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. 2020 Apr 27;9:e55667. doi: 10.7554/eLife.55667

Figure 3. RNA polymerase II is critical for H2A.Z eviction.

(A) Normalized histograms and two-component Gaussian fits for H2A.Z-Halo imaged in cells co-depleted for Rpb1 and Swc5. (B) H2A.Z-Halo distributions in cells co-depleted for Ino80 and Swc5. (C) Spot-On results showing co-depletion of Rpb1 along with Swc5 inhibits H2A.Z eviction. All molecules tracked with JF552 dye.

Figure 3.

Figure 3—figure supplement 1. Efficient nuclear depletion of Swc5 in double anchor-away (SWC5-FRB; RPB1-FRB) strain.

Figure 3—figure supplement 1.

(A, B) Deconvolution fluorescence microscopy shows nuclear depletion of Swc5-FRB-GFP in rapamycin-treated, single anchor-away cells, and in Swc5-FRB-GFP; Rpb1-FRBdouble anchor-away cells. Images of JF646-labeled H2A.Z-Halo locate nuclei. Z-axis steps of 200 µm were captured on a DeltaVision fluorescence microscope and 10 deconvoluted stacks were projected.
Figure 3—figure supplement 2. H2A.Z diffusion histograms in cells for single AA of Rpb1 and Ino80.

Figure 3—figure supplement 2.

(A, B) H2A.Z-Halo distributions in Rpb1-AA strain without (A) and with (B) depletion of Rpb1. (C) Spot-On results for chromatin bound H2A.Z upon Rpb1 depletion. Single AA of Rpb1 causes only a slight increase in chromatin-bound H2A.Z, which is likely due to concomitant inhibition of SWR1’s biochemical activity at reduced concentration of free H2A.Z-H2B dimer (Wang et al., 2016). (D, E) H2A.Z distributions in single anchor-away of Ino80. (F) Spot-On results show Ino80 depletion has no effect on level of chromatin-bound H2A.Z. All molecules tracked with JF552 dye.