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.

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.

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.

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 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.

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

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.

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).

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].

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

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.

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.

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.

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].

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.

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.

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.

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.

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.

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].

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.

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.

Mechanism and regulation of Cdc25/Twine protein destruction in embryonic cell-cycle remodeling.

Background: In Drosophila embryos, the midblastula transition (MBT) dramatically remodels the cell cycle during the 14(th) interphase. Before the MBT, each cycle is composed of only a short S phase and mitosis. At the MBT, S phase is dramatically lengthened by the onset of late replication, and a G2 phase is introduced.

Different cyclin types collaborate to reverse the S-phase checkpoint and permit prompt mitosis.

Precise timing coordinates cell proliferation with embryonic morphogenesis. As Drosophila melanogaster embryos approach cell cycle 14 and the midblastula transition, rapid embryonic cell cycles slow because S phase lengthens, which delays mitosis via the S-phase checkpoint. We probed the contributions of each of the three mitotic cyclins to this timing of interphase duration.

Embryonic onset of late replication requires Cdc25 down-regulation.

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.

Barriers to male transmission of mitochondrial DNA in sperm development.

Across the eukaryotic phylogeny, offspring usually inherit their mitochondrial genome from only one of two parents: in animals, the female. Although mechanisms that eliminate paternally derived mitochondria from the zygote have been sought, the developmental stage at which paternal transmission of mitochondrial DNA is restricted is unknown in most animals. Here, we show that the mitochondria of mature Drosophila sperm lack DNA, and we uncover two processes that eliminate mitochondrial DNA during spermatogenesis.