GSE109078
Casein Kinase 2 Mediated Phosphorylation of Spt6 Modulates Histone dynamics and Regulates Spurious Transcription (ChIP-Seq)
CK2 is an essential protein kinase implicated in various cellular processes. In this study, we address a potential role of this kinase in chromatin modulations associated with transcription. We found that CK2 depletion from yeast cells leads to replication-independent increase of histone H3K56 acetylation and global activation of H3 turnover in coding regions. This suggests a positive role of CK2 in maintenance/recycling of the histone H3/H4 tetramers during transcription. Interestingly, strand-specific RNA-seq analyses show that CK2 inhibits global cryptic promoters driving both sense and antisense transcription. This further indicates a role of CK2 in the modulation of chromatin during transcription. Next, we showed that CK2 interacts with the major histone chaperone Spt6, and phosphorylates it in vivo and in vitro. CK2 phosphorylation of Spt6 is required for its cellular levels, for the suppression of histone H3 turnover and for the inhibition of spurious transcription. Finally, we show that CK2 and Spt6 phosphorylation sites are important to various transcriptional responses suggesting that cryptic intragenic and antisense transcript production may have an impact on cell adaptation to environmental cues. Altogether, our data indicate that CK2 mediated phosphorylation of Spt6 regulates chromatin dynamics associated with transcription, and prevents aberrant transcription. Overall design: Cells from CK2ts mutant strain and its respective WT were grown at 30°C in YP-Raffinose, arrested in G1 and then 2% galactose was added to induce Flag-H3 synthesis and shifted to 37ºC for 1h. Cultures were cross-linked by adding formaldehyde to a final concentration of 1%. Yeast lysates were generate by bead beating with glass beads of 0.5?mm diameter, and chromatin solubilization was performed by sonication (average DNA fragment size of 350-500 bp). Immunoprecipitation (IP) of Rpb1 was performed using 2-3 µg of the 8WG16 anti-CTD antibody per IP (Covance, MMS-126R). The Flag Immunoprecipitations were done using 10 µL of anti-Flag agarose beads per IP (Sigma, A2220). ChIP-seq experiments were performed fallowing following TruSeq DNA illumina protocol and Illumina next generation sequencing was performed as 50 base pairs single-end reads (McGill University and Génome Québec Innovation Centre, Montréal, Canada). The sequenced reads were aligned to the S-cerevisiae genome assembly R64-1-1 (GCA_000146045.2) using bowtie v0.12.8 (Langmead et al., 2009). Coverage was calculated using bedtools (http://bedtools.readthedocs.io/) (Quinlan et al., 2010) and was normalized into reads per million (RPM).