Role of nucleosome modification, composition, and position in specification of replication origins. Neha P. Paranjape, Jun Liu, Brian R. Calvi. Department of Biology, Indiana University, Bloomington, IN.

   Metazoans initiate DNA replication from many sites in the genome called origins. Origins are binding sites for a pre-Replicative Complex (pre-RC) of proteins that is then activated in S phase to initiate replication. It is currently unclear, however, how genomic loci are selected to be pre-RC binding sites and active origins. Metazoan replication origins lack a DNA consensus sequence and show remarkable developmental plasticity. We use developmental gene amplification- a specialized replication program in the Drosophila ovary - as a model system to study how origins are specified in development. Amplification involves re-replication from origins at six specific loci in follicle cells late in Drosophila oogenesis, which results in an increase in the DNA copy number of genes required for rapid eggshell synthesis. We have found that hyperacetylation of nucleosomes on multiple lysines contributes to activation of amplicon origins. Moreover, the level of acetylation at the six amplicon loci correlates with their different levels of amplification. Genomic location analysis from a number of labs has revealed a correlation between pre-RC binding sites, nucleosome depleted regions (NDRs) and enrichment for histone variants H3.3 and H2Av. Using MNase-seq and ChIP-qPCR, we have found that amplicon origins are also NDRs and enriched for the nucleosome variants H3.3 and H2Av. At the well-defined amplicon DAFC-66D, NDRs and H3.3/H2Av correspond to regions that are essential for origin function. Analysis of H3.3 mutant strains indicated, however, that H3.3 is not required for origin activity. Moreover, our data suggest that, although essential origin elements are NDRs, this nucleosome depletion is not sufficient to specify origin location or timing. We will also describe an unbiased genetic screen to identify new attributes of the epigenome that influence origin function. Thus, the amplicon model system provides a unique opportunity to discover how different chromatin features contribute to differential origin activity in development.