Great strides are continually made in showing how the initiation of individual replication forks is regulated, and the next frontier is to understand how DNA replication is coordinated with transcription and chromatin structure. In particular, little is known about the mechanisms and functions of DNA replication control during vertebrate development, when transcription and chromatin structure are highly dynamic. This gap in knowledge is an important problem because, until it is filled, the roles for DNA replication in developmental disorders and cancers associated with epigenetic or DNA replication deregulation will be largely incomprehensible. Every cell type replicates its genome in a unique spatiotemporal pattern that changes with transcription and epigenetic modifications. The applicant has established a tractable zebrafish model that allows easy measurement and manipulation of replication timing in the developing embryo as epigenetic marks are added, as transcription begins, and as cells differentiate. The overall objective of this grant is to define the mechanisms of replication timing changes in the zebrafish embryo. The research proposal seeks to complete three specific aims: 1) Determine the function of Rif1 in the developmental control of replication timing and chromatin structure; 2) Determine whether individual genes drive domain-wide replication timing changes; and 3) Determine how epigenetic targeting of replication initiation factors drives replication timing patterns.
In Aim 1, the applicant will build on their data showing that Rif1 is required for an early-to-late replication timing change of a nearly 50 Mb genomic segment (Chr4q). The applicant will use their established replication timing assays as well as RNAseq and ChipSeq to test whether heterochromatinization of Chr4q requires the Rif1-dependent timing switch. Experiments in Aim 2 will test whether an individual gene can act in cis to drive domain-wide replication changes. The applicant will induce genetic and epigenetic modifications to test whether a model replication- timing switching gene (nr2f2) is necessary and sufficient for a timing change across a 1.6 Mb genomic domain. Work in Aim 3 will test whether early replication of acetylated chromatin depends on a physical interaction, which the applicant discovered, between a key replication initiation protein (TICRR) and a histone acetylation ?reader?. Replication timing will be profiled in human cells and zebrafish in which TICRR is mutated to prevent that interaction. The proposed research is innovative because it will be the first using a true in vivo vertebrate embryo model to investigate how DNA replication is coordinated with dynamic transcriptional and epigenetic changes. This work will be significant because it will answer fundamental questions about how and why spatiotemporal DNA replication patterns change during development. Given the functional interplay between DNA replication and epigenetic changes, these studies will ultimately improve understanding of a wide-range of diseases associated with epigenetic deregulation.
The proposed research is relevant to public health because the discovery of evolutionarily conserved mechanisms of DNA replication control will ultimately increase understanding of how defects in DNA replication cause cancer and human developmental disorders. Thus, the project is relevant to the mission of the NIH to seek fundamental knowledge about the nature of living systems; thereby enhancing health, lengthening life, and reducing illness and disability.
| Sansam, Courtney G; Pietrzak, Katarzyna; Majchrzycka, Blanka et al. (2018) A mechanism for epigenetic control of DNA replication. Genes Dev 32:224-229 |
| Siefert, Joseph C; Georgescu, Constantin; Wren, Jonathan D et al. (2017) DNA replication timing during development anticipates transcriptional programs and parallels enhancer activation. Genome Res 27:1406-1416 |
| Alomer, Reem M; da Silva, Eulália M L; Chen, Jingrong et al. (2017) Esco1 and Esco2 regulate distinct cohesin functions during cell cycle progression. Proc Natl Acad Sci U S A 114:9906-9911 |
