Analysis of Chromosomal Insulators and Insulator Binding Proteins

Louisiana State University, Baton Rouge LA

Investigators

Abstract

The goal of this research is to understand how nuclear and chromosomal context establish and maintain patterns of gene expression. The focus is on chromatin domain insulators (also called boundary elements) because the role they play in regulating enhancer promoter communication suggests they might play a broader role in overall chromosome organization. Insulators are DNA elements that define possible regulatory interactions within genomes. They block enhancer-promoter communication, but only when located between the enhancer and promoter. Insulators are thought to allow adjacent genes to be differentially regulated, preventing cross-talk by partitioning them into functionally independent domains. One aim of this project is to study insulator function using genetic tools. Knockout mutations in the single gene encoding these two proteins and in the 32A portion of the gene have been generated by homologous recombination. In addition, flies with a GAL4-inducible transgene encoding a dominant-negative BEAF protein have been generated. The hypothesis that the BEAF proteins play a role in maintaining patterns of gene expression will be tested using gene microarrays with RNA isolated from mutant versus wild-type animals. Knockout flies will also be used in chromatin mapping experiments to test the hypothesis that BEAF plays a role in global chromatin organization. Using BEAF-fluorescent protein fusions in the BEAF knockout background, BEAF localization and dynamics will be studied. The dominant negative transgene was used in a screen to identify genetic interactions. Follow-up experiments will be conducted to gain insight into molecular mechanisms of BEAF function. The second aim is to test the hypothesis that insulators play key roles in linking genome organization to gene expression. A genomic map of BEAF binding sites will be constructed by hybridizing ChIP DNA to genome tiling arrays. This data will be combined with the gene expression microarray analysis of the first aim, to test the hypothesis that BEAF-dependent insulators separate differentially regulated adjacent genes from each other and play a role in their differential regulation. These experiments are an important test of the role of insulators in relationship to global genomic function, and will provide insight into molecular mechanisms of insulator function. An integral goal of this study is to train the next generation of scientists. To this end, graduate and undergraduate students, including those from underrepresented minority groups, will participate in the research activity.