Abstract Accurate replication of the genome during cell proliferation is necessary for normal animal development and homeostasis. Disruption of the regulation or fidelity of replication contributes to many human pathologies, particularly cancer. Thus, a complete understanding of the mechanisms governing genome replication is paramount to human health. Replication of large genomes like that found in human cells requires the initiation of bi-directional DNA synthesis at thousands of individual locations on each chromosome. It also requires cell cycle-regulated synthesis of large amounts of histone proteins to package newly replicated DNA into chromatin. This project will focus on how chromatin assembly and organization influences genome replication during animal development. The basic building block of chromatin is the nucleosome, an octamer of histone proteins encompassed by ~147 base pairs of DNA. Most histone proteins within chromatin are synthesized each S phase of the cell cycle from replication-dependent histone genes, which encode the only eukaryotic mRNAs that end in a stem loop rather than a poly A tail. The genes encoding all five RD-histone proteins are clustered in metazoan genomes, and transcription and pre-mRNA processing factors required for histone mRNA biosynthesis are organized into a nuclear body (the Histone Locus Body or HLB) that assembles at these gene clusters. We will determine the requirements for the coordinate synthesis of the RD-histone mRNAs using both biochemical and genetic approaches in Drosophila, with a particular focus on the role that the HLB plays in histone transcription and pre-mRNA processing. Each core histone protein has an N-terminal tail that protrudes from the nucleosome core and is subject to a variety of chemical modifications (e.g. methylation, acetylation, and phosphorylation) that modulate chromatin organization and thus influence all aspects of genome function, including DNA replication. We have developed a method in Drosophila for engineering any desired histone tail mutation, providing us a means of preventing modification of specific histone residues and thus of manipulating chromatin organization in a way not available in any other animal. This genetic approach will be combined with cell biological and next generation DNA sequencing methods to determine how chromatin organization modulates DNA replication throughout the entire genome, thereby addressing a major question in the field. Our Drosophila experimental paradigm permits the in vivo interrogation of these fundamental processes in gene expression and DNA replication in ways that are unavailable in other experimental systems.