Tracking epigenetic histone modifications in single cells using Fab-based live endogenous modification labeling - PubMed (original) (raw)

Monitoring the levels of histone H3 modifications in living cells. (A) Schematic drawing showing how modification levels are monitored in living cells using Fab. The nuclear concentration of Fab, or the intensity ratio of nucleus:cytoplasm, reflects the level of the target modification. (B–D) Monitoring H3K27ac levels in living U2OS cells. Cells loaded with FabH3K27ac-Cy3 were imaged every 15 min, and TSA was added (Movie 5 in

Supplementary Data

). Shown are pseudo-color images (B) and the fluorescent intensity ratio of nucleus:cytoplasm (C and D); (C) the average (n > 8); (D) the value of nucleus:cytoplasm at 2 h normalized to its value at 0 h in individual cells. Asterisks indicate P < 0.005 by two-tailed Student’s _t-_test; actual _P_-values are: 1 nM, 0.178; 3.3 nM, 0.166; 10 nM, 0.00016; 33 nM, 0.0023; 100 nM, 0.00078; 333 nM, 0.0022; and 1000 nM, 0.0000037. (E–H) Effects of TSA on H3K9 acetylation and methylation levels in HeLa cells. Cells were loaded with FabH3K9ac-Cy5, PCNA-Cy3 and FabH3K9me2-488 (E, F, H) or FabH3K9me1-488 (G), imaged every 15 min, and TSA (3.3 µM) was added. (E) Fluorescent images (Movie 6 in

Supplementary Data

). (F and G) The intensity ratio of nucleus:cytoplasm (averages with SD; n = 10). (H) The ratio of chromatin-bound:free FabH3K9me2 in individual cells. _P_-values by two-tailed Student’s _t_-test are indicated. (I and J) Monitoring H3K9ac and H3K9me2 levels after the removal of TSA. HeLa cells loaded with FabH3K9me2-488 and FabH3K9ac-Cy3 were imaged every 30 min, TSA (3.3 µM) was added at time point 0 and removed 3 h later. (I) Pseudo-color images of fluorescence intensity (see Movie 7 in

Supplementary Data

for gray scale and merged images). (J) The intensity ratio of nucleus:cytoplasm (averages with SD; n = 10). Bars, 10 µm.