Delineation of the molecular mechanisms underlying DNA replication initiation and changes in gene copy number during Drosophila development

Abstract

The study of differential DNA replication programs in Drosophila has provided important insight into the molecular control of replication initiation and fork progression during development. We investigated the mechanisms by which binding of the origin recognition complex (ORC) and replication fork inhibition give rise to locally underreplicated regions in Drosophila polyploid tissues. We identified copy number changes genome-wide in two additional polyploid tissues and compared our results to three previously profiled larval tissues. These results revealed a high level of tissue-specificity in the number of underreplicated sites within a given tissue but also highlighted the conservation of the locations of many of these underreplicated regions across tissues. By mapping ORC binding sites in the larval fat body, we found that the repression of replication initiation is a common mechanism of underreplication in polytene tissues. Our ORC localization studies also suggest that underreplication zones are hard-wired across tissues and that differential underreplication of these zones is dependent upon variation in fork progression across these regions. We then utilized the Drosophila amplicons in follicle cells (DAFCs) as a model replication system to dissect the molecular mechanisms underlying the activation of individual replication origins. Repression of the DAFC-22B origin is not achieved through changes in subnuclear localization but rather through effects of the surrounding chromatin. We identified two novel genomic sites at which origin activity is modulated directly by the surrounding chromatin environment. At one site, the surrounding chromatin promotes one additional round of origin firing at a specific developmental time point. At the other site, origin activity is repressed by the surrounding chromatin through inhibition of the localization of the MCM2-7 helicase complex. Origin repression at this site is not correlated with the establishment of heterochromatin, raising the possibility that the activity of individual replication origins are regulated by the chromatin environment on a greater, conformational level. Finally, we dissected the requirement of transcription in the activation of the DAFC-62D origin. Surprisingly, transcription is not required in cis for origin activation. These results indicate the requirement of a trans-acting factor specifically at this site and highlight the diversity of mechanisms that control metazoan origin activation.