Volume 10, Issue 4, Pages 935-942 (October 2002) Essential Role for the SANT Domain in the Functioning of Multiple Chromatin Remodeling Enzymes  Laurie A. Boyer, Michael R. Langer, Kimberly A. Crowley, Song Tan, John M. Denu, Craig L. Peterson  Molecular Cell  Volume 10, Issue 4, Pages 935-942 (October 2002) DOI: 10.1016/S1097-2765(02)00634-2

Figure 1 SANT Domains Play Key Roles for Chromatin Remodeling Enzymes In Vivo The amino acid sequences of the SANT domains are shown at the top of each panel. Deletions are indicated by black bars beneath the sequence. All other residues targeted for mutagenesis are identified by asterisks. (A) The Swi3p SANT domain is required for SWI/SNF function in vivo. Strains were grown to mid log phase in YEPD and analyzed for β-galactosidase expression from an integrated HO-lacZ reporter (Stern et al., 1984). Analyses were performed in triplicate and values were averaged. Values varied by <15%. Activity was normalized to percentages of wild-type levels. Strains used were CY666 (wild-type), CY667 (swi3-), CY669 (ΔSANT), CY671 (W546A), CY674 (C561A), CY675 (R564A), CY676 (F565A), CY694 (R565E), CY695 (Q560A), and CY697 (W546A, F565A). (B) The SANT domain is essential for RSC function. Strains harbored a deletion in RSC8/SWH3 and contained a wild-type copy of RSC8/SWH3 on a URA3-CEN plasmid (CY885). CY885 was then transformed with a control LEU2-CEN plasmid (RS315) or LEU2-CEN plasmid containing a copy of one of the following: wild-type RSC8/SWH3 (RS315-RSC8), rsc8/swh3 K353A (CP817), or rsc8/swh3 ΔSANT (CP816). Strains were initially grown on YEPD and replica plated onto either selective media or media containing 5-fluoroorotic acid (FOA) and grown for 5 days at 30°C. (C) Ada2p function requires a functional SANT domain. Strains were grown to mid log phase in YEPD and analyzed for β-galactosidase activity from an integrated HO-lacZ reporter. Analyses were performed in triplicate and values were averaged. Values varied by <15%. Activity was normalized to percentages of wild-type levels. Strains used were CY733 (wild-type), CY718 (H103E), CY719 (Δ98-102), and CY884 (Δ97-106). Molecular Cell 2002 10, 935-942DOI: (10.1016/S1097-2765(02)00634-2)

Figure 2 The SANT Domain Is Required for the Activity of Gcn5p-Containing HAT Complexes (A) Western blotting was used to probe the subunit composition of peak SAGA fractions isolated from wild-type and Ada2Δ97-106 strains. (B) In vitro acetylation assays using nucleosomal substrates. Assays were performed using a 6-fold range in concentration of Gcn5p-containing HAT complexes (0.5, 1.5, 3 μl). Gcn5p-containing HAT complexes were incubated with oligonucleosomes and 3H-acetyl CoA and subjected to 18% SDS-PAGE. Gels were stained with Coomassie brilliant blue to visualize histones, destained, soaked in ENHANCE (NEN), and analyzed by autoradiography. Data shown are representative of at least three independent experiments and multiple HAT purifications. (C) GST binding reactions were performed with SAGA fractions isolated from CY733 (wild-type) and CY884 (Ada2Δ97-106). Equivalent amounts of SAGA were incubated with glutathione agarose beads bound with GST-H3 (amino acids 1-46) or GST as control. Binding reactions were analyzed by SDS-PAGE and immunoblotting with antisera to Ada3p or Gcn5p. 100% of the bound fraction, 50% of the input, and 50% of the unbound fractions were analyzed. Similar results were seen in at least five independent experiments. Molecular Cell 2002 10, 935-942DOI: (10.1016/S1097-2765(02)00634-2)

Figure 3 Ada2p SANT Domain Stimulates Catalytic Activity of Gcn5p (A) Purification of recombinant Gcn5p, Gcn5p/Ada2p, and Gcn5p/Ada2pΔSANT. Shown are Western blots of Ni2+-NTA agarose eluates obtained by fractionation of bacterial lysates. (B) H3 peptide saturation curves for Gcn5p (open circles), Gcn5p/Ada2p (closed squares), and Gcn5p/Ada2pΔSANT (open diamonds). (C) Core histone saturation curves for Gcn5p (open circles), Gcn5p/Ada2p (closed squares), and Gcn5p/Ada2pΔSANT (open diamonds). (D) Plots of initial velocity versus [Nucleosomes] for Gcn5p (open circles) and Gcn5p/Ada2p (closed squares). Reactions were performed using 100 μM AcCoA at 24 ± 1°C, in 5 mM DTT, 50 mM Tris (pH 7.5). Initial rates (nM product formed per second per Gcn5p equivalent, Eeq) were determined and analyzed via fits to the Michaelis-Menten equation (displayed curves). The Gcn5p enzyme equivalent, Eeq, was determined from quantitative Western blot titration of the three Gcn5p preparations. Steady state parameters for the various forms of Gcn5p were obtained from duplicate, triplicate, or quadruplicate experiments and are summarized in Table 1. Stock concentrations of histones and nucleosomes were calculated separately after complete acetylation of substrates when excess Gcn5p was employed. This ensured accurate determination of initial substrate concentrations as reported in the x axis of each plot. Molecular Cell 2002 10, 935-942DOI: (10.1016/S1097-2765(02)00634-2)