Figure 1. Tethering LSD2 to the alphoid^tetO HAC decreases the H3K4m2 levels.

(a) Schematic drawings of the tetR-fusion constructs. (b) Schematic of the alphoid^tetO DNA array, derived from Nakano et al.²⁷. (c) Immunofluorescence images of 1C7 cells expressing the indicated tetR-fusion proteins and staining for H3K4me2. Arrowheads depict the HAC as determined by the EYFP signal. Scale bar, 10 μm. (d) Quantification of fluorescence signals of HAC-associated H3K4me2 staining in individual cells transfected as in C plotted as arbitrary fluorescence units (AFU). Solid bars indicate the medians of three independent experiments and error bars represent the s.e.m. (e) ChIP analysis in 1C7 cells expressing tetR-EYFP-LSD2^WT (top) or tetR-EYFP-LSD2^E412AK661A (bottom) using the indicated antibodies. Data represents the levels of the indicated epigenetic marks in the presence of dox (grey bars) and after 3 days of dox washout (white bars). The alphoid^tetO HAC centromere (tetO), endogenous chromosome 21 centromere (chr21), the blasticidin resistance gene (bsr) and the degenerate satellite type-II (Sat2) repeats were assessed. Values were normalized to the chromosome 21 centromere and data represent the mean and s.d. of three independent experiments. Asterisks indicate a significant difference (P<0.05; Mann–Whitney's test).