DNA replication times the cell cycle and contributes to the mid-blastula transition in Drosophila embryos

Department of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, CA 94143
The Journal of Cell Biology (Impact Factor: 9.83). 10/2009; 187(1):7-14. DOI: 10.1083/jcb.200906191
Source: PubMed


We examined the contribution of S phase in timing cell cycle progression during Drosophila embryogenesis using an approach that deletes S phase rather than arresting its progress. Injection of Drosophila Geminin, an inhibitor of replication licensing, prevented subsequent replication so that the following mitosis occurred with uninemic chromosomes, which failed to align. The effect of S phase deletion on interphase length changed with development. During the maternally regulated syncytial blastoderm cycles, deleting S phase shortened interphase, and deletion of the last of blastoderm S phase (cycle 14) induced an extra synchronous division and temporarily deferred mid-blastula transition (MBT) events. In contrast, deleting S phase after the MBT in cycle 15 did not dramatically affect mitotic timing, which appears to retain its dependence on developmentally programmed zygotic transcription. We conclude that normal S phase and replication checkpoint activities are important timers of the undisturbed cell cycle before, but not after, the MBT.

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Available from: Patrick H O'Farrell
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    • "Embryonic cells replicate their entire genome in a short time period from a large number of randomly placed origins (Blumenthal et al. 1974). This pattern changes at the mid-blastula transition when S-phase length increases and the replication program is established (Newport and Kirschner 1982; Hyrien et al. 1995; McCleland et al. 2009). The analysis of replication timing in pluripotent stem cells undergoing differentiation has revealed that different cell-type-specific lineages change their gene expression pattern and chromatin architecture as well as their pattern of replication timing (Hiratani et al. 2010; Ryba et al. 2010; Chandra et al. 2012). "
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    ABSTRACT: DNA replication is a dynamic process that occurs in a temporal order along each of the chromosomes. A consequence of the temporally coordinated activation of replication origins is the establishment of broad domains (>100 kb) that replicate either early or late in S phase. This partitioning of the genome into early and late replication domains is important for maintaining genome stability, gene dosage, and epigenetic inheritance; however, the molecular mechanisms that define and establish these domains are poorly understood. The modENCODE Project provided an opportunity to investigate the chromatin features that define the Drosophila replication timing program in multiple cell lines. The majority of early and late replicating domains in the Drosophila genome were static across all cell lines; however, a small subset of domains was dynamic and exhibited differences in replication timing between the cell lines. Both origin selection and activation contribute to defining the DNA replication program. Our results suggest that static early and late replicating domains were defined at the level of origin selection (ORC binding) and likely mediated by chromatin accessibility. In contrast, dynamic domains exhibited low ORC densities in both cell types, suggesting that origin activation and not origin selection governs the plasticity of the DNA replication program. Finally, we show that the male-specific early replication of the X chromosome is dependent on the dosage compensation complex (DCC), suggesting that the transcription and replication programs respond to the same chromatin cues. Specifically, MOF-mediated hyperacetylation of H4K16 on the X chromosome promotes both the up-regulation of male-specific transcription and origin activation.
    Full-text · Article · Jul 2014 · Genome Research
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    • "We note that, sex-lethal, which plays a critical role in the fundamental biological process of dosage compensation in Drosophila, also exhibits a quick shutdown, at a time similar to that of hb shutdown [65]. Metazoan embryos share a common feature in that they start with a series of rapid, synchronous cell divisions, followed by a sudden slowing in division and onset of asynchronous divisions and morphogenic movements [66]–[68]. This period is referred to as the mid-blastula transition (MBT), which corresponds to the cycle 14A interphase in Drosophila [66]–[68]. "
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    ABSTRACT: Anterior-posterior (AP) patterning in the Drosophila embryo is dependent on the Bicoid (Bcd) morphogen gradient. However, most target genes of Bcd also require additional inputs to establish their expression domains, reflective of the operation of a cross-regulatory network and contributions of other maternal signals. This is in contrast to hunchback (hb), which has an anterior expression domain driven by an enhancer that appears to respond primarily to the Bcd input. To gain a better understanding of the regulatory logic of the AP patterning network, we perform quantitative studies that specifically investigate the dynamics of hb transcription during development. We show that Bcd-dependent hb transcription, monitored by the intron-containing nascent transcripts near the P2 promoter, is turned off quickly-on the order of a few minutes-upon entering the interphase of nuclear cycle 14A. This shutdown contrasts with earlier cycles during which active hb transcription can persist until the moment when the nucleus enters mitosis. The shutdown takes place at a time when the nuclear Bcd gradient profile in the embryo remains largely intact, suggesting that this is a process likely subject to control of a currently unknown regulatory mechanism. We suggest that this dynamic feature offers a window of opportunity for hb to faithfully interpret, and directly benefit from, Bcd gradient properties, including its scaling properties, to help craft a robust AP patterning outcome.
    Full-text · Article · Apr 2013 · PLoS ONE
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    • "PCNA associates with DNA polymerase at replication forks, and GFP-tagged PCNA accumulates into visible foci during replication. The formation of these foci requires replication, as none are observed in embryos that are prevented from licensing their origins and initiating replication (McCleland et al. 2009). Thus, we used GFP-PCNA foci as an approximation of the progress of replication. "
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    ABSTRACT: The Drosophila midblastula transition (MBT), a major event in embryogenesis, remodels and slows the cell cycle. In the pre-MBT cycles, all genomic regions replicate simultaneously in rapid S phases that alternate with mitosis, skipping gap phases. At the MBT, down-regulation of Cdc25 phosphatase and the resulting inhibitory phosphorylation of the mitotic kinase Cdk1 create a G2 pause in interphase 14. However, an earlier change in interphase 14 is the prolongation of S phase. While the signals modifying S phase are unknown, the onset of late replication-where replication of constitutively heterochromatic satellite sequences is delayed-extends S-phase 14. We injected Cdc25 mRNA to bypass the developmentally programmed down-regulation of Cdc25 at the MBT. Introduction of either Cdc25 isoform (String or Twine) or enhanced Cdk1 activity triggered premature replication of late-replicating sequences, even after their specification, and thereby shortened S phase. Reciprocally, reduction of Cdk1 activity by knockdown of mitotic cyclins extended pre-MBT S phase. These findings suggest that high Cdc25 and Cdk1 contribute to the speed of the rapid, pre-MBT S phases and that down-regulation of these activities plays a broader role in MBT-associated changes than was previously suspected.
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