Abstract Chromatin was first defined by Flemming in 1882 as the `substance in the cell nucleus which is readily stained' that `is refractile to digestion'. The term stuck, and now is taken to refer to DNA (the readily stained component) and the associated proteins that make it refractile to digestion. It has become clear over the past twenty years that chromatin structure is vastly more dynamic than previously suspected. Regulation of chromatin structure frequently regulates the ability of proteins to access DNA, and in this manner plays a key role in regulating transcription, replication and recombination of the genome. The basic structural component of chromatin, the nucleosome, plays a central role in this regulation. The position of nucleosomes on the DNA can be changed, nucleosomes can be evicted, and nucleosomes can contain various histone variants and can be covalently modified in different ways, allowing them to have differential abilities to interact with the regulatory machinery. The dynamic nature of chromatin has been intensively studied for the past fifteen years, with much of the resultant information pertaining to the type and extent of covalent modification of the nucleosome. Information on the location of nucleosomes and on the full spectrum of proteins that might be involved in regulating a specific genomic locus has lagged behind this flood of information on covalent alterations to chromatin. The purpose of the experiments described in this application is to investigate how the location of nucleosomes can be altered in vitro and in vivo and to determine the full spectrum of proteins that might be involved in these types of regulation on a given genomic locus. Three Aims are proposed: Aim 1 will characterize nucleosome remodeling in vitro by examining individual remodeled products and through structural approaches; Aim 2 will characterize structural changes to chromatin during regulation of a set of genes in cultured human cells; and Aim 3 will develop technology to catalog proteins and RNA that bind to specific loci.