Fig. 3: The effect of peptides representing the C-terminus of H2A.Z.1 on the stability, nuclear localization, and nuclease digestability of different nucleosomes.
From: Epigenetic modulation via the C-terminal tail of H2A.Z
A Stability of H2A.Z nucleosomes, measured as the residual ZAbA IF in permeabilized HeLa nuclei exposed to 1 M NaCl in the presence of different length peptides (C6-C9) compared to the control (no peptide). ~600 G1 nuclei were measured in each sample by LSC. Means +/− SD of 3 biological replicates are shown. B Representative CLSM images showing the localization of H2A.Z recognized by ZAbA and of H3K9me3 co-labeled with H2A.Z, in HeLa nuclei treated with C9 or the scrambled control peptide (SCR). C Colocalization analysis of H2A.Z and H3K9me3 on CLSM images from the experiment of (B). The MCC values representing the fraction of H2A.Z overlapping H3K9me3 are shown. Box-and-whisker plot was created from data of ~30 nuclei. D, E Global nickase sensitivity of HeLa nuclei after treatment with C9 or SCR, measuring DNA content based on SYBR Gold staining (D) or halo size (E), by LSC. Means +/− SD of 4 biological replicates are shown; events were collected from the whole cell population. F–H Global MNase sensitivity of HeLa nuclei after treatment with SCR or C9, measuring DNA content (F; SYBR Gold-staining), H3K9me3 (G) and H3K4me3 IF (H) by LSC. Means +/− SD of 4 biological replicates are shown; events were collected from the whole cell population. I Contouring and calculation of mean pixel intensities at the nuclear periphery or in the center of HeLa nuclei used in the experiments of (J–L). J Mean pixel intensities of PI-stained DNA measured at the periphery (PE) or in the center (CE) of HeLa nuclei after SCR or C9 treatment and without peptide addition (CTRL). Box-and-whisker plot was created from the data of 20 nuclei. K, L CLSM measurement of MNase sensitivity of SCR-, C9-pretreated and control HeLa nuclei, measuring PI-stained DNA in the center or at the periphery. The mean pixel intensities normalized to 0 U/ml MNase are shown; error bars represent the SEM of 20 nuclei.
