Figure 6. The Cak1 kinase functions in the Ctk1/Set2/Rpd3C(S) pathway that suppresses cryptic initiation by RNA polymerase II.

a) Subset of Type I TGMs that share the Cak1-Set2 positive genetic interaction. For a complete picture of all Type I TGMs see Supplementary Figure 4. b) Tetrad analysis and serial 10-fold dilution spot testing demonstrates that the slow growth of the cak1-DAmP allele is strongly suppressed by set2Δ or eaf3Δ. c) CAK1 suppresses cryptic transcription initiation. The GAL1-FLO8-HIS3 reporter (Nourani et al., 2006) is described in Methods. The HIS3 gene product is only produced when transcription aberrantly initiates from a cryptic promoter within FLO8, as when chromatin structure in the transcribing gene is disrupted (Carrozza et al., 2005; Joshi and Struhl, 2005; Keogh et al., 2005). His3 expression was monitored by 10-fold dilution spotting of the indicated strains onto synthetic complete (SC) medium +/− histidine with galactose/raffinose (2%/1% respectively) as the carbon source. The lower panels employed a series of cak1-temperature sensitive alleles (Espinoza et al., 1998) at their semi-permissive temperature (SC, 32°C, 48hrs; -HIS, 32°C, 96hrs). d) Possible schematic of this pathway controlling intergenic chromatin fidelity. e) The MAP kinase Fus3 regulates cryptic initiation. All strains contain a GAL1_Sp-FLO8::HIS reporter, similar to that in Figure 6c (see Methods). Reporter strains were pinned in duplicate onto SC-HIS with galactose/raffinose as the carbon source, incubated at 30°C and photographed on days indicated. Panels are standardized to facilitate cross-comparison of reporter expression in each strain/time-point.