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. 2018 May 25;9:2080. doi: 10.1038/s41467-018-04455-7

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© The Author(s) 2018

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 6 — The interactions between H3.3K27M and Ezh2 stabilize Ezh2 on chromatin. a Cumulative distribution of displacements for HaloTag-Ezh2^Catalytic in HEK293T cells expressing H3.3-FLAG (N = 42 cells, n = 2365 displacements) or H3.3K27M-FLAG (N = 36 cells, n = 4120 displacements). The cumulative distributions were fitted with three populations. Ezh2^Catalytic contains only the catalytic lobe (Fig. 3a). Fitted parameters are shown in Supplementary Table 7. b Fraction of the chromatin-bound population (F₁) obtained from a. Results are means ± SD. c Survival probability distribution of the dwell times for HaloTag-Ezh2^Catalytic in HEK293T cells expressing H3.3-FLAG (N = 56 cells, n = 1412 displacements) or H3.3K27M-FLAG (N = 46 cells, n = 1042 displacements). The distributions were fitted with a two-component exponential decay model. Fitted parameters are shown in Supplementary Table 8. d–f Fraction (d), residence time (e), and search time (f) of the long-lived population (F_1sb) for HaloTag-Ezh2^Catalytic in HEK293T cells expressing H3.3-FLAG or H3.3K27M-FLAG. Results are means ± SD. g In vitro single-molecule binding assay. FLAG-HaloTag-Ezh2 (F-HT-Ezh2) was expressed stably in Ezh2^─/─ mES cells and labeled by JF₆₄₆ dyes. FLAG-HaloTag-Ezh2-PRC2 was one-step enriched by FLAG antibody and loaded into the flow cells. Nucleosome labeled with Alexa Fluor®488 was tethered to the surface of a coverslip that had been passivated and functionalized with NeutrAvidin. The position of individual nucleosomes was recorded (green image). We followed the duration of FLAG-HaloTag-Ezh2-PRC2 on nucleosome (magenta image). The colocalization of nucleosome and FLAG-HaloTag-Ezh2 indicates a binding event (overlay image). Arrowheads indicate binding events. Scale bar, 5.0 µm. h Survival probability distribution of the dwell times for FLAG-HaloTag-Ezh2-PRC2 on H3.3-nucleosome (n = 99 trajectories) and on H3.3K27M-nucleosome (n = 115 trajectories). The distributions were fitted with a one-component exponential decay model. i Residence time of FLAG-HaloTag-Ezh2-PRC2 on H3.3-nucleosome and on H3.3K27M-nucleosome. Results are means ± SD from three biological replicates