Figure 4.

Phosphorylation by CK2 regulates Spt6 chromatin function. (A) Mutation of CK2 phosphosites in Spt6 leads to spt- phenotype. SPT6 WT and spt6-7SA (Serine to Alanine) strains containing the Lys2-128δ SPT reporter allele were serially diluted and spotted on synthetic complete (SC) or SC medium lacking lysine (SC-Lys) at 30 or 39°C. The spt6-7SA allele is thermosensitive and has an spt- phenotype. (B) The spt6-7SA is not able to suppress spurious transcription from FLO8 cryptic promoter. WT and spt6Δ strains expressing SPT6 WT or spt6-7SA containing pGAL1-FLO8-HIS3 spurious transcription reporter were serially diluted and spotted on synthetic complete (SC) medium, or SC medium lacking histidine (SC-His) with Galactose as a carbon source. The spt6-7SA allele growth in absence of histidine indicates intragenic cryptic initiation within FLO8 coding region. (C) The spt6-7SA mutant is associated with cryptic transcription. Cells expressing either SPT6 WT or spt6-7SA were grown in YPD at 37°C. Total RNA was isolated and analyzed by Northern blot with a probe specific for 3′-end of FLO8. SCR1 served as loading control. (D) Replication-independent histone H3K56ac level is increased at coding regions of genes in spt6-7SA mutant. ChIP assays assessing the H3K56 acetylation level in G1-arrested cells expressing Spt6 or the mutated Spt6-7SA version. Cells were G1-arrested and subsequently grown for 2 h at 30 or 37°C. The values shown represent the average and standard errors of three independent experiments measuring H3K56ac levels (IP/Input) relative to histone H3 occupancy (IP/Input). Coding regions of different genes were tested and NoORF is a non-transcribed control locus of chromosome V. (*) P value < 0.05; (**) P value < 0.01; (***) P value<0.001.