Simultaneous Profiling of DNA Accessibility and Gene Expression Dynamics with ATAC-Seq and RNA-Seq.

This chapter describes sequencing-based methods for profiling dynamic changes in DNA accessibility and gene expression in Saccharomyces cerevisiae. Assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-Seq) is a powerful technique for identifying nucleosome-free regions of the genome. Combining ATAC-Seq with RNA Sequencing (RNA-Seq) is a rapid approach for studying the relationship between genome structure and changes in global patterns of gene expression from a single experiment. A laboratory protocol is presented for these methods as well as examples of typical results and visualizations.

An estradiol-inducible promoter enables fast, graduated control of gene expression in fission yeast.

The fission yeast Schizosaccharomyces pombe lacks a diverse toolkit of inducible promoters for experimental manipulation. Available inducible promoters suffer from slow induction kinetics, limited control of expression levels and/or a requirement for defined growth medium. In particular, no S. pombe inducible promoter systems exhibit a linear dose-response, which would allow expression to be tuned to specific levels. We have adapted a fast, orthogonal promoter system with a large dynamic range and a linear dose response, based on ß-estradiol-regulated function of the human oestrogen receptor, for use in S. pombe. We show that this promoter system, termed Z3 EV, turns on quickly, can reach a maximal induction of 20-fold, and exhibits a linear dose response over its entire induction range, with few off-target effects. We demonstrate the utility of this system by regulating the mitotic inhibitor Wee1 to create a strain in which cell size is regulated by ß-estradiol concentration. This promoter system will be of great utility for experimentally regulating gene expression in fission yeast. Copyright © 2017 John Wiley & Sons, Ltd.