A transcriptional insulator element, the su(Hw) binding site, protects a chromosomal DNA replication origin from position effects

Abstract

Eukaryotic chromosomes are organized into domains of activity for both transcription and DNA replication. Transcriptional "border," or "insulator," elements have been implicated in mediating the organization of transcriptional domains. However, the DNA sequence elements which might demarcate domains of DNA replication activity are unknown. su(Hw) protein binding sites [su(Hw)BSs] are potent transcriptional insulator elements which can block enhancer action, as well as positive and negative chromosomal position effects. Here we report that flanking su(Hw)BSs can also create a chromosomal domain permissible for activity of the chorion gene DNA replication origin. During Drosophila oogenesis the chorion (eggshell) gene loci are amplified approximately 80-fold through repeated initiation of DNA replication. The cis-acting amplification control element, on the third chromosome (ACE3), is required for high levels of amplification initiating at the nearby major origin of replication, Ori-beta. A transgenic chorion locus construct containing ACE3 and Ori-beta was able to amplify but was extremely sensitive to position effects: only 7 of 21 independent insertions amplified >10-fold. The inclusion of flanking su(Hw)BSs in the construct dramatically protected DNA replication from position effects: 31 of 31 insertions now amplified >10-fold, and this protection was reduced in a su(Hw) mutant background. Amplification was equal on both sides of the su(Hw)BS, demonstrating that replication fork passage is not significantly impeded by these sites. Inclusion of only a single su(Hw)BS in the construct did not detectably protect the chorion gene DNA replication origin from position effects.