Figure 2.
Figure 2. Pulse-chase detection of macroH2A incorporation into the inactive X chromosome territory. HEK 293T cells stably expressing SNAP-tagged macroH2A were synchronized and pulse-chase labeled as shown in Fig. 1 B. (A) Image analysis of SNAP-tagged macroH2A incorporation on the two inactive X chromosomes (Xi) in HEK 293T cells during S-G2 phase. The top images show (from left) the images of preexisting (Oregon Green, green), newly incorporated (TMR, red) SNAP-macroH2A, and EdU labeling. The lower left image shows the merged images of preexisting and newly incorporated SNAP-macroH2A. In the lower right panel, we show the signal intensities measured along the white dashed lines in the images of preexisting and newly incorporated (TMR, red) SNAP-macroH2A, and EdU labeling. The green, red, and magenta lines show the signal intensities of preexisting and newly incorporated macroH2A and EdU, per pixel, respectively. The levels of intensities were obtained using Color Profiler in ImageJ. (B) The same types of images as in A but during G1 phase. (C) We illustrate the areas measured. NUC-Xi is the entire area of the nucleus excluding the two inactive X chromosomes (Xi). (D) The signal intensities of preexisting macroH2A in NUC-xi and Xi, showing the increased signal in the Xi territories in both S-G2 and G1. (E) A comparison of the signal intensities of newly incorporated macroH2A between the NUC-Xi and Xi subnuclear domains in S/G2 and in G1. With the minimal signal in S–G2, no enrichment of the Xi territories occurs, but significant enrichment is found in G1. (F) The signal intensities of EdU incorporation in the NUC-Xi and Xi domains. In D and E, each intensity per pixel was normalized by Hoechst intensity to take into account local chromatin compaction. In D–F, the number above the data points indicates the mean value, P values were determined using unpaired t tests (*, P < 0.04; **, P < 0.001). Error bars represent standard deviation from the number of single cells that indicated on each dataset in Fig. 1 D. We conclude that the inactive X chromosome is preferentially targeted for macroH2A deposition compared with the rest of the nucleus.

Pulse-chase detection of macroH2A incorporation into the inactive X chromosome territory. HEK 293T cells stably expressing SNAP-tagged macroH2A were synchronized and pulse-chase labeled as shown in Fig. 1 B. (A) Image analysis of SNAP-tagged macroH2A incorporation on the two inactive X chromosomes (Xi) in HEK 293T cells during S-G2 phase. The top images show (from left) the images of preexisting (Oregon Green, green), newly incorporated (TMR, red) SNAP-macroH2A, and EdU labeling. The lower left image shows the merged images of preexisting and newly incorporated SNAP-macroH2A. In the lower right panel, we show the signal intensities measured along the white dashed lines in the images of preexisting and newly incorporated (TMR, red) SNAP-macroH2A, and EdU labeling. The green, red, and magenta lines show the signal intensities of preexisting and newly incorporated macroH2A and EdU, per pixel, respectively. The levels of intensities were obtained using Color Profiler in ImageJ. (B) The same types of images as in A but during G1 phase. (C) We illustrate the areas measured. NUC-Xi is the entire area of the nucleus excluding the two inactive X chromosomes (Xi). (D) The signal intensities of preexisting macroH2A in NUC-xi and Xi, showing the increased signal in the Xi territories in both S-G2 and G1. (E) A comparison of the signal intensities of newly incorporated macroH2A between the NUC-Xi and Xi subnuclear domains in S/G2 and in G1. With the minimal signal in S–G2, no enrichment of the Xi territories occurs, but significant enrichment is found in G1. (F) The signal intensities of EdU incorporation in the NUC-Xi and Xi domains. In D and E, each intensity per pixel was normalized by Hoechst intensity to take into account local chromatin compaction. In D–F, the number above the data points indicates the mean value, P values were determined using unpaired t tests (*, P < 0.04; **, P < 0.001). Error bars represent standard deviation from the number of single cells that indicated on each dataset in Fig. 1 D. We conclude that the inactive X chromosome is preferentially targeted for macroH2A deposition compared with the rest of the nucleus.

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