Fig 2: HIRA collaborates with SRCAP to deposit H2A.Z on genome. (A) Meta-analysis (left) and heatmap (right) show Srcap reads density around summit (upper) and TSS (below) within a ±3 kb window. (B) Representative loci on Slc25a42 gene show the enrichment of H2A.Z and Srcap. (C) ChIP-qPCR validation of H2A.Z and Srcap enrichment at promoter of Slc35a42 gene. Error bars represent data from three independent experiments. (D) Venn diagram shows the overlap between H2A.Z, Hira and Srcap peaks. (E) Western blot shows the interaction among endogenous HA-Srcap, Hira and Ubn1 from nuclear lysates. Ino80, Daxx, H2A.Z and H3.3 are also detected. (F) Top panel showed schematic presentation of full length and truncation mutants of Hira subunit. Bottom panel showed the biochemical interactions between Hira truncations and SRCAP complex are analyzed by GST pull-down coupled with western blot analyses. (G) Heatmaps (upper) show H2A.Z reads density around H2A.Z peak summits (upper) in R1 WT, Hira-KO and Hira-truncation mutant expressing cells within a ±3 kb window. Representative loci (bottom) show the enrichment of H2A.Z. (H) ChIP-qPCR of H2A.Z, Srcap and Flag at representative genes Nrg2 and Spata4 in the rescued overexpressed cell lines. Error bars represent data from three independent experiments.

Fig 3: H2A.Z deposition requires both ANP32E and ATP in the RhIP assay.(A) Scheme of cell fractionation (left). HeLa cells were treated with non-ionic detergent and the supernatant (soluble fraction), which is removed from permeabilized cells, and the pellet, which corresponds to permeabilized cells, were separated (insoluble fraction). Distributions of SRCAP, TIP60 and ANP32E were investigated by western blotting (right). Histone H3 and GAPDH served as the chromatin and cytoplasmic protein controls, respectively. (B) Preparation of the ANP32E-knockdown cellular extract. HeLa cells were transfected with siRNA against ANP32E. After 72 hr, the ANP32E-knockdown cellular extract was prepared from the cells. The ANP32E-knockdown was confirmed by western blotting (upper). Loading control with the membrane stained with CBB (lower). (C) Scheme of RhIP-ChIP-seq of H2A.Z using the ANP32E-knockdown cellular extract. (D) Enrichment of incorporated H2A.Z with the control or ANP32E-knockdown extract. Each chromatin region was previously annotated by the chromHMM, as follows. TssA: active TSS, Enh: enhancers, and Ins: insulator. (E) Scheme of RhIP-ChIP-seq of H2A.Z using the ATP-depleted cellular extract. (F) Enrichment of incorporated H2A.Z with the ATP-depleted or ATP-supplemented extract. Each chromatin region was the same as in (D).

Fig 4: Defect of HIRA complex results in H2A.Z decrease on genome wide. (A) Schematic diagram of genome editing in defective cell lines. Hira-, Ubn2- and H3.3-knockout cell lines and Ubn1/2 double mutants were generated using CRISPR-Cas9 system. For subsequent experiments, 6 × HA tag was added to the N-terminus of Srcap in R1 wild type and above defective cell lines. (B) Western blot respectively shows the proteins levels of Hira (a), Ubn1&Ubn2 (b), and H3.3 (c) in the defective cell lines. (C) Meta-analysis (left) and heatmap (right) show H2A.Z reads density around summit (upper) and TSS (below) in R1 WT, Hira-KO, Ubn2-KO/Ubn1-KD, Ubn1/2-DM and H3.3-KO cells within a ±3 kb window. (D) Venn diagram shows the overlap of H2A.Z down-regulated peaks between the above defective cell lines. (E) The reduced H2A.Z peaks in either Hira-KO or Ubn2-KO/Ubn1-KD cell lines are defined as HIRA complex regulated (HR) peaks (23 530 peaks). The 5426 HR peaks overlapping with Ubn1/2-DM cell line is defined as HIRA-regulated and H3.3-dependent (HIRA-H3.3D). The other 18 104 HR peaks are HIRA-regulated and H3.3-independent (HIRA-H3.3ID).