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. 2019 Mar 19;8:e43008. doi: 10.7554/eLife.43008

Figure 2. Reb1 binding induces nucleosome structural change.

(A) Nucleosome structure (Davey et al., 2002) containing the internal FRET pair used in this study. Cy3 is attached to the 5′ end of the DNA NPS and adjacent to the Reb1 binding site (blue). The octamer is labeled with Cy5 at H2A(K119C). When fully wrapped, the nucleosome is in a high FRET state. (B) Nucleosome FRET efficiency measurements while titrating Reb1 with the nucleosome constructs: P3 (red), P8 (blue), P13 (green), P18 (gold), or no binding site (P–, black). Reb1 titrations with P3, P8, and P13 nucleosomes fit to binding isotherms with S1/2 Reb1–Nuc P3 FRET = 7.9 ± 1.3 nM, S1/2 Reb1–Nuc P8 FRET = 2.4 ± 0.3 nM, S1/2 Reb1–Nuc P13 FRET = 101.5 ± 19.1 nM. We do not observe a significant ΔFRET for P18 and P– nucleosomes. (C) Comparison of the S1/2 values obtained from EMSA and FRET experiments. For P3 and P8 nucleosomes, the FRET S1/2 values are in close agreement to the EMSA S1/2 values, indicating that ΔFRET is a measure of Reb1 binding to nucleosomes.

Figure 2.

Figure 2—figure supplement 1. Reb1-induced nucleosome ΔFRET is not the result of structural changes in the H2A C-terminal domain.

Figure 2—figure supplement 1.

(A) Nucleosome states representing one explanation for the observed Reb1-induced ΔFRET, which is that Reb1 distorts the H2A C-terminal domain. (B) Nucleosome structure (Davey et al., 2002) with the Reb1-binding site in blue, Cy3 in green and Cy5 that is attached to H3(V35C) in red. Cy3 and Cy5 undergo efficient energy transfer when the nucleosome is fully wrapped. (C) Plot of the FRET efficiency of these nucleosomes when Reb1 is titrated. We observe a similar decrease in FRET for Cy5-H3(V35C) (cyan) nucleosomes as for Cy5-H2A(K119C) (blue). This result demonstrates that the Reb1-dependent ΔFRET does not result from structural changes in H2A and instead is due to a structural change affecting the DNA and the entire octamer.
Figure 2—figure supplement 2. Reb1 does not trap nucleosomes in a repositioned state.

Figure 2—figure supplement 2.

(A) Nucleosome states that represent a second possible explanation for the Reb1-dependent change in FRET, which is that Reb1 binds to repositioned nucleosomes that expose its binding site. To test this, we prepared 167-bp nucleosomes containing a Cy3 fluorophore and a 20-bp linker opposite the P8 Reb1-binding site. The octamer was labeled at H2A(K119C). (B) Cy5 image of the EMSA determining that Reb1 binds these nucleosomes. (C) A plot of the FRET efficiency from these nucleosomes as Reb1 is titrated. The FRET efficiency does not decrease as Reb1 is titrated over the concentration range in which it binds nucleosomes. This result proves that the Reb1-dependent ΔFRET is not due to nucleosome repositioning.