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. 2008 Apr 15;14(4):507–522. doi: 10.1016/j.devcel.2008.02.001

Figure 3.

Figure 3

Targeting a Transcriptional Activator or Silencer into the HAC Kinetochore Induces HAC Loss

(A) The HAC cell-by-cell stability assay. Coding regions of proteins to be tested were cloned into a vector that also expresses a puromycin resistance marker. Nontransfected cells were killed by puromycin and the remaining population was analyzed by FISH to quantify the HAC retention after 11–12 d of culture. To correct for variability in transfection and killing efficiency, all values were normalized to the results of transfection with empty vector bearing puromycin resistance (gray bars in lower panel). Lower panel: expression of transcriptional activators (tTA, tTA3, tTA4) and silencer tTS causes significant destabilization of the HAC with efficiency tTS > tTA > tTA3,tTA4 (n = 2–5). Constructs yielding results indistinguishable from the control have a value on the ordinate of 1.0. Addition of doxycycline, which prevents TetR from binding TetO, blocked the inactivation of HAC kinetochore by tTA and tTS (shaded bars).

(B) Quantitative HAC stability assay using real-time PCR. The proteins to be tested were expressed using retroviral vectors. Virus-infected cells were maintained in medium containing neomycin ± doxycycline. After 30 d postinfection (left panel) (or additionally, 7 or 14 d for tTS or tTS mutant expressing cells, middle and right panels), the relative copy number of the alphoidtetO array was quantitated by real-time PCR. Numbers above the bars indicate the HAC loss rate (R) calculated using the formula: N30 = N0 × (1−R)30. Asterisk indicates HAC loss rate between 7 and 14 d after tTS binding. Cells expressing the tetR protein (tetR) or infected with empty vector (pFB-Neo) showed no HAC instability after 30 d of culture. The KAP-1 interaction deficient mutant (tTS mutant, right blue bars) failed to induce HAC instability over 14 d. Error bars indicate SD (n = 3).

(C and C′) The HAC (detected by FISH, green in [C′]) fails to segregate with the bulk chromosomes (stained with DAPI, grayscale in [C], blue in [C′]) in anaphase cells expressing the tTA transactivator. Size bar = 5 μm.

(D and D′) Nanonucleus revealed by DAPI staining (D) contains the HAC (D′), as revealed by FISH with the BAC probe (colors as in [C′]). HACs are indicated by arrows.