ChIP Seq Analysis of H3K4me3 in Wild Type and Set1 S228A S228E mutants
Cells need to coordinate gene expression with their metabolic states to maintain cell homeostasis and growth. However, how cells transduce nutrient availability to appropriate gene expression response via histone modifications remains poorly understood. Here,we reported cla4 catalyzed Set1 phosphorylaton regulates cell cycle and histone gene transcription. Overall design: ChIP-Seq analysis. Yeast strain were grown in 200 ml YPD  media at 30°C until OD600 of ~0.7-1.0. The crosslinking was performed in 1% formaldehyde and quenched by adding 10 ml of 2.5 M glycine. Cells were harvested, washed once   with cold TBS + PMSF, lysed in FA-SDS buffer (40 mM HEPES-KOH pH7.5, 1 mM EDTA pH8.0, 0.1% SDS, 1% Triton X-100, 0.1% Na deoxycholate, 1 mM PMSF, 2 µg/ml leupeptin, 1 µg/ml pepstatin A, protease inhibitor cocktail, phosphatase inhibitor cocktail). Chromatin was sonicated to an average size of ~500 bp and then subjected to immunoprecipitation with antibodies pre-bound to Protein G Dynabeads (Invitrogen) at 4°C overnight. The beads were washed successively with FA buffer, FA buffer + 1 M NaCl, FA buffer + 0.5 M NaCl, TEL buffer (10 mM Tris pH8.0, 1 mM EDTA, 0.25 M LiCl, 1% NP-40, 1% Na deoxycholate) and TE buffer (10 mM Tris pH7.4, 1 mM EDTA). The eluted DNA/protein complex was treated with 20 µg Proteinase K at 55°C for 1 h followed by treatment at 65°C overnight. After removing RNA by RNase (Roche), the DNA was purified with ethanol precipitation and sequenced . Yeast strains WT (BY4741 in Open Biosystem) and Rif1-FLAG were the in same group, yeast strains Sen1-FLAG and Glc7-FLAG cultured both in YPD medium and SD-C medium were the other group.