Project summary- Much of the control of gene expression is exerted at two points: assembly of the RNA polymerase II (Pol II) preinitiation complex at promoters and the successful advance of Pol II into productive elongation. Pol II promoters are accompanied by an immediately downstream (+1) nucleosome typically located just beyond the likely boundary of the promoter. It is often speculated that promoter activity is controlled by this distinctive local chromatin architecture, which includes histone modifications and histone variants specific to promoter-proximal nucleosomes. However, the roles that adjacent nucleosomes play in the assembly of the transcription complex remain poorly understood. After transcription initiates Pol II complexes elongate very slowly, eventually encountering the +1 nucleosome while associating with positive and negative factors. Most of these complexes terminate while a minority advance into effective elongation and traverse the nucleosome. The extent to which complexes achieve full elongation competence is a major determinant of overall promoter strength, but it is not known how the +1 nucleosome barrier modulates the crucial competition between maturation into productive elongation and termination. Our overall aim is to biochemically dissect Pol II initiation and early elongation to understand the mechanistic basis for distinct outcomes for Pol II complexes within the local chromatin context. We are approaching these questions using a nuclear extract based in vitro transcription system in which transcription complex assembly and early elongation are challenged by a +1 nucleosome positioned at a series of precise downstream locations. These nucleosomes can include the histone modifications and histone variants typically found near promoters. Our current results already provide important and novel insights into both the negative and positive effects of +1 nucleosomes on transcription complex assembly. These effects depend significantly on promoter class (presence or absence of the TATA element) and on promoter-enriched histone modifications whose function is currently unknown. In the studies proposed here, we will substantially extend the present findings to uncover the mechanistic basis for the control of Pol II transcription complex assembly by the +1 nucleosome. We have also found with extract-based transcription that both the advance into productive elongation and termination can be studied in single reactions. This will allow us for the first time to uncover the mechanisms and factors that competitively drive both relevant fates for Pol II immediately following initiation from a promoter in the context of an appropriately located and modified +1 nucleosome.