Project Summary/Abstract Our current understanding of chromatin transcription has been limited by our knowledge of the factors that are involved in this process. In this regard, we have identified a novel nucleosome destabilization factor, termed NDF, that can destabilize nucleosomes and facilitate transcription elongation through nucleosomes. In this proposal, we will investigate the function of NDF in chromatin dynamics and RNA Polymerase II transcription elongation using biochemistry, genomics, bioinformatics and structural biology approaches. In Specific Aim 1, we will develop and employ new methods and strategies to study the molecular mechanism of NDF-mediated nucleosome destabilization and RNA Polymerase II transcription. We will use ultra-high-resolution optical tweezer as well as cryo-electron microscopy to characterize the dynamics of NDF-mediated nucleosome destabilization. In Specific Aim 2, we will examine how NDF is recruited to the transcribed regions of active genes in cells. We will assess whether other cellular factors, other than H3K36me3 histones, contribute to the recruitment of NDF. In Specific Aim 3, we will determine how genes lacking NDF are transcribed, and investigate protein factors that are related to NDF. The studies proposed here should significantly increase our understanding of the mechanisms of Pol II transcription elongation through chromatin.