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1.
FIGURE 3.

FIGURE 3. From: NuA4-dependent Acetylation of Nucleosomal Histones H4 and H2A Directly Stimulates Incorporation of H2A.Z by the SWR1 Complex.

The SWR1 complex incorporates H2A.Z-H2B dimers into native chromatin in an ATP-dependent manner. A, shown is an experimental scheme of in vitro histone swapping assays. Immobilized native chromatin is incubated with the SWR1 complex for 30 min to allow binding. ATP and recombinant H2A.Z-H2B dimers are then added after washes to remove unbound SWR1. B, native chromatin immobilized on paramagnetic beads was bound by purified SWR1 complex and incubated in the presence or absence of ATP. After washes, incorporated H2A.Z was analyzed by SDS-PAGE and Western blotting with anti-Htz1 (yeast H2A.Z). Anti-H3 and H2B signals are used as chromatin loading/dimer stoichiometry controls.

Mohammed Altaf, et al. J Biol Chem. 2010 May 21;285(21):15966-15977.
2.
FIGURE 4.

FIGURE 4. From: NuA4-dependent Acetylation of Nucleosomal Histones H4 and H2A Directly Stimulates Incorporation of H2A.Z by the SWR1 Complex.

NuA4-mediated acetylation of chromatin stimulates the histone exchange ability of SWR1 complex. A, shown is an experimental scheme of the histone swapping assay with prior acetylation by the NuA4 complex. Immobilized chromatin is preacetylated with NuA4 followed by washes to remove acetyl-CoA and the addition of the SWR1 complex. ATP and H2A.Z-H2B dimers are added last after washes to remove unbound SWR1. B, NuA4 enhances SWR1 histone exchange activity. Immobilized chromatin was preacetylated with NuA4 followed by the addition of SWR1 and H2A.Z-H2B dimers. Beads were loaded on 15% gel and probed with anti-Htz1 (yH2A.Z) antibodies. Anti-H3 and H2B are used as chromatin loading/dimer stoichiometry controls. C, stimulation of SWR1 activity by NuA4 requires acetyl-CoA. Reactions were incubated with NuA4 in the presence or absence of acetyl-CoA followed by washes and SWR1 addition. After incubation with H2A.Z-H2B dimers and ATP, the beads were washed and analyzed as in B, except that an anti-H4 signal was used as chromatin loading control. More SWR1 activity was used in lanes 2–4 compared with 5–6.

Mohammed Altaf, et al. J Biol Chem. 2010 May 21;285(21):15966-15977.
3.
FIGURE 5.

FIGURE 5. From: NuA4-dependent Acetylation of Nucleosomal Histones H4 and H2A Directly Stimulates Incorporation of H2A.Z by the SWR1 Complex.

Acetylation of either H2A or H4 tail by NuA4 is sufficient to enhance the incorporation of H2A.Z by SWR1. Native chromatin purified from wild type (A), H2A K4R/K7R/K13R (B), and H4 K5Q/K8Q/K12Q/K16Q (C) strains was used as a substrate for in vitro histone exchange assays as in Fig. 4. The same amount of NuA4 complex is used in lanes 5 and 6, but 2-fold more was used in lane 7. The washed beads were analyzed by Western blotting using antibodies against Htz1 (yH2A.Z)), H2B (dimer stoichiometry control), hyperacetyl-H4 (to visualize NuA4 action), and total H4 (chromatin loading control). Note that anti-hyperacetyl-H4 (penta) recognizes both AcH4 and AcH2A in yeast (27). In B, third panel, the signal detected above AcH4 is not AcH2A but the remaining signal of Htz1 from previous screening of the membrane. Also a Western artifact masks part of the H2B band signal in lane 6.

Mohammed Altaf, et al. J Biol Chem. 2010 May 21;285(21):15966-15977.
4.
FIGURE 2.

FIGURE 2. From: NuA4-dependent Acetylation of Nucleosomal Histones H4 and H2A Directly Stimulates Incorporation of H2A.Z by the SWR1 Complex.

Preparation of native chromatin substrate for histone exchange assays. Micrococcal nuclease (MNase)-treated yeast native chromatin was fractionated on Superose 6 gel filtration column. Fractions were analyzed for DNA content on agarose gel with ethidium bromide staining (A) and protein content on SDS-PAGE with Coomassie staining (B). The DNA marker is a 1-kb ladder/pBR322 digested with HinfI, and the mononucleosome size is indicated on the left (1N). Core histone bands are identified on the right. M, molecular mass marker. C, shown is schematic representation of chromatin biotinylation and binding to paramagnetic beads. Pol, polymerase; RT, room temperature. D, silver stain of biotinylated native chromatin bound to streptavidin magnetic beads next to known concentrations of purified human native chromatin is shown. tH3 refers to a common truncated histone H3 in yeast, lacking the first 21 amino acids.

Mohammed Altaf, et al. J Biol Chem. 2010 May 21;285(21):15966-15977.
5.
FIGURE 1.

FIGURE 1. From: NuA4-dependent Acetylation of Nucleosomal Histones H4 and H2A Directly Stimulates Incorporation of H2A.Z by the SWR1 Complex.

Specificity of the NuA4 complex on recombinant histones and native yeast chromatin. Purified complexes from Swr1-FLAG (A) and Epl1-TAP (B) strains were loaded on 8% SDS-PAGE and visualized by silver staining. Swc4 and Eaf2 are the same protein present in both complexes along Yaf9, Arp4, and Act1. Asterisks denote nonspecific proteins obtained with the respective FLAG and TAP purification protocols. Note that a strong nonspecific band migrates close to Swc3 after FLAG purification and that Bdf1-expected migration is put in parentheses because of an apparent substoichiometry compared with other SWR1 subunits. Histone acetyltransferase assays with purified NuA4 complex were performed with recombinant yeast histones (C) and native yeast chromatin (D). Chromatin acetylation levels were measured by loading the assays on 15% SDS-PAGE followed by fluorography and autoradiography. The monomeric recombinant H2A.Z protein is different than the others because it contains an HA epitope (lane 7). The asterisk indicates the streptavidin band coming from the magnetic beads linked to the reconstituted recombinant chromatin (lanes 1 and 2, it migrates at the same position as H4).

Mohammed Altaf, et al. J Biol Chem. 2010 May 21;285(21):15966-15977.
6.
FIGURE 7.

FIGURE 7. From: NuA4-dependent Acetylation of Nucleosomal Histones H4 and H2A Directly Stimulates Incorporation of H2A.Z by the SWR1 Complex.

Bdf1-depleted SWR1 complex is not stimulated by NuA4-mediated acetylation of chromatin. A, affinity-purified Bdf1 is associated with core histones, and a small portion is associated with SWR1 components. TAP Bdf1 was loaded on gel and stained with silver nitrate. Purified NuA4 complex (Epl1-TAP) was also loaded for comparison. NuA4 and SWR1 components are identified. TEV, tobacco etch virus. B, shown is a purification protocol to obtain SWR1 complex that is depleted of Bdf1. WCE, whole cell extracts; FT, flow-through. C, Western analysis of Bdf1-depleted SWR1 complex. FLAG-purified SWR1 complex was analyzed before and after going through IgG-Sepharose to remove Bdf1-TAP from the sample. Anti-FLAG and anti-TAP antibodies were used as well as purified SWR1 and Bdf1 complexes as controls. D, purified Bdf1-depleted SWR1 complex was loaded on gel and silver-stained. E, NuA4 does not enhance histone exchange activity of Bdf1-depleted SWR1 complex. Immobilized chromatin was preacetylated (or not) with NuA4 and acetyl-CoA followed by washes and the addition of SWR1, H2A.Z-H2B dimers, and ATP (as in Fig. 4A). Washed beads were analyzed by Western blotting using antibodies against Htz1 (yH2A.Z), H2B, hyperacetyl-H4, and total H4. F, shown is a model of functional cooperation between NuA4 and SWR1 complexes in vivo. NuA4 is targeted to a promoter by a DNA-bound transcription factor and creates a region of chromatin acetylated on H4 and H2A tails, which is recognized by Bdf1 bromodomains, allowing the associated SWR1 complex to preferentially exchange histone H2A-H2B dimers with H2A.Z-H2B in the local nucleosomes. This mechanism could be involved in presetting the chromatin structure on the promoter of highly inducible genes before gene activation, e.g. PHO5 (31).

Mohammed Altaf, et al. J Biol Chem. 2010 May 21;285(21):15966-15977.
7.
FIGURE 6.

FIGURE 6. From: NuA4-dependent Acetylation of Nucleosomal Histones H4 and H2A Directly Stimulates Incorporation of H2A.Z by the SWR1 Complex.

Histone H4 and H2A acetylation-defective mutants show decreased incorporation of H2A.Z at specific chromosomal loci in vivo, and their combination is lethal for the cell. H2A.Z (Htz1) enrichment at the promoters of SGF29 (−273 to −60 bp from transcription start site) (A), ABP1 (−310 to −29) (B), 10 kb from TelVIR (C), and 12 kb from TelVL (D) was analyzed by chromatin immunoprecipitations. Putative variation in nucleosome occupancy was corrected by analyzing in parallel total histone H3 signals at the same locations. H2A.Z enrichment was measured as a ratio of Htz1 to H3 immunoprecipitation/input (values range from 0.2 to 0.3 Htz1/H3 ratio in wild type cells). Data are presented as a relative change compared with isogenic wild type strain (set to 1). Standard errors are based on two to four independent experiments. E, a yeast strain with all core histone genes deleted and covered by a URA3 vector containing a single copy of H4, H3, H2A, and H2B genes was transformed with a Leu2 vector expressing wild type histones or histones mutated on the lysine residues targeted by the NuA4 complex in vivo (H4 Lys-5/8/12 and H2A Lys-3/7/13) to arginines or glutamines. Exponentially growing cells were then serially diluted (10-fold) and spotted on control plates lacking leucine and uracil or on 5-fluoro-orotic acid (5′FOA) plates to get rid of the URA3 expressing vector.

Mohammed Altaf, et al. J Biol Chem. 2010 May 21;285(21):15966-15977.

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