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

Binding of transcription factors (TFs) promotes the subsequent recruitment of coactivators and preinitiation complexes to initiate eukaryotic transcription, but this time course is usually not visualized. It is commonly assumed that recruited factors eventually co-reside in a higher-order structure, allowing distantly bound TFs to activate transcription at core promoters. We use live imaging of endogenously tagged proteins, including the pioneer TF Zelda, the coactivator dBrd4, and RNA polymerase II (RNAPII), to define a cascade of events upstream of transcriptional initiation in early Drosophila embryos. These factors are sequentially and transiently recruited to discrete clusters during activation of non-histone genes. Zelda and the acetyltransferase dCBP nucleate dBrd4 clusters, which then trigger pre-transcriptional clustering of RNAPII. Subsequent transcriptional elongation disperses clusters of dBrd4 and RNAPII. Our results suggest that activation of transcription by eukaryotic TFs involves a succession of distinct biomolecular condensates that culminates in a self-limiting burst of transcription.

Coordinating transcription and replication to mitigate their conflicts 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 hypothesize that collision-avoidance mechanisms safeguard this early transcription. Real-time imaging of endogenously tagged RNA polymerase II (RNAPII) and a reporter for nascent transcripts in unperturbed embryos shows clustering of RNAPII at around 2 min after mitotic exit, followed by progressive dispersal as associated nascent transcripts accumulate later in interphase. Abrupt inhibition of various steps in DNA replication, including origin licensing, origin firing, and polymerization, suppresses post-mitotic RNAPII clustering and transcription in nuclear cycles. We propose that replication dependency defers the onset of transcription so that RNAPII transcribes behind advancing replication forks. The resulting orderly progression can explain how early embryos circumvent transcription-replication conflicts to express essential developmental genes.

Mitigation of age-dependent accumulation of defective mitochondrial genomes.

Unknown processes promote the accumulation of mitochondrial DNA (mtDNA) mutations during aging. Accumulation of defective mitochondrial genomes is thought to promote the 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 health span. 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 enhance purifying selection sustained higher genome quality, retained more vigorous climbing activity, and lost fewer dopaminergic neurons. A pharmacological agent thought to enhance 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 well-being in a Drosophila model of heteroplasmic mitochondrial disease.

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 midblastula 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.

Interphase-arrested Drosophila embryos activate zygotic gene expression and initiate mid-blastula transition events 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. A threshold N/C has been widely proposed to activate zygotic genome transcription and onset of morphogenesis at the mid-blastula transition (MBT). To test whether a threshold N/C is required for these events, we blocked N/C increase by down-regulating cyclin/Cdk1 to arrest early cell cycles in Drosophila. Embryos that were arrested two cell cycles prior to the normal MBT activated widespread transcription of the zygotic genome including genes previously described as N/C dependent. Zygotic transcription of these genes largely retained features of their regulation in space and time. Furthermore, zygotically regulated post-MBT events such as cellularization and gastrulation movements occurred in these cell cycle-arrested embryos. These results are not compatible with models suggesting that these MBT events are directly coupled to N/C. Cyclin/Cdk1 activity normally declines in tight association with increasing N/C and is regulated by N/C. By experimentally promoting the decrease in cyclin/Cdk1, we uncoupled MBT from N/C increase, arguing that N/C-guided down-regulation of cyclin/Cdk1 is sufficient for genome activation and MBT.

A Genome-wide Screen Reveals that Reducing Mitochondrial DNA Polymerase Can Promote Elimination of Deleterious Mitochondrial Mutations.

A mutant mitochondrial genome arising amid the pool of mitochondrial genomes within a cell must compete with existing genomes to survive to the next generation. Even weak selective forces can bias transmission of one genome over another to affect the inheritance of mitochondrial diseases and guide the evolution of mitochondrial DNA (mtDNA). Studies in several systems suggested that purifying selection in the female germline reduces transmission of detrimental mitochondrial mutations [1-7]. In contrast, some selfish genomes can take over despite a cost to host fitness [8-13]. Within individuals, the outcome of competition is therefore influenced by multiple selective forces. The nuclear genome, which encodes most proteins within mitochondria, and all external regulators of mitochondrial biogenesis and dynamics can influence the competition between mitochondrial genomes [14-18], yet little is known about how this works. Previously, we established a Drosophila line transmitting two mitochondrial genomes in a stable ratio enforced by purifying selection benefiting one genome and a selfish advantage favoring the other [8]. Here, to find nuclear genes that impact mtDNA competition, we screened heterozygous deletions tiling ∼70% of the euchromatic regions and examined their influence on this ratio. This genome-wide screen detected many nuclear modifiers of this ratio and identified one as the catalytic subunit of mtDNA polymerase gene (POLG), tam. A reduced dose of tam drove elimination of defective mitochondrial genomes. This study suggests that our approach will uncover targets for interventions that would block propagation of pathogenic mitochondrial mutations.

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 naïve genome. In many organisms, repetitive DNA is packaged into constitutive heterochromatin that is marked by di/trimethylation 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 appear only later, when rapid embryonic cell cycles slow down at the midblastula transition (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.

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, down-regulation of cyclin-dependent kinase 1 (Cdk1) activity initiates this slowing by delaying replication of heterochromatic satellite sequences and extending S phase. We found that Cdk1 activity inhibited the chromatin association of Rap1 interacting factor 1 (Rif1), a candidate repressor of replication. Furthermore, Rif1 bound selectively to satellite sequences following Cdk1 down-regulation 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, cell division cycle 7 (Cdc7). Our work shows that Rif1 and S phase kinases compose a replication timer controlling first the developmental onset of late replication and then the precise schedule of replication within S phase. In addition, we describe how onset of late replication fits into the progressive maturation of heterochromatin during development.

Sophisticated lessons from simple organisms: appreciating the value of curiosity-driven research.

For hundreds of years, biologists have studied accessible organisms such as garden peas, sea urchins collected at low tide, newt eggs, and flies circling rotten fruit. These organisms help us to understand the world around us, attracting and inspiring each new generation of biologists with the promise of mystery and discovery. Time and time again, what we learn from such simple organisms has emphasized our common biological origins by proving to be applicable to more complex organisms, including humans. Yet, biologists are increasingly being tasked with developing applications from the known, rather than being allowed to follow a path to discovery of the as yet unknown. Here, we provide examples of important lessons learned from research using selected non-vertebrate organisms. We argue that, for the purpose of understanding human disease, simple organisms cannot and should not be replaced solely by human cell-based culture systems. Rather, these organisms serve as powerful discovery tools for new knowledge that could subsequently be tested for conservation in human cell-based culture systems. In this way, curiosity-driven biological research in simple organisms has and will continue to pay huge dividends in both the short and long run for improving the human condition.

The Mitochondrial DNA Polymerase Promotes Elimination of Paternal Mitochondrial Genomes.

Mitochondrial DNA (mtDNA) is typically inherited from only one parent [1-3]. In animals, this is usually the mother. Maternal inheritance is often presented as the passive outcome of the difference in cytoplasmic content of egg and sperm; however, active programs enforce uniparental inheritance at two levels, eliminating paternal mitochondrial genomes or destroying mitochondria delivered to the zygote by the sperm [4-13]. Both levels operate in Drosophila [8, 12, 13]. As sperm formation begins, hundreds of doomed mitochondrial genomes are visualized within the two huge mitochondria of each spermatid. These genomes abruptly disappear during spermatogenesis. Genome elimination, which is not in the interests of the restricted genomes, is directed by nuclear genes. Mutation of EndoG, which encodes a mitochondria-targeted endonuclease, retarded elimination [8]. Here, we show that knockdown of the nuclear-encoded mtDNA polymerase (Pol γ-α), Tamas, produces a more complete block of mtDNA elimination. Tamas is found in large particles that localize to mtDNA during genome elimination. We discount a simple possible mechanism by showing that the 3'-exonuclease function of the polymerase is not needed. While DNA elimination is a surprising function for DNA polymerase, it could provide a robust nexus for nuclear control of mitochondrial genome copy number, since use of common interactions for elimination and replication might limit options for the mitochondrial genome to escape restriction. We suggest that the DNA polymerase may play this role more widely and that inappropriate activation of its elimination ability might underlie association of DNA loss syndromes with mutations of the human mtDNA polymerase [14-16].

Timing the Drosophila Mid-Blastula Transition: A Cell Cycle-Centered View.

At the mid-blastula transition (MBT), externally developing embryos refocus from increasing cell number to elaboration of the body plan. Studies in Drosophila reveal a sequence of changes in regulators of Cyclin:Cdk1 that increasingly restricts the activity of this cell cycle kinase to slow cell cycles during early embryogenesis. By reviewing these events, we provide an outline of the mechanisms slowing the cell cycle at and around the time of MBT. The perspectives developed should provide a guiding paradigm for the study of other MBT changes as the embryo transits from maternal control to a regulatory program centered on the expression of zygotic genes.

Selfish drive can trump function when animal mitochondrial genomes compete.

Mitochondrial genomes compete for transmission from mother to progeny. We explored this competition by introducing a second genome into Drosophila melanogaster to follow transmission. Competitions between closely related genomes favored those functional in electron transport, resulting in a host-beneficial purifying selection. In contrast, matchups between distantly related genomes often favored those with negligible, negative or lethal consequences, indicating selfish selection. Exhibiting powerful selfish selection, a genome carrying a detrimental mutation displaced a complementing genome, leading to population death after several generations. In a different pairing, opposing selfish and purifying selection counterbalanced to give stable transmission of two genomes. Sequencing of recombinant mitochondrial genomes showed that the noncoding region, containing origins of replication, governs selfish transmission. Uniparental inheritance prevents encounters between distantly related genomes. Nonetheless, in each maternal lineage, constant competition among sibling genomes selects for super-replicators. We suggest that this relentless competition drives positive selection, promoting change in the sequences influencing transmission.

TALE-light imaging reveals maternally guided, H3K9me2/3-independent emergence of functional heterochromatin in Drosophila embryos.

Metazoans start embryogenesis with a relatively naïve genome. The transcriptionally inert, late-replicating heterochromatic regions, including the constitutive heterochromatin on repetitive sequences near centromeres and telomeres, need to be re-established during development. To explore the events initiating heterochromatin formation and examine their temporal control, sequence specificity, and immediate regulatory consequence, we established a live imaging approach that enabled visualization of steps in heterochromatin emergence on specific satellite sequences during the mid-blastula transition (MBT) in Drosophila. Unexpectedly, only a subset of satellite sequences, including the 359-base-pair (bp) repeat sequence, recruited HP1a at the MBT. The recruitment of HP1a to the 359-bp repeat was dependent on HP1a's chromoshadow domain but not its chromodomain and was guided by maternally provided signals. HP1a recruitment to the 359-bp repeat was required for its programmed shift to later replication, and ectopic recruitment of HP1a was sufficient to delay replication timing of a different repeat. Our results reveal that emergence of constitutive heterochromatin follows a stereotyped developmental program in which different repetitive sequences use distinct interactions and independent pathways to arrive at a heterochromatic state. This differential emergence of heterochromatin on various repetitive sequences changes their replication order and remodels the DNA replication schedule during embryonic development.

Selections that isolate recombinant mitochondrial genomes in animals.

Homologous recombination is widespread and catalyzes evolution. Nonetheless, its existence in animal mitochondrial DNA is questioned. We designed selections for recombination between co-resident mitochondrial genomes in various heteroplasmic Drosophila lines. In four experimental settings, recombinant genomes became the sole or dominant genome in the progeny. Thus, selection uncovers occurrence of homologous recombination in Drosophila mtDNA and documents its functional benefit. Double-strand breaks enhanced recombination in the germline and revealed somatic recombination. When the recombination partner was a diverged Drosophila melanogaster genome or a genome from a different species such as Drosophila yakuba, sequencing revealed long continuous stretches of exchange. In addition, the distribution of sequence polymorphisms in recombinants allowed us to map a selected trait to a particular region in the Drosophila mitochondrial genome. Thus, recombination can be harnessed to dissect function and evolution of mitochondrial genome.

Growing an Embryo from a Single Cell: A Hurdle in Animal Life.

A requirement that an animal be able to feed to grow constrains how a cell can grow into an animal, and it forces an alternation between growth (increase in mass) and proliferation (increase in cell number). A growth-only phase that transforms a stem cell of ordinary proportions into a huge cell, the oocyte, requires dramatic adaptations to help a nucleus direct a 10(5)-fold expansion of cytoplasmic volume. Proliferation without growth transforms the huge egg into an embryo while still accommodating an impotent nucleus overwhelmed by the voluminous cytoplasm. This growth program characterizes animals that deposit their eggs externally, but it is changed in mammals and in endoparasites. In these organisms, development in a nutritive environment releases the growth constraint, but growth of cells before gastrulation requires a new program to sustain pluripotency during this growth.

Cyclin B3 is a mitotic cyclin that promotes the metaphase-anaphase transition.

The timing mechanism for mitotic progression is still poorly understood. The spindle assembly checkpoint (SAC), whose reversal upon chromosome alignment is thought to time anaphase [1-3], is functional during the rapid mitotic cycles of the Drosophila embryo; but its genetic inactivation had no consequence on the timing of the early mitoses. Mitotic cyclins-Cyclin A, Cyclin B, and Cyclin B3-influence mitotic progression and are degraded in a stereotyped sequence [4-11]. RNAi knockdown of Cyclins A and B resulted in a Cyclin B3-only mitosis in which anaphase initiated prior to chromosome alignment. Furthermore, in such a Cyclin B3-only mitosis, colchicine-induced SAC activation failed to block Cyclin B3 destruction, chromosome decondensation, or nuclear membrane re-assembly. Injection of Cyclin B proteins restored the ability of SAC to prevent Cyclin B3 destruction. Thus, SAC function depends on particular cyclin types. Changing Cyclin B3 levels showed that it accelerated progress to anaphase, even in the absence of SAC function. The impact of Cyclin B3 on anaphase initiation appeared to decline with developmental progress. Our results show that different cyclin types affect anaphase timing differently in the early embryonic divisions. The early-destroyed cyclins-Cyclins A and B-restrain anaphase-promoting complex/cyclosome (APC/C) function, whereas the late-destroyed cyclin, Cyclin B3, stimulates function. We propose that the destruction schedule of cyclin types guides mitotic exit by affecting both Cdk1 and APC/C, whose activities change as each cyclin type is lost.

From egg to gastrula: how the cell cycle is remodeled during the Drosophila mid-blastula transition.

Many, if not most, embryos begin development with extremely short cell cycles that exhibit unusually rapid DNA replication and no gap phases. The commitment to the cell cycle in the early embryo appears to preclude many other cellular processes that only emerge as the cell cycle slows just prior to gastrulation at a major embryonic transition known as the mid-blastula transition (MBT). As reviewed here, genetic and molecular studies in Drosophila have identified changes that extend S phase and introduce a post-replicative gap phase, G2, to slow the cell cycle. Although many mysteries remain about the upstream regulators of these changes, we review the core mechanisms of the change in cell cycle regulation and discuss advances in our understanding of how these might be timed and triggered. Finally, we consider how the elements of this program may be conserved or changed in other organisms.

Transmission of mitochondrial mutations and action of purifying selection in Drosophila melanogaster.

It is not known how selection affects mutations in the multiple copies of the mitochondrial genome. We transferred cytoplasm between D. melanogaster embryos carrying mitochondrial mutations to create heteroplasmic lines transmitting two mitochondrial genotypes. Increased temperature imposed selection against a temperature-sensitive mutation affecting cytochrome oxidase, driving decreases in the abundance of the mutant genome over successive generations. Selection did not influence the health or fertility of the flies but acted during midoogenesis to influence competition between the genomes. Mitochondria might incur an advantage through selective localization, survival or proliferation, yet timing and insensitivity to park mutation suggest that preferential proliferation underlies selection. Selection drove complete replacement of the temperature-sensitive mitochondrial genome by a wild-type genome but also stabilized the multigenerational transmission of two genomes carrying complementing detrimental mutations. While they are so balanced, these stably transmitted mutations have no detrimental phenotype, but their segregation could contribute to disease phenotypes and somatic aging.