An integrated structural model of the DNA damage-responsive H3K4me3 binding WDR76:SPIN1 complex with the nucleosome.

Serial capture affinity purification (SCAP) is a powerful method to isolate a specific protein complex. When combined with cross-linking mass spectrometry and computational approaches, one can build an integrated structural model of the isolated complex. Here, we applied SCAP to dissect a subpopulation of WDR76 in complex with SPIN1, a histone reader that recognizes trimethylated histone H3 lysine4 (H3K4me3). In contrast to a previous SCAP analysis of the SPIN1:SPINDOC complex, histones and the H3K4me3 mark were enriched with the WDR76:SPIN1 complex. Next, interaction network analysis of copurifying proteins and microscopy analysis revealed a potential role of the WDR76:SPIN1 complex in the DNA damage response. Since we detected 149 pairs of cross-links between WDR76, SPIN1, and histones, we then built an integrated structural model of the complex where SPIN1 recognized the H3K4me3 epigenetic mark while interacting with WDR76. Finally, we used the powerful Bayesian Integrative Modeling approach as implemented in the Integrative Modeling Platform to build a model of WDR76 and SPIN1 bound to the nucleosome.

Regulators of rDNA array morphology in fission yeast.

Nucleolar morphology is a well-established indicator of ribosome biogenesis activity that has served as the foundation of many screens investigating ribosome production. Missing from this field of study is a broad-scale investigation of the regulation of ribosomal DNA morphology, despite the essential role of rRNA gene transcription in modulating ribosome output. We hypothesized that the morphology of rDNA arrays reflects ribosome biogenesis activity. We established GapR-GFP, a prokaryotic DNA-binding protein that recognizes transcriptionally-induced overtwisted DNA, as a live visual fluorescent marker for quantitative analysis of rDNA organization in Schizosaccharomyces pombe. We found that the morphology-which we refer to as spatial organization-of the rDNA arrays is dynamic throughout the cell cycle, under glucose starvation, RNA pol I inhibition, and TOR activation. Screening the haploid S. pombe Bioneer deletion collection for spatial organization phenotypes revealed large ribosomal protein (RPL) gene deletions that alter rDNA organization. Further work revealed RPL gene deletion mutants with altered rDNA organization also demonstrate resistance to the TOR inhibitor Torin1. A genetic analysis of signaling pathways essential for this resistance phenotype implicated many factors including a conserved MAPK, Pmk1, previously linked to extracellular stress responses. We propose RPL gene deletion triggers altered rDNA morphology due to compensatory changes in ribosome biogenesis via multiple signaling pathways, and we further suggest compensatory responses may contribute to human diseases such as ribosomopathies. Altogether, GapR-GFP is a powerful tool for live visual reporting on rDNA morphology under myriad conditions.

Protein supersaturation powers innate immune signaling.

Innate immunity protects us in youth but turns against us as we age. The reason for this tradeoff is unclear. Seeking a thermodynamic basis, we focused on death fold domains (DFDs), whose ordered polymerization has been stoichiometrically linked to innate immune signal amplification. We hypothesized that soluble ensembles of DFDs function as phase change batteries that store energy via supersaturation and subsequently release it through nucleated polymerization. Using imaging and FRET-based cytometry to characterize the phase behaviors of all 109 human DFDs, we found that the hubs of innate immune signaling networks encode large nucleation barriers that are intrinsically insulated from cross-pathway activation. We showed via optogenetics that supersaturation drives signal amplification and that the inflammasome is constitutively supersaturated in vivo. Our findings reveal that the soluble "inactive" states of adaptor DFDs function as essential, yet impermanent, kinetic barriers to inflammatory cell death, suggesting a thermodynamic driving force for aging.

Pathologic polyglutamine aggregation begins with a self-poisoning polymer crystal.

A long-standing goal of amyloid research has been to characterize the structural basis of the rate-determining nucleating event. However, the ephemeral nature of nucleation has made this goal unachievable with existing biochemistry, structural biology, and computational approaches. Here, we addressed that limitation for polyglutamine (polyQ), a polypeptide sequence that causes Huntington's and other amyloid-associated neurodegenerative diseases when its length exceeds a characteristic threshold. To identify essential features of the polyQ amyloid nucleus, we used a direct intracellular reporter of self-association to quantify frequencies of amyloid appearance as a function of concentration, conformational templates, and rational polyQ sequence permutations. We found that nucleation of pathologically expanded polyQ involves segments of three glutamine (Q) residues at every other position. We demonstrate using molecular simulations that this pattern encodes a four-stranded steric zipper with interdigitated Q side chains. Once formed, the zipper poisoned its own growth by engaging naive polypeptides on orthogonal faces, in a fashion characteristic of polymer crystals with intramolecular nuclei. We further show that self-poisoning can be exploited to block amyloid formation, by genetically oligomerizing polyQ prior to nucleation. By uncovering the physical nature of the rate-limiting event for polyQ aggregation in cells, our findings elucidate the molecular etiology of polyQ diseases.

Diseases that typically occur later in life, such as Alzheimer's, are often caused by specific proteins clumping together into structures known as amyloids. Once the process starts, amyloids will continue to form, leading to worse symptoms that cannot be cured. The best way to treat these diseases is therefore to stop amyloids from arising in the first place. Amyloids initially develop by proteins coming together to create an unstable structure referred to as the nucleus. The instability of the nucleus means it cannot be observed directly, making it hard to study this nucleation process. To overcome this, Kandola, Venkatesan et al. investigated the simplest protein known to form an amyloid ­ polyglutamine, which is made up of a chain of repeating building blocks known as amino acids. Polyglutamine forms only one type of amyloid which is associated with nine neurodegenerative diseases, including Huntington's disease. However, it only does this when its chain of amino acids exceeds a certain length, suggesting that a specific structure may be required for nucleation to begin. Kandola, Venkatesan et al. made alternative versions of the polyglutamine protein which each contained slightly different sequences of amino acids that will alter the way the protein folds. They then tested how well these different variants could form amyloids in yeast cells. This revealed that in order to join together into a nucleus, polyglutamine needs to be able to fold into a zipper shape made up of four interlocking strands. The length of the protein required to form this shape is also the same length that causes the amyloid associated with neurodegenerative diseases. Kandola, Venkatesan et al. also found that polyglutamine tends to bind to nuclei that have already formed in a way that hinders their growth. This 'self-poisoning' affect could potentially be exploited as a way to pre-emptively stop amyloids from initially arising. These findings have uncovered a potential therapeutic strategy for blocking amyloid formation that could eventually benefit people with or at risk of developing neurodegenerative diseases linked to polyglutamine. Additionally, this approach provides a blueprint for understanding how other proteins undergo amyloid nucleation, including those responsible for Alzheimer's, Parkinson's, and other diseases.

Lola-I is a promoter pioneer factor that establishes de novo Pol II pausing during development.

While the accessibility of enhancers is dynamically regulated during development, promoters tend to be constitutively accessible and poised for activation by paused Pol II. By studying Lola-I, a Drosophila zinc finger transcription factor, we show here that the promoter state can also be subject to developmental regulation independently of gene activation. Lola-I is ubiquitously expressed at the end of embryogenesis and causes its target promoters to become accessible and acquire paused Pol II throughout the embryo. This promoter transition is required but not sufficient for tissue-specific target gene activation. Lola-I mediates this function by depleting promoter nucleosomes, similar to the action of pioneer factors at enhancers. These results uncover a level of regulation for promoters that is normally found at enhancers and reveal a mechanism for the de novo establishment of paused Pol II at promoters.

rRNA transcription is integral to phase separation and maintenance of nucleolar structure.

Transcription of ribosomal RNA (rRNA) by RNA Polymerase (Pol) I in the nucleolus is necessary for ribosome biogenesis, which is intimately tied to cell growth and proliferation. Perturbation of ribosome biogenesis results in tissue specific disorders termed ribosomopathies in association with alterations in nucleolar structure. However, how rRNA transcription and ribosome biogenesis regulate nucleolar structure during normal development and in the pathogenesis of disease remains poorly understood. Here we show that homozygous null mutations in Pol I subunits required for rRNA transcription and ribosome biogenesis lead to preimplantation lethality. Moreover, we discovered that Polr1a-/-, Polr1b-/-, Polr1c-/- and Polr1d-/- mutants exhibit defects in the structure of their nucleoli, as evidenced by a decrease in number of nucleolar precursor bodies and a concomitant increase in nucleolar volume, which results in a single condensed nucleolus. Pharmacological inhibition of Pol I in preimplantation and midgestation embryos, as well as in hiPSCs, similarly results in a single condensed nucleolus or fragmented nucleoli. We find that when Pol I function and rRNA transcription is inhibited, the viscosity of the granular compartment of the nucleolus increases, which disrupts its phase separation properties, leading to a single condensed nucleolus. However, if a cell progresses through mitosis, the absence of rRNA transcription prevents reassembly of the nucleolus and manifests as fragmented nucleoli. Taken together, our data suggests that Pol I function and rRNA transcription are required for maintaining nucleolar structure and integrity during development and in the pathogenesis of disease.

Shared retinoic acid responsive enhancers coordinately regulate nascent transcription of Hoxb coding and non-coding RNAs in the developing mouse neural tube.

Signaling pathways regulate the patterns of Hox gene expression that underlie their functions in the specification of axial identity. Little is known about the properties of cis-regulatory elements and underlying transcriptional mechanisms that integrate graded signaling inputs to coordinately control Hox expression. Here, we optimized a single molecule fluorescent in situ hybridization (smFISH) technique with probes spanning introns to evaluate how three shared retinoic acid response element (RARE)-dependent enhancers in the Hoxb cluster regulate patterns of nascent transcription in vivo at the level of single cells in wild-type and mutant embryos. We predominately detect nascent transcription of only a single Hoxb gene in each cell, with no evidence for simultaneous co-transcriptional coupling of all or specific subsets of genes. Single and/or compound RARE mutations indicate that each enhancer differentially impacts global and local patterns of nascent transcription, suggesting that selectivity and competitive interactions between these enhancers is important to robustly maintain the proper levels and patterns of nascent Hoxb transcription. This implies that rapid and dynamic regulatory interactions potentiate transcription of genes through combined inputs from these enhancers in coordinating the retinoic acid response.

Pathologic polyglutamine aggregation begins with a self-poisoning polymer crystal.

A long-standing goal of amyloid research has been to characterize the structural basis of the rate-determining nucleating event. However, the ephemeral nature of nucleation has made this goal unachievable with existing biochemistry, structural biology, and computational approaches. Here, we addressed that limitation for polyglutamine (polyQ), a polypeptide sequence that causes Huntington's and other amyloid-associated neurodegenerative diseases when its length exceeds a characteristic threshold. To identify essential features of the polyQ amyloid nucleus, we used a direct intracellular reporter of self-association to quantify frequencies of amyloid appearance as a function of concentration, conformational templates, and rational polyQ sequence permutations. We found that nucleation of pathologically expanded polyQ involves segments of three glutamine (Q) residues at every other position. We demonstrate using molecular simulations that this pattern encodes a four-stranded steric zipper with interdigitated Q side chains. Once formed, the zipper poisoned its own growth by engaging naive polypeptides on orthogonal faces, in a fashion characteristic of polymer crystals with intramolecular nuclei. We further show that self-poisoning can be exploited to block amyloid formation, by genetically oligomerizing polyQ prior to nucleation. By uncovering the physical nature of the rate-limiting event for polyQ aggregation in cells, our findings elucidate the molecular etiology of polyQ diseases.

Serial Capture Affinity Purification and Integrated Structural Modeling of the H3K4me3 Binding and DNA Damage Related WDR76:SPIN1 Complex.

WDR76 is a multifunctional protein involved in many cellular functions. With a diverse and complicated protein interaction network, dissecting the structure and function of specific WDR76 complexes is needed. We previously demonstrated the ability of the Serial Capture Affinity Purification (SCAP) method to isolate specific complexes by introducing two proteins of interest as baits at the same time. Here, we applied SCAP to dissect a subpopulation of WDR76 in complex with SPIN1, a histone marker reader that specifically recognizes trimethylated histone H3 lysine4 (H3K4me3). In contrast to the SCAP analysis of the SPIN1:SPINDOC complex, H3K4me3 was copurified with the WDR76:SPIN1 complex. In combination with crosslinking mass spectrometry, we built an integrated structural model of the complex which revealed that SPIN1 recognized the H3K4me3 epigenetic mark while interacting with WDR76. Lastly, interaction network analysis of copurifying proteins revealed the potential role of the WDR76:SPIN1 complex in the DNA damage response. Teaser: In contrast to the SPINDOC/SPIN1 complex, analyses reveal that the WDR76/SPIN1 complex interacts with core histones and is involved in DNA damage.

S. pombe wtf drivers use dual transcriptional regulation and selective protein exclusion from spores to cause meiotic drive.

Meiotic drivers bias gametogenesis to ensure their transmission into more than half the offspring of a heterozygote. In Schizosaccharomyces pombe, wtf meiotic drivers destroy the meiotic products (spores) that do not inherit the driver from a heterozygote, thereby reducing fertility. wtf drivers encode both a Wtfpoison protein and a Wtfantidote protein using alternative transcriptional start sites. Here, we analyze how the expression and localization of the Wtf proteins are regulated to achieve drive. We show that transcriptional timing and selective protein exclusion from developing spores ensure that all spores are exposed to Wtf4poison, but only the spores that inherit wtf4 receive a dose of Wtf4antidote sufficient for survival. In addition, we show that the Mei4 transcription factor, a master regulator of meiosis, controls the expression of the wtf4poison transcript. This transcriptional regulation, which includes the use of a critical meiotic transcription factor, likely complicates the universal suppression of wtf genes without concomitantly disrupting spore viability. We propose that these features contribute to the evolutionary success of the wtf drivers.

The wtf meiotic driver gene family has unexpectedly persisted for over 100 million years.

Meiotic drivers are selfish elements that bias their own transmission into more than half of the viable progeny produced by a driver+/driver- heterozygote. Meiotic drivers are thought to exist for relatively short evolutionary timespans because a driver gene or gene family is often found in a single species or in a group of very closely related species. Additionally, drivers are generally considered doomed to extinction when they spread to fixation or when suppressors arise. In this study, we examine the evolutionary history of the wtf meiotic drivers first discovered in the fission yeast Schizosaccharomyces pombe. We identify homologous genes in three other fission yeast species, S. octosporus, S. osmophilus, and S. cryophilus, which are estimated to have diverged over 100 million years ago from the S. pombe lineage. Synteny evidence supports that wtf genes were present in the common ancestor of these four species. Moreover, the ancestral genes were likely drivers as wtf genes in S. octosporus cause meiotic drive. Our findings indicate that meiotic drive systems can be maintained for long evolutionary timespans.

Genome-wide quantification of contributions to sexual fitness identifies genes required for spore viability and health in fission yeast.

Numerous genes required for sexual reproduction remain to be identified even in simple model species like Schizosaccharomyces pombe. To address this, we developed an assay in S. pombe that couples transposon mutagenesis with high-throughput sequencing (TN-seq) to quantitatively measure the fitness contribution of nonessential genes across the genome to sexual reproduction. This approach identified 532 genes that contribute to sex, including more than 200 that were not previously annotated to be involved in the process, of which more than 150 have orthologs in vertebrates. Among our verified hits was an uncharacterized gene, ifs1 (important for sex), that is required for spore viability. In two other hits, plb1 and alg9, we observed a novel mutant phenotype of poor spore health wherein viable spores are produced, but the spores exhibit low fitness and are rapidly outcompeted by wild type. Finally, we fortuitously discovered that a gene previously thought to be essential, sdg1 (social distancing gene), is instead required for growth at low cell densities and can be rescued by conditioned medium. Our assay will be valuable in further studies of sexual reproduction in S. pombe and identifies multiple candidate genes that could contribute to sexual reproduction in other eukaryotes, including humans.

A nucleation barrier spring-loads the CBM signalosome for binary activation.

Immune cells activate in binary, switch-like fashion via large protein assemblies known as signalosomes, but the molecular mechanism of the switch is not yet understood. Here, we employed an in-cell biophysical approach to dissect the assembly mechanism of the CARD-BCL10-MALT1 (CBM) signalosome, which governs nuclear transcription factor-κB activation in both innate and adaptive immunity. We found that the switch consists of a sequence-encoded and deeply conserved nucleation barrier to ordered polymerization by the adaptor protein BCL10. The particular structure of the BCL10 polymers did not matter for activity. Using optogenetic tools and single-cell transcriptional reporters, we discovered that endogenous BCL10 is functionally supersaturated even in unstimulated human cells, and this results in a predetermined response to stimulation upon nucleation by activated CARD multimers. Our findings may inform on the progressive nature of age-associated inflammation, and suggest that signalosome structure has evolved via selection for kinetic rather than equilibrium properties of the proteins.

The innate immune system is the body's first line of defence against pathogens. Although innate immune cells do not recognize specific disease-causing agents, they can detect extremely low levels of harmful organisms or substances. In response, they activate signals that lead to inflammation, which tells other cells that there is an infection. Innate immune cells are turned on in a switch-like fashion, becoming active very quickly after interacting with a pathogen. This is due to the action of signalosomes, large complexes made up of several proteins that clump together to form long chains that activate the cell. But how do these large protein complexes assemble quick enough to create the switch-like activation observed in innate immune cells? To answer this question, Rodríguez Gama et al. focused on the CBM signalosome, which is involved in triggering inflammation through the activation of a protein called NF-kB. First, Rodríguez Gama et al. used genetic tools to determine that activating the CBM signalosome drives a switch-like activation of NF-kB in cells. This means that individual cells in a population either become fully activated or not at all in response to minute amounts of harmful substances. Once they had established this, Rodríguez Gama et al. wanted to know which protein in the CBM signalosome was responsible for the switch. They found that one of the proteins in the signalosome, called BCL10, has a 'nucleation barrier' encoded in its sequence. This means that it is very hard for BCL10 to start clumping together, but once it does, the clumps grow on their own. The nucleation barrier describes exactly how hard it is for these clumps to get started, and is determined by how disorganized the protein is. When a pathogen 'stimulates' an immune cell, a tiny template is formed that lowers the nucleation barrier so that BCL10 can then aggregate itself together, leading to the switch-like behaviour observed. The nucleation barrier allows there to be more than enough BCL10 present in the cell at all times ­ ready to clump together at a moment's notice ­ and this permits the cell to detect very low levels of a pathogen. Rodríguez Gama et al. then tested whether BCL10 from other animals also has a nucleation barrier. They found that this feature is conserved from cnidarians, such as corals or jellyfish, to mammals, including humans. This suggests that the use of nucleation barriers to regulate innate immune signalling has existed for a long time throughout evolution. The work by Rodríguez Gama et al. broadens our understanding of how the innate immune system senses and responds to extremely low levels of pathogens. That BCL10 is always ready to clump together suggests it may be a driving force for chronic and age-associated inflammation. Additionally, the findings of Rodríguez Gama et al. also offer insights into how other signalosomes may become activated, and offer the possibility of new drugs aimed at modifying nucleation barriers.

Diverse mating phenotypes impact the spread of wtf meiotic drivers in Schizosaccharomyces pombe.

Meiotic drivers are genetic elements that break Mendel's law of segregation to be transmitted into more than half of the offspring produced by a heterozygote. The success of a driver relies on outcrossing (mating between individuals from distinct lineages) because drivers gain their advantage in heterozygotes. It is, therefore, curious that Schizosaccharomyces pombe, a species reported to rarely outcross, harbors many meiotic drivers. To address this paradox, we measured mating phenotypes in S. pombe natural isolates. We found that the propensity for cells from distinct clonal lineages to mate varies between natural isolates and can be affected both by cell density and by the available sexual partners. Additionally, we found that the observed levels of preferential mating between cells from the same clonal lineage can slow, but not prevent, the spread of a wtf meiotic driver in the absence of additional fitness costs linked to the driver. These analyses reveal parameters critical to understanding the evolution of S. pombe and help explain the success of meiotic drivers in this species.

The fission yeast, Schizosaccharomyces pombe, is a haploid organism, meaning it has a single copy of each of its genes. S. pombe cells generally carry one copy of each chromosome and can reproduce clonally by duplicating these chromosomes and then dividing into two cells. However, when the yeast are starving, they can reproduce sexually. This involves two cells mating by fusing together to create a 'diploid zygote', which contains two copies of each gene. The zygote then undergoes 'meiosis', a special type of cell division in which the zygote first duplicates its genome and then divides twice. This results in four haploid spores which are analogous to sperm and eggs in humans that each contain one copy of the genome. The spores will grow and divide normally when conditions improve. The genes carried by each of the haploid spores depend on the cells that formed the zygote. If the two 'parent' yeast had the same version or 'allele' of a gene, all four spores will have it in their genome. However, if the two parents have different alleles, only 50% of the offspring will carry each version. Although this is usually the case, there are certain alleles, called meiotic drivers, that are transmitted to all offspring even in situations where it is only carried by one parent. Meiotic drivers can be found in many organisms, including mammals, but their behavior is easiest to study in yeast. Meiotic drivers known as killers achieve this by disposing of any 'sister' spores that do not inherit the same allele of this gene. This 'killing' can only happen when only one of the 'parents' carries the driver. This scenario is thought to rarely occur in species that inbreed, as inbreeding leads to both gene copies being the same. However, this does not appear to be the case for S. pombe, which contain a whole family of killer meiotic drivers, the wtf genes, despite also being reported to mainly inbreed. To investigate this contradiction, López Hernández et al. isolated several genetically distinct populations of S.pombe. These isolates were grown together to determine how often the each one would outcross (mate with an individual from a different population) or inbreed. The results found that levels of inbreeding varied between isolates. Next, López Hernández et al. used mathematical modelling and experimental evolution analyses to study how wtf drivers spread amongst these populations. This revealed that wtf genes spread faster in populations with more outcrossing. In some instances, the wtf driver was linked to a gene that could harm the population. In these cases, López Hernández et al. found than inbreeding could purge these drivers and stop them from spreading the dangerous alleles through the population. López Hernández et al. establish a simple experimental system to model driver evolution and experimentally demonstrate how key parameters, such as outcrossing rates, affect the spread of these genes. Understanding how meiotic drivers spread is important, as these systems could potentially be used to modify populations important to humans, such as crops or disease vectors.

Hox genes regulate asexual reproductive behavior and tissue segmentation in adult animals.

Hox genes are highly conserved transcription factors renowned for their roles in the segmental patterning of the embryonic anterior-posterior (A/P) axis. We report functions for Hox genes in A/P tissue segmentation and transverse fission behavior underlying asexual reproduction in adult planarian flatworms, Schmidtea mediterranea. Silencing of each of the Hox family members identifies 5 Hox genes required for asexual reproduction. Among these, silencing of hox3 genes results in supernumerary fission segments, while silencing of post2b eliminates segmentation altogether. The opposing roles of hox3 and post2b in segmentation are paralleled in their respective regulation of fission behavior. Silencing of hox3 increases the frequency of fission behavior initiation while silencing of post2b eliminates fission behavior entirely. Furthermore, we identify a network of downstream effector genes mediating Hox gene functions, providing insight into their respective mechanisms of action. In particular, we resolve roles for post2b and effector genes in the functions of the marginal adhesive organ in fission behavior regulation. Collectively, our study establishes adult stage roles for Hox genes in the regulation of tissue segmentation and behavior associated with asexual reproduction.

The disordered regions of the methyltransferase SETD2 govern its function by regulating its proteolysis and phase separation.

SETD2 is an important methyltransferase that methylates crucial substrates such as histone H3, tubulin, and STAT1 and also physically interacts with transcription and splicing regulators such as Pol II and various hnRNPs. Of note, SETD2 has a functionally uncharacterized extended N-terminal region, the removal of which leads to its stabilization. How this region regulates SETD2 half-life is unclear. Here we show that SETD2 consists of multiple long disordered regions across its length that cumulatively destabilize the protein by facilitating its proteasomal degradation. SETD2 disordered regions can reduce the half-life of the yeast homolog Set2 in mammalian cells as well as in yeast, demonstrating the importance of intrinsic structural features in regulating protein half-life. In addition to the shortened half-life, by performing fluorescence recovery after photobleaching assay we found that SETD2 forms liquid droplets in vivo, another property associated with proteins that contain disordered regions. The phase-separation behavior of SETD2 is exacerbated upon the removal of its N-terminal segment and results in activator-independent histone H3K36 methylation. Our findings reveal that disordered region-facilitated proteolysis is an important mechanism governing SETD2 function.

Mechanistic Inferences From Analysis of Measurements of Protein Phase Transitions in Live Cells.

The combination of phase separation and disorder-to-order transitions can give rise to ordered, semi-crystalline fibrillar assemblies that underlie prion phenomena namely, the non-Mendelian transfer of information across cells. Recently, a method known as Distributed Amphifluoric Förster Resonance Energy Transfer (DAmFRET) was developed to study the convolution of phase separation and disorder-to-order transitions in live cells. In this assay, a protein of interest is expressed to a broad range of concentrations and the acquisition of local density and order, measured by changes in FRET, is used to map phase transitions for different proteins. The high-throughput nature of this assay affords the promise of uncovering sequence-to-phase behavior relationships in live cells. Here, we report the development of a supervised method to obtain automated and accurate classifications of phase transitions quantified using the DAmFRET assay. Systems that we classify as undergoing two-state discontinuous transitions are consistent with prion-like behaviors, although the converse is not always true. We uncover well-established and surprising new sequence features that contribute to two-state phase behavior of prion-like domains. Additionally, our method enables quantitative, comparative assessments of sequence-specific driving forces for phase transitions in live cells. Finally, we demonstrate that a modest augmentation of DAmFRET measurements, specifically time-dependent protein expression profiles, can allow one to apply classical nucleation theory to extract sequence-specific lower bounds on the probability of nucleating ordered assemblies. Taken together, our approaches lead to a useful analysis pipeline that enables the extraction of mechanistic inferences regarding phase transitions in live cells.

Driving integrative structural modeling with serial capture affinity purification.

Streamlined characterization of protein complexes remains a challenge for the study of protein interaction networks. Here we describe serial capture affinity purification (SCAP), in which two separate proteins are tagged with either the HaloTag or the SNAP-tag, permitting a multistep affinity enrichment of specific protein complexes. The multifunctional capabilities of this protein-tagging system also permit in vivo validation of interactions using acceptor photobleaching Förster resonance energy transfer and fluorescence cross-correlation spectroscopy quantitative imaging. By coupling SCAP to cross-linking mass spectrometry, an integrative structural model of the complex of interest can be generated. We demonstrate this approach using the Spindlin1 and SPINDOC protein complex, culminating in a structural model with two SPINDOC molecules docked on one SPIN1 molecule. In this model, SPINDOC interacts with the SPIN1 interface previously shown to bind a lysine and arginine methylated sequence of histone H3. Our approach combines serial affinity purification, live cell imaging, and cross-linking mass spectrometry to build integrative structural models of protein complexes.

The wtf4 meiotic driver utilizes controlled protein aggregation to generate selective cell death.

Meiotic drivers are parasitic loci that force their own transmission into greater than half of the offspring of a heterozygote. Many drivers have been identified, but their molecular mechanisms are largely unknown. The wtf4 gene is a meiotic driver in Schizosaccharomyces pombe that uses a poison-antidote mechanism to selectively kill meiotic products (spores) that do not inherit wtf4. Here, we show that the Wtf4 proteins can function outside of gametogenesis and in a distantly related species, Saccharomyces cerevisiae. The Wtf4poison protein forms dispersed, toxic aggregates. The Wtf4antidote can co-assemble with the Wtf4poison and promote its trafficking to vacuoles. We show that neutralization of the Wtf4poison requires both co-assembly with the Wtf4antidote and aggregate trafficking, as mutations that disrupt either of these processes result in cell death in the presence of the Wtf4 proteins. This work reveals that wtf parasites can exploit protein aggregate management pathways to selectively destroy spores.

Meiotic drivers are genes that break the normal rules of inheritance. Usually, a gene has a 50% chance of passing on to the next generation. Meiotic drivers force their way into the next generation by poisoning the gametes (the sex cells that combine to form a zygote) that do not carry them. Harnessing the power of genetic drivers could allow scientists to spread beneficial genes across populations. One group of meiotic drivers found in fission yeast is called the 'with transposon fission yeast', or 'wtf' gene family. The wtf drivers act during the production of spores, which are the fission yeast equivalent of sperm, and they encode both a poison that can destroy the spores and its antidote. The poison spreads through the sac holding the spores, and can affect all of them, while the antidote only protects the spores that make it. This means that the spores carrying the wtf genes survive, while the rest of the spores are killed. To understand whether it is possible to use the wtf meiotic drivers to spread other genes, perhaps outside of fission yeast, scientists must first establish exactly how the proteins coded for by genes behave. To do this, Nuckolls et al. examined a member of the wtf family called wtf4. Attaching a fluorescent label to the poison and antidote proteins produced by wtf4 made it possible to see what they do. This revealed that the poison clumps, forming toxic aggregates that damage yeast spores. The antidote works by mopping up these aggregates and moving them to the cell's main storage compartment, called the vacuole. Mutations that disrupted the ability of the antidote to interact with the poison or its ability to move the poison into storage stopped the antidote from working. Nuckolls et al. also showed that if genetic engineering was used to introduce wtf4 into a distantly related species of budding yeast the effects of this meiotic driver were the same. This suggests that the wtf genes may be good candidates for future genetic engineering experiments. Engineered systems known as 'gene drives' could spread beneficial genetic traits through populations. This could include disease-resistance genes in crops, or disease-preventing genes in mosquitoes. The wtf genes are small and work independently of other genes, making them promising candidates for this type of system. These experiments also suggest that the wtf genes could be useful for understanding why clumps of proteins are toxic to cells. Future work could explore why clumps of wtf poison kill spores, while clumps of poison plus antidote do not. This could aid research into human ailments caused by protein clumps, such as Huntington's or Alzheimer's disease.

Dramatically diverse Schizosaccharomyces pombe wtf meiotic drivers all display high gamete-killing efficiency.

Meiotic drivers are selfish alleles that can force their transmission into more than 50% of the viable gametes made by heterozygotes. Meiotic drivers are known to cause infertility in a diverse range of eukaryotes and are predicted to affect the evolution of genome structure and meiosis. The wtf gene family of Schizosaccharomyces pombe includes both meiotic drivers and drive suppressors and thus offers a tractable model organism to study drive systems. Currently, only a handful of wtf genes have been functionally characterized and those genes only partially reflect the diversity of the wtf gene family. In this work, we functionally test 22 additional wtf genes for meiotic drive phenotypes. We identify eight new drivers that share between 30-90% amino acid identity with previously characterized drivers. Despite the vast divergence between these genes, they generally drive into >85% of gametes when heterozygous. We also identify three wtf genes that suppress other wtf drivers, including two that also act as autonomous drivers. Additionally, we find that wtf genes do not underlie a weak (64% allele transmission) meiotic driver on chromosome 1. Finally, we find that some Wtf proteins have expression or localization patterns that are distinct from the poison and antidote proteins encoded by drivers and suppressors, suggesting some wtf genes may have non-meiotic drive functions. Overall, this work expands our understanding of the wtf gene family and the burden selfish driver genes impose on S. pombe.