Matched MNase digestions (BY4741 and BY4741/tup1D)
Tup1 stabilizes promoter nucleosome positioning and occupancy at transcriptionally plastic genes.
Summary: High-throughput DNA sequencing has generated genomewide chromatin structure maps with unprecedented resolution and accuracy. Despite technical advances, the future of chromatin mapping studies requires an ability to make accurate comparisons between different chromatin states to enhance our understanding of genome biology. In this study we describe a novel technical approach, based on systematic production and confirmation of Matched MNase Digestions, which enables sequencing technology to identify specific and accurate differences between broadly similar chromatin states. In this case we compare chromatin in the presence and absence of the well-characterized repressor protein in S. cerevisiae, Tup1. Analysis of both wild-type and tup1? chromatin datasets reveals unique organizational themes relating to the function of Tup1.  Regulatory regions bound by Tup1 assume a unique chromatin architecture composed of a wide nucleosome-depleted region and low occupancy and/or unstable nucleosomes, regardless of Tup1’s role in the regulation of downstream expression. Additionally, nucleosome stabilization by Tup1 overlies the binding motifs of many key transcription factors including Tup1 recruiting proteins and stress response factors. Ultimately, identification of unique chromatin features related to Tup1 biology add new insights to our understanding of this co-repressor’s function that would not have been evident without the specificity of our novel mapping approach. Keywords: chromatin, Tup1, nucleosome, MNase-Seq, genomic. Overall Design: Illumina sequencing of mononucleosomal DNA prepared using Matched MNase digestion protocol
Chromatin mapping by MNase-Seq, using a novel technical approach (Matched MNase Digestions)that enables high-throughput sequencing technology to identify specific and accurate differences between broadly similar chromatin states. In this case we compare chromatin structures in the presence and absence of the well-characterized repressor protein in S. cerevisiae, Tup1.