Dynamic co-evolution of transposable elements and the piRNA pathway in African cichlid fishes.

East African cichlid fishes have diversified in an explosive fashion, but the (epi)genetic basis of the phenotypic diversity of these fishes remains largely unknown. Although transposable elements (TEs) have been associated with phenotypic variation in cichlids, little is known about their transcriptional activity and epigenetic silencing. Here, we describe dynamic patterns of TE expression in African cichlid gonads and during early development. Orthology inference revealed an expansion of piwil1 genes in Lake Malawi cichlids, likely driven by PiggyBac TEs. The expanded piwil1 copies have signatures of positive selection and retain amino acid residues essential for catalytic activity. Furthermore, the gonads of African cichlids express a Piwi-interacting RNA (piRNA) pathway that target TEs. We define the genomic sites of piRNA production in African cichlids and find divergence in closely related species, in line with fast evolution of piRNA-producing loci. Our findings suggest dynamic co-evolution of TEs and host silencing pathways in the African cichlid radiations. We propose that this co-evolution has contributed to cichlid genomic diversity.

The protein phosphatase-2A subunit PR130 is involved in the formation of cytotoxic protein aggregates in pancreatic ductal adenocarcinoma cells.

As a major source of cellular serine and threonine phosphatase activity, protein phosphatase-2A (PP2A) modulates signaling pathways in health and disease. PP2A complexes consist of catalytic, scaffolding, and B-type subunits. Seventeen PP2A B-type subunits direct PP2A complexes to selected substrates. It is ill-defined how PP2A B-type subunits determine the growth and drug responsiveness of tumor cells. Pancreatic ductal adenocarcinoma (PDAC) is a disease with poor prognosis. We analyzed the responses of murine and human mesenchymal and epithelial PDAC cells to the specific PP2A inhibitor phendione. We assessed protein levels by immunoblot and proteomics and cell fate by flow cytometry, confocal microscopy, and genetic manipulation. We show that murine mesenchymal PDAC cells express significantly higher levels of the PP2A B-type subunit PR130 than epithelial PDAC cells. This overexpression of PR130 is associated with a dependency of such metastasis-prone cells on the catalytic activity of PP2A. Phendione induces apoptosis and an accumulation of cytotoxic protein aggregates in murine mesenchymal and human PDAC cells. These processes occur independently of the frequently mutated tumor suppressor p53. Proteomic analyses reveal that phendione upregulates the chaperone HSP70 in mesenchymal PDAC cells. Inhibition of HSP70 promotes phendione-induced apoptosis and phendione promotes a proteasomal degradation of PR130. Genetic elimination of PR130 sensitizes murine and human PDAC cells to phendione-induced apoptosis and protein aggregate formation. These data suggest that the PP2A-PR130 complex dephosphorylates and thereby prevents the aggregation of proteins in tumor cells.

The TELOMERE REPEAT BINDING proteins TRB4 and TRB5 function as transcriptional activators of PRC2-controlled genes to regulate plant development.

Plant-specific transcriptional regulators called TELOMERE REPEAT BINDING proteins (TRBs) combine two DNA-binding domains, the GH1 domain, which binds to linker DNA and is shared with H1 histones, and the Myb/SANT domain, which specifically recognizes the telobox DNA-binding site motif. TRB1, TRB2, and TRB3 proteins recruit Polycomb group complex 2 (PRC2) to deposit H3K27me3 and JMJ14 to remove H3K4me3 at gene promoters containing telobox motifs to repress transcription. Here, we demonstrate that TRB4 and TRB5, two related paralogs belonging to a separate TRB clade conserved in spermatophytes, regulate the transcription of several hundred genes involved in developmental responses to environmental cues. TRB4 binds to several thousand sites in the genome, mainly at transcription start sites and promoter regions of transcriptionally active and H3K4me3-marked genes, but, unlike TRB1, it is not enriched at H3K27me3-marked gene bodies. However, TRB4 can physically interact with the catalytic components of PRC2, SWINGER, and CURLY LEAF (CLF). Unexpectedly, we show that TRB4 and TRB5 are required for distinctive phenotypic traits observed in clf mutant plants and thus function as transcriptional activators of several hundred CLF-controlled genes, including key flowering genes. We further demonstrate that TRB4 shares multiple target genes with TRB1 and physically and genetically interacts with members of both TRB clades. Collectively, these results reveal that TRB proteins engage in both positive and negative interactions with other members of the family to regulate plant development through both PRC2-dependent and -independent mechanisms.

The Proteomic Composition and Organization of Constitutive Heterochromatin in Mouse Tissues.

Pericentric heterochromatin (PCH) forms spatio-temporarily distinct compartments and affects chromosome organization and stability. Albeit some of its components are known, an elucidation of its proteome and how it differs between tissues in vivo is lacking. Here, we find that PCH compartments are dynamically organized in a tissue-specific manner, possibly reflecting compositional differences. As the mouse brain and liver exhibit very different PCH architecture, we isolated native PCH fractions from these tissues, analyzed their protein compositions using quantitative mass spectrometry, and compared them to identify common and tissue-specific PCH proteins. In addition to heterochromatin-enriched proteins, the PCH proteome includes RNA/transcription and membrane-related proteins, which showed lower abundance than PCH-enriched proteins. Thus, we applied a cut-off of PCH-unspecific candidates based on their abundance and validated PCH-enriched proteins. Amongst the hits, MeCP2 was classified into brain PCH-enriched proteins, while linker histone H1 was not. We found that H1 and MeCP2 compete to bind to PCH and regulate PCH organization in opposite ways. Altogether, our workflow of unbiased PCH isolation, quantitative mass spectrometry, and validation-based analysis allowed the identification of proteins that are common and tissue-specifically enriched at PCH. Further investigation of selected hits revealed their opposing role in heterochromatin higher-order architecture in vivo.

ZNF524 directly interacts with telomeric DNA and supports telomere integrity.

Telomeres are nucleoprotein structures at the ends of linear chromosomes. In humans, they consist of TTAGGG repeats, which are bound by dedicated proteins such as the shelterin complex. This complex blocks unwanted DNA damage repair at telomeres, e.g. by suppressing nonhomologous end joining (NHEJ) through its subunit TRF2. Here, we describe ZNF524, a zinc finger protein that directly binds telomeric repeats with nanomolar affinity, and reveal base-specific sequence recognition by cocrystallization with telomeric DNA. ZNF524 localizes to telomeres and specifically maintains the presence of the TRF2/RAP1 subcomplex at telomeres without affecting other shelterin members. Loss of ZNF524 concomitantly results in an increase in DNA damage signaling and recombination events. Overall, ZNF524 is a direct telomere-binding protein involved in the maintenance of telomere integrity.

E4F1 and ZNF148 are transcriptional activators of the -57A > C and wild-type TERT promoter.

Point mutations within the TERT promoter are the most recurrent somatic noncoding mutations identified across different cancer types, including glioblastoma, melanoma, hepatocellular carcinoma, and bladder cancer. They are most abundant at -146C > T and -124C > T, and rarer at -57A > C, with the latter originally described as a familial case, but subsequently shown also to occur somatically. All three mutations create de novo E26-specific (ETS) binding sites and result in activation of the TERT gene, allowing cancer cells to achieve replicative immortality. Here, we used a systematic proteomics screen to identify transcription factors preferentially binding to the -146C > T, -124C > T, and -57A > C mutations. Although we confirmed binding of multiple ETS factors to the mutant -146C > T and -124C > T sequences, we identified E4F1 as a -57A > C-specific binder and ZNF148 as a TERT wild-type (WT) promoter binder that showed reduced interaction with the -124C > T allele. Both proteins are activating transcription factors that bind specifically to the -57A > C and WT (at position 124) TERT promoter sequence in corresponding cell lines, and up-regulate TERT transcription and telomerase activity. Our work describes new regulators of TERT gene expression with possible roles in cancer.

Long live the host! Proteomic analysis reveals possible strategies for parasitic manipulation of its social host.

Parasites with complex life cycles often manipulate the phenotype of their intermediate hosts to increase the probability of transmission to their definitive hosts. Infection with Anomotaenia brevis, a cestode that uses Temnothorax nylanderi ants as intermediate hosts, leads to a multiple-fold extension of host lifespan and to changes in behaviour, morphology and colouration. The mechanisms behind these changes are unknown, as is whether the increased longevity is achieved through parasite manipulation. Here, we demonstrate that the parasite releases proteins into its host with functions that might explain the observed changes. These parasitic proteins make up a substantial portion of the proteome of the hosts' haemolymph, and thioredoxin peroxidase and superoxide dismutase, two antioxidants, exhibited the highest abundances among them. The largest part of the secreted proteins could not be annotated, indicating they are either novel or severely altered during recent coevolution to function in host manipulation. We also detected shifts in the hosts' proteome with infection, in particular an overabundance of vitellogenin-like A in infected ants, a protein that regulates division of labour in Temnothorax ants, which could explain the observed behavioural changes. Our results thus suggest two different strategies that might be employed by this parasite to manipulate its host: secreting proteins with immediate influence on the host's phenotype and altering the host's translational activity. Our findings highlight the intricate molecular interplay required to influence the phenotype of a host and point to potential signalling pathways and genes involved in parasite-host communication.

AlexandrusPS: A User-Friendly Pipeline for the Automated Detection of Orthologous Gene Clusters and Subsequent Positive Selection Analysis.

The detection of adaptive selection in a system approach considering all protein-coding genes allows for the identification of mechanisms and pathways that enabled adaptation to different environments. Currently, available programs for the estimation of positive selection signals can be divided into two groups. They are either easy to apply but can analyze only one gene family at a time, restricting system analysis; or they can handle larger cohorts of gene families, but require considerable prerequisite data such as orthology associations, codon alignments, phylogenetic trees, and proper configuration files. All these steps require extensive computational expertise, restricting this endeavor to specialists. Here, we introduce AlexandrusPS, a high-throughput pipeline that overcomes technical challenges when conducting transcriptome-wide positive selection analyses on large sets of nucleotide and protein sequences. The pipeline streamlines 1) the execution of an accurate orthology prediction as a precondition for positive selection analysis, 2) preparing and organizing configuration files for CodeML, 3) performing positive selection analysis using CodeML, and 4) generating an output that is easy to interpret, including all maximum likelihood and log-likelihood test results. The only input needed from the user is the CDS and peptide FASTA files of proteins of interest. The pipeline is provided in a Docker image, requiring no program or module installation, enabling the application of the pipeline in any computing environment. AlexandrusPS and its documentation are available via GitHub (https://github.com/alejocn5/AlexandrusPS).

Mitochondrion of the Trypanosoma brucei long slender bloodstream form is capable of ATP production by substrate-level phosphorylation.

The long slender bloodstream form Trypanosoma brucei maintains its essential mitochondrial membrane potential (ΔΨm) through the proton-pumping activity of the FoF1-ATP synthase operating in the reverse mode. The ATP that drives this hydrolytic reaction has long been thought to be generated by glycolysis and imported from the cytosol via an ATP/ADP carrier (AAC). Indeed, we demonstrate that AAC is the only carrier that can import ATP into the mitochondrial matrix to power the hydrolytic activity of the FoF1-ATP synthase. However, contrary to expectations, the deletion of AAC has no effect on parasite growth, virulence or levels of ΔΨm. This suggests that ATP is produced by substrate-level phosphorylation pathways in the mitochondrion. Therefore, we knocked out the succinyl-CoA synthetase (SCS) gene, a key mitochondrial enzyme that produces ATP through substrate-level phosphorylation in this parasite. Its absence resulted in changes to the metabolic landscape of the parasite, lowered virulence, and reduced mitochondrial ATP content. Strikingly, these SCS mutant parasites become more dependent on AAC as demonstrated by a 25-fold increase in their sensitivity to the AAC inhibitor, carboxyatractyloside. Since the parasites were able to adapt to the loss of SCS in culture, we also analyzed the more immediate phenotypes that manifest when SCS expression is rapidly suppressed by RNAi. Importantly, when performed under nutrient-limited conditions mimicking various host environments, SCS depletion strongly affected parasite growth and levels of ΔΨm. In totality, the data establish that the long slender bloodstream form mitochondrion is capable of generating ATP via substrate-level phosphorylation pathways.

piRNA processing by a trimeric Schlafen-domain nuclease.

Transposable elements are genomic parasites that expand within and spread between genomes1. PIWI proteins control transposon activity, notably in the germline2,3. These proteins recognize their targets through small RNA co-factors named PIWI-interacting RNAs (piRNAs), making piRNA biogenesis a key specificity-determining step in this crucial genome immunity system. Although the processing of piRNA precursors is an essential step in this process, many of the molecular details remain unclear. Here, we identify an endoribonuclease, precursor of 21U RNA 5'-end cleavage holoenzyme (PUCH), that initiates piRNA processing in the nematode Caenorhabditis elegans. Genetic and biochemical studies show that PUCH, a trimer of Schlafen-like-domain proteins (SLFL proteins), executes 5'-end piRNA precursor cleavage. PUCH-mediated processing strictly requires a 7-methyl-G cap (m7G-cap) and a uracil at position three. We also demonstrate how PUCH interacts with PETISCO, a complex that binds to piRNA precursors4, and that this interaction enhances piRNA production in vivo. The identification of PUCH concludes the search for the 5'-end piRNA biogenesis factor in C. elegans and uncovers a type of RNA endonuclease formed by three SLFL proteins. Mammalian Schlafen (SLFN) genes have been associated with immunity5, exposing a molecular link between immune responses in mammals and deeply conserved RNA-based mechanisms that control transposable elements.

The sex-specific factor SOA controls dosage compensation in Anopheles mosquitoes.

The Anopheles mosquito is one of thousands of species in which sex differences play a central part in their biology, as only females need a blood meal to produce eggs. Sex differentiation is regulated by sex chromosomes, but their presence creates a dosage imbalance between males (XY) and females (XX). Dosage compensation (DC) can re-equilibrate the expression of sex chromosomal genes. However, because DC mechanisms have only been fully characterized in a few model organisms, key questions about its evolutionary diversity and functional necessity remain unresolved1. Here we report the discovery of a previously uncharacterized gene (sex chromosome activation (SOA)) as a master regulator of DC in the malaria mosquito Anopheles gambiae. Sex-specific alternative splicing prevents functional SOA protein expression in females. The male isoform encodes a DNA-binding protein that binds the promoters of active X chromosomal genes. Expressing male SOA is sufficient to induce DC in female cells. Male mosquitoes lacking SOA or female mosquitoes ectopically expressing the male isoform exhibit X chromosome misregulation, which is compatible with viability but causes developmental delay. Thus, our molecular analyses of a DC master regulator in a non-model organism elucidates the evolutionary steps that lead to the establishment of a chromosome-specific fine-tuning mechanism.

Proteome changes of fibroblasts and endothelial cells upon incubation with human cytomegalovirus subviral Dense Bodies.

Human cytomegalovirus (HCMV) is a pathogen of high medical relevance. Subviral Dense Bodies (DB) were developed as a vaccine candidate to ameliorate the severe consequences of HCMV infection. Development of such a candidate vaccine for human application requires detailed knowledge of its interaction with the host. A comprehensive mass spectrometry (MS)- based analysis was performed regarding the changes in the proteome of cell culture cells, exposed to DB.

Characterization of two novel proteins involved in mitochondrial DNA anchoring in Trypanosoma brucei.

Trypanosoma brucei is a single celled eukaryotic parasite in the group of the Kinetoplastea. The parasite harbors a single mitochondrion with a singular mitochondrial genome that is known as the kinetoplast DNA (kDNA). The kDNA consists of a unique network of thousands of interlocked circular DNA molecules. To ensure proper inheritance of the kDNA to the daughter cells, the genome is physically linked to the basal body, the master organizer of the cell cycle in trypanosomes. The connection that spans, cytoplasm, mitochondrial membranes and the mitochondrial matrix is mediated by the Tripartite Attachment Complex (TAC). Using a combination of proteomics and RNAi we test the current model of hierarchical TAC assembly and identify TbmtHMG44 and TbKAP68 as novel candidates of a complex that connects the TAC to the kDNA. Depletion of TbmtHMG44 or TbKAP68 each leads to a strong kDNA loss but not missegregation phenotype as previously defined for TAC components. We demonstrate that the proteins rely on both the TAC and the kDNA for stable localization to the interface between these two structures. In vitro experiments suggest a direct interaction between TbmtHMG44 and TbKAP68 and that recombinant TbKAP68 is a DNA binding protein. We thus propose that TbmtHMG44 and TbKAP68 are part of a distinct complex connecting the kDNA to the TAC.

DNA damage repair proteins across the Tree of Life.

Genome maintenance is orchestrated by a highly regulated DNA damage response with specific DNA repair pathways. Here, we investigate the phylogenetic diversity in the recognition and repair of three well-established DNA lesions, primarily repaired by base excision repair (BER) and ribonucleotide excision repair (RER): (1) 8-oxoguanine, (2) abasic site, and (3) incorporated ribonucleotide in DNA in 11 species: Escherichia coli, Bacillus subtilis, Halobacterium salinarum, Trypanosoma brucei, Tetrahymena thermophila, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Caenorhabditis elegans, Homo sapiens, Arabidopsis thaliana, and Zea mays. Using quantitative mass spectrometry, we identified 337 binding proteins across these species. Of these proteins, 99 were previously characterized to be involved in DNA repair. Through orthology, network, and domain analysis, we linked 44 previously unconnected proteins to DNA repair. Our study presents a resource for future study of the crosstalk and evolutionary conservation of DNA damage repair across all domains of life.

RNA-dependent interactome allows network-based assignment of RNA-binding protein function.

RNA-binding proteins (RBPs) form highly diverse and dynamic ribonucleoprotein complexes, whose functions determine the molecular fate of the bound RNA. In the model organism Sacchromyces cerevisiae, the number of proteins identified as RBPs has greatly increased over the last decade. However, the cellular function of most of these novel RBPs remains largely unexplored. We used mass spectrometry-based quantitative proteomics to systematically identify protein-protein interactions (PPIs) and RNA-dependent interactions (RDIs) to create a novel dataset for 40 RBPs that are associated with the mRNA life cycle. Domain, functional and pathway enrichment analyses revealed an over-representation of RNA functionalities among the enriched interactors. Using our extensive PPI and RDI networks, we revealed putative new members of RNA-associated pathways, and highlighted potential new roles for several RBPs. Our RBP interactome resource is available through an online interactive platform as a community tool to guide further in-depth functional studies and RBP network analysis (https://www.butterlab.org/RINE).

The human sperm proteome-Toward a panel for male fertility testing.

BACKGROUND: Although male factor accounts for 40%-50% of unintended childlessness, we are far from fully understanding the detailed causes. Usually, affected men cannot even be provided with a molecular diagnosis. OBJECTIVES: We aimed at a higher resolution of the human sperm proteome for better understanding of the molecular causes of male infertility. We were particularly interested in why reduced sperm count decreases fertility despite many normal-looking spermatozoa and which proteins might be involved. MATERIAL AND METHODS: Applying mass spectrometry analysis, we qualitatively and quantitatively examined the proteomic profiles of spermatozoa from 76 men differing in fertility. Infertile men had abnormal semen parameters and were involuntarily childless. Fertile subjects exhibited normozoospermia and had fathered children without medical assistance. RESULTS: We discovered proteins from about 7000 coding genes in the human sperm proteome. These were mainly known for involvements in cellular motility, response to stimuli, adhesion, and reproduction. Numbers of sperm proteins showing at least threefold deviating abundances increased from oligozoospermia (N = 153) and oligoasthenozoospermia (N = 154) to oligoasthenoteratozoospermia (N = 368). Deregulated sperm proteins primarily engaged in flagellar assembly and sperm motility, fertilization, and male gametogenesis. Most of these participated in a larger network of male infertility genes and proteins. DISCUSSION: We expose 31 sperm proteins displaying deviant abundances under infertility, which already were known before to have fertility relevance, including ACTL9, CCIN, CFAP47, CFAP65, CFAP251 (WDR66), DNAH1, and SPEM1. We propose 18 additional sperm proteins with at least eightfold differential abundance for further testing of their diagnostic potential, such as C2orf16, CYLC1, SPATA31E1, SPATA31D1, SPATA48, EFHB (CFAP21), and FAM161A. CONCLUSION: Our results shed light on the molecular background of the dysfunctionality of the fewer spermatozoa produced in oligozoospermia and syndromes including it. The male infertility network presented may prove useful in further elucidating the molecular mechanism of male infertility.

RNA-DNA hybrids prevent resection at dysfunctional telomeres.

At critically short telomeres, stabilized TERRA RNA-DNA hybrids drive homology-directed repair (HDR) to delay replicative senescence. However, even at long- and intermediate-length telomeres, not subject to HDR, transient TERRA RNA-DNA hybrids form, suggestive of additional roles. We report that telomeric RNA-DNA hybrids prevent Exo1-mediated resection when telomeres become non-functional. We used the well-characterized cdc13-1 allele, where telomere resection can be induced in a temperature-dependent manner, to demonstrate that ssDNA generation at telomeres is either prevented or augmented when RNA-DNA hybrids are stabilized or destabilized, respectively. The viability of cdc13-1 cells is affected by the presence or absence of hybrids accordingly. Telomeric hybrids do not affect the shortening rate of bulk telomeres. We suggest that TERRA hybrids require dynamic regulation to drive HDR at short telomeres; hybrid presence may initiate HDR through replication stress, whereby their removal allows strand resection.

Nematode gene annotation by machine-learning-assisted proteotranscriptomics enables proteome-wide evolutionary analysis.

Nematodes encompass more than 24,000 described species, which were discovered in almost every ecological habitat, and make up >80% of metazoan taxonomic diversity in soils. The last common ancestor of nematodes is believed to date back to ∼650-750 million years, generating a large and phylogenetically diverse group to be explored. However, for most species high-quality gene annotations are incomprehensive or missing. Combining short-read RNA sequencing with mass spectrometry-based proteomics and machine-learning quality control in an approach called proteotranscriptomics, we improve gene annotations for nine genome-sequenced nematode species and provide new gene annotations for three additional species without genome assemblies. Emphasizing the sensitivity of our methodology, we provide evidence for two hitherto undescribed genes in the model organism Caenorhabditis elegans Extensive phylogenetic systems analysis using this comprehensive proteome annotation provides new insights into evolutionary processes of this metazoan group.

Subviral Dense Bodies of Human Cytomegalovirus Induce an Antiviral Type I Interferon Response.

(1) Background: Cells infected with the human cytomegalovirus (HCMV) produce subviral particles, termed dense bodies (DBs), both in-vitro and in-vivo. They are released from cells, comparable to infectious virions, and are enclosed by a membrane that resembles the viral envelope and mediates the entry into cells. To date, little is known about how the DB uptake influences the gene expression in target cells. The purpose of this study was to investigate the impact of DBs on cells, in the absence of a viral infection. (2) Methods: Mass spectrometry, immunoblot analyses, siRNA knockdown, and a CRISPR-CAS9 knockout, were used to investigate the changes in cellular gene expression following a DB exposure; (3) Results: A number of interferon-regulated genes (IRGs) were upregulated after the fibroblasts and endothelial cells were exposed to DBs. This upregulation was dependent on the DB entry and mediated by the type I interferon signaling through the JAK-STAT pathway. The induction of IRGs was mediated by the sensing of the DB-introduced DNA by the pattern recognition receptor cGAS. (4) Conclusions: The induction of a strong type I IFN response by DBs is a unique feature of the HCMV infection. The release of DBs may serve as a danger signal and concomitantly contribute to the induction of a strong, antiviral immune response.

Slow growing behavior in African trypanosomes during adipose tissue colonization.

When Trypanosoma brucei parasites, the causative agent of sleeping sickness, colonize the adipose tissue, they rewire gene expression. Whether this adaptation affects population behavior and disease treatment remained unknown. By using a mathematical model, we estimate that the population of adipose tissue forms (ATFs) proliferates slower than blood parasites. Analysis of the ATFs proteome, measurement of protein synthesis and proliferation rates confirm that the ATFs divide on average every 12 h, instead of 6 h in the blood. Importantly, the population of ATFs is heterogeneous with parasites doubling times ranging between 5 h and 35 h. Slow-proliferating parasites remain capable of reverting to the fast proliferation profile in blood conditions. Intravital imaging shows that ATFs are refractory to drug treatment. We propose that in adipose tissue, a subpopulation of T. brucei parasites acquire a slow growing behavior, which contributes to disease chronicity and treatment failure.