[
International Worm Meeting,
2017]
During DNA replication Half of the genome is duplicated discontinuously on the lagging strand in the form of Okazaki Fragments (OFs). Despite many years of research, the precise location of replication origins, efficiency, and abundance in metazoa remains elusive. We have recently isolated and sequenced OFs from C. elegans embryos and developing larva, and have generated a first comprehensive genome wide map of DNA replication in multi cellular organism1. Aligning sequence reads of OFs to the C. elegans genome reveals the complementary enrichment of Okazaki fragments to either the Watson or Crick strands, which is the hallmark of a replication origin1,2. Importantly, we found that replication origins are associated with transcriptionally active regions of chromatin and replication start sites are strongly associated with histone modifications that define genetic enhancers such as H3K27 acetylation. By mapping DNA replication origins in various embryonic stages, we have demonstrated that origins are pre-defined prior to the onset of zygotic transcription. However, at the time of gastrulation, when zygotic transcription initiates, it does so in proximity of pre- defined replication origins. Replication origins and transcription are correlated until the last wave of embryonic cell division. When cell division ceases, this association breaks down and transcription shifts away from replication initiation sites and anti-correlates with replication origins in differentiated cells. Whilst majority of embryonic cells are terminally differentiated at the time of hatching, 53 somatic blast cells in newly hatched L1 larvae divide post embryonically to generate additional 403 cells in C. elegans hermaphrodites. The majority of the somatic blast cell lineage generate vulva, muscle, hypodermal, and neuronal cells. We have shown that the position and the efficiency of somatic replication origins are distinguishable from the embryonic origins yet remain associated with somatic H3K27 acetylation. Interestingly, genes located in proximity of somatic replication start sites are functionally related to vulva, muscle, hypodermal and neuronal cells. Our data strongly suggest that in rapidly dividing embryonic cells, replication is a fundamental regulator of gene activity, while during notably slower post embryonic cell division, transcriptional activity shapes the DNA replication profile. Currently we are investigating the causal link between transcription and somatic replication. 1.Spatiotemporal coupling and decoupling of gene transcription with DNA replication origins during embryogenesis in C. elegans. Ehsan Pourkarimi, James M Bellush, Iestyn Whitehouse. eLIFE, 2016. 2. Intrinsic coupling of lagging-strand synthesis to chromatin assembly. Duncan J. Smith & Iestyn Whitehouse. Nature, 2012
[
International Worm Meeting,
2015]
In eukaryotes, multiple replication origins initiate DNA synthesis bidirectionally. Half of the genome is duplicated discontinuously on the lagging strand in the form of Okazaki Fragments (OFs). Previously we have purified and sequenced OFs from S. cerevisiae, and have demonstrated their utility in mapping genome wide DNA replication patterns (1,2). Despite many years of research, the precise location efficiency and abundance of replication origins in metazoa remains elusive. Here we capture and sequence OFs from C. elegans and generate a genome wide map of DNA replication.We use a DNA ligation compromised genetic background and purify single stranded OFs from developing embryos. Aligning sequence reads of OFs to the C. elegans genome reveals a characteristic strand bias at specific sites that are approximately 50-100 Kb apart. The complementary enrichment of Okazaki fragments to either the Watson or Crick strands is the hallmark of a replication origin. Additionally we have found that origins of replication in C. elegans are correlated with transcriptionally active regions of chromatin. Using the histone modification data set of modENCODE we found a strong association between replication start sites and acetylation of Histone H3 lysine 27 (H3K27ac). Acetylation of H3K27 is a chromatin mark characteristic of gene enhancer elements. Finally, we show that a partial depletion of CBP/P300 the sole histone H3 acetyltransferase (HAT) responsible for H3K27 acetylation has a profound effect on DNA replication. Our data indicate that the DNA replication program is likely plastic and is matched with the transcriptional program. Ultimately our data show, both DNA replication and gene transcription are likely regulated by similar epigenetic processes. 1. Intrinsic coupling of lagging-strand synthesis to chromatin assemblyDuncan J. Smith& Iestyn Whitehouse. Nature, 20122. Quantitative, genome-wide analysis of eukaryotic replication initiation and termination.McGuffee SR, Smith DJ, Whitehouse I. Mol Cell, 2013.