The budding yeast Saccharomyces cerevisiae is used as model system to understand genomic mechanisms by which chromatin organization is established. The start sites of genes, or promoters, are typically encased in a nucleosome-free region, that is bookended with two well-positioned nucleosomes. This precise organization is standard for most genes, and plays into how these genes are regulated. Therefore a fundamental understanding of gene regulatory mechanisms requires a complete understanding of how such canonical nucleosome organization arises and guides the placement of the transcription machinery. The proposed work will take advantage of biochemical reconstitution of aspects of canonical nucleosome organization on a genomic scale. This will allow individual mechanistic contributions of chromatin organizing factors and their effectors to be explicitly defined. In particular, how ATP-dependent chromatin remodeler complexes recognize DNA features and sequence-specific organizing factors, will be addressed. The organization of chromatin directs the organized assembly of the transcription machinery. Biochemical reconstitution will be used to tease apart selected individual contributions of chromatin organization and activator/repressor binding towards assembly of the transcription machinery on a genomic scale. Chromatin is generally thought to be composed of nucleosome particles containing 2 copies of each of the four core histones. However, it is now becoming clear that various partially assembled nucleosomes or subnucleosomes exist in vivo, and these structures may play critical roles in chromatin dynamics. The existence of these substructures and the histone chaperones that are likely to be involved in their assembly and disassembly will be investigated on a genomic scale.