Amyloid-like Assembly Activates a Phosphatase in the Developing Drosophila Embryo.

Prion-like proteins can assume distinct conformational and physical states in the same cell. Sequence analysis suggests that prion-like proteins are prevalent in various species; however, it remains unclear what functional space they occupy in multicellular organisms. Here, we report the identification of a prion-like protein, Herzog (CG5830), through a multimodal screen in Drosophila melanogaster. Herzog functions as a membrane-associated phosphatase and controls embryonic patterning, likely being involved in TGF-ß/BMP and FGF/EGF signaling pathways. Remarkably, monomeric Herzog is enzymatically inactive and becomes active upon amyloid-like assembly. The prion-like domain of Herzog is necessary for both its assembly and membrane targeting. Removal of the prion-like domain impairs activity, while restoring assembly on the membrane using a heterologous prion-like domain and membrane-targeting motif can restore phosphatase activity. This study provides an example of a prion-like domain that allows an enzyme to gain essential functionality via amyloid-like assembly to control animal development.

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.

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.

Quantifying Nucleation In Vivo Reveals the Physical Basis of Prion-like Phase Behavior.

Protein self-assemblies modulate protein activities over biological timescales that can exceed the lifetimes of the proteins or even the cells that harbor them. We hypothesized that these timescales relate to kinetic barriers inherent to the nucleation of ordered phases. To investigate nucleation barriers in living cells, we developed distributed amphifluoric FRET (DAmFRET). DAmFRET exploits a photoconvertible fluorophore, heterogeneous expression, and large cell numbers to quantify via flow cytometry the extent of a protein's self-assembly as a function of cellular concentration. We show that kinetic barriers limit the nucleation of ordered self-assemblies and that the persistence of the barriers with respect to concentration relates to structure. Supersaturation resulting from sequence-encoded nucleation barriers gave rise to prion behavior and enabled a prion-forming protein, Sup35 PrD, to partition into dynamic intracellular condensates or to form toxic aggregates. Our results suggest that nucleation barriers govern cytoplasmic inheritance, subcellular organization, and proteotoxicity.

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.

Wnt and TGFß coordinate growth and patterning to regulate size-dependent behaviour.

Differential coordination of growth and patterning across metazoans gives rise to a diversity of sizes and shapes at tissue, organ and organismal levels. Although tissue size and tissue function can be interdependent1-5, mechanisms that coordinate size and function remain poorly understood. Planarians are regenerative flatworms that bidirectionally scale their adult body size6,7 and reproduce asexually, via transverse fission, in a size-dependent manner8-10. This model offers a robust context to address the gap in knowledge that underlies the link between size and function. Here, by generating an optimized planarian fission protocol in Schmidtea mediterranea, we show that progeny number and the frequency of fission initiation are correlated with parent size. Fission progeny size is fixed by previously unidentified mechanically vulnerable planes spaced at an absolute distance along the anterior-posterior axis. An RNA interference screen of genes for anterior-posterior patterning uncovered components of the TGFß and Wnt signalling pathways as regulators of the frequency of fission initiation rather than the position of fission planes. Finally, inhibition of Wnt and TGFß signalling during growth altered the patterning of mechanosensory neurons-a neural subpopulation that is distributed in accordance with worm size and modulates fission behaviour. Our study identifies a role for TGFß and Wnt in regulating size-dependent behaviour, and uncovers an interdependence between patterning, growth and neurological function.

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.

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.

Enzymatic modules of the SAGA chromatin-modifying complex play distinct roles in Drosophila gene expression and development.

The Spt-Ada-Gcn5-acetyltransferase (SAGA) chromatin-modifying complex is a transcriptional coactivator that contains four different modules of subunits. The intact SAGA complex has been well characterized for its function in transcription regulation and development. However, little is known about the roles of individual modules within SAGA and whether they have any SAGA-independent functions. Here we demonstrate that the two enzymatic modules of Drosophila SAGA are differently required in oogenesis. Loss of the histone acetyltransferase (HAT) activity blocks oogenesis, while loss of the H2B deubiquitinase (DUB) activity does not. However, the DUB module regulates a subset of genes in early embryogenesis, and loss of the DUB subunits causes defects in embryogenesis. ChIP-seq (chromatin immunoprecipitation [ChIP] combined with high-throughput sequencing) analysis revealed that both the DUB and HAT modules bind most SAGA target genes even though many of these targets do not require the DUB module for expression. Furthermore, we found that the DUB module can bind to chromatin and regulate transcription independently of the HAT module. Our results suggest that the DUB module has functions within SAGA and independent functions.

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.

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 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.

A suppressor of a wtf poison-antidote meiotic driver acts via mimicry of the driver's antidote.

Meiotic drivers are selfish alleles that subvert gametogenesis to increase their transmission into progeny. Drivers impose a fitness cost, putting pressure on the genome to evolve suppressors. Here we investigate the wtf gene family from Schizosaccharomyces pombe, previously shown to contain meiotic drivers in wild isolates. We discovered that wtf13 found in lab stocks is a meiotic driver. wtf13 kills spores that do not inherit it by generating both a diffusible poison and a spore-specific antidote. Additionally, we demonstrate that wtf13 is suppressed by another wtf gene, wtf18-2, that arose spontaneously in the lab and makes only an antidote. Wtf18-2 does not act indiscriminately to prevent spore destruction. Instead, it rescues only the spores that inherit wtf18-2. In this way, wtf18-2 selfishly gains a transmission advantage of its own while dampening the drive of wtf13. This establishes a novel paradigm for meiotic drive suppressors and provides insight into the mechanisms and evolution of drive systems.

Dynamic regulation of Nanog and stem cell-signaling pathways by Hoxa1 during early neuro-ectodermal differentiation of ES cells.

Homeobox a1 (Hoxa1) is one of the most rapidly induced genes in ES cell differentiation and it is the earliest expressed Hox gene in the mouse embryo. In this study, we used genomic approaches to identify Hoxa1-bound regions during early stages of ES cell differentiation into the neuro-ectoderm. Within 2 h of retinoic acid treatment, Hoxa1 is rapidly recruited to target sites that are associated with genes involved in regulation of pluripotency, and these genes display early changes in expression. The pattern of occupancy of Hoxa1 is dynamic and changes over time. At 12 h of differentiation, many sites bound at 2 h are lost and a new cohort of bound regions appears. At both time points the genome-wide mapping reveals that there is significant co-occupancy of Nanog (Nanog homeobox) and Hoxa1 on many common target sites, and these are linked to genes in the pluripotential regulatory network. In addition to shared target genes, Hoxa1 binds to regulatory regions of Nanog, and conversely Nanog binds to a 3' enhancer of Hoxa1 This finding provides evidence for direct cross-regulatory feedback between Hoxa1 and Nanog through a mechanism of mutual repression. Hoxa1 also binds to regulatory regions of Sox2 (sex-determining region Y box 2), Esrrb (estrogen-related receptor beta), and Myc, which underscores its key input into core components of the pluripotential regulatory network. We propose a model whereby direct inputs of Nanog and Hoxa1 on shared targets and mutual repression between Hoxa1 and the core pluripotency network provides a molecular mechanism that modulates the fine balance between the alternate states of pluripotency and differentiation.

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.

Ubiquitination and degradation of the hominoid-specific oncoprotein TBC1D3 is regulated by protein palmitoylation.

Expression of the hominoid-specific oncoprotein TBC1D3 promotes enhanced cell growth and proliferation by increased activation of signal transduction through several growth factors. Recently we documented the role of CUL7 E3 ligase in growth factors-induced ubiquitination and degradation of TBC1D3. Here we expanded our study to discover additional molecular mechanisms that control TBC1D3 protein turnover. We report that TBC1D3 is palmitoylated on two cysteine residues: 318 and 325. The expression of double palmitoylation mutant TBC1D3:C318/325S resulted in protein mislocalization and enhanced growth factors-induced TBC1D3 degradation. Moreover, ubiquitination of TBC1D3 via CUL7 E3 ligase complex was increased by mutating the palmitoylation sites, suggesting that depalmitoylation of TBC1D3 makes the protein more available for ubiquitination and degradation. The results reported here provide novel insights into the molecular mechanisms that govern TBC1D3 protein degradation. Dysregulation of these mechanisms in vivo could potentially result in aberrant TBC1D3 expression and promote oncogenesis.

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.

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.