Volume 12, Issue 6, Pages 1599-1606 (December 2003) Histone H2A/H2B Dimer Exchange by ATP-Dependent Chromatin Remodeling Activities  Michael Bruno, Andrew Flaus, Chris Stockdale, Chantal Rencurel, Helder Ferreira, Tom Owen-Hughes  Molecular Cell  Volume 12, Issue 6, Pages 1599-1606 (December 2003) DOI: 10.1016/S1097-2765(03)00499-4

Figure 1 The Movement of Nucleosomes beyond DNA Ends Is Expected to Destabilize the Association of One Histone Dimer (A) Nucleosome positioning on the 219 bp MMTV LTR-derived DNA fragment 54A18 was monitored by site directed mapping. Nucleosomes (100 nM) initially positioned at +70 (lane 1) were transformed to a series of new upstream cleavage sites following incubation with RSC (6 nM) and 1 mM Mg:ATP for 20, 40, and 80 min (lanes 2–4). (B) Native gel electrophoresis of samples in (A) shows increased mobility after RSC action, consistent with the relocation toward DNA ends (lanes 1–4). (C) Extended electrophoresis of mapping products from (A) with G ladder and deduced nucleosome positions up to 38 bp beyond the end of the DNA fragment. (D) Orthogonal views of nucleosome with 30 bp DNA removed from one edge. Histone-DNA contacts stabilizing the association of one histone dimer (yellow) are lost. (E) Chromatin assembled onto the 219 bp 54A18 fragment and the 116 bp 0A-31 fragment was purified by native gel electrophoresis and the histone content assessed by SDS PAGE. Molecular Cell 2003 12, 1599-1606DOI: (10.1016/S1097-2765(03)00499-4)

Figure 2 ATP-Dependent Histone H2A/H2B Dimer Transfer by the RSC Complex Nucleosomes (200 nM) comprising Cy5 labeled 54A18 DNA were incubated in the presence or absence of a 4-fold molar excess of tetramer on Cy5 labeled 0A0 DNA, 7 nM RSC, and 1 mM Mg:ATP as indicated. (A) shows the Cy5 signal from DNA and (B) the Oregon Green labeled histones. Transfer of Oregon Green fluorescence from the remodeled 54A18 nucleosomes onto 0A0/tetramer is detected in the presence of RSC and ATP (lane 7). Transfer also occurs when Oregon green is attached to H2B, but not when it is attached to H4 (lanes 9 and 11). (C) monitors the fate of Oregon Green H2A during remodeling by RSC (20 nM) for 0, 10s, 30s, 1.5 min, 5 min, 15 min, 45 min, and 120 min in lanes 1–8, respectively. The signal is lost from the initial position and appears at intermediate locations before accumulating as a more discrete species that is likely to represent nucleosomes at and beyond the end of the fragment (see Figure 1). The majority of the H2A/H2B transfer onto 0A0 occurs late in the reaction. The temporal relationship between the different species is illustrated in (D). In order to confirm that attachment of dyes did not affect the positioning or movement of nucleosomes, a comparison of the variant octamers was made (Supplemental Figure S1). Molecular Cell 2003 12, 1599-1606DOI: (10.1016/S1097-2765(03)00499-4)

Figure 3 Dimer Exchange by Other ATP-Dependent Remodeling Activities (A) Dimer exchange assays were performed using 54A18 nucleosomes (200 nM) labeled with Oregon Green on H2A and a 4-fold molar excess of 0A0/tetramer (as described in Figure 2), 1 mM Mg:ATP, and increasing concentrations of Drosophila ISWI protein (0, 50 and 100 nM, lanes 1–3, respectively) and yeast SWI/SNF complex (0, 14, and 28 nM, lanes 4–6, respectively). (B) The IOC2 containing ISW1b and IOC3 containing ISW1a complexes were also tested for dimer exchange activity as in (A). Lanes 1 to 4 contain 0, 3.5, 7, and 35 nM ISW1b complex and 1 mM Mg:ATP as indicated. Lanes 6–9 contain 0, 3.5, 7, and 35 nM ISW1a complex and 1 mM Mg:ATP. Lanes 5 and 10 contain 35 nM ISW1b and ISW1a but no Mg:ATP. ISW1b causes an increase in nucleosome mobility consistent with the relocation of nucleosomes toward DNA ends and a modest exchange of dimer. ISW1a causes a subtle decrease in nucleosome mobility consistent with the movement of nucleosomes to a more central location and does not significantly increase dimer exchange. Molecular Cell 2003 12, 1599-1606DOI: (10.1016/S1097-2765(03)00499-4)

Figure 4 Dimer Transfer on Multinucleosome Templates (A) In order to assay dimer exchange between nucleosomes, a 10-mer tandem repeat of a 177 bp NucA positioning sequence was reconstituted with wild-type histone octamer, and used as an acceptor (10× NucA array) in exchange reactions. Remodeling reactions were performed in the presence of 200 nM Oregon Green labeled 54A18 nucleosome, 17.5 nM RSC, and 1 mM Mg:ATP as indicated and the products resolved on a 1% agarose 1×TBE gel. Dimer transfer was observed in the presence of 0.8 μM acceptor (lanes 3 and 4) and 4 μM (lanes 5 and 6), but not observed in its absence (lanes 1 and 2). (B) Circular plasmid DNA (5 kb) reconstituted with Oregon Green H2A labeled histone octamer (800 nM) was incubated with a 8 μM 10× NucA array in the presence of 1 mM Mg:ATP and increasing concentrations of RSC complex (0, 3.5, 7, and 35 nM RSC, lanes 1, 2, 3, and 4, respectively). Histone H4 occupancy was monitored by preparing an identical series of reactions as lanes 1–5 except that Oregon Green was labeled at H4 on the reconstituted plasmid (lanes 6–10). (C) The integrity of plasmid DNA was monitored during the course of remodeling in the presence of RSC complex. Chromatin assembled plasmid DNA was incubated with RSC complex as in (B). Following remodeling protein was removed by digestion with proteinase K and the plasmid DNA analyzed by agarose gel electrophoresis. No linear DNA could be detected. Molecular Cell 2003 12, 1599-1606DOI: (10.1016/S1097-2765(03)00499-4)