Decoding the Replication Timing Program in Zebrafish: The Role of Rif1

Deregulated DNA replication is a major contributor to human developmental disorders and cancer, yet our understanding of how replication is coordinated with changes in transcription and chromatin structure is limited. Our lab has employed the zebrafish model to investigate the mechanisms driving changes in the replication timing program during development. Previous studies have identified changes in replication timing patterns from the onset of zygotic transcription through gastrulation in zebrafish embryos. The protein Rif1 is crucial for replication timing in a wide range of eukaryotes, yet its role in establishing the replication timing program and chromatin structure during early vertebrate development is not well understood. Using Rif1 mutant zebrafish and performing RNA sequencing and whole-genome replication timing analysis, we found that Rif1 mutants were viable but had a defect in female sex determination. Interestingly, Rif1 loss primarily affected DNA replication timing after gastrulation, while its impact on transcription was more pronounced during zygotic genome activation. Our results indicate that Rif1 has distinct roles in regulating DNA replication and transcription at different stages of development. Overall design: Replication timing data was generated for wild type and rif1 mutant zebrafish embryos at different developmental stages (preMBT, Shield, Bud, and 24 hpf) using our RepliTimer pipeline (https://github.com/SansamLab/RepliTimer/tree/v1.0.11; DOI 10.5281/zenodo.6426313). The data was based on the copy-number variations between filtered G1 and S-phase whole-genome sequencing read counts.