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. 2018 May 29;7:e34100. doi: 10.7554/eLife.34100

Figure 4. The sequence of DNA 24–39 bp from the dyad can restrict or permit shifts in H2B(S53C) cross-linking upon Chd1 binding.

(A) Schematic representation of the nucleosome, highlighting sequence elements of the canonical Widom 601 sequence that are altered in 601 variants. (B) The 24–39 bp segment of the canonical 601 is responsible for allowing or blocking Chd1-dependent shifts in H2B(S53C) cross-linking. Shown are cross-linking experiments for the canonical Widom 601 and three 601 variants, carried out with 150 nM 40N40 nucleosomes and 600 nM Chd1 under apo conditions. Schematic cartoons indicate the region of 601 altered for each nucleosome variant. (C) Replacing the 24–39 bp segment on the TA-rich side of the 601 with random DNA sequences allows Chd1 binding (apo condition) to stimulate a shift in H2B(S53C) cross-linking on the TA-rich side. Each gel in B and C is representative of 4 or more experiments. The DNA sequences for all 601 variants are given in Figure 1—figure supplement 2. Similar experiments to those carried out in B and C but in other nucleotide conditions, are shown in Figure 4—figure supplement 1.

Figure 4.

Figure 4—figure supplement 1. Effects of Chd1 binding in different nucleotide states on H2B(S53C) histone mapping of 601 variants.

Figure 4—figure supplement 1.

Histone H2B(S53C) mapping reactions carried out in the presence or absence of 600 nM Chd1 in ADP, AMP-PNP, and ADP·BeF3– conditions. Gels are representative of 4 or more experiments, and correspond to the 601 variants given in Figure 4B (A) or Figure 4C (B).
Figure 4—figure supplement 2. Distinct TA-poor and TA-rich Chd1 cross-linking patterns are transposed on the 601[swap SHL2.5/3.5] nucleosome.

Figure 4—figure supplement 2.

(A) Chd1(V721C) cross-linking reactions on the Widom 601 and 601[swap SHL2.5/3.5] nucleosomes in AMP-PNP and ADP·BeF3– conditions. The Widom 601 nucleosome has reduced cross-linking on the TA-poor side when compared to the TA-rich side, whereas the 601[swap SHL2.5/3.5] nucleosome reverses this trend with diminished cross-linking on the TA-rich side compared to the TA-poor side. Each trace represents the intensity profile of the corresponding NCP gel lane, obtained using ImageJ. (B) Quantification of Chd1(V721C) cross-linking data. The area of each peak was calculated in ImageJ (RRID: SCR_003070), scaled according to background peaks, and normalized against a 601[swap SHL2.5/3.5] sample in ADP·BeF3– . Error bars signify the standard deviations from three or more experiments. The differences in Chd1 cross-linking in AMP-PNP for canonical 601 versus 601[swap SHL2.5/3.5] are statistically significant (** p-value < 0.01, *** p-value ≤ 0.001).