Regulation of alternative polyadenylation by the C2H2-zinc-finger protein Sp1.

Alternative polyadenylation (APA) enhances gene regulatory potential by increasing the diversity of mRNA transcripts. 3' UTR shortening through APA correlates with enhanced cellular proliferation and is a widespread phenomenon in tumor cells. Here, we show that the ubiquitously expressed transcription factor Sp1 binds RNA in vivo and is a common repressor of distal poly(A) site usage. RNA sequencing identified 2,344 genes (36% of the total mapped mRNA transcripts) with lengthened 3' UTRs upon Sp1 depletion. Sp1 preferentially binds the 3' UTRs of such lengthened transcripts and inhibits cleavage at distal sites by interacting with the subunits of the core cleavage and polyadenylation (CPA) machinery. The 3' UTR lengths of Sp1 target genes in breast cancer patient RNA-seq data correlate with Sp1 expression levels, implicating Sp1-mediated APA regulation in modulating tumorigenic properties. Taken together, our findings provide insights into the mechanism for dynamic APA regulation by unraveling a previously unknown function of the DNA-binding transcription factor Sp1.

Identifying human pre-mRNA cleavage and polyadenylation factors by genome-wide CRISPR screens using a dual fluorescence readthrough reporter.

Messenger RNA precursors (pre-mRNA) generally undergo 3' end processing by cleavage and polyadenylation (CPA), which is specified by a polyadenylation site (PAS) and adjacent RNA sequences and regulated by a large variety of core and auxiliary CPA factors. To date, most of the human CPA factors have been discovered through biochemical and proteomic studies. However, genetic identification of the human CPA factors has been hampered by the lack of a reliable genome-wide screening method. We describe here a dual fluorescence readthrough reporter system with a PAS inserted between two fluorescent reporters. This system enables measurement of the efficiency of 3' end processing in living cells. Using this system in combination with a human genome-wide CRISPR/Cas9 library, we conducted a screen for CPA factors. The screens identified most components of the known core CPA complexes and other known CPA factors. The screens also identified CCNK/CDK12 as a potential core CPA factor, and RPRD1B as a CPA factor that binds RNA and regulates the release of RNA polymerase II at the 3' ends of genes. Thus, this dual fluorescence reporter coupled with CRISPR/Cas9 screens reliably identifies bona fide CPA factors and provides a platform for investigating the requirements for CPA in various contexts.

Functional characterization of RebL1 highlights the evolutionary conservation of oncogenic activities of the RBBP4/7 orthologue in Tetrahymena thermophila.

Retinoblastoma-binding proteins 4 and 7 (RBBP4 and RBBP7) are two highly homologous human histone chaperones. They function in epigenetic regulation as subunits of multiple chromatin-related complexes and have been implicated in numerous cancers. Due to their overlapping functions, our understanding of RBBP4 and 7, particularly outside of Opisthokonts, has remained limited. Here, we report that in the ciliate protozoan Tetrahymena thermophila a single orthologue of human RBBP4 and 7 proteins, RebL1, physically interacts with histone H4 and functions in multiple epigenetic regulatory pathways. Functional proteomics identified conserved functional links for Tetrahymena RebL1 protein as well as human RBBP4 and 7. We found that putative subunits of multiple chromatin-related complexes including CAF1, Hat1, Rpd3, and MuvB, co-purified with RebL1 during Tetrahymena growth and conjugation. Iterative proteomics analyses revealed that the cell cycle regulatory MuvB-complex in Tetrahymena is composed of at least five subunits including evolutionarily conserved Lin54, Lin9 and RebL1 proteins. Genome-wide analyses indicated that RebL1 and Lin54 (Anqa1) bind within genic and intergenic regions. Moreover, Anqa1 targets primarily promoter regions suggesting a role for Tetrahymena MuvB in transcription regulation. RebL1 depletion inhibited cellular growth and reduced the expression levels of Anqa1 and Lin9. Consistent with observations in glioblastoma tumors, RebL1 depletion suppressed DNA repair protein Rad51 in Tetrahymena, thus underscoring the evolutionarily conserved functions of RBBP4/7 proteins. Our results suggest the essentiality of RebL1 functions in multiple epigenetic regulatory complexes in which it impacts transcription regulation and cellular viability.

Assessment of a method to characterize antibody selectivity and specificity for use in immunoprecipitation.

Antibodies are used in multiple cell biology applications, but there are no standardized methods to assess antibody quality-an absence that risks data integrity and reproducibility. We describe a mass spectrometry-based standard operating procedure for scoring immunoprecipitation antibody quality. We quantified the abundance of all the proteins in immunoprecipitates of 1,124 new recombinant antibodies for 152 chromatin-related human proteins by comparing normalized spectral abundance factors from the target antigen with those of all other proteins. We validated the performance of the standard operating procedure in blinded studies in five independent laboratories. Antibodies for which the target antigen or a member of its known protein complex was the most abundant protein were classified as 'IP gold standard'. This method generates quantitative outputs that can be stored and archived in public databases, and it represents a step toward a platform for community benchmarking of antibody quality.

Interaction landscape of membrane-protein complexes in Saccharomyces cerevisiae.

Macromolecular assemblies involving membrane proteins (MPs) serve vital biological roles and are prime drug targets in a variety of diseases. Large-scale affinity purification studies of soluble-protein complexes have been accomplished for diverse model organisms, but no global characterization of MP-complex membership has been described so far. Here we report a complete survey of 1,590 putative integral, peripheral and lipid-anchored MPs from Saccharomyces cerevisiae, which were affinity purified in the presence of non-denaturing detergents. The identities of the co-purifying proteins were determined by tandem mass spectrometry and subsequently used to derive a high-confidence physical interaction map encompassing 1,726 membrane protein-protein interactions and 501 putative heteromeric complexes associated with the various cellular membrane systems. Our analysis reveals unexpected physical associations underlying the membrane biology of eukaryotes and delineates the global topological landscape of the membrane interactome.

Risk estimates for complex disorders: comparing personal genome testing and family history.

PURPOSE: Personal genome testing allows the identification of single-nucleotide polymorphisms associated with an increased risk for common complex disorders. An area of concern in the use of personal genome testing is how risk estimates generated differ from traditional measures of risk (e.g., family history analysis). We sought to analyze the concordance of risk estimates generated by family history analysis and by personal genome testing. METHODS: Risk categorizations for 20 complex conditions included in Navigenics personal genome testing were compared with risk categorization estimates derived from family history assessment using the kappa (κ) statistic. RESULTS: The only conditions showing slight agreement between risk assessment methods were Alzheimer disease (κ = 0.131), breast cancer (κ = 0.154), and deep vein thrombosis (κ = 0.201) in females, and colon cancer (κ = 0.124) in males. Eighty-six individuals (11.4%) were found to have additional genetic risks not assessed by personal genome testing after family and medical history assessment, including 38 individuals with family histories suggestive of hereditary cancer syndromes. CONCLUSION: Discordance between personal genome testing and family history risk estimates suggests that these methods may provide independent information that could be used in a complementary manner. Results also support that eliciting family history adds value to overall risk assessment for individuals undergoing personal genome testing.

DAnCER: disease-annotated chromatin epigenetics resource.

Chromatin modification (CM) is a set of epigenetic processes that govern many aspects of DNA replication, transcription and repair. CM is carried out by groups of physically interacting proteins, and their disruption has been linked to a number of complex human diseases. CM remains largely unexplored, however, especially in higher eukaryotes such as human. Here we present the DAnCER resource, which integrates information on genes with CM function from five model organisms, including human. Currently integrated are gene functional annotations, Pfam domain architecture, protein interaction networks and associated human diseases. Additional supporting evidence includes orthology relationships across organisms, membership in protein complexes, and information on protein 3D structure. These data are available for 962 experimentally confirmed and manually curated CM genes and for over 5000 genes with predicted CM function on the basis of orthology and domain composition. DAnCER allows visual explorations of the integrated data and flexible query capabilities using a variety of data filters. In particular, disease information and functional annotations are mapped onto the protein interaction networks, enabling the user to formulate new hypotheses on the function and disease associations of a given gene based on those of its interaction partners. DAnCER is freely available at http://wodaklab.org/dancer/.

Omicron Spike Protein is Vulnerable to Reduction.

SARS-CoV-2 virus spike (S) protein is an envelope protein responsible for binding to the ACE2 receptor, driving subsequent entry into host cells. The existence of multiple disulfide bonds in the S protein makes it potentially susceptible to reductive cleavage. Using a tri-part split luciferase-based binding assay, we evaluated the impacts of chemical reduction on S proteins from different virus variants and found that those from the Omicron family are highly vulnerable to reduction. Through manipulation of different Omicron mutations, we found that alterations in the receptor binding module (RBM) are the major determinants of this vulnerability. Specifically we discovered that Omicron mutations facilitate the cleavage of C480-C488 and C379-C432 disulfides, which consequently impairs binding activity and protein stability. The vulnerability of Omicron S proteins suggests a mechanism that can be harnessed to treat specific SARS-CoV-2 strains.

Multilevel interrogation of H3.3 reveals a primordial role in transcription regulation.

BACKGROUND: Eukaryotic cells can rapidly adjust their transcriptional profile in response to molecular needs. Such dynamic regulation is, in part, achieved through epigenetic modifications and selective incorporation of histone variants into chromatin. H3.3 is the ancestral H3 variant with key roles in regulating chromatin states and transcription. Although H3.3 has been well studied in metazoans, information regarding the assembly of H3.3 onto chromatin and its possible role in transcription regulation remain poorly documented outside of Opisthokonts. RESULTS: We used the nuclear dimorphic ciliate protozoan, Tetrahymena thermophila, to investigate the dynamics of H3 variant function in evolutionarily divergent eukaryotes. Functional proteomics and immunofluorescence analyses of H3.1 and H3.3 revealed a highly conserved role for Nrp1 and Asf1 histone chaperones in nuclear influx of histones. Cac2, a putative subunit of H3.1 deposition complex CAF1, is not required for growth, whereas the expression of the putative ortholog of the H3.3-specific chaperone Hir1 is essential in Tetrahymena. Our results indicate that Cac2 and Hir1 have distinct localization patterns during different stages of the Tetrahymena life cycle and suggest that Cac2 might be dispensable for chromatin assembly. ChIP-seq experiments in growing Tetrahymena show H3.3 enrichment over the promoters, gene bodies, and transcription termination sites of highly transcribed genes. H3.3 knockout followed by RNA-seq reveals large-scale transcriptional alterations in functionally important genes. CONCLUSION: Our results provide an evolutionary perspective on H3.3's conserved role in maintaining the transcriptional landscape of cells and on the emergence of specialized chromatin assembly pathways.

Survival-based CRISPR genetic screens across a panel of permissive cell lines identify common and cell-specific SARS-CoV-2 host factors.

SARS-CoV-2 depends on host cell components for infection and replication. Identification of virus-host dependencies offers an effective way to elucidate mechanisms involved in viral infection and replication. If druggable, host factor dependencies may present an attractive strategy for anti-viral therapy. In this study, we performed genome wide CRISPR knockout screens in Vero E6 cells and four human cell lines including Calu-3, UM-UC-4, HEK-293 and HuH-7 to identify genetic regulators of SARS-CoV-2 infection. Our findings identified only ACE2, the cognate SARS-CoV-2 entry receptor, as a common host dependency factor across all cell lines, while other host genes identified were largely cell line specific, including known factors TMPRSS2 and CTSL. Several of the discovered host-dependency factors converged on pathways involved in cell signalling, immune-related pathways, and chromatin modification. Notably, the chromatin modifier gene KMT2C in Calu-3 cells had the strongest impact in preventing SARS-CoV-2 infection when perturbed.

OrthoNets: simultaneous visual analysis of orthologs and their interaction neighborhoods across different organisms.

MOTIVATION: Protein interaction networks contain a wealth of biological information, but their large size often hinders cross-organism comparisons. We present OrthoNets, a Cytoscape plugin that displays protein-protein interaction (PPI) networks from two organisms simultaneously, highlighting orthology relationships and aggregating several types of biomedical annotations. OrthoNets also allows PPI networks derived from experiments to be overlaid on networks extracted from public databases, supporting the identification and verification of new interactors. Any newly identified PPIs can be validated by checking whether their orthologs interact in another organism. AVAILABILITY: OrthoNets is freely available at http://wodaklab.org/orthonets/.

Brain abnormalities in patients with Beckwith-Wiedemann syndrome.

Beckwith-Wiedemann syndrome (BWS) is an overgrowth disorder with variability in clinical manifestations and molecular causes. In most cases, patients with BWS have normal development. Cases with developmental delay are usually attributed to neonatal hypoglycemia or chromosome abnormalities involving copy number variation for genes beyond the critical BWS region at 11p15.5. Brain abnormalities have not previously been recognized within the BWS phenotypic spectrum. We report on seven cases of BWS associated with posterior fossa abnormalities. Of these, two cases presented with Blake's pouch cyst, two with Dandy-Walker variant (DWV; hypoplasia of the inferior part of the vermis), one with Dandy-Walker malformation (DWM) and one with a complex of DWM, dysgenesis of the corpus callosum and brain stem abnormality. In all these cases, molecular findings involved the centromeric imprinted domain on chromosome locus 11p15.5, which includes imprinting center 2 (IC2) and the imprinted growth suppressor gene, CDKN1C. Three cases had loss of methylation at IC2, two had CDKN1C mutations, and one had loss of methylation at IC2 and a microdeletion. In one case no mutation/methylation abnormality was detected. These findings together with previously reported correlations suggest that genes in imprinted domain 2 at 11p15.5 are involved in normal midline development of several organs including the brain. Our data suggest that brain malformations may present as a finding within the BWS phenotype when the molecular etiology involves imprinted domain 2. Brain imaging may be useful in identifying such malformations in individuals with BWS and neurodevelopmental issues.

Global landscape of protein complexes in the yeast Saccharomyces cerevisiae.

Identification of protein-protein interactions often provides insight into protein function, and many cellular processes are performed by stable protein complexes. We used tandem affinity purification to process 4,562 different tagged proteins of the yeast Saccharomyces cerevisiae. Each preparation was analysed by both matrix-assisted laser desorption/ionization-time of flight mass spectrometry and liquid chromatography tandem mass spectrometry to increase coverage and accuracy. Machine learning was used to integrate the mass spectrometry scores and assign probabilities to the protein-protein interactions. Among 4,087 different proteins identified with high confidence by mass spectrometry from 2,357 successful purifications, our core data set (median precision of 0.69) comprises 7,123 protein-protein interactions involving 2,708 proteins. A Markov clustering algorithm organized these interactions into 547 protein complexes averaging 4.9 subunits per complex, about half of them absent from the MIPS database, as well as 429 additional interactions between pairs of complexes. The data (all of which are available online) will help future studies on individual proteins as well as functional genomics and systems biology.

SARS-CoV-2 nucleocapsid protein binds host mRNAs and attenuates stress granules to impair host stress response.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleocapsid (N) protein is essential for viral replication, making it a promising target for antiviral drug and vaccine development. SARS-CoV-2 infected patients exhibit an uncoordinated immune response; however, the underlying mechanistic details of this imbalance remain obscure. Here, starting from a functional proteomics workflow, we cataloged the protein-protein interactions of SARS-CoV-2 proteins, including an evolutionarily conserved specific interaction of N with the stress granule resident proteins G3BP1 and G3BP2. N localizes to stress granules and sequesters G3BPs away from their typical interaction partners, thus attenuating stress granule formation. We found that N binds directly to host mRNAs in cells, with a preference for 3' UTRs, and modulates target mRNA stability. We show that the N protein rewires the G3BP1 mRNA-binding profile and suppresses the physiological stress response of host cells, which may explain the imbalanced immune response observed in SARS-CoV-2 infected patients.

Challenges and rewards of interaction proteomics.

The recent explosion of high throughput experimental technologies for characterizing protein interactions has generated large amounts of data describing interactions between thousands of proteins and producing genome scale views of protein assemblies. The systems level views afforded by these data hold great promise of leading to new knowledge but also involve many challenges. Deriving meaningful biological conclusions from these views crucially depends on our understanding of the approximation and biases that enter into deriving and interpreting the data. The challenges and rewards of interaction proteomics are reviewed here using as an example the latest comprehensive high throughput analyses of protein interactions in yeast.

Up-to-date catalogues of yeast protein complexes.

Gold standard datasets on protein complexes are key to inferring and validating protein-protein interactions. Despite much progress in characterizing protein complexes in the yeast Saccharomyces cerevisiae, numerous researchers still use as reference the manually curated complexes catalogued by the Munich Information Center of Protein Sequences database. Although this catalogue has served the community extremely well, it no longer reflects the current state of knowledge. Here, we report two catalogues of yeast protein complexes as results of systematic curation efforts. The first one, denoted as CYC2008, is a comprehensive catalogue of 408 manually curated heteromeric protein complexes reliably backed by small-scale experiments reported in the current literature. This catalogue represents an up-to-date reference set for biologists interested in discovering protein interactions and protein complexes. The second catalogue, denoted as YHTP2008, comprises 400 high-throughput complexes annotated with current literature evidence. Among them, 262 correspond, at least partially, to CYC2008 complexes. Evidence for interacting subunits is collected for 68 complexes that have only partial or no overlap with CYC2008 complexes, whereas no literature evidence was found for 100 complexes. Some of these partially supported and as yet unsupported complexes may be interesting candidates for experimental follow up. Both catalogues are freely available at: http://wodaklab.org/cyc2008/.

The evolutionary landscape of the chromatin modification machinery reveals lineage specific gains, expansions, and losses.

Model organisms such as yeast, fly, and worm have played a defining role in the study of many biological systems. A significant challenge remains in translating this information to humans. Of critical importance is the ability to differentiate those components where knowledge of function and interactions may be reliably inferred from those that represent lineage-specific innovations. To address this challenge, we use chromatin modification (CM) as a model system for exploring the evolutionary properties of their components in the context of their known functions and interactions. Collating previously identified components of CM from yeast, worm, fly, and human, we identified a "core" set of 50 CM genes displaying consistent orthologous relationships that likely retain their interactions and functions across taxa. In addition, we catalog many components that demonstrate lineage specific expansions and losses, highlighting much duplication within vertebrates that may reflect an expanded repertoire of regulatory mechanisms. Placed in the context of a high-quality protein-protein interaction network, we find, contrary to existing views of evolutionary modularity, that CM complex components display a mosaic of evolutionary histories: a core set of highly conserved genes, together with sets displaying lineage specific innovations. Although focused on CM, this study provides a template for differentiating those genes which are likely to retain their functions and interactions across species. As such, in addition to informing on the evolution of CM as a system, this study provides a set of comparative genomic approaches that can be generally applied to any biological systems.

Local coherence in genetic interaction patterns reveals prevalent functional versatility.

MOTIVATION: Epistatic or genetic interactions, representing the effects of mutating one gene on the phenotypes caused by mutations in one or more distinct genes, can be very helpful for uncovering functional relationships between genes. Recently, the epistatic miniarray profiles (E-MAP) method has emerged as a powerful approach for identifying such interactions systematically. For E-MAP data analysis, hierarchical clustering is used to partition genes into groups on the basis of the similarity between their global interaction profiles, and the resulting descriptions assign each gene to only one group, thereby ignoring the multifunctional roles played by most genes. RESULTS: Here, we present the original local coherence detection (LCD) algorithm for identifying groups of functionally related genes from E-MAP data in a manner that allows individual genes to be assigned to more than one functional group. This enables investigation of the pleiotropic nature of gene function. The performance of our algorithm is illustrated by applying it to two E-MAP datasets and an E-MAP-like in silico dataset for the yeast Saccharomyces cerevisiae. In addition to recapitulating the majority of the functional modules and many protein complexes reported previously, our algorithm uncovers many recently documented and novel multifunctional relationships between genes and gene groups. Our algorithm hence represents a valuable tool for uncovering new roles for genes with annotated functions and for mapping groups of genes and proteins into pathways.

The Med31 Conserved Component of the Divergent Mediator Complex in Tetrahymena thermophila Participates in Developmental Regulation.

Mediator is a large protein complex required for basal and regulated expression of most RNA polymerase II (RNAP II)-transcribed genes, in part due to its interaction with and phosphorylation of the conserved C-terminal domain (CTD) of Rpb1 [1, 2]. Mediator has been implicated in many aspects of gene expression including chromatin looping [3], higher-order chromatin folding [4], mRNA processing [5] and export [6], and transcriptional memory [7]. Mediator is thought to have played a major role during eukaryotic diversification [8, 9], although its function remains unknown in evolutionarily deep branching eukaryotes lacking canonical CTD heptad repeats. We used the ciliate protozoan Tetrahymena thermophila as a model organism whose genome encodes a highly divergent Rpb1 lacking canonical CTD heptad repeats. We endogenously tagged the Med31 subunit of the Mediator complex and performed affinity purification coupled with mass spectrometry (AP-MS) to identify Mediator subunits. We found that Med31 physically interacts with a large number of proteins (>20), several of which share similarities to canonical Mediator subunits in yeast and humans as well as Tetrahymena-specific proteins. Furthermore, Med31 ChIP-seq analysis suggested a global role for Mediator in transcription regulation. We demonstrated that MED31 knockdown in growing Tetrahymena results in the ectopic expression of developmental genes important for programmed DNA rearrangements. In addition, indirect immunofluorescence revealed Med31 localization in meiotic micronuclei, implicating Mediator in RNAPII-dependent ncRNA transcription. Our results reveal structural and functional insights and implicate Mediator as an ancient cellular machinery for transcription regulation with a possible involvement in global transcription of ncRNAs.

GenePro: a Cytoscape plug-in for advanced visualization and analysis of interaction networks.

MOTIVATION: Analyzing the networks of interactions between genes and proteins has become a central theme in systems biology. Versatile software tools for interactively displaying and analyzing these networks are therefore very much in demand. The public-domain open software environment Cytoscape has been developed with the goal of facilitating the design and development of such software tools by the scientific community. RESULTS: We present GenePro, a plugin to Cytoscape featuring a set of versatile tools that greatly facilitates the visualization and analysis of protein networks derived from high-throughput interactions data and the validation of various methods for parsing these networks into meaningful functional modules. AVAILABILITY: The GenePro plugin is available at the website http://genepro.ccb.sickkids.ca.