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.

The parasite intraerythrocytic cycle and human circadian cycle are coupled during malaria infection.

During infections with the malaria parasites Plasmodium vivax, patients exhibit rhythmic fevers every 48 h. These fever cycles correspond with the time the parasites take to traverse the intraerythrocytic cycle (IEC). In other Plasmodium species that infect either humans or mice, the IEC is likely guided by a parasite-intrinsic clock [Rijo-Ferreiraet al., Science 368, 746-753 (2020); Smith et al., Science 368, 754-759 (2020)], suggesting that intrinsic clock mechanisms may be a fundamental feature of malaria parasites. Moreover, because Plasmodium cycle times are multiples of 24 h, the IECs may be coordinated with the host circadian clock(s). Such coordination could explain the synchronization of the parasite population in the host and enable alignment of IEC and circadian cycle phases. We utilized an ex vivo culture of whole blood from patients infected with P. vivax to examine the dynamics of the host circadian transcriptome and the parasite IEC transcriptome. Transcriptome dynamics revealed that the phases of the host circadian cycle and the parasite IEC are correlated across multiple patients, showing that the cycles are phase coupled. In mouse model systems, host-parasite cycle coupling appears to provide a selective advantage for the parasite. Thus, understanding how host and parasite cycles are coupled in humans could enable antimalarial therapies that disrupt this coupling.

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.

BLMP-1/Blimp-1 regulates the spatiotemporal cell migration pattern in C. elegans.

Spatiotemporal regulation of cell migration is crucial for animal development and organogenesis. Compared to spatial signals, little is known about temporal signals and the mechanisms integrating the two. In the Caenorhabditis elegans hermaphrodite, the stereotyped migration pattern of two somatic distal tip cells (DTCs) is responsible for shaping the gonad. Guidance receptor UNC-5 is necessary for the dorsalward migration of DTCs. We found that BLMP-1, similar to the mammalian zinc finger transcription repressor Blimp-1/PRDI-BF1, prevents precocious dorsalward turning by inhibiting precocious unc-5 transcription and is only expressed in DTCs before they make the dorsalward turn. Constitutive expression of blmp-1 when BLMP-1 would normally disappear delays unc-5 transcription and causes turn retardation, demonstrating the functional significance of blmp-1 down-regulation. Correct timing of BLMP-1 down-regulation is redundantly regulated by heterochronic genes daf-12, lin-29, and dre-1, which regulate the temporal fates of various tissues. DAF-12, a steroid hormone receptor, and LIN-29, a zinc finger transcription factor, repress blmp-1 transcription, while DRE-1, the F-Box protein of an SCF ubiquitin ligase complex, binds to BLMP-1 and promotes its degradation. We have therefore identified a gene circuit that integrates the temporal and spatial signals and coordinates with overall development of the organism to direct cell migration during organogenesis. The tumor suppressor gene product FBXO11 (human DRE-1 ortholog) also binds to PRDI-BF1 in human cell cultures. Our data suggest evolutionary conservation of these interactions and underscore the importance of DRE-1/FBXO11-mediated BLMP-1/PRDI-BF1 degradation in cellular state transitions during metazoan development.

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.

The cell-cycle transcriptional network generates and transmits a pulse of transcription once each cell cycle.

Multiple studies have suggested the critical roles of cyclin-dependent kinases (CDKs) as well as a transcription factor (TF) network in generating the robust cell-cycle transcriptional program. However, the precise mechanisms by which these components function together in the gene regulatory network remain unclear. Here we show that the TF network can generate and transmit a "pulse" of transcription independently of CDK oscillations. The premature firing of the transcriptional pulse is prevented by early G1 inhibitors, including transcriptional corepressors and the E3 ubiquitin ligase complex APCCdh1. We demonstrate that G1 cyclin-CDKs facilitate the activation and accumulation of TF proteins in S/G2/M phases through inhibiting G1 transcriptional corepressors (Whi5 and Stb1) and APCCdh1, thereby promoting the initiation and propagation of the pulse by the TF network. These findings suggest a unique oscillatory mechanism in which global phase-specific transcription emerges from a pulse-generating network that fires once-and-only-once at the start of the cycle.

Reconciling conflicting models for global control of cell-cycle transcription.

Models for the control of global cell-cycle transcription have advanced from a CDK-APC/C oscillator, a transcription factor (TF) network, to coupled CDK-APC/C and TF networks. Nonetheless, current models were challenged by a recent study that concluded that the cell-cycle transcriptional program is primarily controlled by a CDK-APC/C oscillator in budding yeast. Here we report an analysis of the transcriptome dynamics in cyclin mutant cells that were not queried in the previous study. We find that B-cyclin oscillation is not essential for control of phase-specific transcription. Using a mathematical model, we demonstrate that the function of network TFs can be retained in the face of significant reductions in transcript levels. Finally, we show that cells arrested at mitotic exit with non-oscillating levels of B-cyclins continue to cycle transcriptionally. Taken together, these findings support a critical role of a TF network and a requirement for CDK activities that need not be periodic.