PROJECT SUMMARY Chromatin states encode epigenetic information and must be stably transmitted to daughter cells to maintain gene expression and cell identity. How the information contained within the chromatin structure and histone modifications is transmitted to daughter cells during cell division remains largely unknown. My long-term goal is to understand the mechanisms of epigenetic inheritance, with particular focus on nucleosome dynamics. In this proposal, I will investigate how histone (H3-H4)2 tetramers, a critical component of nucleosomes (the basic unit of chromatin) are transferred from parental to newly-synthesized DNA and assembled into nucleosomes during the S phase of the cell cycle. Previous studies have shed light on nucleosome assembly of new (H3-H4)2 tetramers. However, the process whereby parental (H3-H4)2 tetramers are assembled into nucleosomes on replicating DNA strands has not received sufficient attention due to the lack of suitable methods. Recently, we have developed eSPAN (enrichment and sequencing of protein-associated nascent DNA), a novel technology that enables, for the first time, the analysis of protein binding to the leading and lagging strands of replicating DNA. Using this method, we have observed that the parental histone (H3-H4)2 tetramers prefer lagging strands, supporting a non-random segregation model. Importantly, we have also found that the DNA polymerase ε and DNA helicase may play a role in the parental histone (H3-H4)2 tetramer transfer process. Based on these significant preliminary results, I hypothesize that parental (H3-H4)2 tetramers are assembled into nucleosomes by histone chaperones, chromatin remodeling complexes and DNA replication machinery. To test this hypothesis, I will identify the proteins involved in parental histone (H3-H4)2 segregation during DNA replication (Aim 1); and investigate the molecular mechanism whereby polymerase ε and the DNA helicase contribute to parental nucleosome assembly (Aim 2). The proposed studies will elucidate the mechanisms of the parental (H3-H4)2 tetramer transfer process. Mutations in, or aberrant regulation of, proteins involved in chromatin replication, such as histone chaperones, histone modification enzymes, and chromatin remodeling complexes, have been directly linked to breast, gastrointestinal, and prostate cancers. Thus, the parental histone transfer and assembly mechanisms I aim to discover and characterize in this study will not only provide novel insights into the fundamental process of epigenetic inheritance but also create new opportunities for human cancer therapy.