A major part of eukaryotic nuclear DNA is organized into nucleosomes that are not distributed randomly, but occupy defined positions with respect to the DNA sequence. Such nucleosome positioning is fundamental to all genomic processes as it regulates the accessibility of DNA. For example, a transcription factor binding site may reside in a nucleosome free region and be readily bound by its corresponding factor in one cell but may be incorporated in a nucleosome and therefore be inaccessible in a different cell. We wish to understand which factors determine nucleosome positioning, especially how DNA sequence is read out and translated into nucleosome positioning, and how positioned nucleosomes in one biological state become remodeled into an altered organization in a different state. This is relevant, for example, in promoter regions where nucleosome organization primes or represses transcriptional activity. We use the unicellular yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe as in vivo and in vitro model. As our speciality, we established the first genome-wide reconstitution system that allows the biochemical characterization of factors and their mechanisms in nucleosome positioning and remodeling.