Fig 1: DAXX adds a de novo H3.3K9me3 deposition pathway to the histone chaperone networkDuring histone supply ASF1 handles H3.1/2/3–H4 dimers and forms several histone-dependent co-chaperone complexes with other histone chaperones, notably ASF1 channels H3 variants toward distinct deposition complexes on chromatin. We identified a new ASF1-centered histone supply pathway to SPT2 that is H3 variant independent, as well as an upstream role for ASF1 in delivering H3.3 histones to DAXX, and an H3.1 variant specificity in TONSL. We found DAXX facilitates the catalysis of H3.3K9me3 through SETDB1 and SUV39H1 methyltransferase recruitment prior to histone deposition on chromatin. DAXX-bound H3.3–H4 recruits SETDB1 and SUV39H1, and the interaction of DAXX with SETDB1 is additionally dependent on SUMOylation. Other factors involved in heterochromatin establishment are differentially dependent on ATRX (e.g., the ChAHP complex) and SUMOylation (e.g., SMCHD1-LRIF1), and we speculate that this represents alternative pathways for H3K9me3 deposition supporting de novo heterochromatin silencing at distinct genomic locations.
Fig 2: Validation of previously uncharacterized histone-dependent interactors(A) Bubble plot showing histone-dependent interactors across triple SILAC IP-MS interactomes. Proteins are referred to by human UniProt protein identification code. Data generated from n = 4 biological replicates. Colors represent Log2 SILAC ratios (HBM/WT), and radii represent p values. See also Table S1.(B) Pull-downs of Strep-HA-tagged histone chaperones WT, HBM, B domain mutant (BDM), or ATRX-binding mutant (ABM) compared with control purifications (-) from soluble cell extracts probed by western blot. Representative of n = 2 biological replicates.(C) “Local” AlphaFold prediction of SPT2 (yellow) and ASF1A (magenta) histone-binding domains bound to H3.1–H4 (red and cyan) depicting the high-confidence regions of the full-length AlphaFold prediction.(D) Predicted alignment error (PAE) plot showing confidence of residue contacts in the full-length SPT2–H3.1–H4–ASF1A “global” AlphaFold prediction. Red dashed lines indicate high-confidence interactions between protein chains in the “local” prediction shown (left). See also Figures 3A–3C.(E) Alignment of local AlphaFold prediction of SPT2–H3.1–H4–ASF1A (colored as C) to the crystal structure of SPT2–(H3.2–H4)2 (white, PDB: 5BS7, with H3.2–H4 omitted for clarity). See also Figures S3D and S3E.
Supplier Page from Abcam for Anti-SPTY2D1 antibody