DNA replication is a tightly regulated process involving multiple pathways to ensure replication occurs only once per cell cycle. The ecdysone response element (EcRE) situated at the origin of the Sciara II/9A locus in salivary glands directly adjacent to the binding site of the origin recognition complex (ORC) may represent a novel steroid hormone regulatory mechanism controlling replication. Preliminary data has shown that ecdysone treatment induces premature amplification of the II/9A gene locus. The present study will focus on defining the role of ecdysone and its hormone receptor, EcR, in the regulation of DNA replication. The in vivo occupancy of the EcR at the Sciara II/9A origin EcRE at specific stages of larval development will be analyzed through chromatin immunoprecipitation (ChIP) analysis. Following the EcRE occupancy studies, coimmunoprecipitation and ChIP assays will be used to characterize protein interactions between Sciara EcR and proteins of the replication machinery. Yeast protein hybrid analysis will be employed to identify and analyze protein constituents found in complex with ScEcR. Sciara DNA amplification could be a paradigm for hormonally sensitive cancers (e.g. breast and prostate).
