Epigenetic regulation of DNA replication in Drosophila melanogaster

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  • Armstrong, Robin Leigh
    • School of Medicine, Curriculum in Genetics and Molecular Biology
Abstract
  • How origins of DNA replication are specified and activated in the context of an intact metazoan genome remains poorly understood. In contrast to Saccharomyces cerevisiae, replication initiation in metazoan genomes is not directed by well-defined sequence motifs. Rather, local chromatin environments have emerged as potential regulators of replication, yielding early and late replicating regions of the genome. Transcriptionally active, accessible euchromatin typically replicates early during S phase, whereas transcriptionally repressive, inaccessible heterochromatin typically replicates late. Current models of replication posit a stochastic process in which a higher density of specified origins in euchromatin compared to heterochromatin increases the probability of replication initiation, resulting in the earlier replication of euchromatin relative to heterochromatin. Despite strong genome-wide correlations between replication and chromatin, a true causal relationship between the two has yet to be determined. We investigated how chromatin organization impacts replication in Drosophila using our genetic platform in which endogenous histone genes are replaced with transgenic histone genes encoding mutations that prevent modification of specific histone residues. To explore the relationship between euchromatin and replication, we implemented a whole-genome sequencing method to produce genome-wide replication timing profiles. We analyzed the X Chromosome, which in Drosophila is 2-fold more transcriptionally active, replicates earlier, and is hyper-acetylated at H4K16 in XY males relative to XX females. H4K16R mutation prevents transcriptional hyper-activation and earlier replication of the male X chromosome, consistent with the notion that transcription promotes early replication. To determine whether perturbation of heterochromatin affects late replication, we generated replication profiles from H3K9R mutant tissue. Despite well-known correlations between late replication and heterochromatin, perturbation of heterochromatin structure through H3K9R mutation does not result in large-scale changes in replication timing suggesting critical regulation beyond chromatin structure. To identify other contributors to replication timing control, we explored the relative contributions of cell lineage, cell cycle, and the trans-acting factor Rif1. We identified that cell lineage, rather than changes in cell cycle status, drive replication timing programs. Furthermore, Rif1 regulates replication timing in a tissue-specific manner supporting the notion that additional mechanisms beyond chromatin structure are key regulators of replication of metazoan genomes.
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Advisor
  • Duronio, Robert J
  • McKay, Daniel J
  • MacAlpine, David M
  • Cook, Jeanette G
  • Hirsch, Matthew L
Degree
  • Doctor of Philosophy
Degree granting institution
  • University of North Carolina at Chapel Hill Graduate School
Graduation year
  • 2020
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