Investigating the Role of Insulators in linking Gene Regulation and Nuclear Organization

Louisiana State University, Baton Rouge LA

Investigators

Abstract

Intellectual merit: Chromosome organization and chromatin dynamics play important roles in nuclear processes such as gene regulation. However, understanding of most mechanisms that link nuclear organization to function remains rudimentary. Chromatin domain insulators (also called boundary elements) might link gene regulation to nuclear organization. Insulators are thought to partition chromosomes into functionally independent domains, preventing cross-talk so that genes in adjacent domains can be differentially regulated. But little is known about molecular mechanisms by which insulators function. The goal of this research project is to gain insight into molecular mechanisms by focusing on a particular insulator-binding protein in Drosophila melanogaster (fruit flies), the Boundary Element-Associated Factor, BEAF. Various approaches will be used to identify important domains in BEAF, and to identify proteins that interact with BEAF. The relationship of these proteins to BEAF will then be characterized. Previous results from the laboratory of the PI found that BEAF usually binds adjacent to promoters and plays a role in keeping the associated promoters active. Experiments will be performed to determine if promoter activity correlates with BEAF-dependent insulator activity, and to determine if interacting proteins affect BEAF-associated promoter activity. Some models of insulator function hypothesize that insulators are involved in nuclear organization by mediating the formation of chromatin loops. There is evidence that BEAF mediates chromatin loop formation. To explore this in more depth, networks of looping interactions will be determined for six BEAF binding regions using circular chromosome conformation capture (4C) assays with high throughput DNA sequencing. The proportion of loop partners that are insulators, promoters, enhancers, or other sequences will be determined, as will the distances over which looping occurs. Results will be validated by two-color fluorescence in situ hybridization and chromosome conformation capture (3C) assays. Identification of interaction partners of BEAF and the role of BEAF and these proteins in promoter activity will provide insight into molecular mechanisms of insulator function. The looping experiments address the role of insulators in nuclear organization. Together, these experiments will provide insight into the relationship between nuclear organization and gene regulation. Broader impacts: An integral goal of research in the lab of the PI is to train the next generation of scientists. Participants in this research project will include both graduate and undergraduate students, including members of traditionally underrepresented groups. At least two undergraduates usually work in the lab each semester and in the summer. They participate in cloning, PCR (mutagenesis, sequencing, inverse PCR to localize transgene insertion sites), fly genetics, microscopy, and protein biochemistry. Most have been females, minorities or both. Several have gone on to careers in medicine or life sciences research. The PI has also served as a local Science Fair judge, mentored two high school science fair projects, and is recruiting another high school student. One project won second place in its category in statewide competition. PhD students and associated undergraduates will be trained during this project. The research will enhance teaching by the PI of junior and senior level molecular cell biology courses, and of a graduate seminar course entitled Chromatin, Nuclear Organization and Gene Expression developed by the PI based on his research expertise. Research results will be disseminated at conferences and by publication. The PI will continue to make reagents developed in his laboratory, such as fly stocks, available to the research community. Thus this research will have significant scientific and educational impacts.