Fig 5. Tup1 depletion stabilizes the opaque state even at 37°C.

(A) Time course of Wor1 and Tup1 protein levels in opaque cells after shift from room temperature to 37°C. Overnight cultures of opaque cells of a strain carrying both Wor1-FLAG and Tup1-HA (HLY4541) were inoculated, grown to mid-log phase, then shifted to 37°C and grown for the indicated times. Protein level was assessed by Western blot as described. (B) ChIP of Wor1-FLAG and Tup1-HA at the WOR1 promoter in opaque cells shifted to room temperature or 37°C. Overnight cultures of opaque cells of a strain carrying both Wor1-FLAG and Tup1-HA (HLY4541) and an untagged control strain (JYC1) were diluted in SCD and grown to log phase at room temperature. Cultures were divided and incubated at either room temperature or 37°C for one hour, formaldehyde cross-linked, and harvested for ChIP. Enrichment is presented as a ratio of the -4kb region of the WOR1 promoter IP (bound/input) over an ADE2 control region IP (bound/input) of the tagged strain, further normalized to the control strain. Values are the average of three independent ChIP experiments with error bars representing the s.d. (C) Tup1 depletion in opaque cells at room temperature and 37°C. Opaque pMET3-TUP1 cells were grown in SCD with or without methionine at either room temperature or 37°C for 24hr. Expression levels of the indicated genes were measured by qPCR and normalized to ACT1. Average expression level of three independent qPCR experiments are plotted with error bars representing the s.d. Samples were also taken at the indicated times, washed three times with H₂O, and plated onto SCD Met- plates to assess phase switching.