Regulation of Collective Metastasis by Nanolumenal Signaling.

Collective metastasis is defined as the cohesive migration and metastasis of multicellular tumor cell clusters. Disrupting various cell adhesion genes markedly reduces cluster formation and colonization efficiency, yet the downstream signals transmitted by clustering remain largely unknown. Here, we use mouse and human breast cancer models to identify a collective signal generated by tumor cell clusters supporting metastatic colonization. We show that tumor cell clusters produce the growth factor epigen and concentrate it within nanolumina-intercellular compartments sealed by cell-cell junctions and lined with microvilli-like protrusions. Epigen knockdown profoundly reduces metastatic outgrowth and switches clusters from a proliferative to a collective migratory state. Tumor cell clusters from basal-like 2, but not mesenchymal-like, triple-negative breast cancer cell lines have increased epigen expression, sealed nanolumina, and impaired outgrowth upon nanolumenal junction disruption. We propose that nanolumenal signaling could offer a therapeutic target for aggressive metastatic breast cancers.

Genetic Screen for Cell Fitness in High or Low Oxygen Highlights Mitochondrial and Lipid Metabolism.

Human cells are able to sense and adapt to variations in oxygen levels. Historically, much research in this field has focused on hypoxia-inducible factor (HIF) signaling and reactive oxygen species (ROS). Here, we perform genome-wide CRISPR growth screens at 21%, 5%, and 1% oxygen to systematically identify gene knockouts with relative fitness defects in high oxygen (213 genes) or low oxygen (109 genes), most without known connection to HIF or ROS. Knockouts of many mitochondrial pathways thought to be essential, including complex I and enzymes in Fe-S biosynthesis, grow relatively well at low oxygen and thus are buffered by hypoxia. In contrast, in certain cell types, knockout of lipid biosynthetic and peroxisomal genes causes fitness defects only in low oxygen. Our resource nominates genetic diseases whose severity may be modulated by oxygen and links hundreds of genes to oxygen homeostasis.

Redox Chemistry in the Genome: Emergence of the [4Fe4S] Cofactor in Repair and Replication.

Many DNA-processing enzymes have been shown to contain a [4Fe4S] cluster, a common redox cofactor in biology. Using DNA electrochemistry, we find that binding of the DNA polyanion promotes a negative shift in [4Fe4S] cluster potential, which corresponds thermodynamically to a ∼500-fold increase in DNA-binding affinity for the oxidized [4Fe4S]3+ cluster versus the reduced [4Fe4S]2+ cluster. This redox switch can be activated from a distance using DNA charge transport (DNA CT) chemistry. DNA-processing proteins containing the [4Fe4S] cluster are enumerated, with possible roles for the redox switch highlighted. A model is described where repair proteins may signal one another using DNA-mediated charge transport as a first step in their search for lesions. The redox switch in eukaryotic DNA primases appears to regulate polymerase handoff, and in DNA polymerase δ, the redox switch provides a means to modulate replication in response to oxidative stress. We thus describe redox signaling interactions of DNA-processing [4Fe4S] enzymes, as well as the most interesting potential players to consider in delineating new DNA-mediated redox signaling networks.

The piRNA Response to Retroviral Invasion of the Koala Genome.

Antisense Piwi-interacting RNAs (piRNAs) guide silencing of established transposons during germline development, and sense piRNAs drive ping-pong amplification of the antisense pool, but how the germline responds to genome invasion is not understood. The KoRV-A gammaretrovirus infects the soma and germline and is sweeping through wild koalas by a combination of horizontal and vertical transfer, allowing direct analysis of retroviral invasion of the germline genome. Gammaretroviruses produce spliced Env mRNAs and unspliced transcripts encoding Gag, Pol, and the viral genome, but KoRV-A piRNAs are almost exclusively derived from unspliced genomic transcripts and are strongly sense-strand biased. Significantly, selective piRNA processing of unspliced proviral transcripts is conserved from insects to placental mammals. We speculate that bypassed splicing generates a conserved molecular pattern that directs proviral genomic transcripts to the piRNA biogenesis machinery and that this "innate" piRNA response suppresses transposition until antisense piRNAs are produced, establishing sequence-specific adaptive immunity.

Circulating Tumor Cell Clustering Shapes DNA Methylation to Enable Metastasis Seeding.

The ability of circulating tumor cells (CTCs) to form clusters has been linked to increased metastatic potential. Yet biological features and vulnerabilities of CTC clusters remain largely unknown. Here, we profile the DNA methylation landscape of single CTCs and CTC clusters from breast cancer patients and mouse models on a genome-wide scale. We find that binding sites for stemness- and proliferation-associated transcription factors are specifically hypomethylated in CTC clusters, including binding sites for OCT4, NANOG, SOX2, and SIN3A, paralleling embryonic stem cell biology. Among 2,486 FDA-approved compounds, we identify Na+/K+ ATPase inhibitors that enable the dissociation of CTC clusters into single cells, leading to DNA methylation remodeling at critical sites and metastasis suppression. Thus, our results link CTC clustering to specific changes in DNA methylation that promote stemness and metastasis and point to cluster-targeting compounds to suppress the spread of cancer.

Combinatorial Control of Plant Specialized Metabolism: Mechanisms, Functions, and Consequences.

Plants constantly perceive internal and external cues, many of which they need to address to safeguard their proper development and survival. They respond to these cues by selective activation of specific metabolic pathways involving a plethora of molecular players that act and interact in complex networks. In this review, we illustrate and discuss the complexity in the combinatorial control of plant specialized metabolism. We hereby go beyond the intuitive concept of combinatorial control as exerted by modular-acting complexes of transcription factors that govern expression of specialized metabolism genes. To extend this discussion, we also consider all known hierarchical levels of regulation of plant specialized metabolism and their interfaces by referring to reported regulatory concepts from the plant field. Finally, we speculate on possible yet-to-be-discovered regulatory principles of plant specialized metabolism that are inspired by knowledge from other kingdoms of life and areas of biological research.

Polymerization of ZBTB transcription factors regulates chromatin occupancy.

BCL6, an oncogenic transcription factor (TF), forms polymers in the presence of a small-molecule molecular glue that stabilizes a complementary interface between homodimers of BCL6's broad-complex, tramtrack, and bric-à-brac (BTB) domain. The BTB domains of other proteins, including a large class of TFs, have similar architectures and symmetries, raising the possibility that additional BTB proteins self-assemble into higher-order structures. Here, we surveyed 189 human BTB proteins with a cellular fluorescent reporter assay and identified 18 ZBTB TFs that show evidence of polymerization. Through biochemical and cryoelectron microscopy (cryo-EM) studies, we demonstrate that these ZBTB TFs polymerize into filaments. We found that BTB-domain-mediated polymerization of ZBTB TFs enhances chromatin occupancy within regions containing homotypic clusters of TF binding sites, leading to repression of target genes. Our results reveal a role of higher-order structures in regulating ZBTB TFs and suggest an underappreciated role for TF polymerization in modulating gene expression.

Oxygen toxicity causes cyclic damage by destabilizing specific Fe-S cluster-containing protein complexes.

Oxygen is toxic across all three domains of life. Yet, the underlying molecular mechanisms remain largely unknown. Here, we systematically investigate the major cellular pathways affected by excess molecular oxygen. We find that hyperoxia destabilizes a specific subset of Fe-S cluster (ISC)-containing proteins, resulting in impaired diphthamide synthesis, purine metabolism, nucleotide excision repair, and electron transport chain (ETC) function. Our findings translate to primary human lung cells and a mouse model of pulmonary oxygen toxicity. We demonstrate that the ETC is the most vulnerable to damage, resulting in decreased mitochondrial oxygen consumption. This leads to further tissue hyperoxia and cyclic damage of the additional ISC-containing pathways. In support of this model, primary ETC dysfunction in the Ndufs4 KO mouse model causes lung tissue hyperoxia and dramatically increases sensitivity to hyperoxia-mediated ISC damage. This work has important implications for hyperoxia pathologies, including bronchopulmonary dysplasia, ischemia-reperfusion injury, aging, and mitochondrial disorders.

Next-Generation Sequencing of T and B Cell Receptor Repertoires from COVID-19 Patients Showed Signatures Associated with Severity of Disease.

We profiled adaptive immunity in COVID-19 patients with active infection or after recovery and created a repository of currently >14 million B and T cell receptor (BCR and TCR) sequences from the blood of these patients. The B cell response showed converging IGHV3-driven BCR clusters closely associated with SARS-CoV-2 antibodies. Clonality and skewing of TCR repertoires were associated with interferon type I and III responses, early CD4+ and CD8+ T cell activation, and counterregulation by the co-receptors BTLA, Tim-3, PD-1, TIGIT, and CD73. Tfh, Th17-like, and nonconventional (but not classical antiviral) Th1 cell polarizations were induced. SARS-CoV-2-specific T cell responses were driven by TCR clusters shared between patients with a characteristic trajectory of clonotypes and traceability over the disease course. Our data provide fundamental insight into adaptive immunity to SARS-CoV-2 with the actively updated repository providing a resource for the scientific community urgently needed to inform therapeutic concepts and vaccine development.

Inheritance of H3K9 methylation regulates genome architecture in Drosophila early embryos.

Constitutive heterochromatin is essential for transcriptional silencing and genome integrity. The establishment of constitutive heterochromatin in early embryos and its role in early fruitfly development are unknown. Lysine 9 trimethylation of histone H3 (H3K9me3) and recruitment of its epigenetic reader, heterochromatin protein 1a (HP1a), are hallmarks of constitutive heterochromatin. Here, we show that H3K9me3 is transmitted from the maternal germline to the next generation. Maternally inherited H3K9me3, and the histone methyltransferases (HMT) depositing it, are required for the organization of constitutive heterochromatin: early embryos lacking H3K9 methylation display de-condensation of pericentromeric regions, centromere-centromere de-clustering, mitotic defects, and nuclear shape irregularities, resulting in embryo lethality. Unexpectedly, quantitative CUT&Tag and 4D microscopy measurements of HP1a coupled with biophysical modeling revealed that H3K9me2/3 is largely dispensable for HP1a recruitment. Instead, the main function of H3K9me2/3 at this developmental stage is to drive HP1a clustering and subsequent heterochromatin compaction. Our results show that HP1a binding to constitutive heterochromatin in the absence of H3K9me2/3 is not sufficient to promote proper embryo development and heterochromatin formation. The loss of H3K9 HMTs and H3K9 methylation alters genome organization and hinders embryonic development.

Hyperactive CDK2 Activity in Basal-like Breast Cancer Imposes a Genome Integrity Liability that Can Be Exploited by Targeting DNA Polymerase ε.

Knowledge of fundamental differences between breast cancer subtypes has driven therapeutic advances; however, basal-like breast cancer (BLBC) remains clinically intractable. Because BLBC exhibits alterations in DNA repair enzymes and cell-cycle checkpoints, elucidation of factors enabling the genomic instability present in this subtype has the potential to reveal novel anti-cancer strategies. Here, we demonstrate that BLBC is especially sensitive to suppression of iron-sulfur cluster (ISC) biosynthesis and identify DNA polymerase epsilon (POLE) as an ISC-containing protein that underlies this phenotype. In BLBC cells, POLE suppression leads to replication fork stalling, DNA damage, and a senescence-like state or cell death. In contrast, luminal breast cancer and non-transformed mammary cells maintain viability upon POLE suppression but become dependent upon an ATR/CHK1/CDC25A/CDK2 DNA damage response axis. We find that CDK1/2 targets exhibit hyperphosphorylation selectively in BLBC tumors, indicating that CDK2 hyperactivity is a genome integrity vulnerability exploitable by targeting POLE.

Maintaining Iron Homeostasis Is the Key Role of Lysosomal Acidity for Cell Proliferation.

The lysosome is an acidic multi-functional organelle with roles in macromolecular digestion, nutrient sensing, and signaling. However, why cells require acidic lysosomes to proliferate and which nutrients become limiting under lysosomal dysfunction are unclear. To address this, we performed CRISPR-Cas9-based genetic screens and identified cholesterol biosynthesis and iron uptake as essential metabolic pathways when lysosomal pH is altered. While cholesterol synthesis is only necessary, iron is both necessary and sufficient for cell proliferation under lysosomal dysfunction. Remarkably, iron supplementation restores cell proliferation under both pharmacologic and genetic-mediated lysosomal dysfunction. The rescue was independent of metabolic or signaling changes classically associated with increased lysosomal pH, uncoupling lysosomal function from cell proliferation. Finally, our experiments revealed that lysosomal dysfunction dramatically alters mitochondrial metabolism and hypoxia inducible factor (HIF) signaling due to iron depletion. Altogether, these findings identify iron homeostasis as the key function of lysosomal acidity for cell proliferation.

Sequence-Independent Self-Assembly of Germ Granule mRNAs into Homotypic Clusters.

mRNAs enriched in membraneless condensates provide functional compartmentalization within cells. The mechanisms that recruit transcripts to condensates are under intense study; however, how mRNAs organize once they reach a granule remains poorly understood. Here, we report on a self-sorting mechanism by which multiple mRNAs derived from the same gene assemble into discrete homotypic clusters. We demonstrate that in vivo mRNA localization to granules and self-assembly within granules are governed by different mRNA features: localization is encoded by specific RNA regions, whereas self-assembly involves the entire mRNA, does not involve sequence-specific, ordered intermolecular RNA:RNA interactions, and is thus RNA sequence independent. We propose that the ability of mRNAs to self-sort into homotypic assemblies is an inherent property of an messenger ribonucleoprotein (mRNP) that is augmented under conditions that increase RNA concentration, such as upon enrichment in RNA-protein granules, a process that appears conserved in diverse cellular contexts and organisms.

Estimating the reproduction number and transmission heterogeneity from the size distribution of clusters of identical pathogen sequences.

Quantifying transmission intensity and heterogeneity is crucial to ascertain the threat posed by infectious diseases and inform the design of interventions. Methods that jointly estimate the reproduction number R and the dispersion parameter k have however mainly remained limited to the analysis of epidemiological clusters or contact tracing data, whose collection often proves difficult. Here, we show that clusters of identical sequences are imprinted by the pathogen offspring distribution, and we derive an analytical formula for the distribution of the size of these clusters. We develop and evaluate an inference framework to jointly estimate the reproduction number and the dispersion parameter from the size distribution of clusters of identical sequences. We then illustrate its application across a range of epidemiological situations. Finally, we develop a hypothesis testing framework relying on clusters of identical sequences to determine whether a given pathogen genetic subpopulation is associated with increased or reduced transmissibility. Our work provides tools to estimate the reproduction number and transmission heterogeneity from pathogen sequences without building a phylogenetic tree, thus making it easily scalable to large pathogen genome datasets.

Exotic self-assembly of hard spheres in a morphometric solvent.

The self-assembly of spheres into geometric structures, under various theoretical conditions, offers valuable insights into complex self-assembly processes in soft systems. Previous studies have utilized pair potentials between spheres to assemble maximum contact clusters in simulations and experiments. The morphometric approach to solvation free energy that we utilize here goes beyond pair potentials; it is a geometry-based theory that incorporates a weighted combination of geometric measures over the solvent accessible surface for solute configurations in a solvent. In this paper, we demonstrate that employing the morphometric model of solvation free energy in simulating the self-assembly of sphere clusters results, under most conditions, in the previously observed maximum contact clusters. Under other conditions, it unveils an assortment of extraordinary sphere configurations, such as double helices and rhombohedra. These exotic structures arise specifically under conditions where the interactions take multibody potentials into account. This investigation establishes a foundation for comprehending the diverse range of geometric forms in self-assembled structures, emphasizing the significance of the morphometric approach in this context.

Photomolecular effect: Visible light interaction with air-water interface.

Although water is almost transparent to visible light, we demonstrate that the air-water interface interacts strongly with visible light via what we hypothesize as the photomolecular effect. In this effect, transverse-magnetic polarized photons cleave off water clusters from the air-water interface. We use 14 different experiments to demonstrate the existence of this effect and its dependence on the wavelength, incident angle, and polarization of visible light. We further demonstrate that visible light heats up thin fogs, suggesting that this process can impact weather, climate, and the earth's water cycle and that it provides a mechanism to resolve the long-standing puzzle of larger measured clouds absorption to solar radiation than theory could predict based on bulk water optical constants. Our study suggests that the photomolecular effect should happen widely in nature, from clouds to fogs, ocean to soil surfaces, and plant transpiration and can also lead to applications in energy and clean water.

Specialization of the Drosophila nuclear export family protein Nxf3 for piRNA precursor export.

The PIWI-interacting RNA (piRNA) pathway is a conserved small RNA-based immune system that protects animal germ cell genomes from the harmful effects of transposon mobilization. In Drosophila ovaries, most piRNAs originate from dual-strand clusters, which generate piRNAs from both genomic strands. Dual-strand clusters use noncanonical transcription mechanisms. Although transcribed by RNA polymerase II, cluster transcripts lack splicing signatures and poly(A) tails. mRNA processing is important for general mRNA export mediated by nuclear export factor 1 (Nxf1). Although UAP56, a component of the transcription and export complex, has been implicated in piRNA precursor export, it remains unknown how dual-strand cluster transcripts are specifically targeted for piRNA biogenesis by export from the nucleus to cytoplasmic processing centers. Here we report that dual-strand cluster transcript export requires CG13741/Bootlegger and the Drosophila nuclear export factor family protein Nxf3. Bootlegger is specifically recruited to piRNA clusters and in turn brings Nxf3. We found that Nxf3 specifically binds to piRNA precursors and is essential for their export to piRNA biogenesis sites, a process that is critical for germline transposon silencing. Our data shed light on how dual-strand clusters compensate for a lack of canonical features of mature mRNAs to be specifically exported via Nxf3, ensuring proper piRNA production.

Nxf3: a middleman with the right connections for unspliced piRNA precursor export.

RNA export is tightly coupled to splicing in metazoans. In the Drosophila germline, precursors for the majority of Piwi-interacting RNAs (piRNAs) are unspliced. In this issue of Genes & Development, Kneuss and colleagues (pp. 1208-1220) identify Nxf3 as a novel germline-specific export adapter for such unspliced transcripts. Their findings reveal the sequence of events leading from its role at the site of transcription to delivery of the cargo to cytoplasmic piRNA biogenesis sites.

piRNA-Guided Genome Defense: From Biogenesis to Silencing.

PIWI-interacting RNAs (piRNAs) and their associated PIWI clade Argonaute proteins constitute the core of the piRNA pathway. In gonadal cells, this conserved pathway is crucial for genome defense, and its main function is to silence transposable elements. This is achieved through posttranscriptional and transcriptional gene silencing. Precursors that give rise to piRNAs require specialized transcription and transport machineries because piRNA biogenesis is a cytoplasmic process. The ping-pong cycle, a posttranscriptional silencing mechanism, combines the cleavage-dependent silencing of transposon RNAs with piRNA production. PIWI proteins also function in the nucleus, where they scan for nascent target transcripts with sequence complementarity, instructing transcriptional silencing and deposition of repressive chromatin marks at transposon loci. Although studies have revealed numerous factors that participate in each branch of the piRNA pathway, the precise molecular roles of these factors often remain unclear. In this review, we summarize our current understanding of the mechanisms involved in piRNA biogenesis and function.

Facile and dynamic cleavage of every iron-sulfide bond in cuboidal iron-sulfur clusters.

Nature employs weak-field metalloclusters to support a wide range of biological processes. The most ubiquitous metalloclusters are the cuboidal Fe-S clusters, which are comprised of Fe sites with locally high-spin electronic configurations. Such configurations enhance rates of ligand exchange and imbue the clusters with a degree of structural plasticity that is increasingly thought to be functionally relevant. Here, we examine this phenomenon using isotope tracing experiments. Specifically, we demonstrate that synthetic [Fe4S4] and [MoFe3S4] clusters exchange their Fe atoms with Fe2+ ions dissolved in solution, a process that involves the reversible cleavage and reformation of every Fe-S bond in the cluster core. This exchange is facile-in most cases occurring at room temperature on the timescale of minutes-and documented over a range of cluster core oxidation states and terminal ligation patterns. In addition to suggesting a highly dynamic picture of cluster structure, these results provide a method for isotopically labeling pre-formed clusters with spin-active nuclei, such as 57Fe. Such a protocol is demonstrated for the radical S-adenosyl-l-methionine enzyme, RlmN.