Files in this Data Supplement:
Fig. S1. HAC chromatin unfolding induced by tethering of tetR-EYFP-VP16. A-C) Immunofluorescence images of 1C7 cells expressing tetR-EYFP-VP16 and stained for CENP-A one day after transfection. Images show examples of large-scale unfolding of the alphoidtetO HAC (green, panel 1). Arrowheads depict the HAC, and point to the HAC-associated CENP-A signal (red, panel 2) in the insets. For CENP-A, both insets show the same area, with the inset on the left displaying signal brightness linearly increased past saturation in the imaging software. Merged images (panel 3) represent overlay of EYFP signal with antibody and DAPI staining (blue). Scale bar: 5 µm. D) 1C7 cells were transfected with the indicated constructs and fixed after two days. The amount of fusion constructs bound to the HAC was quantified based on the associated EYFP signal. Solid lines indicate the median.
Fig. S2. Tethering of VP16 facilitates active displacement of CENP-A. A) 1C7 cells were transfected and treated with nocodazole as indicated in the schematic diagram. The doubling time of 1C7 cells is approximately 20 hours. G2 cells (large nuclei, single HAC signal, chromatin not condensed into chromosomes) expressing the indicated tetR fusion constructs (green, panel 1) were analyzed by immunofluorescence staining for CENP-A (red, panel 2). Merged images (panel 3) represent the overlay of EYFP signal with antibody and DAPI staining (blue). B) HAC-associated CENP-A signals in individual cells (A) were quantified and are plotted. Tethering of VP16 causes a significant reduction of CENP-A levels at the HAC prior to mitotic progression. Solid lines indicate the median. C) An alternative protocol is used to arrest transfected cells in prometaphase. EYFP signals first become apparent 8 hours after transfection (data not shown), allowing to arrest the majority of cells prior to their first division in the presence of tethered fusion construct. Images show arrested cells expressing either of the fusion constructs (green, panel 1). Arrows in insets point to HAC sister kinetochores. Scale bars: 5µm.
