Biology subjects

O'Farrell, P. H.

Publications and source records attributed to O'Farrell, P. H..

4 recordsLinked to original sources

bioRxiv2018

Rapid embryonic cell cycles defer the establishment of heterochromatin by Eggless/SetDB1 in Drosophila

Acquisition of chromatin modifications during embryogenesis distinguishes different regions of an initially naive genome. In many organisms, repetitive DNA is packaged into constitutive heterochromatin that is marked by di/tri methylation of histone H3K9 and the associated protein HP1a. These modifications enforce the unique epigenetic properties of heterochromatin. However, in the early Drosophila melanogaster embryo the heterochromatin lacks these modifications which only appear later when rapid embryonic cell cycles slow down at the Mid-Blastula Transition or MBT. Here we focus on the initial steps restoring heterochromatic modifications in the embryo. We describe the JabbaTrap, a technique for inactivating maternally provided proteins in embryos. Using the JabbaTrap we reveal a major requirement for the methyltransferase Eggless/SetDB1 in the establishment of heterochromatin. In contrast, other methyltransferases contribute minimally. Live-imaging reveals that endogenous Eggless gradually accumulates on chromatin in interphase, but then dissociates in mitosis and its accumulation must restart in the next cell cycle. Cell cycle slowing as the embryo approaches the MBT permits increasing accumulation and action of Eggless at its targets. Experimental manipulation of interphase duration shows that cell cycle speed regulates Eggless. We propose that developmental slowing of the cell cycle times embryonic heterochromatin formation.

developmental biology↗

bioRxiv2017

Rif1 prolongs the embryonic S phase at the Drosophila mid-blastula transition

In preparation for dramatic morphogenetic events of gastrulation, rapid embryonic cell cycles slow at the Mid-Blastula Transition, MBT. In Drosophila melanogaster embryos, downregulation of Cdk1 activity initiates this slowing by delaying replication of satellite sequences and extending S phase. We found that Cdk1 inhibited the chromatin association of Rif1, a candidate repressor of replication. Furthermore, Rif1 bound selectively to satellite sequences following Cdk1 downregulation at the MBT. In the next S phase, Rif1 dissociated from different satellites in an orderly schedule that anticipated their replication. Rif1 lacking potential phosphorylation sites failed to dissociate and dominantly prevented completion of replication. Loss of Rif1 in mutant embryos shortened the post-MBT S phase, and rescued embryonic cell cycles disrupted by depletion of the S phase-promoting kinase, Cdc7. Thus, Drosophila Rif1 mediates the MBT extension of S phase and functionally interacts with S phase promoting kinases to introduce a replication-timing program.

developmental biology↗

bioRxiv2017

Interphase-Arrested Drosophila Embryos Initiate Mid-Blastula Transition At A Low Nuclear-Cytoplasmic Ratio

Externally deposited eggs begin development with an immense cytoplasm and a single overwhelmed nucleus. Rapid mitotic cycles restore normality as the ratio of nuclei to cytoplasm (N/C) increases. At the 14th cell cycle in Drosophila embryos, the cell cycle slows, transcription increases, and morphogenesis begins at the Mid-Blastula Transition (MBT). To explore the role of N/C in MBT timing, we blocked N/C-increase by downregulating cyclin/Cdk1 to arrest early cell cycles. Embryos arrested in cell cycle 12 cellularized, initiated gastrulation movements and activated transcription of genes previously described as N/C dependent. Thus, occurrence of these events is not directly coupled to N/C-increase. However, N/C might act indirectly. Increasing N/C promotes cyclin/Cdk1 downregulation which otherwise inhibits many MBT events. By experimentally inducing downregulation of cyclin/Cdk1, we bypassed this input of N/C-increase. We describe a regulatory cascade wherein the increasing N/C downregulates cyclin/Cdk1 to promote increasing transcription and the MBT.\n\nImpact statementBy showing that cell-cycle arrest allows early Drosophila embryos to progress to later stages, this work eliminates numerous models for embryonic timing and shows the dominating influence of cell-cycle slowing.

developmental biology↗

bioRxiv2016

DNA-polymerase guided elimination of paternal mitochondrial genomes:An escape-proof obstacle to their transmission

Mitochondrial DNA is predominantly inherited from only one parent. In animals this is usually the mother. This program is not in the interest of the paternal mitochondrial genome whose potential to contribute to future generations is restricted. However, in a dramatic example of genetic conflict, nuclear programs ensure the outcome. Two large mitochondria extend the length of Drosophila sperm tails. The hundreds of nucleoids in these mitochondria vanish during spermatogenesis eliminating their potential for transmission. Our previous work showed that mutational inactivation of EndoG, a nuclear encoded mitochondrial endonuclease, slows elimination of mitochondrial genomes. Here, we show that knockdown of the nuclearly encoded mitochondrial DNA polymerase, Tamas, produces a much more complete block of mtDNA loss. Recruitment of Tamas to the nucleoid at the time of its disappearance suggests a direct contribution to the elimination, but the 3'-exonuclease function of the polymerase is not needed. While DNA elimination is a surprising function for DNA polymerase, its use to restrict paternal genomes provides a strategy that cannot easily be evaded by the mitochondrial genome without compromising its replication.

developmental biology↗