Biology subjects

O'Farrell, P. H.

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

11 recordsLinked to original sources

bioRxiv2025

A mitotic bookmark coordinates transcription and replication

Collisions between advancing replication forks and elongating transcripts pose a universal threat. During the rapid nuclear division cycles in early Drosophila embryos, coordinating transcription and replication is critical to reduce the risk of collisions. In each cycle, replication begins immediately after mitosis, while transcription starts 3 minutes later, overlapping with replication for the remainder of interphase. We previously showed that transcription depends on the coactivator Brd4, which forms hubs at active genes. Here, we show that Brd4 persists on mitotic chromosomes as bookmarks of transcriptional activity and, upon anaphase entry, recruits the replication activator Cdc7 to specify early-replicating genomic regions in the following interphase. Additionally, Cdc7 activity removes Brd4 bookmarks such that post-mitotic transcription occurs only after a new round of Brd4 hub assembly. Early initiation of replication while deferring initiation of transcription is proposed to allow unimpeded transcriptional elongation behind advancing replication forks. Supporting this, inhibiting Cdc7 delayed replication, stabilized Brd4 bookmarks, and resulted in premature transcription with elongation defects. We propose that Cdc7 triggers a functional switch in Brd4 that enforces temporal ordering of the initiation of transcription and replication, thereby minimizing collisions. This switching process might underlie the widespread correlation between transcriptional activity and early replication.

cell biology↗

bioRxiv2024

Cell cycle-regulated transcriptional pausing of Drosophila replication-dependent histone genes

Coordinated expression of replication-dependent (RD) histones genes occurs within the Histone Locus Body (HLB) during S phase, but the molecular steps in transcription that are cell cycle regulated are unknown. We report that Drosophila RNA Pol II promotes HLB formation and is enriched in the HLB outside of S phase, including G1-arrested cells that do not transcribe RD histone genes. In contrast, the transcription elongation factor Spt6 is enriched in HLBs only during S phase. Proliferating cells in the wing and eye primordium express full-length histone mRNAs during S phase but express only short nascent transcripts in cells in G1 or G2 consistent with these transcripts being paused and then terminated. Full-length transcripts are produced when Cyclin E/Cdk2 is activated as cells enter S phase. Thus, activation of transcription elongation by Cyclin E/Cdk2 and not recruitment of RNA pol II to the HLB is the critical step that links histone gene expression to cell cycle progression in Drosophila.

cell biology↗

bioRxiv2024

Selection promotes age-dependent degeneration of the mitochondrial genome

Somatic mutations in mitochondrial genomes (mtDNA) accumulate exponentially during aging. Using single cell sequencing, we characterize the spectrum of age-accumulated mtDNA mutations in mouse and human liver and identify directional forces that accelerate the accumulation of mutations beyond the rate predicted by a neutral model. "Driver" mutations that give genomes a replicative advantage rose to high cellular abundance and carried along "passenger" mutations, some of which are deleterious. In addition, alleles that alter mtDNA-encoded proteins selectively increased in abundance overtime, strongly supporting the idea of a "destructive" selection that favors genomes lacking function. Overall, this combination of selective forces acting in hepatocytes promotes somatic accumulation of mutations in coding regions of mtDNA that are otherwise conserved in evolution. We propose that these selective processes could contribute to the population prevalence of mtDNA mutations, accelerate the course of heteroplasmic mitochondrial diseases and promote age-associated erosion of the mitochondrial genome.

genomics↗

bioRxiv2022

Stepwise modifications of transcriptional hubs link pioneer factor activity to a burst of transcription

Eukaryotic transcription begins with the binding of transcription factors (TFs), which promotes the subsequent recruitment of coactivators and pre-initiation complexes. It is commonly assumed that these factors eventually co-reside in a higher-order structure, allowing distantly bound TFs to activate transcription at core promoters. Here we performed live imaging of endogenously tagged proteins, including the pioneer TF Zelda, the coactivator dBrd4, and RNA polymerase II (RNAPII), in early Drosophila embryos. We show that these factors are sequentially and transiently recruited to discrete clusters during activation of non-histone genes. We present evidence that Zelda acts with the acetyltransferase dCBP to nucleate dBrd4 hubs, which then trigger pre-transcriptional clustering of RNAPII; continuous transcriptional elongation then disperses clusters of dBrd4 and RNAPII. Our results suggest that activation of transcription by eukaryotic TFs involves a succession of distinct biochemical complexes that culminate in a self-limiting burst of transcription.

cell biology↗

bioRxiv2022

Mitigation of the age-dependent decline in mitochondrial genome integrity

Unknown processes promote accumulation of mitochondrial DNA mutations during aging. Accumulation of defective mitochondrial genomes is thought to promote progression of heteroplasmic mitochondrial diseases and degenerative changes with natural aging. We used a heteroplasmic Drosophila model to test 1) whether purifying selection acts to limit the abundance of deleterious mutations during development and aging, 2) whether quality control pathways contribute to purifying selection, 3) whether activation of quality control can mitigate accumulation of deleterious mutations, and 4) whether improved quality control improves healthspan. We show that purifying selection operates during development and growth, but is ineffective during aging. Genetic manipulations suggest that a quality control process known to enforce purifying selection during oogenesis also suppresses accumulation of a deleterious mutation during growth and development. Flies with nuclear genotypes that enhanced purifying selection sustained higher genome quality, retained more vigorous climbing activity and lost fewer dopaminergic neurons. Pharmacological enhancement of quality control produced similar benefits. Importantly, similar pharmacological treatment of aged mice reversed age-associated accumulation of a deleterious mtDNA mutation. Our findings reveal dynamic maintenance of mitochondrial genome fitness, and reduction in the effectiveness of purifying selection during life. Importantly, we describe interventions that mitigate and even reverse age-associated genome degeneration in flies and in mice. Furthermore, mitigation of genome degeneration improved wellbeing in a Drosophila model of heteroplasmic mitochondrial disease. Significance StatementIn contrast to the orderly segregation of nuclear DNA, mitochondrial genomes compete for replication and segregation. The abundance of mutants that emerge in a cell depends on their success in this competition. We show that quality control mechanisms put deleterious mutations at a disadvantage, but these mechanisms become ineffective during aging. The resulting rise in mutant genomes, which compromises vigor, can be suppressed using genetic backgrounds that enhance quality control. Feeding kinetin to adult Drosophila or mice also reduced the load of mutant mitochondrial genomes. This pharmacological reversal of the age associated deterioration of mtDNA quality suggests possible therapies to alleviate mitochondrial diseases and normal aging.

cell biology↗

bioRxiv2022

Temporal control of late replication and coordination of origin firing by self-stabilizing Rif1-PP1 hubs in Drosophila

In the metazoan S phase, coordinated firing of clusters of origins replicates different parts of the genome in a temporal program. Despite advances, neither the mechanism controlling timing nor that coordinating firing of multiple origins is fully understood. Rif1, an evolutionarily conserved inhibitor of DNA replication, recruits protein phosphatase 1 (PP1) and counteracts firing of origins by S-phase kinases. During the mid-blastula transition (MBT) in Drosophila embryos, Rif1 forms subnuclear hubs at each of the large blocks of satellite sequences and delays their replication. Each Rif1 hub disperses abruptly just prior to the replication of the associated satellite sequences. Here, we show that the level of activity of the S-phase kinase, DDK, accelerated this dispersal program, and that the level of Rif1-recruited PP1 retarded it. Further, Rif1-recruited PP1 supported chromatin association of nearby Rif1. This influence of nearby Rif1 can create a "community effect" counteracting kinase-induced dissociation such that an entire hub of Rif1 undergoes switch-like dispersal at characteristic times that shift in response to the balance of Rif1-PP1 and DDK activities. We propose a model in which the spatiotemporal program of late replication in the MBT embryo is controlled by self-stabilizing Rif1-PP1 hubs, whose abrupt dispersal synchronizes firing of associated late origins. SIGNIFICANCESeventy years ago, it was discovered that large domains of the eukaryotic genome replicate at different times. Detailed descriptions left significant questions unresolved. How are the many origins in the large domains coordinated to fire in unison? What distinguishes different domains and gives rise to a temporal program? When Drosophila embryos first establish late replication, an inhibitor of DNA replication, Rif1, forms hubs over domains of late replicating DNA. Rif1 recruits protein phosphatase 1 (PP1), which prevents kinases from dispersing Rif1 hubs or activating associated origins. When kinase activity eventually exceeds a hub-specific threshold, the self-stabilization of Rif1-PP1 breaks down, hubs disperse abruptly, and all associated origins are free to initiate.

cell biology↗

bioRxiv2022

Mandatory coupling of zygotic transcription to DNA replication in early Drosophila embryos

Collisions between transcribing RNA polymerases and DNA replication forks are disruptive. The threat of collisions is particularly acute during the rapid early embryonic cell cycles of Drosophila when S phase occupies the entirety of interphase. We hypothesized that collision-avoidance mechanisms safeguard the onset of zygotic transcription in these cycles. To explore this hypothesis, we used real-time imaging of transcriptional events at the onset of each interphase. Endogenously tagged RNA polymerase II (RNAPII) abruptly formed clusters before nascent transcripts accumulated, indicating recruitment prior to transcriptional engagement. Injection of inhibitors of DNA replication prevented RNAPII clustering, blocked formation of foci of the pioneer factor Zelda, and largely prevented expression of transcription reporters. Knockdown of Zelda or the histone acetyltransferase CBP prevented RNAPII cluster formation except at the replication-dependent (RD) histone gene locus. We suggest a model in which the passage of replication forks allows Zelda and a distinct pathway at the RD histone locus to reconfigure chromatin to nucleate RNAPII clustering and promote transcriptional initiation. The replication dependency of these events defers initiation of transcription and ensures that RNA polymerases transcribe behind advancing replication forks. The resulting coordination of transcription and replication explains how early embryos circumvent collisions and promote genome stability.

cell biology↗

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↗