Purification of H2A.Z complex from human cells. (A) Schematic summary of purification of H2A.Z complex. Nuclear extracts from H2A.Z-expressing cells were first fractionated by P11 cation exchange column with BC buffer as indicated. The P11 1.0 and 1.2 M KCl fractions containing ectopic H2A.Z were combined and further purified with anti-FLAG affinity chromatography as described under ‘Materials and Methods’. The purified proteins were separated in 4–20% gradient SDS–PAGE and subjected to western blot analysis with anti-FLAG and -HA antibodies. Lane 1, mock purification with nuclear extracts prepared from regular HeLa cells; lane 2, FLAG/HA-H2A.Z immunoprecipitation with nuclear extracts prepared from H2A.Z expressing cells. (B) Mass spectrometric analysis of H2A.Z-associated polypeptides. After a large-scale isolation of H2A.Z complex, the purified polypeptides were resolved in 4–20% gradient SDS–PAGE. The protein bands, that were not present in the control lane, were excised, and identified by mass spectrometric analysis. Lane 1, the proteins purified from control HeLa cells; lane 2, the proteins purified from H2A.Z expressing cells. The positions of the molecular mass markers (in kDa) are indicated on the left. (C) Western blot analysis of the purified H2A.Z complex. H2A.Z complex was separated by 4–20% gradient SDS-PAGE, transferred to nitrocellulose, and probed by western analysis with antibodies as indicated on the left. Lane 1, mock-purified control; lane 2, H2A.Z complex. (D) Acetylation of H2A histone octamer and H2A nucleosome by H2A.Z complex. Recombinant histone octamer and nucleosome were subjected to HAT assay with [³H]-acetyl-CoA and the H2A.Z complex. HAT reactions were analyzed by 15% SDS–PAGE with subsequent fluorography. Lanes 1 and 4, buffer control; lanes 2 and 5, mock-purified control; lanes 3 and 6, H2A.Z complex. (E) H2A/H4-targeted acetylation of H2A nucleosome by H2A.Z complex. HAT assays were as in (D), but with nucleosomes and cold acetyl-CoA. HAT reactions were analyzed by western blot analysis with the indicated antibodies. Lane 1, mock-purified control; lane 2, H2A.Z complex. (F) Acetylation of H2A.Z histone octamer and H2A.Z nucleosome by H2A.Z complex. Assays were identical to (D), except that H2A.Z containing octamer and nucleosome were used as substrates. Lanes 1 and 4, buffer control; lanes 2 and 5, mock-purified control; lanes 3 and 6, H2A.Z complex.

Stimulatory effect of H2A acetylation on H2A.Z exchange. (A) ATP hydrolysis by H2A.Z-S and H2A.Z-B complexes. ATPase activities of the H2A.Z-S and H2A.Z-B complexes were checked with [γ-³²P] ATP in the presence or absence of nucleosomes. ATPse assays were normalized to the level of FLAG/HA-H2A.Z in the complexes. Lanes 1 and 2, buffer control; lanes 3 and 4, H2A.Z-S complex; lanes 5 and 6, H2A.Z-B complex. (B) Schematic summary of H2A.Z exchange assay. Nucleosomes were reconstituted with 5′ biotinylated 207 bp 601 DNA fragments and immobilized on streptavidin-conjugated Dynabeads. Immobilized nucleosomes were incubated with either free FLAG-H2A.Z-H2B dimers or the H2A.Z subcomplexes containing FLAG/HA-H2A.Z-H2B dimers. Nucleosomal incorporation of H2A.Z was determined by western blot analysis with anti-FLAG antibody. (C) H2A.Z exchange by H2A.Z-S and H2A.Z-B complexes in vitro. Immobilized nucleosomes were incubated with either free FLAG-H2A.Z-H2B dimers (lanes 4 and 7) or the H2A.Z complexes containing FLAG/HA-H2A.Z-H2B dimers (lanes 5, 6, 8 and 9) in the presence or absence of ATP for 60 min. After extensive washing of the immobilized nucleosomes, H2A.Z incorporation was determined by western blot analysis with anti-FLAG antibody (first panel). Relative intensity of bands was quantified using Phosphorimager (Bio-Rad). For analysis of free unincorporated H2A.Z, supernatant was collected before the washing step and 25% of the supernatant was analyzed by western blot (third panel). Nucleosomal H3 was used as an internal loading control (second panel). The amount of H2A.Z-S and H2A.Z-B complexes was normalized based on the level of FLAG/HA-H2A.Z present in the complexes. Results shown are from a single experiment and are representative of three independent experiments. Lanes 1–3, input (50%) of free FLAG-H2A.Z-H2B dimer, FLAG/HA-H2A.Z-H2B dimer in H2A.Z-S complex and FLAG/HA-H2A.Z-H2B dimer in H2A.Z-B complex. (D) H2A.Z exchange into acetylated nucleosomes in vitro. Exchange assay was identical to (C), but nucleosomes were acetylated with the initial H2A.Z complex before immobilizing on magnetic beads and ATP was included in all reactions. (E) Preferential exchange of H2A.Z for acetylated H2A. After H2A.Z exchange reaction, equal amounts of nucleosomes were analyzed by western blot analyses with anti-FLAG (α-FLAG), anti-H3 (α-H3), anti-acetyl-H2A (α-Ac-H2A) and anti-H2A antibodies (α-H2A). Equal loading of nucleosomes was confirmed by western blotting for nucleosomal H3 (α-H3). (F) Requirement of ATP hydrolysis for H2A.Z exchange. In vitro exchange reactions were identical to lanes 7–9 in (D), but in the absence (lanes 1–3 and lanes 7–9) or presence (lanes 4–6) of ATP or pre-treatment of the H2A.Z complexes by 0.5 U apyrase (lane 10–12).

Exchange of H2A.Z-H2B dimer by TIP48 and TIP49. (A) ATPase activity of recombinant TIP48 and TIP49. ATPase assay was performed with recombinant TIP48/TIP49 and [γ-³²P] ATP, and ATP hydrolysis was examined by 12% polyacrylamide gel (19:1) containing 7 M urea in a 1× TBE buffer. Lane 1, buffer control; lane2, recombinant TIP48; lane 3, recombinant TIP49; lane 4, recombinant TIP48/TIP49 complex. (B) H2A.Z exchange by TIP48, TIP49 or TIP48/TIP49 complexes in vitro. H2A.Z exchange activity of recombinant TIP48 and/or TIP49 was checked by using unmodified (lanes 1–4) or pre-acetylated (lanes 5–12) nucleosomes in the presence (lanes 3, 4, 7 and 8–12) or absence (lanes 1, 2, 5 and 6) of ATP as described in ‘Materials and Methods’ section. Nucleosomal incorporation of His-H2A.Z was analyzed by western blot analysis with anti-His antibody (α-His, upper panel). Free unincorporated H2A.Z was determined by western blot analysis of the supernatant (25%) with anti-His antibody (α-His, lower panel). Nucleosomal H3 was used as an internal loading control. Results are representative of three independent experiments. Lanes 1, 3, 5, 7 and 9–12 are reactions without TIP48 and/or TIP49. (C) Effect of H2A acetylation on H2A.Z exchange by TIP48/TIP49 complexes. In vitro exchange assay was identical to lanes 7–8 in Figure 6B, but using nucleosomes containing intact (lanes 1 and 2) or K5 mutated (lanes 3 and 4) H2A. Nucleosomal incorporation of His-H2A.Z was analyzed by western blot analysis with anti-His antibody (α-His). Nucleosomal H3 (α-H3) was used as an internal loading control. (D) Specific action of TIP48/TIP49 complex on H2A.Z exchange. In vitro exchange assays were performed with acetylated nucleosomes as in Figure 6B, using h:H2A.Z/H2B dimer (lanes 2 and 3), h:H2A.X/H2B dimer (lanes 5 and 6) and f:H2A/H2B dimer (lanes 8 and 9). Nucleosomal incorporation of dimers was analyzed by western blot analysis with the indicated antibodies (upper panel). Free unincorporated dimers were determined by western blot analysis of the supernatant (25%) with the indicated antibodies (lower panel). Nucleosomal H3 was used as an internal loading control (second panel). Results are representative of three independent experiments. Lanes 1, 4 and 7, input (25%) of free dimers. (E) Positive effect of acetylation of H2A-K5 on interaction of TIP49 with nucleosome. The unmodified (lanes 3, 6, 7, 9 and 11) or acetylated (lanes 10 and 12) nucleosomes containing wild type (lanes 6, 7, 9 and 10) or K5 mutated (lanes 11 and 12) H2A were immobilized on magnetic beads and incubated with FLAG-TIP48 (lane 6), FLAG-TIP49 (lane 7) or FLAG-TIP48/TIP49 complex (lanes 9–12). The interaction of TIP48/TIP49 with nucleosomes was analyzed by immunoblot with anti-FLAG antibody. The immobilized 5′ biotinylated 207 bp DNA fragments containing 601 nucleosome positioning sequence (lane 2) was also included to determine relative DNA binding affinity of TIP48/TIP49. Histone H3 was used as a loading control. Results are representative of three independent experiments. Lane 1, input of FLAG-TIP48; lane 2, input of FLAG-TIP49; lane 5, input of FLAG-TIP48/TIP49 complex.

Isolation of H2A.Z subcomplexes. (A) Glycerol gradient fractionation of H2A.Z subcomplexes. The H2A.Z complex was separated by glycerol gradient centrifugation as describe under ‘Materials and Methods’ section. Total 32 fractions were collected from the top to the bottom, and analyzed by 4–20% gradient SDS–PAGE and silver staining. Each fraction was also subjected to western blot analysis with anti-FLAG antibody. Asterisks indicate the ectopic H2A.Z. H2A.Z-S and H2A.Z-B indicate the H2A.Z-small (S) and H2A.Z-big (B) complexes, respectively. (B) Western blot analysis of the purified H2A.Z subcomplexes. Aliquots of fractions corresponding to H2A.Z-S and H2A.Z-B complexes were separated by 4–20% SDS–PAGE and analyzed by immunoblot with the indicated antibodies. The initial H2A.Z complex shown in the Figure 1B was used as input. (C) Mass spectrometric analysis of H2A.Z subcomplexes. H2A.Z-S and H2A.Z-B complexes were separated by 4–20% SDS–PAGE followed by silver staining and identification of the bands by tandem mass spectrometry. Protein size markers are indicated on the left. Lane 1, H2A.Z-S complex; lane 2, H2A.Z-B complex. (D) HAT assay of H2A.Z subcomplexes. Assays were identical to Figure 1D, except that H2A.Z-S (lane 3) and H2A.Z-B complexes (lane 4) described in (C) were used. Lane 1, buffer control; lane 3, mock-purified control; lane 5, initial H2A.Z complex.

Dominant role of H2A-K5 acetylation for H2A.Z exchange. (A) HAT assay with recombinant nucleosomes containing wild-type or mutant H4. HAT assay was identical to Figure 1E, but using nucleosomes reconstituted with intact (lanes 1 and 2) or lysine mutated (lanes 3 and 4) H4. Reaction products were subjected to western blot analysis with an antibody recognizing acetylated H4. (B) Minimal effect of H4 acetylation on H2A.Z exchange. In vitro exchange assay was identical to Figure 3D, but using nucleosomes reconstituted with intact (lanes 1–6) or lysine mutated (lanes 7–9) H4. Nucleosomes were unmodified (lanes 1–3) or acetylated (lanes 4–9) by TIP60 in the initial H2A.Z complex. Nucleosomal incorporation of H2A.Z was analyzed by western blot analysis with anti-FLAG antibody (α-FLAG). Equal loading of nucleosomes was confirmed by western blotting for nucleosomal H3 (α-H3). (C) HAT assay with recombinant nucleosomes containing wild-type or mutant H2A. HAT assay was identical to Figure 4A, but using nucleosomes reconstituted with intact (lanes 1, 2, 5 and 6), K5/K9 mutated (lanes 3 and 4) or K5 mutated (lanes 7 and 8) H2A. HAT reactions were subjected to immunoblot analysis with antibodies recognizing acetylated K5 or K9 of H2A. (D) Significant effect of H2A acetylation on H2A.Z exchange. In vitro exchange assay was identical to Figure 4B, but using nucleosomes containing intact (lanes 1–6), K5/K9 mutated (lanes 7–9) or K5 mutated (lanes 10–12) H2A. Again, histone H3 was used as loading control.

Requirement of TIP48/TIP49 for H2A.Z deposition into chromatin in vivo. (A) Knockdown of TIP49 using TIP49 siRNA. 293T cells were transfected with TIP49-targeted siRNA (Si-Tip49) or negative control siRNA (Si-NC), and repression of TIP49 expression was confirmed by RT–qPCR and western blotting. The mRNA levels of TIP49 were normalized to the β-actin mRNA, which was unaffected by TIP49 siRNA transfection. Whole-cell lysates of the cells transfected with TIP49 or negative control siRNA were analyzed by immunoblot with anti-TIP49 and anti-actin antibodies. Error bars indicate the mean ± SD of results from three independent experiments. (B) Repressive effect of TIP49 knockdown on H2A.Z incorporation. 293T cells were initially depleted TIP49 for 48 h and transfected with FLAG-H2A.Z (f:H2A.Z, Lanes 1 and 2), FLAG-H2A.X (f:H2A.X, lanes 3 and 4) or FLAG-H2A (f:H2A, lanes 5 and 6) for 48 h. Chromatin was isolated from the transfected cells, and the amount of chromatin was normalized by western blot analysis of nucleosomal histone H3 (lower panel). Nucleosomal incorporations of f:H2A.Z, f:H2A.X and f:H2A were analyzed by western blot analysis with anti-FLAG antibody (α-FLAG). Results are representative of three independent experiments. (C) TIP48/TIP49-dependent incorporation of H2A.Z at the p21 promoter region. ChIP analysis of the p21 promoter was performed as described in ‘Materials and Methods’ section. Lines (A–H) under the diagram of p21 promoter region indicate segments used for qPCR. Error bars indicate the mean ± SD of results from three independent experiments. The white boxes and black box indicate two known p53 response elements (p53RE1 and p53RE2) and TATA box, respectively. (D) Summary of TIP48/TIP49-induced H2A.Z exchange. TIP60 is recruited to target gene promoters for localized acetylation of histone H2A at K5, and this acetylation mark acts as a signal for the recruitment of TIP48 and TIP49. TIP48 and TIP49 then facilitate promoter-targeted exchange of H2A.Z to establish platform that would favor the action of chromatin remodeling factors to regulate transcription.