Volume 6, Issue 5, Pages 1037-1048 (November 2000) Histone Acetylation and hSWI/SNF Remodeling Act in Concert to Stimulate V(D)J Cleavage of Nucleosomal DNA  Jongbum Kwon, Katrina B. Morshead, Jeffrey R. Guyon, Robert E. Kingston, Marjorie A. Oettinger  Molecular Cell  Volume 6, Issue 5, Pages 1037-1048 (November 2000) DOI: 10.1016/S1097-2765(00)00102-7

Figure 1 RAG1/2 Binding to Tailless Nucleosomes (A) DNA templates used for assembling the nucleosomes and expected sizes of nicked and cleaved products are shown. Asterisk, position of the 32P label; triangle, RSS; TG, two copies of nucleosome positioning sequence. The predicted position of the dyad is indicated. (B) Gel shift in Ca2+ of 12/TP1 tailless nucleosome (Nuc-T). Triangle indicates increasing concentrations of HMG1 (1.2, 2.5, 5.0, and 10.0 μg/ml, lanes 2–5 and 1.2, 2.5, and 5.0 μg/ml lanes 13–15) and RAG1 (1, 2, and 4 μg/ml). Where the presence of RAG1 or RAG2 is indicated by a plus sign, the concentration is 1 μg/ml. (C) The binding of RAG1/2 to 12 or 23RSS at two different translational positions in tailless nucleosomes is shown. Reactions were carried out in Ca2+ or Mn2+ as indicated and in the presence (plus) or absence (minus) of HMG1 (1.2 μg/ml). A novel band, most of whose DNA content is hairpin (data not shown, see discussion of acetylated 12TP3 nucleosome), is marked with an asterisk. The positions of naked DNA (DNA), unbound tailless nucleosome (Nuc-T), and tailless nucleosomes complexed with RAG proteins with and without HMG1 (Nuc-T + RAG1/2 [+ HMG1]) are shown. (D) The mobility of free DNA complexed with RAG proteins in the presence of HMG1, and the mobility of the tailless nucleosome/RAG1/2/HMG1 complex is shown. A vertical line marks the position of the band shift. Molecular Cell 2000 6, 1037-1048DOI: (10.1016/S1097-2765(00)00102-7)

Figure 2 V(D)J Cleavage on Tailless Nucleosomes (A) Analysis of 12RSS substrates. Denaturing gel analysis of cleavage of naked DNA (DNA) and tailless nucleosomes (Nuc-T) is shown. The level of hairpin is 5% in lane 6 and 18% in lane 12. (B) Analysis of 23RSS substrates. Less DNA was loaded in lane 3 than in the other lanes. The percent hp is <0.01% in lane 6 and 21% in lane 12. (C) Native gel analysis of glycerol gradient fractions of the tailless nucleosomes used in the cleavage assays shown in (A) and (B). The amount of free DNA is less than 0.8%. The level of free DNA present in each sample remained the same after incubation under cleavage reaction conditions in the absence of the proteins (not shown). S, substrate; hp, hairpin; n, nick. Molecular Cell 2000 6, 1037-1048DOI: (10.1016/S1097-2765(00)00102-7)

Figure 3 V(D)J Cleavage of Hyperacetylated Nucleosomes (A) Top, SDS and acid-urea gel analysis of the core histones purified from butyrate-treated (Ac) and nontreated (no label) HeLa cells. The acetylation status of histone H4 is indicated. Bottom, native gel analysis of the nucleosomes incubated with the reaction buffer under the same conditions as used in (B). The percentage of free DNA is indicated. (B) Comparison of V(D)J cleavage of hyperacetylated and nonacetylated nucleosomes using the direct extraction method. All reactions contain RAG1/2 with (plus) or without (minus) HMG1. The cleavage of the corresponding free DNA (D) is shown in the first lane of each set of acetylated nucleosomes (N). The level of cleavage (determined as the percentage of hairpin relative to the sum of hairpinned, nicked, and remaining uncleaved substrate) in the presence of HMG1 is as follows: lane 3, 1.0%; lane 6, 4.3%; lane 9, <0.01%; lane 12, 4.1%; lane 14, 0.4%; lane 16, 1.1%; lane 18, <0.01%; and lane 20, 0.8%. The level of hairpin for cleavage of the free DNA is in the range of 45%–55%. Positions of the cleavage products are designated as in Figure 2 for the acetylated panel, and hairpin bands for the standard cores are marked by a closed arrow. Open arrows in lane 9 designate noncanonical cleavage products. (C) “Gel excision analysis” of cleavage of hyperacetylated (Ac) and nonacetylated (minus) nucleosomes. All reactions contain RAG1/2 and HMG1. Left, preparative native gel of the nucleosomal cleavage reactions. The hairpin products from the internal free DNA are marked (hp1–4). The “hairpin*” species of the 12/TP3 acetylated nucleosome (see text) is indicated by an asterisk in lane 4. Right, denaturing gel analysis of DNA isolated from the nucleosome bands (Nuc) from the native gel at left (1′, analysis of Nuc band from lane 1 at left, etc.) The percent hairpin follows: lane 1′, 0.24%; lane 2′, 0.63%; lane 3′, 0.50%; lane 4′, 0.47% (nick = 2.92%); lanes 5′ and 6′, <0.01%; lane 7′, 0.37%; and lane 8′, 2.5%. Brackets indicate the positions in which 23/TP1 hairpin and nick would migrate. All other labels are as in Figure 2. Molecular Cell 2000 6, 1037-1048DOI: (10.1016/S1097-2765(00)00102-7)

Figure 4 Stimulation of Nucleosomal V(D)J Cleavage by hSWI/SNF Remodeling (A) Top left, Coomassie-stained SDS gel of purified FLAG-tagged hSWI/SNF complex. Right, DNase I digestion analysis of nuclesomes subjected to hSWI/SNF remodeling. The diagnostic 10 bp repeat pattern, indicative of rotationally phased nucleosome, is indicated by horizontal bars. Bottom left, detection of remodeled 12/TP1 nucleosomes by gel shift. Positions of the remodeled (rNuc) and unremodeled (core) nucleosomes are indicated. Another band with a slower mobility was frequently seen, and this species disappears upon glycerol gradient purification (see Figure 5A for an example). (B) The effect of hSWI/SNF remodeling on the cleavage of 12/TP1 and 12/TP3 nucleosomal DNA was assessed using the gel excision method. Shown is the denaturing gel analysis of DNA isolated from standard core or remodeled nucleosome bands excised from a preparative native gel (not shown). Lanes 1–7 come from the same exposure of the same gel as is the case for lanes 8–14. The percent hairpin in the presence of HMG1 is as follows: lane 3, 0.17%; lane 5, 2.1%; lane 7, 2.6%; lane 10, 0.16%; lane 12, 3.5%; lane 14, 4.5%. (C) The effect of hSWI/SNF on the cleavage of 23RSS nucleosomes. Analysis was as in (B). The lanes for each nucleosome come from the same exposure of the same gel. The cleavage of 23/TP1 free DNA is shown in the very first lane marked “D”. The percent hp is as follows: lane 3, <0.01%; lane 5, <0.01%; lane 10, 0.27%; lane 12, 1.9%; and lane 14, 2.5%. (D) Free DNA present in nucleosome fractions does not contaminate the nucleosomal bands (see text for details). 12/TP3 nucleosome (containing 2% free DNA) was mixed with either 10% 23/TP2 free DNA (lanes 1–3) or 5% gel-purified 23/TP2 hairpin (hp4) (lanes 4–6). The position where the hairpin from the control free DNA (hp4) would migrate is indicated. All other labels are as in Figure 2. Molecular Cell 2000 6, 1037-1048DOI: (10.1016/S1097-2765(00)00102-7)

Figure 5 V(D)J Cleavage on Purified Remodeled Nucleosomes (A) The native gel of the glycerol gradient fractions for the hSWI/SNF remodeling reaction of the 12/TP1 nucleosome is shown. Remodeled nucleosomes (rNuc) are present in the early fractions (bottom) where free DNA is barely detectable. (B) Cleavage of purified remodeled nucleosomes was analyzed by the direct extraction method. Remodeled nucleosomes (rNuc) and the corresponding free DNA (D) were incubated with the indicated proteins under standard conditions. The denaturing gel for the extracted DNA is shown. The percent hp is as follows: lane 4, 8.5%; lane 8, 9.0%; lane 12, 0.3%; and lane 16, 6.0%. The level of hairpin from free DNA cleavage is in the range of 40%–50%. (C) Native gel analysis of buffer-incubated nucleosomes. The amount of dissociated free DNA is shown as a percentage of the free DNA relative to the sum of the other bands. Molecular Cell 2000 6, 1037-1048DOI: (10.1016/S1097-2765(00)00102-7)

Figure 6 Acetylation Further Facilitates V(D)J Cleavage of hSWI/SNF-Remodeled Nucleosomal DNA (A) Preparative native gels used for analysis by the gel excision method (lanes 1–16). Standard cores (no label) and hyperacetylated nucleosomes (Ac) were incubated with RAG1/2 and hSWI/SNF plus ATP in the absence (minus) or presence (plus) of HMG1, and the reaction products were separated on a native gel. Asterisks in lanes 8 and 16 indicate the position of “hairpin*” bands. (B) The denaturing gel analysis of DNA isolated from the core nucleosome bands of the gel shown in (A). The percent hp is as follows: lane 2′, 2.1%; lane 4′, 4.1%; lane 6′, 4.8%; lane 8′, 12%; lane 10′, <0.01%; lane 12′, <0.01%; lane 14′, 1.9%; and lane 16′, 9.4%. Labeling is as in Figure 2 and Figure 4. Molecular Cell 2000 6, 1037-1048DOI: (10.1016/S1097-2765(00)00102-7)