Extravillous trophoblast cell lineage development is associated with active remodeling of the chromatin landscape.

The extravillous trophoblast cell lineage is a key feature of placentation and successful pregnancy. Knowledge of transcriptional regulation driving extravillous trophoblast cell development is limited. Here, we map the transcriptome and epigenome landscape as well as chromatin interactions of human trophoblast stem cells and their transition into extravillous trophoblast cells. We show that integrating chromatin accessibility, long-range chromatin interactions, transcriptomic, and transcription factor binding motif enrichment enables identification of transcription factors and regulatory mechanisms critical for extravillous trophoblast cell development. We elucidate functional roles for TFAP2C, SNAI1, and EPAS1 in the regulation of extravillous trophoblast cell development. EPAS1 is identified as an upstream regulator of key extravillous trophoblast cell transcription factors, including ASCL2 and SNAI1 and together with its target genes, is linked to pregnancy loss and birth weight. Collectively, we reveal activation of a dynamic regulatory network and provide a framework for understanding extravillous trophoblast cell specification in trophoblast cell lineage development and human placentation.

Meiotic nuclear pore complex remodeling provides key insights into nuclear basket organization.

Nuclear pore complexes (NPCs) are large proteinaceous assemblies that mediate nuclear compartmentalization. NPCs undergo large-scale structural rearrangements during mitosis in metazoans and some fungi. However, our understanding of NPC remodeling beyond mitosis remains limited. Using time-lapse fluorescence microscopy, we discovered that NPCs undergo two mechanistically separable remodeling events during budding yeast meiosis in which parts or all of the nuclear basket transiently dissociate from the NPC core during meiosis I and II, respectively. Meiosis I detachment, observed for Nup60 and Nup2, is driven by Polo kinase-mediated phosphorylation of Nup60 at its interface with the Y-complex. Subsequent reattachment of Nup60-Nup2 to the NPC core is facilitated by a lipid-binding amphipathic helix in Nup60. Preventing Nup60-Nup2 reattachment causes misorganization of the entire nuclear basket in gametes. Strikingly, meiotic nuclear basket remodeling also occurs in the distantly related fission yeast, Schizosaccharomyces pombe. Our study reveals a conserved and developmentally programmed aspect of NPC plasticity, providing key mechanistic insights into the nuclear basket organization.

Quantitative analysis of nuclear pore complex organization in Schizosaccharomyces pombe.

The number, distribution, and composition of nuclear pore complexes (NPCs) in the nuclear envelope varies between cell types and changes during cellular differentiation and in disease. To understand how NPC density and organization are controlled, we analyzed the NPC number and distribution in the fission yeast Schizosaccharomyces pombe using structured illumination microscopy. The small size of yeast nuclei, genetic features of fungi, and our robust image analysis pipeline allowed us to study NPCs in intact nuclei under multiple conditions. Our data revealed that NPC density is maintained across a wide range of nuclear sizes. Regions of reduced NPC density are observed over the nucleolus and surrounding the spindle pole body (SPB). Lem2-mediated tethering of the centromeres to the SPB is required to maintain NPC exclusion near SPBs. These findings provide a quantitative understanding of NPC number and distribution in S. pombe and show that interactions between the centromere and the nuclear envelope influences local NPC distribution.

Comprehensive structure and functional adaptations of the yeast nuclear pore complex.

Nuclear pore complexes (NPCs) mediate the nucleocytoplasmic transport of macromolecules. Here we provide a structure of the isolated yeast NPC in which the inner ring is resolved by cryo-EM at sub-nanometer resolution to show how flexible connectors tie together different structural and functional layers. These connectors may be targets for phosphorylation and regulated disassembly in cells with an open mitosis. Moreover, some nucleoporin pairs and transport factors have similar interaction motifs, which suggests an evolutionary and mechanistic link between assembly and transport. We provide evidence for three major NPC variants that may foreshadow functional specializations at the nuclear periphery. Cryo-electron tomography extended these studies, providing a model of the in situ NPC with a radially expanded inner ring. Our comprehensive model reveals features of the nuclear basket and central transporter, suggests a role for the lumenal Pom152 ring in restricting dilation, and highlights structural plasticity that may be required for transport.

High-Throughput Identification of Nuclear Envelope Protein Interactions in Schizosaccharomyces pombe Using an Arrayed Membrane Yeast-Two Hybrid Library.

The nuclear envelope (NE) contains a specialized set of integral membrane proteins that maintain nuclear shape and integrity and influence chromatin organization and gene expression. Advances in proteomics techniques and studies in model organisms have identified hundreds of proteins that localize to the NE. However, the function of many of these proteins at the NE remains unclear, in part due to a lack of understanding of the interactions that these proteins participate in at the NE membrane. To assist in the characterization of NE transmembrane protein interactions we developed an arrayed library of integral and peripheral membrane proteins from the fission yeast Schizosaccharomyces pombe for high-throughput screening using the split-ubiquitin based membrane yeast two -hybrid system. We used this approach to characterize protein interactions for three conserved proteins that localize to the inner nuclear membrane: Cut11/Ndc1, Lem2 and Ima1/Samp1/Net5. Additionally, we determined how the interaction network for Cut11 is altered in canonical temperature-sensitive cut11-ts mutants. This library and screening approach is readily applicable to characterizing the interactomes of integral membrane proteins localizing to various subcellular compartments.

Factors promoting nuclear envelope assembly independent of the canonical ESCRT pathway.

The nuclear envelope (NE) undergoes dynamic remodeling to maintain NE integrity, a process involving the inner nuclear membrane protein LEM2 recruiting CHMP7/Cmp7 and then ESCRT-III. However, prior work has hinted at CHMP7/ESCRT-independent mechanisms. To identify such mechanisms, we studied NE assembly in Schizosaccharomyces japonicus, a fission yeast that undergoes partial mitotic NE breakdown and reassembly. S. japonicus cells lacking Cmp7 have compromised NE sealing after mitosis but are viable. A genetic screen identified mutations that promote NE integrity in cmp7Δ cells. Unexpectedly, loss of Lem2 or its interacting partner Nur1 suppressed cmp7Δ defects. In the absence of Cmp7, Lem2 formed aggregates that appear to interfere with ESCRT-independent NE sealing. A gain-of-function mutation implicated a membrane and ESCRT-III regulator, Alx1, in this alternate pathway. Additional results suggest a potentially general role for unsaturated fatty acids in NE integrity. These findings establish the existence of mechanisms for NE sealing independent of the canonical ESCRT pathway.

TgDrpC, an atypical dynamin-related protein in Toxoplasma gondii, is associated with vesicular transport factors and parasite division.

Dynamin-related proteins (Drps) are involved in diverse processes such as organelle division and vesicle trafficking. The intracellular parasite Toxoplasma gondii possesses three distinct Drps. TgDrpC, whose function remains unresolved, is unusual in that it lacks a conserved GTPase Effector Domain, which is typically required for function. Here, we show that TgDrpC localizes to cytoplasmic puncta; however, in dividing parasites, TgDrpC redistributes to the growing edge of the daughter cells. By conditional knockdown, we determined that loss of TgDrpC stalls division and leads to rapid deterioration of multiple organelles and the IMC. We also show that TgDrpC interacts with proteins that exhibit homology to those involved in vesicle transport, including members of the adaptor complex 2. Two of these proteins, a homolog of the adaptor protein 2 (AP-2) complex subunit alpha-1 and a homolog of the ezrin-radixin-moesin (ERM) family proteins, localize to puncta and associate with the daughter cells. Consistent with the association with vesicle transport proteins, re-distribution of TgDrpC to the IMC during division is dependent on post-Golgi trafficking. Together, these results support that TgDrpC contributes to vesicle trafficking and is critical for stability of parasite organelles and division.

To Make a Long Spindle Short: Nuclear Envelope Breakdown during Meiosis.

In fission yeast, the nuclear envelope (NE) remains intact during mitosis and meiosis I but is compromised during meiosis II. In this issue of Cell Reports, Flor-Parra et al. (2018) demonstrate that this NE alteration regulates meiosis II spindle disassembly and the ploidy of meiotic products.

TgTKL1 Is a Unique Plant-Like Nuclear Kinase That Plays an Essential Role in Acute Toxoplasmosis.

In the protozoan parasite Toxoplasma gondii, protein kinases have been shown to play key roles in regulating parasite motility, invasion, replication, egress, and survival within the host. The tyrosine kinase-like (TKL) family of proteins are an unexplored set of kinases in Toxoplasma Of the eight annotated TKLs in the Toxoplasma genome, a recent genome-wide loss-of-function screen showed that six are important for tachyzoite fitness. By utilizing an endogenous tagging approach, we showed that these six T. gondii TKLs (TgTKLs) localize to various subcellular compartments, including the nucleus, the cytosol, the inner membrane complex, and the Golgi apparatus. To gain insight into the function of TKLs in Toxoplasma, we first characterized TgTKL1, which contains the plant-like enhanced disease resistance 1 (EDR1) domain and localizes to the nucleus. TgTKL1 knockout parasites displayed significant defects in progression through the lytic cycle; we show that the defects were due to specific impairment of host cell attachment. Transcriptomics analysis identified over 200 genes of diverse functions that were differentially expressed in TgTKL1 knockout parasites. Importantly, numerous genes implicated in host cell attachment and invasion were among those most significantly downregulated, resulting in defects in microneme secretion and processing. Significantly, all of the mice inoculated intraperitoneally with TgTKL1 knockout parasites survived the infection, suggesting that TgTKL1 plays an essential role in acute toxoplasmosis. Together, these findings suggest that TgTKL1 mediates a signaling pathway that regulates the expression of multiple factors required for parasite virulence, underscoring the potential of this kinase as a novel therapeutic target.IMPORTANCEToxoplasma gondii is a protozoan parasite that can cause chronic and life-threatening disease in mammals; new drugs are greatly needed for treatment. One attractive group of drug targets consists of parasite kinases containing unique features that distinguish them from host proteins. In this report, we identify and characterize a previously unstudied kinase, TgTKL1, that localizes to the nucleus and contains a domain architecture unique to plants and protozoa. By disrupting TgTKL1, we showed that this kinase is required for the proper expression of hundreds of genes, including many that are required for the parasite to gain entry into the host cell. Specifically, parasites lacking TgTKL1 have defects in host cell attachment, resulting in impaired growth in vitro and a complete loss of virulence in mice. This report provides insight into the importance of the parasite tyrosine kinase-like kinases and establishes TgTKL1 as a novel and essential virulence factor in Toxoplasma.

Characterization of Plasmodium Atg3-Atg8 Interaction Inhibitors Identifies Novel Alternative Mechanisms of Action in Toxoplasma gondii.

Protozoan parasites, including the apicomplexan pathogens Plasmodium falciparum (which causes malaria) and Toxoplasma gondii (which causes toxoplasmosis), infect millions of people worldwide and represent major human disease burdens. Despite their prevalence, therapeutic strategies to treat infections caused by these parasites remain limited and are threatened by the emergence of drug resistance, highlighting the need for the identification of novel drug targets. Recently, homologues of the core autophagy proteins, including Atg8 and Atg3, were identified in many protozoan parasites. Importantly, components of the Atg8 conjugation system that facilitate the lipidation of Atg8 are required for both canonical and parasite-specific functions and are essential for parasite viability. Structural characterization of the P. falciparum Atg3-Atg8 (PfAtg3-Atg8) interaction has led to the identification of compounds that block this interaction. Additionally, many of these compounds inhibit P. falciparum growth in vitro, demonstrating the viability of this pathway as a drug target. Given the essential role of the Atg8 lipidation pathway in Toxoplasma, we sought to determine whether three PfAtg3-Atg8 interaction inhibitors identified in the Medicines for Malaria Venture Malaria Box exerted a similar inhibitory effect in Toxoplasma While all three inhibitors blocked Toxoplasma replication in vitro at submicromolar concentrations, they did not inhibit T. gondii Atg8 (TgAtg8) lipidation. Rather, high concentrations of two of these compounds induced TgAtg8 lipidation and fragmentation of the parasite mitochondrion, similar to the effects seen following starvation and monensin-induced autophagy. Additionally, we report that one of the PfAtg3-Atg8 interaction inhibitors induces Toxoplasma egress and provide evidence that this is mediated by an increase in intracellular calcium in response to drug treatment.

TgPRELID, a Mitochondrial Protein Linked to Multidrug Resistance in the Parasite Toxoplasma gondii.

New drugs to control infection with the protozoan parasite Toxoplasma gondii are needed as current treatments exert toxic side effects on patients. Approaches to develop novel compounds for drug development include screening of compound libraries and targeted inhibition of essential cellular pathways. We identified two distinct compounds that display inhibitory activity against the parasite's replicative stage: F3215-0002, which we previously identified during a compound library screen, and I-BET151, an inhibitor of bromodomains, the "reader" module of acetylated lysines. In independent studies, we sought to determine the targets of these two compounds using forward genetics, generating resistant mutants and identifying the determinants of resistance with comparative genome sequencing. Despite the dissimilarity of the two compounds, we recovered resistant mutants with nonsynonymous mutations in the same domain of the same gene, TGGT1_254250, which we found encodes a protein that localizes to the parasite mitochondrion (designated TgPRELID after the name of said domain). We found that mutants selected with one compound were cross resistant to the other compound, suggesting a common mechanism of resistance. To further support our hypothesis that TgPRELID mutations facilitate resistance to both I-BET151 and F3215-0002, CRISPR (clustered regularly interspaced short palindromic repeat)/CAS9-mediated mutation of TgPRELID directly led to increased F3215-0002 resistance. Finally, all resistance mutations clustered in the same subdomain of TgPRELID. These findings suggest that TgPRELID may encode a multidrug resistance factor or that I-BET151 and F3215-0002 have the same target(s) despite their distinct chemical structures. IMPORTANCE We report the discovery of TgPRELID, a previously uncharacterized mitochondrial protein linked to multidrug resistance in the parasite Toxoplasma gondii. Drug resistance remains a major problem in the battle against parasitic infection, and understanding how TgPRELID mutations augment resistance to multiple, distinct compounds will reveal needed insights into the development of new therapies for toxoplasmosis and other related parasitic diseases.

TgATAT-Mediated α-Tubulin Acetylation Is Required for Division of the Protozoan Parasite Toxoplasma gondii.

Toxoplasma gondii is a widespread protozoan parasite that causes potentially life-threatening opportunistic disease. New inhibitors of parasite replication are urgently needed, as the current antifolate treatment is also toxic to patients. Microtubules are essential cytoskeletal components that have been selectively targeted in microbial pathogens; further study of tubulin in Toxoplasma may reveal novel therapeutic opportunities. It has been noted that α-tubulin acetylation at lysine 40 (K40) is enriched during daughter parasite formation, but the impact of this modification on Toxoplasma division and the enzyme mediating its delivery have not been identified. We performed mutational analyses to provide evidence that K40 acetylation stabilizes Toxoplasma microtubules and is required for parasite replication. We also show that an unusual Toxoplasma homologue of α-tubulin acetyltransferase (TgATAT) is expressed in a cell cycle-regulated manner and that its expression peaks during division. Disruption of TgATAT with CRISPR/Cas9 ablates K40 acetylation and induces replication defects; parasites appear to initiate mitosis yet exhibit incomplete or improper nuclear division. Together, these findings establish the importance of tubulin acetylation, exposing a new vulnerability in Toxoplasma that could be pharmacologically targeted. IMPORTANCE Toxoplasma gondii is an opportunistic parasite that infects at least one-third of the world population. New treatments for the disease (toxoplasmosis) are needed since current drugs are toxic to patients. Microtubules are essential cellular structures built from tubulin that show promise as antimicrobial drug targets. Microtubules can be regulated by chemical modification, such as acetylation on lysine 40 (K40). To determine the role of K40 acetylation in Toxoplasma and whether it is a liability to the parasite, we performed mutational analyses of the α-tubulin gene. Our results indicate that parasites cannot survive without K40 acetylation unless microtubules are stabilized with a secondary mutation. Additionally, we identified the parasite enzyme that acetylates α-tubulin (TgATAT). Genetic disruption of TgATAT caused severe defects in parasite replication, further highlighting the importance of α-tubulin K40 acetylation in Toxoplasma and its promise as a potential new drug target.

Dynamic and Combinatorial Landscape of Histone Modifications during the Intraerythrocytic Developmental Cycle of the Malaria Parasite.

A major obstacle in understanding the complex biology of the malaria parasite remains to discover how gene transcription is controlled during its life cycle. Accumulating evidence indicates that the parasite's epigenetic state plays a fundamental role in gene expression and virulence. Using a comprehensive and quantitative mass spectrometry approach, we determined the global and dynamic abundance of histones and their covalent post-transcriptional modifications throughout the intraerythrocytic developmental cycle of Plasmodium falciparum. We detected a total of 232 distinct modifications, of which 160 had never been detected in Plasmodium and 88 had never been identified in any other species. We further validated over 10% of the detected modifications and their expression patterns by multiple reaction monitoring assays. In addition, we uncovered an unusual chromatin organization with parasite-specific histone modifications and combinatorial dynamics that may be directly related to transcriptional activity, DNA replication, and cell cycle progression. Overall, our data suggest that the malaria parasite has a unique histone modification signature that correlates with parasite virulence.

The little elongation complex functions at initiation and elongation phases of snRNA gene transcription.

The small nuclear RNA (snRNA) genes have been widely used as a model system for understanding transcriptional regulation due to the unique aspects of their promoter structure, selectivity for either RNA polymerase (Pol) II or III, and because of their unique mechanism of termination that is tightly linked with the promoter. Recently, we identified the little elongation complex (LEC) in Drosophila that is required for the expression of Pol II-transcribed snRNA genes. Here, using Drosophila and mammalian systems, we provide genetic and molecular evidence that LEC functions in at least two phases of snRNA transcription: an initiation step requiring the ICE1 subunit, and an elongation step requiring ELL.