The Drosophila Fourth Chromosome: Gene Expression in the Context of Repetitious DNA

Washington University, Saint Louis MO

Investigators

Abstract

Intellctual Mert: The DNA of the eukaryotic genome is packaged by association with the histones and other chromosomal proteins into alternative forms: the more condensed form, heterochromatin, inhibits gene expression while a more open form, euchromatin, facilitates gene expression. Eukaryotic genomes have been greatly expanded over evolutionary time by the retention of repetitious sequences, primarily copies of transposable elements (DNA transposons and retroviruses - TEs). Where these sequences are present at high density, they are packaged as heterochromatin, inhibiting expression of these elements. The long-term goal of this project is to understand how genes can be expressed from a domain with high levels of silenced repetitious sequence. Work in the fruit fly Drosophila melanogaster has shown that many genes normally resident in euchromatin exhibit a variegating phenotype (due to silencing of the gene in some of the cells where it should be active) when inserted into heterochromatin by rearrangement or transposition. The small fourth chromosome (Muller F element), which is largely heterochromatic, contains ~80 genes, both housekeeping and developmentally regulated genes. In higher eukaryotes, specifically mammals, the long chromosome arms that contain genes appear to be structured like the F element, with ~30% TE repeats. Thus how genes can function in this environment, while repetitious elements are silenced, is a general issue. The goal here is to understand the combination of characteristics that enables fourth chromosome genes to function in a chromatin context where euchromatic genes variegate (are silenced). The investigators postulate the presence of one or more key sequence features or motifs organizing and/or protecting the fourth chromosome genes. To test this hypothesis, they will identify 'landing pad' sites in the D. melanogaster fourth chromosome where a fourth chromosome gene is appropriately expressed, but an hsp70-white gene gives a variegating phenotype, and test whether this is determined by the 5' region of the gene (the transcription start site with ~1 kb flanking sequence, both upstream and downstream). Establishing this system will allow one to identify the minimal features that can drive full expression of any reporter in a fourth chromosome heterochromatic site. As a complement to this work, the investigators plan to use an in silico approach to find sequences in other species that are similar to the sequences identified in the D. melanogaster F element. This comparative bioinformatics approach will be facilitated by carrying out ChIP-seq experiments to find binding sites for RNA polymerase II (marking the start site for transcription) in two Drosophila species which are at a suitable evolutionary distance for finding sequence motifs, D biarmipes and D. elegans. By testing the findings gained from one approach with the methodology of the other approach, the investigators expect to gain a better understanding of the global regulation of different chromosomal regions and of the sequence elements needed to drive expression of genes in heterochromatin. Broader Impacts: This project is a collaborative effort with the Genomics Education Partnership (GEP), a group of over 80 faculty who are using Drosophila genomics to bring students into the research community. The GEP undergraduate students will be responsible for generating high quality annotations for the genes on the Muller F elements of D. biarmipes and D. elegans. The student annotations will facilitate the search for common features/motifs on the F element using a comparative genomics approach. Undergraduates will also take charge of looking at the impact of Su(var) and E(var) mutations on various reporters, testing sensitivity of the "synthetic fourth chromosome gene" constructs to mutations in appropriate chromosomal proteins. The postdoctoral fellow supported here will become an integral member of GEP, and will become very skilled at working with undergraduates in a research program.