Fig 2: CDAN1 interacts with ASF1 through multiple motifs.a,b, Structural models of CDAN1 B-domain interactions with a, ASF1A-1 (BDA1) and b, ASF1A-2 (BDA2), colored as in Fig. 3. c, Sequence alignments of CDAN1 B-domains (BD, top) and H3 mimic helices (HMH, bottom) with other ASF1 interactors or histone H3. CDAN1 sequences are colored according to Consurf63 conservation values as indicated. d, Structural model of the CDAN1 HMH interaction with ASF1A-1 (HMHA1) superposed with the corresponding X. laevis histone H3 helix (transparent gray; residues 121-136, PDB 2IO5). e, Alphafold3 model of the putative CDAN1 HMH interaction with ASF1A-2 (HMHA2) superposed with the H3 helix (transparent gray).

Fig 3: CDAN1 and CDIN1 assemble together with ASF1 in the cytosol.a, Endogenous CDAN1 and CDIN1 assemble with ASF1. Flp-In 293 T-REx cells without or with a C-terminal HaloTag-FLAG (HF) tag on endogenous CDIN1 or CDAN1 were lysed (input), subjected to anti-FLAG immunoprecipitations (IP), and analyzed by SDS-PAGE and immunoblotting; representative of 3 independent replicates. Note: the CDAN1 antibody raised against a C-terminal epitope does not recognize CDAN1-HF. Where applicable, different protein populations recognized by the same antibody are labeled. b, CDIN1 stability requires CDAN1. Parental, CDIN1-HF, or CDAN1-HF cells were treated without or with 500 nM HaloPROTAC3 (HP3) or an inactive enantiomer (ent-HP3) for 72 hr, lysed, and analyzed by SDS-PAGE and immunoblotting; representative of at least 3 independent replicates. c, CDAN1 and CDIN1 are cytosolic. Parental, CDIN1-HF, or CDAN1-HF cell lysates (total) were fractionated to separate cytosolic and nuclear contents and analyzed by SDS-PAGE and immunoblotting; representative of 3 independent replicates. d, Representative live-cell images of parental, CDIN1-HF, and CDAN1-HF cells labeled with JFX650 HaloTag ligand and Hoechst. Scale bar, 10 μm.

Fig 4: ASF1A and ASF1B have different CDAN1 binding requirements.a, Mutations of key residues in the canonical CDAN1 B-domain (BDA2 mutated to B×) and H3 mimic helix (HMHA1 mutated to H×) (top) and scheme of ASF1A and ASF1B N-terminal domain (NTD) swaps analyzed. b, ASF1B interaction with CDAN1 is more reliant on HMHA1 than ASF1A. Flp-In 293 T-REx cells transiently transfected with Strep-tagged CDAN1 (ST-CDAN1) variants without or with mutations in BDA2 (B×) and/or HMHA1 (H×) were lysed (input) and subjected to Strep-Tactin pulldowns (PD) before analysis by SDS-PAGE and immunoblotting; representative of 3 independent replicates. c, The indicated ST-CDAN1 variants were co-transfected with FLAG-tagged ASF1A (F-ASF1A, left) or ASF1B (F-ASF1B, right) with the indicated N-terminal domain (NTD). Cell lysates before (input) or after Strep PD were analyzed by SDS-PAGE and immunoblotting; representative of 2 independent replicates.

Fig 5: CDAN1 complexes contain multiple copies of each subunit.a, Strep-tagged CDAN1 (ST-CDAN1), FLAG-tagged CDIN1 (F-CDIN1), and HA-tagged ASF1A were overexpressed in Expi293 cells by transient transfection for 72 hr. The cells were then lysed, and the post-nuclear supernatants (PNS) were subjected to anti-FLAG immunoprecipitation (FLAG IP) followed by Strep-Tactin pulldowns (Strep PD) to purify the CDAN1-CDIN1-ASF1A (C:C:A) complex. The input, flow-through (FT), and elution (Elu.) samples were analyzed by SDS-PAGE and Coomassie staining (top) or immunoblotting (bottom); representative of at least 2 independent replicates. Arrowheads denote histones. b, As in a, except with overexpression of ST-CDAN1 and FLAG-tagged ASF1A (F-ASF1A) to purify the CDAN1-ASF1A (C:A) complex. c, Size exclusion chromatography with multi-angle light scattering (SEC-MALS) of the C:C:A and C:A complexes determined molecular weights consistent with 2 copies of CDAN1, 3 copies of ASF1A, and, in the C:C:A complex, 2 copies CDIN1. The predicted molecular weights of these stoichiometries are indicated.