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. 2022 Jul 21;33(9):br15. doi: 10.1091/mbc.E21-06-0283

FIGURE 2:

FIGURE 2:

Histone H3K4 is acetylated from S/G2 until midprophase during cell cycle progression. (A) Histone H3 K4ac level in lysates of U2OS either not synchronized (DMSO), arrested in G1/S with mimosine, or in mitosis with colcemid. (B) ChIP analysis where chromatin was immunoprecipitated with histone H3K4ac antibodies and probed by real-time PCR with primers that recognize four unique α-satellite sequences on the specified chromosomes. Note the increase in K4 acetylation signal in S/G2 (G2) relative to G1 and M phase. Experiment was performed twice. For statistical analysis an Anova test was applied; p value = 1.1⋅10–5; Tukey test was performed for pairwise comparisons; ***P < 0.001. (C) Immunofluorescence analysis of histone H3K4ac shows high level of H3K4ac in prophase cells (white arrowhead) n = (25, 14) but reduced H3K4ac signal during prometaphase (white carets) n = (25, 14); p value1 = (4.488⋅10–7, 2.564⋅10–5). For statistical analysis Welch’s t test was applied; ***P < 0.001. Red cells stained with anti-H3K4ac; green cells stained with anti-CENP-C; scale bar, 3 µm. The two blue-colored dots in the quantification represent the two experimental replicates. (D) The histone H3K4ac mark is removed from centromeres in midprophase. Images of an example of early and late prophase cells that were defined by the distance of CENP-C foci (plot on the right); the white mark in the merged inset shows a typical line portrayed in the intensity profiles in graphs below; scale bar, 3 µm; inset bar, 0.3 µm. Line graphs below images represent the fluorescent intensities of a 2.5-µm line drawn perpendicular to the axis of sister kinetochores are tiled for cells in either early or late prophase. Line graphs were normalized between 0 and 1, and the CENP-C peaks were centered; CENP-C (red, n = 12) and histone H3K4 (green, n = 12). A white line on the chromosome scheme next to line graphs highlights the region taken for analysis. There was enough CENP-C in inner centromere regions in late prophase chromosomes to define a central peak.