Figure 5.
Mediator as a chromatin receptor for Scc2C
(A) Tethering of GBP-Scc2C to Med14-GFP bypasses the need for Scc2. Cells expressing the indicated components were streaked on to YPD medium containing 3-indoleacetic acid (IAA) to degrade the Scc2degron. The Sth1 subunit of RSC is included as a positive control.
(B) Tethering GBP-Scc2C to Med14-GFP reconstitutes cohesin loading. Cells were synchronized in G1 with alpha-factor, Scc2degron was depleted with IAA, and cells were released into nocodazole-imposed mitotic arrest. Cohesin levels were assessed by ChIP against HA-tagged Scc1 followed by quantitative real-time PCR at three chromosome arms and two centromere-adjacent cohesin binding sites and a negative control site. Means and SEM of three independent experiments are shown.
(C) Validation of Med14degron in the presence of auxin (IAA). 10-fold serial dilutions of yeast strains used to examine sister chromatid cohesion, containing a lacO-LacI-GFP marked pericentromere, arm, or telomere site and either WT or degron tagged Med14, were spotted onto YPD+IAA to deplete Med14degron. Depletion of Med14 results in diminished cell growth.
(D) Med14 protein is degraded in sister chromatid cohesion strains after the addition of auxin. Cells were synchronized in G1 with alpha-factor, Med14degron was depleted with IAA, and cells were released into nocodazole-imposed G2/M arrest. Cells were pelleted and WCE prepared and analyzed for the depletion of Med14 by immunoblot.
(E) Mediator promotes sister chromatid cohesion. Cells were synchronized in G1 with alpha-factor, Med14degron was depleted with IAA, and cells were released into nocodazole-imposed mitotic arrest. Cells were fixed and DAPI stained, and the percent of large-budded cells with a single DAPI mass containing 2 GFP spots, indicating loss of sister chromatid cohesion, was scored. A representative image of a cell with cohesion (1 spot) and loss of cohesion (2 spots) is shown. Means and SEM of three independent experiments are shown. A minimum of 100 cells were scored for each replicate; WT CEN, ARM, TEL n = 523, 443, 570 and Med14 degron CEN, ARM, TEL n = 603, 576, 637; ∗∗∗∗ p <.0001; two-way ANOVA test.
See also Figure S6.