Project description:ChIP-on chip assays to measure the change in histone H3 K36 trimethylation over the yeast genome in wild-type yeast strains. Two color experiment.WT cells. Biological replicates=3 per IP per cell type.
Project description:Project abstract : The trimethylation of histone H3 lysine 4 (H3K4me3) is a crucial factor in defining the promoter regions of active genes in all eukaryotes ranging from Saccharomyces cerevisiae (yeast) to humans. In budding yeast, this trimethylation process facilitated by the Set1 complex results in H3K4me3 requiring a prior mono-ubiquitination at the histone H2BK123 residue (H2Bub) by E2 enzyme Rad6 and E3 enzyme Bre1. A previous in vitro study suggested that ubiquitinated H2B directly facilitates H3K4me3. However, even low levels of global H2Bub is sufficient for the required H3K4me3 in yeast cells, thereby indicating that other factors resulting in the H2Bub-dependent H3K4me3 remain unknown. This study revealed the high level of correlation of H3K4me3 with chromatin recruitment of Rad6 at the genome-wide level. Rad6 is confirmd to interact and co-localize with Swd2/Cps35, a key factor for the H2Bub-dependent H3K4me3 in genes with high levels of H3K4me3 and intronic genes rather than non-intronic genes. This study therefore provides a mechanistic insight of the H2Bub-Rad6- Swd2/Cps35-H3K4me3 axis and its potential role in RNA biogenesis.
Project description:In cells lacking the histone methyltransferase Set2, initiation of RNA polymerase II transcription occurs inappropriately within the protein-coding regions of genes, rather than being restricted to the proximal promoter. Here, we mapped the transcripts produced in an S. cerevisiae strain lacking Set2, and applied rigorous statistical methods to identify sites of cryptic transcription at high resolution. Wild type (BY4741) and set2â (BY4741) strains were grown at 30°C in YPD (1% yeast extract, 2% peptone, 2% dextrose) to an OD600 of 0.6-0.8. For each of the three replicates, Total RNA was extracted by acid-phenol method (Xiao et al. 2003). Double-stranded cDNA was prepared using an Invitrogen SuperScript⢠(Cat No. 11917-010) primed with Oligo(dt) and random hexamers. For each replicate, the wt and set2â cDNA were independetly fluorescently labeled and comparatively hybridized to high-resolution 385K Saccharomyces cerevisiae CGH arrays (2005-08-16_SCER_WG_CGH) with Tm-normalized probes. In one of the replicates, assignment of the fluorescent label was reversed.
Project description:Effect of FLO8 or MSS11 deletion and -overexpression on yeast transcript profiles compared to wild type in laboratory yeast strains Σ1278b and S288c.
