Protein-intrinsic properties and context-dependent effects regulate pioneer factor binding and function.

Chromatin is a barrier to the binding of many transcription factors. By contrast, pioneer factors access nucleosomal targets and promote chromatin opening. Despite binding to target motifs in closed chromatin, many pioneer factors display cell-type-specific binding and activity. The mechanisms governing pioneer factor occupancy and the relationship between chromatin occupancy and opening remain unclear. We studied three Drosophila transcription factors with distinct DNA-binding domains and biological functions: Zelda, Grainy head and Twist. We demonstrated that the level of chromatin occupancy is a key determinant of pioneering activity. Multiple factors regulate occupancy, including motif content, local chromatin and protein concentration. Regions outside the DNA-binding domain are required for binding and chromatin opening. Our results show that pioneering activity is not a binary feature intrinsic to a protein but occurs on a spectrum and is regulated by a variety of protein-intrinsic and cell-type-specific features.

Localization of the Drosophila pioneer factor GAF to subnuclear foci is driven by DNA binding and required to silence satellite repeat expression.

The eukaryotic genome is organized to enable the precise regulation of gene expression. This organization is established as the embryo transitions from a fertilized gamete to a totipotent zygote. To understand the factors and processes that drive genomic organization, we focused on the pioneer factor GAGA factor (GAF) that is required for early development in Drosophila. GAF transcriptionally activates the zygotic genome and is localized to subnuclear foci. This non-uniform distribution is driven by binding to highly abundant GA repeats. At GA repeats, GAF is necessary to form heterochromatin and silence transcription. Thus, GAF is required to establish both active and silent regions. We propose that foci formation enables GAF to have opposing transcriptional roles within a single nucleus. Our data support a model in which the subnuclear concentration of transcription factors acts to organize the nucleus into functionally distinct domains essential for the robust regulation of gene expression.

Protein-intrinsic properties and context-dependent effects regulate pioneer-factor binding and function.

Chromatin is a barrier to the binding of many transcription factors. By contrast, pioneer factors access nucleosomal targets and promote chromatin opening. Despite binding to target motifs in closed chromatin, many pioneer factors display cell-type specific binding and activity. The mechanisms governing pioneer-factor occupancy and the relationship between chromatin occupancy and opening remain unclear. We studied three Drosophila transcription factors with distinct DNA-binding domains and biological functions: Zelda, Grainy head, and Twist. We demonstrated that the level of chromatin occupancy is a key determinant of pioneering activity. Multiple factors regulate occupancy, including motif content, local chromatin, and protein concentration. Regions outside the DNA-binding domain are required for binding and chromatin opening. Our results show that pioneering activity is not a binary feature intrinsic to a protein but occurs on a spectrum and is regulated by a variety of protein-intrinsic and cell-type-specific features.

Cell-type-specific chromatin occupancy by the pioneer factor Zelda drives key developmental transitions in Drosophila.

During Drosophila embryogenesis, the essential pioneer factor Zelda defines hundreds of cis-regulatory regions and in doing so reprograms the zygotic transcriptome. While Zelda is essential later in development, it is unclear how the ability of Zelda to define cis-regulatory regions is shaped by cell-type-specific chromatin architecture. Asymmetric division of neural stem cells (neuroblasts) in the fly brain provide an excellent paradigm for investigating the cell-type-specific functions of this pioneer factor. We show that Zelda synergistically functions with Notch to maintain neuroblasts in an undifferentiated state. Zelda misexpression reprograms progenitor cells to neuroblasts, but this capacity is limited by transcriptional repressors critical for progenitor commitment. Zelda genomic occupancy in neuroblasts is reorganized as compared to the embryo, and this reorganization is correlated with differences in chromatin accessibility and cofactor availability. We propose that Zelda regulates essential transitions in the neuroblasts and embryo through a shared gene-regulatory network driven by cell-type-specific enhancers.

Activity of Plasmodium vivax promoter elements in Plasmodium knowlesi, and a centromere-containing plasmid that expresses NanoLuc throughout the parasite life cycle.

BACKGROUND: Plasmodium knowlesi is now the major cause of human malaria in Malaysia, complicating malaria control efforts that must attend to the elimination of multiple Plasmodium species. Recent advances in the cultivation of P. knowlesi erythrocytic-stage parasites in vitro, transformation with exogenous DNA, and infection of mosquitoes with gametocytes from culture have opened up studies of this pathogen without the need for resource-intensive and costly non-human primate (NHP) models. For further understanding and development of methods for parasite transformation in malaria research, this study examined the activity of various trans-species transcriptional control sequences and the influence of Plasmodium vivax centromeric (pvcen) repeats in plasmid-transfected P. knowlesi parasites. METHODS: In vitro cultivated P. knowlesi parasites were transfected with plasmid constructs that incorporated Plasmodium vivax or Plasmodium falciparum 5' UTRs driving the expression of bioluminescence markers (firefly luciferase or Nanoluc). Promoter activities were assessed by bioluminescence, and parasites transformed with human resistant allele dihydrofolate reductase-expressing plasmids were selected using antifolates. The stability of transformants carrying pvcen-stabilized episomes was assessed by bioluminescence over a complete parasite life cycle through a rhesus macaque monkey, mosquitoes, and a second rhesus monkey. RESULTS: Luciferase expression assessments show that certain P. vivax promoter regions, not functional in the more evolutionarily-distant P. falciparum, can drive transgene expression in P. knowlesi. Further, pvcen repeats may improve the stability of episomal plasmids in P. knowlesi and support detection of NanoLuc-expressing elements over the full parasite life cycle from rhesus macaque monkeys to Anopheles dirus mosquitoes and back again to monkeys. In assays of drug responses to chloroquine, G418 and WR9910, anti-malarial half-inhibitory concentration (IC50) values of blood stages measured by NanoLuc activity proved comparable to IC50 values measured by the standard SYBR Green method. CONCLUSION: All three P. vivax promoters tested in this study functioned in P. knowlesi, whereas two of the three were inactive in P. falciparum. NanoLuc-expressing, centromere-stabilized plasmids may support high-throughput screenings of P. knowlesi for new anti-malarial agents, including compounds that can block the development of mosquito- and/or liver-stage parasites.

GAF is essential for zygotic genome activation and chromatin accessibility in the early Drosophila embryo.

Following fertilization, the genomes of the germ cells are reprogrammed to form the totipotent embryo. Pioneer transcription factors are essential for remodeling the chromatin and driving the initial wave of zygotic gene expression. In Drosophila melanogaster, the pioneer factor Zelda is essential for development through this dramatic period of reprogramming, known as the maternal-to-zygotic transition (MZT). However, it was unknown whether additional pioneer factors were required for this transition. We identified an additional maternally encoded factor required for development through the MZT, GAGA Factor (GAF). GAF is necessary to activate widespread zygotic transcription and to remodel the chromatin accessibility landscape. We demonstrated that Zelda preferentially controls expression of the earliest transcribed genes, while genes expressed during widespread activation are predominantly dependent on GAF. Thus, progression through the MZT requires coordination of multiple pioneer-like factors, and we propose that as development proceeds control is gradually transferred from Zelda to GAF.

Most cells in an organism share the exact same genetic information, yet they still adopt distinct identities. This diversity emerges because only a selection of genes is switched on at any given time in a cell. Proteins that latch onto DNA control this specificity by activating certain genes at the right time. However, to perform this role they first need to physically access DNA: this can be difficult as the genetic information is tightly compacted so it can fit in a cell. A group of proteins can help to unpack the genome to uncover the genes that can then be accessed and activated. While these 'pioneer factors' can therefore shape the identity of a cell, much remains unknown about how they can work together to do so. For instance, the pioneer factor Zelda is essential in early fruit fly development, as it enables the genetic information of the egg and sperm to undergo dramatic reprogramming and generate a new organism. Yet, it was unclear whether additional helpers were required for this transition. Using this animal system, Gaskill, Gibson et al. identified GAGA Factor as a protein which works with Zelda to open up and reprogram hundreds of different sections along the genome of fruit fly embryos. This tag-team effort started with Zelda being important initially to activate genes; regulation was then handed over for GAGA Factor to continue the process. Without either protein, the embryo died. Getting a glimpse into early genetic events during fly development provides insights that are often applicable to other animals such as fish and mammals. Ultimately, this research may help scientists to understand how things can go wrong in human embryos.

H3 K27M and EZHIP Impede H3K27-Methylation Spreading by Inhibiting Allosterically Stimulated PRC2.

Diffuse midline gliomas and posterior fossa type A ependymomas contain the recurrent histone H3 lysine 27 (H3 K27M) mutation and express the H3 K27M-mimic EZHIP (CXorf67), respectively. H3 K27M and EZHIP are competitive inhibitors of Polycomb Repressive Complex 2 (PRC2) lysine methyltransferase activity. In vivo, these proteins reduce overall H3 lysine 27 trimethylation (H3K27me3) levels; however, residual peaks of H3K27me3 remain at CpG islands (CGIs) through an unknown mechanism. Here, we report that EZHIP and H3 K27M preferentially interact with PRC2 that is allosterically activated by H3K27me3 at CGIs and impede its spreading. Moreover, H3 K27M oncohistones reduce H3K27me3 in trans, independent of their incorporation into the chromatin. Although EZHIP is not found outside placental mammals, expression of human EZHIP reduces H3K27me3 in Drosophila melanogaster through a conserved mechanism. Our results provide mechanistic insights for the retention of residual H3K27me3 in tumors driven by H3 K27M and EZHIP.

Total synthesis of (+)-pilosinine via a stereodivergent conjugate addition strategy.

In recent work, asymmetric conjugate addition reactions to chiral 4-phenyl-N-enoyl-1,3-oxazolidinones have been shown to give different stereochemical outcomes depending on the conditions employed. Through the application of stereodivergent reaction conditions, the total synthesis of (+)-pilosinine and the formal synthesis of (-)-pilosinine has been completed from a single enantiomer of the 1,3-oxazolidi-none auxiliary.

Establishment of chromatin accessibility by the conserved transcription factor Grainy head is developmentally regulated.

The dramatic changes in gene expression required for development necessitate the establishment of cis-regulatory modules defined by regions of accessible chromatin. Pioneer transcription factors have the unique property of binding closed chromatin and facilitating the establishment of these accessible regions. Nonetheless, much of how pioneer transcription factors coordinate changes in chromatin accessibility during development remains unknown. To determine whether pioneer-factor function is intrinsic to the protein or whether pioneering activity is developmentally modulated, we studied the highly conserved, essential transcription factor Grainy head (Grh). Prior work established that Grh is expressed throughout Drosophila development and is a pioneer factor in the larva. We demonstrated that Grh remains bound to mitotic chromosomes, a property shared with other pioneer factors. By assaying chromatin accessibility in embryos lacking maternal and/or zygotic Grh at three stages of development, we discovered that Grh is not required for chromatin accessibility in early embryogenesis, in contrast to its essential functions later in development. Our data reveal that the pioneering activity of Grh is temporally regulated and likely influenced by additional factors expressed at a given developmental stage.

Plasmodium vivax chloroquine resistance links to pvcrt transcription in a genetic cross.

Mainstay treatment for Plasmodium vivax malaria has long relied on chloroquine (CQ) against blood-stage parasites plus primaquine against dormant liver-stage forms (hypnozoites), however drug resistance confronts this regimen and threatens malaria control programs. Understanding the basis of P. vivax chloroquine resistance (CQR) will inform drug discovery and malaria control. Here we investigate the genetics of P. vivax CQR by a cross of parasites differing in drug response. Gametocytogenesis, mosquito infection, and progeny production are performed with mixed parasite populations in nonhuman primates, as methods for P. vivax cloning and in vitro cultivation remain unavailable. Linkage mapping of progeny surviving >15 mg/kg CQ identifies a 76 kb region in chromosome 1 including pvcrt, an ortholog of the Plasmodium falciparum CQR transporter gene. Transcriptional analysis supports upregulated pvcrt expression as a mechanism of CQR.

Continued Activity of the Pioneer Factor Zelda Is Required to Drive Zygotic Genome Activation.

Reprogramming cell fate during the first stages of embryogenesis requires that transcriptional activators gain access to the genome and remodel the zygotic transcriptome. Nonetheless, it is not clear whether the continued activity of these pioneering factors is required throughout zygotic genome activation or whether they are only required early to establish cis-regulatory regions. To address this question, we developed an optogenetic strategy to rapidly and reversibly inactivate the master regulator of genome activation in Drosophila, Zelda. Using this strategy, we demonstrate that continued Zelda activity is required throughout genome activation. We show that Zelda binds DNA in the context of nucleosomes and suggest that this allows Zelda to occupy the genome despite the rapid division cycles in the early embryo. These data identify a powerful strategy to inactivate transcription factor function during development and suggest that reprogramming in the embryo may require specific, continuous pioneering functions to activate the genome.

Artemisinin resistance phenotypes and K13 inheritance in a Plasmodium falciparum cross and Aotus model.

Concerns about malaria parasite resistance to treatment with artemisinin drugs (ARTs) have grown with findings of prolonged parasite clearance t1/2s (>5 h) and their association with mutations in Plasmodium falciparum Kelch-propeller protein K13. Here, we describe a P. falciparum laboratory cross of K13 C580Y mutant with C580 wild-type parasites to investigate ART response phenotypes in vitro and in vivo. After genotyping >400 isolated progeny, we evaluated 20 recombinants in vitro: IC50 measurements of dihydroartemisinin were at similar low nanomolar levels for C580Y- and C580-type progeny (mean ratio, 1.00; 95% CI, 0.62-1.61), whereas, in a ring-stage survival assay, the C580Y-type progeny had 19.6-fold (95% CI, 9.76-39.2) higher average counts. In splenectomized Aotus monkeys treated with three daily doses of i.v. artesunate, t1/2 calculations by three different methods yielded mean differences of 0.01 h (95% CI, -3.66 to 3.67), 0.80 h (95% CI, -0.92 to 2.53), and 2.07 h (95% CI, 0.77-3.36) between C580Y and C580 infections. Incidences of recrudescence were 57% in C580Y (4 of 7) versus 70% in C580 (7 of 10) infections (-13% difference; 95% CI, -58% to 35%). Allelic substitution of C580 in a C580Y-containing progeny clone (76H10) yielded a transformant (76H10C580Rev) that, in an infected monkey, recrudesced regularly 13 times over 500 d. Frequent recrudescences of ART-treated P. falciparum infections occur with or without K13 mutations and emphasize the need for improved partner drugs to effectively eliminate the parasites that persist through the ART component of combination therapy.

Infection of mosquitoes from in vitro cultivated Plasmodium knowlesi H strain.

In vitro studies of sexual blood stages of the most fatal malaria species, Plasmodium falciparum, have revealed key processes by which gametocytes develop and transmit infection from humans to anopheline mosquitoes. However, most malaria cases outside sub-Saharan Africa are caused by other Plasmodium spp., frequently Plasmodium vivax and Plasmodium knowlesi, a zoonotic parasite of macaque monkeys. Gametocytes of P. vivax and P. knowlesi exhibit distinct morphology, faster development, and a shorter life span compared with gametocytes of P. falciparum, reflecting the evolutionary separation and biological differences of these species. Unlike P. falciparum, P. vivax cannot be cultivated in vitro, necessitating access to infected primates for laboratory studies. In contrast, P. knowlesi asexual stages have been successfully adapted to cultures in macaque and human red blood cells, but these stages have not been reported to produce gametocytes infective to mosquitoes. Here, we show that gametocyte production and sporadic, low-level mosquito infectivity of a P. knowlesi strain was not improved by application of a "crash" method commonly used to induce gametocytes in P. falciparum cultures. However, Percoll-gradient purified schizonts from this strain yielded highly synchronised populations that, in three of six experiments, produced infections at an average rate of 0.97-9.1 oocysts in Anopheles dirus mosquitoes. Oocyst counts were most abundant in mosquitoes that were fed from the synchronised cultures 36 h after schizont purification. Gametocytes in these cultures occurred at low prevalence and were difficult to observe. Transcription from orthologs of P. falciparum gametocyte-specific markers did not correlate with infectivity of the P. knowlesi parasites to mosquitoes. The ability to infect mosquitoes from in vitro-cultivated P. knowlesi will support research on the unique features of this emerging pathogen and facilitate comparative studies of transmission by the different human malarias.

Comparison of two methods for transformation of Plasmodium knowlesi: Direct schizont electroporation and spontaneous plasmid uptake from plasmid-loaded red blood cells.

Human infections from Plasmodium knowlesi present challenges to malaria control in Southeast Asia. P. knowlesi also offers a model for other human malaria species including Plasmodium vivax. P. knowlesi parasites can be cultivated in the laboratory, and their transformation is standardly performed by direct electroporation of schizont-infected red blood cells (RBCs) with plasmid DNA. Here we show that the efficiency of direct electroporation is exquisitely dependent on developmental age of the schizonts. Additionally, we show that transformation of P. knowlesi can be achieved without direct electroporation by using the parasite's ability to infect and take up DNA from plasmid-loaded RBCs. Transformation with plasmid-loaded RBCs does not require labor-intensive preparations of schizont-infected RBCs as for direct electroporation, and parasite damage from high voltage discharge is avoided. Further studies of the mechanism of spontaneous DNA uptake may suggest strategies for improved transformation and provide insights into the transport pathways of apicomplexans.