Investigation of Rap1 binding occupancy in global H3-depletion condition [H3MNase_ChIPseq]
We constructed the inducible H3-depleted and FLAG-tag of the repressor activator protein 1 (Rap1) yeasts to investigate the depleted effect of nucleosomal H3 on DNA-binding profiles of Rap1 and resulting transcriptional changes. The H3 inducible shut-off strain (named “DGS200.1” or “H3i” strain) was prepared from the wildtype YEF473A strain by knocking out the HHT1 gene (encoding H3), and replacing the native promoter of the HHT2 gene (the other H3-encoding gene) with an inducible Gal1 promoter. Conditional depletion of H3 was induced by culturing the H3i strain in the YPGal liquid medium (1% yeast extract, 2% peptone, 2% galactose) and transferring to the YPD medium (1% yeast extract, 2% peptone, 2% dextrose) for three hours. WT yeast were also cultured and switched the growth media in the same way as controls. DNA-binding profiles of H3 and Rap1 were determined using chromatin immunoprecipitation followed by next-generation sequencing (ChIP-seq), and the changes in transcriptional levels were investigated using RNA-seq. Overall design: Genome-wide nucleosomal H3 binding was investigated using ChIP-seq to study effects of H3 depletion in WT and H3i (conditional H3 shut-off) yeasts