the best model for the Pcf1(200-335)-Pcf3(1-408) complex corresponding the module of SpCAF-1 including the seven WD repeats of …
Figure 1—figure supplement 5
Experimental and fitted SAXS data for SpCAF-1 and SpCAF-1−H3−H4.
(A) Experimental and fitted SAXS profile intensity (I) as a function of the momentum transfer (q) for SpCAF-1 (black circles) and for the best model generated by the Dadimodo Software (Rudenko et …
Figure 2 with 2 supplements
The acidic ED domain binds histones alone and in the full SpCAF-1 complex.
(A) SEC profile and the SDS-PAGE purity of SpCAF-1−H3−H4 histones at 150 mM NaCl and 1 M NaCl. (B) Mapping of the interaction between SpCAF-1(15N-Pcf1) and SpH3−H4 histones, using the intensities …
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Figure 2—source data 1
Uncropped SDS PAGE gels presented in Figure 2A and Figure 2—figure supplement 2B.
- https://cdn.elifesciences.org/articles/91461/elife-91461-fig2-data1-v1.pdf
Figure 2—figure supplement 1
The acidic ED domain binds histones alone and in the full CAF-1 complex.
(A) Left panel: Overlay of the NMR 1H-15N SOFAST-HMQC spectrum of S. pombe CAF-1(15N-Pcf1) alone (magenta) and after addition of S. pombe H3−H4 (1:1) (black). middle panel: Overlay of the 1H-15N …
Figure 2—figure supplement 2
Structure prediction of the module Pcf1(352-383)−H3−H4 from SpCAF-1 and purification of reconstituted CAF-1 complexes WT and mutants.
(A) Predicted Alignment Error plot (PAE) calculated by AlphaFold2 for the best model for the Pcf1(352-383)−H3(60–136)−H4(25–103). (B) SDS-PAGE electrophoresis of the reconstituted CAF-1 complexes WT …
Figure 3 with 4 supplements
Pcf1_KER is the main DNA binding domain of SpCAF-1.
(A) EMSA with SpCAF-1 and 40 dsDNA (1 µM) revealed with SYBR SAFE staining. (B) MicroScale thermophoresis (MST) fitted curves of SpCAF-1 WT and mutants with 40 bp dsDNA. (C) Upper panel: Modeled …
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Figure 3—source data 1
Uncropped SDS PAGE gels presented in Figure 3 and Figure 3—figure supplements 1–4.
- https://cdn.elifesciences.org/articles/91461/elife-91461-fig3-data1-v1.pdf
Figure 3—figure supplement 1
Structural characterization of the Pcf1_KER domain.
(A) EMSA with SpCAF-1 and a ladder of 20–100 bp dsDNA fragments revealed with SYBR SAFE staining. (B) Circular dichroism (CD) spectrum of the Pcf1_KER domain (56-170) at 20 °C. The measured signal …
Figure 3—figure supplement 2
Binding of WT and mutants Pcf1_KER and SpCAF-1 to DNA analysed by EMSA.
(A) EMSA showing Pcf1_KER binding to a 20–100 bp ladder of ds DNA fragments. (B) EMSA showing Pcf1_KER-PIP binding to a 40 bp dsDNA. (C) CD spectrum of the mutated Pcf1_KER* at 20 °C. (D–F) EMSA …
Figure 3—figure supplement 3
Binding of WT and mutants SpCAF-1 to histones H3-H4 analyzed by NMR.
(A) Left panel: Overlay of the 1H-15N SOFAST-HMQC spectrum of the WT SpCAF-1(15N-Pcf1) (magenta) and the SpCAF-1(15N-Pcf1-KER*) (purple). Right panel: Overlay of the 1H-15N SOFAST-HMQC spectrum of …
Figure 3—figure supplement 4
Pcf1_WHD domain adopts a WHD fold similar to Sc C-terminal domain of Cac1, but does not bind DNA alone.
(A) Left panel: 1H-15N HSQC spectrum of the Pcf1_WHD domain at 10 °C. Assignments are indicated. The large spectral dispersion of the HN signals is consistent with a fully folded domain Right panel: …
Figure 4 with 2 supplements
The SpCAF-1-KER* mutant is affected for PCNA binding.
(A) EMSA showing interactions of purified SpCAF-1 (at the indicated concentrations), with or without recombinant SpPCNA (3 µM) in the presence and absence of 40 bp dsDNA (1 µM), revealed with …
Figure 4—figure supplement 1
Characterization of the Pcf1 PIP motif.
(A) ITC thermograms and data fitting for the indicated peptides upon titration of SpPCNA. The sequence of the three peptides is indicated as well as the canonical consensus for a PIP motif (h means …
Figure 4—figure supplement 2
EMSA showing interactions of purified CAF-1 mutated complexes with DNA and PCNA.
EMSA showing interactions of purified CAF-1 mutated complexes (at the indicated concentrations), with or without recombinant SpPCNA (3 µM) in the presence and absence of 40 bp dsDNA (1 µM). For each …
Figure 5 with 1 supplement
Efficient nucleosome assembly by SpCAF-1 in vitro requires interactions with H3−H4, DNA and PCNA, and the C-terminal WHD domain.
(A) Experimental scheme depicting the nucleosome assembly assay to monitor the efficiency of the SpCAF-1 complex. A plasmid with UV lesions (black star) when incubated in Xenopus HSE extracts …
Figure 5—figure supplement 1
Western blot analysis of mock- and p150-depleted HSE.
Xenopus p150 and p60 CAF-1 are shown for 0.25, 0.5, 1, and 3 µL of HSE as indicated. β-actin is used as a loading control. Stars on the p60 indicate non-specific bands. M, molecular weight markers …
Figure 6 with 1 supplement
Association of CAF-1 with histone modulates PCNA interaction in vivo and foci formation.
(A) Anti-FLAG Pulldown to address PCNA−CAF-1 interaction in vivo in indicated strains. (B) Quantification of bound PCNA from (A). (C) Simultaneous acquisition of Pcf2-GFP in WT (red arrow) and pcf1 …
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Figure 6—source data 1
Uncropped blots presented in Figure 6A.
- https://cdn.elifesciences.org/articles/91461/elife-91461-fig6-data1-v1.pdf
Figure 6—figure supplement 1
Association of CAF-1 with histone is coupled to PCNA interaction in vivo.
(A) Expression levels of the FLAG-Pcf1 in indicated strains from total extracts. PCNA was used as loading control. (B) Nitrocellulose membrane from Figure 6A stained with Red ponceau. (C) Example of …
Figure 7 with 1 supplement
The WHD domain of SpCAF-1 specifies CAF-1 function.
(A) Top panel: Schematic representation of the silencing assay used. Otr: outer repeats; imr: inner repeats; cnt1: central core of the centromere 1. Bottom panel: Serial fivefold dilution of …
Figure 7—figure supplement 1
the pcf1-ED* mutation confers a synthetic growth defect when combined with hip1∆.
Spores of the indicated genotypes were streaked onto a YEA agar plate and grown at 30 °C for 3 days.
Author response image 1
Quantitative analysis of interaction with DNA by EMSA.
a: quantification of the amount of bound DNA for the Pcf1_KER domain (blue points with error bars). The fit with a KD model is shown as a dashed line, and the fit with a Hill model with a solid …
Author response image 2
Tables
Table 1
Experimental affinities of different SpCAF-1 constructs with a 40 bp dsDNA measured by MicroScale thermophoresis (MST) fitted with the Hill model (Tso et al., 2018).
| Construct | EC50 (µM) | Hill coeff., h |
|---|---|---|
| Pcf1_KER | 1.1±0.2 | 3.3±0.5 |
| Pcf1_KER* | 12.2±0.7 | 1.5±0.3 |
| Pcf1_KER-PIP | 1.9±0.3 | 5.2±0.9 |
| Pcf1_WHD | Not detected | Not detected |
| SpCAF-1 WT | 0.7±0.1 | 2.7±0.2 |
| SpCAF-1-ΔWHD | 0.7±0.1 | 2.3±0.3 |
| SpCAF-1-KER* | 2.8±0.4 | 1.3±0.3 |
| SpCAF-1-ED* | 1.0±0.1 | 2.3±0.1 |
| SpCAF-1-PIP* | 0.7±0.1 | 2.7±0.3 |
Table 2
Interactions parameter with SpPCNA measured by isothermal microcalorimetry (ITC).
| Ligand | Kd (μM) | ΔG (kCal.M–1) | N* | ΔH (kCal.M–1) | -TΔS (kCal.M–1) |
|---|---|---|---|---|---|
| Pcf1_PIP | 7.1±1.3 | –6.9±0.1 | 0.97±0.08 | –2.9±0.2 | –0.39±0.3 |
| Pcf1_PIP* | Not detected | ND | ND | ND | ND |
| Pcf1_KER-PIP | 0.7±0.2 | –8.2±0.2 | 0.64±0.04 | +2.9 ± 0.6 | –11.2±0.8 |
| Pcf1_KER*-PIP | 7.1±1.5 | –6.9±1.2 | 0.7±0.2 | +1.0 ± 0.5 | –7.9±0.7 |
| Pcf1_KER-PIP* | Not detected | ND | ND | ND | ND |
| Cdc27_PIP | 3.5±0.3 | –7.3±0.1 | 0.9±0.1 | –4.8±0.02 | –2.4±0.1 |
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*The stoichiometry (N) is calculate as a molar ratio of monomeric PCNA.
Additional files
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MDAR checklist
- https://cdn.elifesciences.org/articles/91461/elife-91461-mdarchecklist1-v1.pdf
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Supplementary file 1
Supplementary tables.
(A) Experimental information and modelling of SAXS data. (B) Yeast strains used in this study.
- https://cdn.elifesciences.org/articles/91461/elife-91461-supp1-v1.docx
