Fig. 1

Overview of the system. A Sketch representing the chromosome 7 with its centromere. Tandem α-satellite repeats of D7Z1 region are represented with the binding of the TALE-effector fusion protein. Chromatin organization with CENP-A and kinetochore assembly with microtubule attachments, characterizes the core centromere during mitosis. The H3K9me3 epigenetic mark and HP1 protein are the hallmark of the pericentromeric heterochromatin. The CPC, interacting with HP1, is essential to correct erroneous microtubule attachments. B Sketch of the different constructs of TALE-fusion proteins used in this study. C Western blot against HA revealing TALE-demethylase (~ 210 kDa), TALE-GFP (~ 110 kDa) and α-tubulin (loading control). The lanes 1, 2 and 5 correspond to transient transfection of cells with the plasmid coding for TALE-demethylase, TALE-demethylase inactive and TALE-GFP, respectively. The + dox (lanes 3 and 6) and − dox (lanes 4 and 7) correspond to stable cell lines with or without doxycycline induction of the TALE-fusion proteins. Due to the different levels of TALE expression between the two systems (transfection and induction), 3 µg of total proteins were loaded for transient transfections of TALE-GFP, 30 µg for TALE-KDM4 and TALE-KDM4-H188A while 90 µg were used for the inducible cell lines. D U2OS cells expressing either the TALE-demethylase (top), its point mutant (middle) or the TALE-GFP (bottom). Cells were stained by IF-FISH, TALE proteins are visualized using an anti-HA antibody, CENP-A using an anti-CENP-A antibody and the D7Z1 region using a specific probe generated at the lab. DNA was stained using Hoechst. We note that the TALE-GFP foci are often more intense than those of TALE-demethylases. This effect is due to the higher expression of the TALE-GFP but also to the double revelation of TALE-GFP by the anti-HA antibody and the endogenous GFP in the same channel. A single focal plane is shown for each cell. Scale bar, 10 µm