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

Washington University, Saint Louis MO

Investigators

Abstract

The chromosomes of eukaryotes contain tightly packed regions known as heterochromatin. Genes in these regions are typically silenced; however, some genes in heterochromatic regions are actively expressed. The aim of this project is to address this long-standing and important question in the field of gene regulation: what is the mechanism that allows certain genes located in heterochromatic regions to be expressed? This work is a collaborative effort with the students and faculty of the Genomics Education Partnership. Undergraduate students from over 60 colleges and universities will analyze heterochromatic DNA sequences in the fourth chromosome of several fruit fly species. The goal of this work is to identify potential regulatory motifs for gene expression in heterochromatic DNA. This process will allow the students to develop basic bioinformatics skills, participate in genomics research, and be contributors (and co-authors) on the resulting publications. The fourth chromosome of Drosophila melanogaster, with a 30% repeat density, is largely heterochromatic; as illustrated by the variegating phenotype of an hsp70-white reporter gene at most insertion sites on the fourth chromosome. This silencing appears to be due to packaging in a heterochromatic form. While high levels of HP1a and H3K9me2/3 are found across the body of fourth chromosome genes, their transcription start sites (TSSs) consistently show depletion of these silencing marks. This project hase identified a "landing pad" site (a MiMIC line) on the fourth chromosome where an inserted hsp70-white transgene gives a variegated phenotype, while a fourth chromosome gene (Rad23) tagged with a fluorescent marker shows full expression. Gene fragments between these constructs will be swapped to determine what features of the fourth chromosome gene drive full expression in this heterochromatic environment, with an initial focus on the region around the TSS. The goal of this project is to determine the sequence elements necessary and sufficient to drive full expression of hsp70-white at this chromatin site, and conversely those changes that would result in variegation of Rad23 (Elements involved in transcription elongation may prove to be as important as the TSS, and will not be neglected). Key motifs identified by the bioinformatics approach will be tested in this system, and vice-versa. The chromatin structure of the various lines will be checked by both genetic and biochemical approaches (analysis of the impact of Su(var) mutations; ChIP-PCR experiments). These studies will lead to a better understanding of heterochromatic gene regulation, and identification of motifs (some of which may be novel) that influence gene expression in repeat-rich domains that are also common in the genomes of higher eukaryotes.