Chromosomal Organization Mediated By Insulators

University Of Tennessee Knoxville, Knoxville TN

Investigators

Abstract

Insulators or chromatin boundaries are phylogenetically widespread DNA sequences bound by proteins that block enhancer-promoter interactions and function as molecular barriers against spreading of heterochromatin through the chromatin fiber. These properties suggest that insulators may be involved in the establishment and maintenance of functional chromatin domains. In Drosophila, the gypsy insulator is found at more than 500 sites uniformly distributed in all chromosomes. Mutations in genes encoding gypsy insulator proteins are lethal or may have important phenotypic consequences such as female sterility. Maintenance of functional chromatin boundaries is therefore required for proper somatic and germ-line development. Evidence emerging from the study of the Drosophila gypsy insulator suggests that chromatin domains mediated by insulators result from the interactions between neighboring insulators bound proteins and the subsequent looping out of the intervening DNA sequences. The important notion that insulators, or chromatin boundaries, mediate the higher level organization of interphasic chromosomal DNA and that this organization influences the transcriptional status of genes is widely accepted but lacks direct experimental evidence. Preliminary data from the laboratory of Dr Labrador provides the foundation for a testable model allowing the experimental assessment of the role that endogenous insulators may play in chromatin organization and gene expression regulation. This project is aimed at performing the first large-scale functional analysis of Drosophila gypsy endogenous insulators in vivo. Three specific aims are suggested to achieve this goal: (1) generate a precise experimental map of endogenous insulator sites along a large region of a chromosome. (2) Assess the functional properties of endogenous gypsy insulators found in this region and (3) determine the role that these insulators have in transcriptional regulation of the genes contained in the region. Gene activity is regulated by interactions between activator or repressor proteins with DNA sequences called enhancers or repressors located at or near genes. A third type of DNA sequences only recently described but present in genomes from yeasts to humans, is capable of blocking the communication between enhancers or repressors with the target genes. These sequences are called insulators and control gene activity through DNA-protein interactions and protein-protein interactions. Results from this project will identify for the first time a large number of contiguous genomic insulators simultaneously with their associated genes in the fruit fly. The role of insulators in the control of the activity of those genes will be determined. Results will provide experimental evidence on the important question of the role that insulators, as well as genome structure, play in the control of gene activity in eukaryotic genomes. Dr. Labrador is committed to making research experience available to undergraduates, thus facilitating the process of learning, introducing them to the process of creating knowledge, and possibly enticing them to pursue a career in research. He believes this project is particularly suitable for these goals, as it involves genetic analysis along with a variety of molecular and cell biology techniques that are not easy to grasp from standard lectures and textbooks. In addition, as part of the Hispanic minority himself, Dr. Labrador is committed to recruiting minority undergraduates to his research group, with the prospect of actively promoting the incorporation of underrepresented minorities into the scientific community.