The extremely long DNA molecules that comprise eukaryotic genomes are wrapped, folded, and looped to compact and organize the DNA for its essential activities, including gene expression, DNA replication, and DNA repair. Exactly how the spatial architecture of chromosomes regulates DNA processes is still poorly understood, and relatively few involved factors have been identified. DNA replication origins are subject to epigenetic regulation of their activity resulting in differential replication timings and origin efficiencies during S phase. This level of control over replication origins helps ensure an appropriate level of origin activity for genome stability, however, the molecular mechanism(s) responsible for this regulation have remained obscure. We discovered the budding yeast Fox proteins, Forkhead 1 (Fkh1) and Forkhead 2 (Fkh2) as key regulators of replication origin initiation timing, required for most early origin firing across the yeast genome. Fkh1 and/or Fkh2 (Fkh1/2) bind to specific sequences at some origins (called Fkh-activated origins). Recent studies indicate that a key function of Fkh1/2 is to recruit Dbf4-dependent kinase (DDK), which is required for replication origin initiation. This project is geared toward fully understanding the cell cycle-regulated binding of Fkh1/2 with origins and Fkh1/2 interactions with DDK. We have identified a potential dimerization motif in Fkh1/2 that is required for its function in origin regulation but not in other functions like transcriptional regulation. We will determine the function of this motif in origin regulation. Fkh1 establishes the origin-timing program in G1 phase and this correlates with re-localization of Fkh1-activated origins from the late-replicating nuclear periphery to an early-replicating interior environment of the nucleus. The exact significance of these movements is unclear but likely represent the assembly of origin clusters that will transform into replication factories. Therefore, this system provides a novel and powerful opportunity to elucidate fundamental mechanisms of chromosomal dynamics, which is a major goal of this proposal. We will use genetic approaches to eliminate function of candidate regulator proteins to dissect the molecular events associated with origin dynamics and replication initiation. We will also develop new tools for better analysis of protein- DNA interactions. Finally, we will perform experiments to strip away epigenetic layers of origin regulation to reveal the underlying sequence-based determinants of origin firing. We will determine the impact on genome stability of these layers of regulation. These studies have strong potential to reveal novel molecular events at the DNA level governing the dynamics of chromosomes and their essential genetic elements, such as replication origins.
The establishment and maintenance of stable epigenetic states is fundamental to normal cellular differentiation and homeostasis. This proposal investigates a novel mechanism of epigenetic regulation of DNA replication origins, which are fundamental elements required for chromosome duplication and stability. These studies have implications for understanding the mechanisms that cause developmental defects and cancers as well as basic biology.
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