Volume 13, Issue 2, Pages 265-277 (January 2004) The Histone-Fold Protein Complex CHRAC-15/17 Enhances Nucleosome Sliding and Assembly Mediated by ACF  Iwao Kukimoto, Sarah Elderkin, Margaret Grimaldi, Thomas Oelgeschläger, Patrick D Varga-Weisz  Molecular Cell  Volume 13, Issue 2, Pages 265-277 (January 2004) DOI: 10.1016/S1097-2765(03)00523-9

Figure 1 CHRAC-15/17 Interacts with the N Terminus of hACF1 (A) Domain structure of hACF1 and the GST-fusion proteins of fragments of hACF1 (in brackets position of amino acids within hACF1). On the right: SDS-polyacrylamide gel stained with Coomassie brilliant blue of the purified GST-fusion proteins; the GST-fusion proteins are indicated with asterisks. A closed circle indicates the position of contaminating bacterial DnaK protein. (B) Pull-down assay with GST-fusion proteins of fragments of hACF1 depicted in (A) and the purified CHRAC-15/17 complex. Analysis was by Western blotting using anti-p17 and anti-p15 antibodies. (C) Pull-down assay of hACF1 using the individual histone-fold proteins. hACF1-Flag was incubated with GST, GST-p17, or GST-p17/p15, and pulled down with glutathione-sepharose beads, or incubated with S-agarose alone (resin), resin-bound S-p15, or resin-bound S-p15/GST-p17. Bound hACF1-Flag was analyzed by Western blotting with anti-Flag antibody. 10% of input protein is shown. (D) The interaction of the full-length hACF1 (hACF1-Flag), N terminus-deleted hACF1 (ΔWAC-hACF1-Flag), SNF2H (Flag-hSNF2H), and ACF complexes of these two hACF1 proteins with p15-p17 was tested using GST-p17/p15 and glutathione-Sepharose beads in a pull-down assay. Analysis was by Western blotting with anti-Flag antibody. 10% of input proteins are shown in the left panel. Molecular Cell 2004 13, 265-277DOI: (10.1016/S1097-2765(03)00523-9)

Figure 2 The CHRAC-15/17 Complex Enhances the Nucleosome Sliding Activity of ACF (A) ACF-mediated nucleosome sliding from end to center positions in the absence or presence of p15-p17 at different salt concentrations. End-positioned nucleosomes were incubated with hACF1 (200 fmol) and SNF2H (380 fmol) at 30°C for 1 hr. Different concentrations of KCl (80, 100, 120, or 160 mM) were included in the reactions as indicated. The addition of the p15-p17 complex (1 pmol) and ATP (1 mM) is indicated. Analysis of reaction products was with 4.5% polyacrylamide gel electrophoresis. Running positions of free DNA, end-positioned nucleosome, and center-positioned nucleosome are indicated on the right side of the gel. Quantification of the results in terms of percentage of moved nucleosomes under the different conditions is in the right panel. (B) Analysis of nucleosome positions before and after the sliding by ACF in the presence of p15-p17. Micrococcal nuclease resistant DNA fragments derived from nucleosomes before and after the reaction with hACF1-SNF2H in the presence of p15-p17 were digested with diagnostic restriction enzymes and resulting DNA fragments were analyzed with a 6% polyacrylamide gel. The restriction enzyme sites and the nucleosome positions obtained are illustrated at the right side. (C) Titration of p15-p17 on ACF-mediated nucleosome sliding. Increasing amounts of p15-p17 (lanes 2–6 and 8–12; 125, 250, 500, 1000, and 2000 fmol, respectively) were added to the nucleosome sliding reaction by hACF1 (200 fmol) and SNF2H (380 fmol) with or without ATP. The reaction was at 30°C for 1 hr. Quantification of the results in terms of percentage of moved nucleosomes depending on the amount of p15-p17 is in the right panel. (D) Time course analysis of the sliding reaction in the absence or presence of p15-p17. End-positioned nucleosomes (40 fmol) were incubated with the ACF (20 or 40 fmol) in 80 mM KCl at 30°C in the absence or presence of p15-p17 (500 fmol). The reactions were stopped by addition of competitor chromatin and analyzed by 4.5% polyacrylamide gel electrophoresis. Quantification of results in terms of percentage of moved nucleosomes at different time points is shown. The upper panel depicts the kinetics at the very early time points of the same experiment as the lower panel. (E) Complex formation between CHRAC-15 and CHRAC-17 is required to enhance ACF-mediated nucleosome sliding. End-positioned nucleosomes were incubated with the ACF complex (40 fmol) at 30°C for 30 min in the absence or the presence of p15-p17, p15, or p17 (each 500 fmol) with or without ATP. Molecular Cell 2004 13, 265-277DOI: (10.1016/S1097-2765(03)00523-9)

Figure 3 The CHRAC-15/17 Complex Interaction with hACF1Is Required to Facilitate ACF-Mediated Nucleosome Sliding (A) End-positioned nucleosomes were incubated with the full-length hACF1-Flag or the N terminus-deleted mutant, ΔWAC-hACF1-Flag (each of 80 fmol) and Flag-SNF2H (80 fmol) with increasing amounts of p15-p17 (lanes 3 and 7; 250 fmol, lanes 4 and 8; 500 fmol, lanes 5 and 9; 1000 fmol) at 30°C for 1 hr. Analysis was with 4.5% polyacrylamide gel electrophoresis. Running positions of free DNA, end-positioned nucleosome, and center-positioned nucleosome are indicated. (B) The effect of p15-p17 on the nucleosome sliding mediated by ΔWAC-hACF1-Flag at different salt concentrations. The end-positioned nucleosomes were incubated either with the full-length hACF1-Flag or the N terminus-deleted mutant, ΔWAC-hACF1-Flag (each 200 fmol) and Flag-SNF2H (380 fmol) in the absence or presence of p15-p17 (1 pmol) in indicated salt concentrations at 30°C for 1 hr. (C) ΔWAC-hACF1-Flag has the same activity as hACF1-Flag in ATP-dependent nucleosome sliding in the absence of p15-p17. Increasing amounts of the ACF complexes (lanes 2 and 7; 40 fmol, lanes 3 and 8; 80 fmol, lanes 4 and 9; 160 fmol, lanes 5 and 10; 320 fmol, lanes 6 and 11; 640 fmol) consisting of the full-length (hACF1-Flag) or the N terminus-deleted hACF1 (ΔWAC-hACF1-Flag) and SNF2H (Flag-hSNF2H) were incubated in 80 mM KCl for 1 hr at 30°C with end-positoned nucleosomes in the presence of ATP. The right panel shows a quantification of the results. Molecular Cell 2004 13, 265-277DOI: (10.1016/S1097-2765(03)00523-9)

Figure 4 The CHRAC-15/17 Complex Stabilizes the Interaction of ACF with End-Positioned Nucleosomes (A) End position nucleosomes were incubated with increasing amounts of p15-p17 (lanes 1–4; 0, 1, 2, and 4 pmol) or increasing amounts of reconstituted ACF complex (lanes 5–9 and 10–14; 0, 40, 80, 160, and 320 fmol) in the presence or absence of 1 pmol p15-p17 without ATP. Analysis was by 4.25% polyacrylamide gel electrophoresis. Running positions of free DNA, end position nucleosomes, and shifted product (asterisk) are indicated on the right-hand side of the gel. The right panel shows the quantification of free DNA and shifted band. (B) The CHRAC-15/17 complex has no effect on the interaction of hACF1-SNF2H with center-positioned nucleosomes. After the end-positioned nucleosomes (lane 1) were moved to the center position with 300 fmol ACF (lane 2), increasing additional amounts of ACF (lanes 3 and 6; 300 fmol, lanes 4 and 7; 600 fmol, and lanes 5 and 8; 1200 fmol) were added to the reactions in the presence or absence of 1 pmol p15-p17. Analysis was by 4.5% polyacrylamide gel electrophoresis. Running positions of free DNA and center-positioned nucleosome are indicated. (C) The CHRAC-15/17 complex does not facilitate ΔWAC-hACF1-Flag binding to the end-positioned nucleosomes. End-positioned nucleosomes were incubated either with hACF1-Flag or ΔWAC-hACF1-Flag (each 200 fmol), and Flag-hSNF2H (380 fmol) in the presence of p15-p17 (1 pmol) at 30°C for 5 min in the absence of ATP. Analysis was by 4.5% polyacrylamide gel electrophoresis. Running positions of free DNA, end-positioned nucleosome, and shifted product (asterisk) are indicated on the right side of the gel. Molecular Cell 2004 13, 265-277DOI: (10.1016/S1097-2765(03)00523-9)

Figure 5 ACF Interacts Only with the Complex Formed by the Histone-Fold Domains of p15-p17 (A) The histone-fold proteins, p17, p15, and p12, with their domain structure. Sequence alignment between p15 and p12 is shown below. Shaded boxes indicate identical amino acids. The charged tail region of p15 and putative histone-fold domains are single and double underlined, respectively. (B) 15% SDS-polyacrylamide gel stained with Coomassie brilliant blue of full-length p15-p17 complex, the C-terminal-deleted p15-p17 complexes, and p17-p12 complex. The recombinant p15 proteins contain N-terminal His- and S-tags (Novagen) and a C-terminal HA-tag. The recombinant p12 protein has N-terminal His- and S-tags. (C) Interaction of hACF1-SNF2H with histone-fold protein complexes. hACF1-SNF2H complex was incubated with GST-fused histone-fold protein complexes, pulled down by glutathione-Sepharose beads, and analyzed by Western blotting with an anti-Flag antibody. 20% of input proteins are shown. Molecular Cell 2004 13, 265-277DOI: (10.1016/S1097-2765(03)00523-9)

Figure 6 The C-Terminal Tails of the CHRAC-15/17 Complex Are Required to Enhance ACF-Mediated Nucleosome Sliding (A) End-positioned nucleosomes were incubated with hACF1-Flag (200 fmol) and Flag-hSNF2H (380 fmol) in the presence of GST (100 ng), GST-fused p17/p15 complexes, and GST-fused p17/p12 (100 ng in the amount of GST-p17) in 80 mM KCl at 30°C for 1 hr with or without ATP. Analysis was by 4.5% polyacrylamide gel electrophoresis. Running positions of free DNA, end-positioned nucleosome, and center-positioned nucleosome are indicated on the right side of the gel. (B) DNA band shift assay with p15-p17, C-terminal-deleted-p17-p15 and p17-p12 complexes. The radio labeled DNA probe was incubated with increasing amounts (lanes 2, 5, 8, 11, 14, 17; 50 ng, lanes 3, 6, 9, 12, 15, and 18; 100 ng, lanes 4, 7, 10, 13, 16, and 19; 200 ng in GST-p17) of GST, GST-fused p17/p15 complexes and GST-fused p17/p12. Analysis was by 4.5% polyacrylamide gel electrophoresis. Running position of free DNA is indicated on the right side of the gel. Molecular Cell 2004 13, 265-277DOI: (10.1016/S1097-2765(03)00523-9)

Figure 7 Histone-Fold Protein Complexes Enhance ACF-Mediated Nucleosome Assembly (A) Chromatin assembly reactions were carried out with the indicated amounts of ACF with or without p15-p17 (1 pmol). The addition of ATP and ATP-regenerating system is indicated. The reaction products were partially digested with two different concentrations of micrococcal nuclease. DNA was visualized by EtBr staining after agarose gel electrophoresis; marker, 123 bp ladder. (B) Titration of p15-p17 on ACF-mediated nucleosome assembly. Reactions were performed with ACF (250 fmol) and lambda DNA (16 fmol) with increasing amounts of p15-p17 as indicated. (C) Effect of individual p15 and p17 on ACF-mediated nucleosome assembly. Reactions were carried out with ACF (250 fmol) and p15-p17, p15, or p17 (each 1 pmol). (D) Effects of C-terminal-deleted mutants of p17-p15 complexes on ACF-mediated nucleosome assembly. Reactions were carried out with ACF (150 fmol) alone (lane 2) or with increasing amounts of the full-length p15-p17 (lanes 3–5; 1, 2, and 5 pmol), p15-p17ΔC (lanes 6 and 7; 2 and 5 pmol), p15ΔC-p17 (lanes 8 and 9; 2 and 5 pmol), or p15ΔC-p17ΔC (lanes 10 and 11; 2 and 5 pmol). (E) The effect of p12-p17 on ACF-mediated nucleosome assembly. Reactions were with 250 fmol ACF with or without 1 pmol p15-p17 or p12-p17. (F) The effect of NC2 on ACF-mediated nucleosome assembly. Reactions were performed with ACF (250 fmol) with or without p15-p17 (1 pmol) or NC2 (6 pmol). Molecular Cell 2004 13, 265-277DOI: (10.1016/S1097-2765(03)00523-9)