Figure 2. Chd1 binding in apo and ADP conditions shifts nucleosomal DNA on the TA-poor side of the 601 sequence.

(A) Molecular representation of a 601 nucleosome (Makde et al., 2010), indicating sites of cysteine substitution for site-specific cross-linking. Blue dotted ovals indicate the regions bound by the Chd1 ATPase motor at each SHL2 site (Farnung et al., 2017; Nodelman et al., 2017; Sundaramoorthy et al., 2018). (B) Histone H2B(S53C) cross-linking reactions in the presence and absence of Chd1. PyMOL representation shows cross-links on the TA-poor side of the nucleosome that occur for the nucleosome alone (cyan) and in the presence of Chd1 (red). Cross-linking was performed using 150 nM canonical 601 nucleosomes with 40 bp flanking DNA on each side (40N40), in the presence or absence of 600 nM Chd1. Cross-linked products were separated on urea denaturing gels and visualized by FAM or Cy5 fluorescence. Numbering refers to the distances of cross-linked sites from the 601 dyad (bp). (C) Histone H2A(G28C) cross-linking reactions, performed as described in B. In the PyMOL representation, sites of cross-linking that occur on the complementary strand are indicated by gray spheres with numbering in square brackets (see Figure 2—figure supplement 2B for +41 and +50 cross-linking sites). (D) Histone H2B(T87C) cross-linking reactions, performed as described in B. As for (C), cross-links that occur on the complementary strand [+37/+38] are shown as gray spheres in the PyMOL representation (Figure 2—figure supplement 2C). (E) Histone H3(M120C) cross-linking reactions, performed as described in B. All cross-linking experiments are representative of 3 or more experiments. Extended gel images are shown in Figure 2—figure supplement 2D.

Figure 2—figure supplement 1. Titration of Chd1 for H2B(S53C) cross-linking reaction.

Increasing amounts of Chd1 produces a novel H2B(S53C) cross-link on the TA-poor side of the 601. In nucleotide-free conditions, Chd1 was added to final concentrations of 50, 150, 300, 600, 1200, and 2400 nM in the presence of 150 nM 40N40 nucleosome.

Figure 2—figure supplement 2. Extended gel images for histone mapping of Widom 601 nucleosomes.

Scans of urea denaturing gels, shown in Figure 2B–E for cross-linking reactions using 40N40 nucleosomes (canonical 601) containing H2B(S53C) (A), H2A(G28C) (B), H2B(T87C) (C), or H3(M120C) (D). Numbering represents the distance (nt) from the 601 dyad. Each reaction contained 600 nM Chd1 and 150 nM nucleosomes.

Figure 2—figure supplement 3. Treatment with hexokinase confirms that ADP is sufficient for supporting a Chd1-dependent shift in H2B(S53C) cross-linking.

Scans of urea denaturing gels for H2B(S53C) cross-linking reactions with 40N40 nucleosomes (canonical Widom 601) in the presence and absence of Chd1. ADP stocks used for this experiment were either untreated or pretreated with hexokinase and glucose to remove any contaminating ATP.

Figure 2—figure supplement 4. Histone mapping with other single cysteine histone variants.

Scans of urea denaturing gels for cross-linking reactions of 40N40 nucleosomes containing H2B(R30C), H2B(T85C), or H2A(A45C) variants. Note that the PyMOL representation of the H2B(T85C) cross-linking sites is flipped over to show the TA-rich side. Numbering represents observed or expected (parentheses) cross-links from the dyad.