Intellectual merit: The first step in gene expression (RNA synthesis, or transcription) of the vast majority of eukaryotic genes is regulated by enhancers, which are DNA sequences that bind specific proteins and activate transcription over large distances. Enhancer-target communication (ETC) leads to direct interaction of enhancers with the target promoters, the sites of transcription initiation, via formation of a DNA loop. ETC can be prevented by insulators, which are DNA elements that form alternative DNA loops and can isolate enhancers from the targets. Thus, gene regulation in human cells involves formation of DNA loops of variable sizes. The mechanisms of efficient communication between enhancers and promoters and the mechanisms by which such communication is prevented by insulators remain unknown. In particular, neither structural nor dynamic aspects of DNA looping have been rationalized. Recently, it has been shown that DNA loops bound to histone proteins are a highly efficient communication device. These observations indicate that ETC could constitute a critical step in gene regulation and raise the following important questions: What elements of the DNA-histone complexes mediate efficient ETC, and how can ETC be inhibited or facilitated by various factors? In this project, a highly purified and functionally active experimental system has been established that allows quantitative analysis of the rate of ETC in the DNA-histone complexes. This system will be used as a tool to analyze structural and dynamic properties of the DNA-histone complexes that dictate the rate of ETC. The objectives of this research are to evaluate the mechanisms of efficient distant communication and inhibition of communication by insulators and to identify factors affecting distant communication and evaluate their effect on the dynamics of the DNA-histone complexes. Broader impacts: This project will provide an enhanced learning experience for several undergraduate and two graduate students involved in the project. They will participate weekly in four different meetings; namely (1) laboratory meetings in which they will have the opportunity to present their own research every 2-3 months, (2) laboratory discussion of recent publications on chromatin structure/dynamics/function, (3) problem solving sessions with a small group of students and senior researchers that focuses on technical challenges, and (4) Departmental Progress Reports where all Departmental students present their research annually. This project is well suited for student participation, as it provides a solid conceptual foundation and utilizes methodologies applicable to many areas of research, and at the same time is paradigm-driven, providing an intuitive transition from the classroom to the bench. The laboratory regularly trains underrepresented minority undergraduates (within the UMDNJ Summer Research Program). New technologies/strategies for analysis of structure/dynamics of the DNA-histone complexes will be developed. Some of them will be of general use in the field of biology. The project is a multi- disciplinary study to examine a complex biological question, and incorporates recent advances in computational science and structural biology at the interface between chemistry, physics and biology.
