Summary Chromatin in active regions of eukaryotic genomes undergoes extensive disruption and replacement of nucleosomes. New models suggest that nucleosome dynamics are critical to both increase DNA accessibility, to limit transcription factor binding, and to protect the genome from damage. The long-term goal of this project is to define the components involved in nucleosome dynamics and how these are important for genome function. Nucleosome replacement in all animals uses the conserved histone H3.3 variant, and the ATRX/Xnp chromatin remodeler has been implicated in H3.3 deposition into chromatin. We have developed methods to track H3.3 deposition in Drosophila, and model examples of nucleosome-depleted chromatin. In this proposal, we use these tools in combination with mutations in key factors to characterize the response of cells to chromatin gaps A second approach in this proposal uses a new method to visualize alternative occupancies of transcription factors and nucleosomes across the genome. In Aim 1, we will define the importance of Drosophila ATRX/Xnp and associated factors for rebuilding nucleosomes at disrupted chromatin. In Aim 2 we examine Polycomb Response Elements as natural examples of nucleosome-depleted chromatin. We analyze the role of ATRX/Xnp and other chromatin remodelers in driving cycles of nucleosome displacement and replacement. Finally, we determine the importance of nucleosome dynamics for factor binding in regulatory elements. These studies have broad relevance for understanding the integration of sequence-specific DNA-binding proteins, nucleosome remodeling, and intrinsic sequence features in dynamic chromatin regions.