The histone variant H2A.Z promotes splicing of weak introns.

Multiple lines of evidence implicate chromatin in the regulation of premessenger RNA (pre-mRNA) splicing. However, the influence of chromatin factors on cotranscriptional splice site usage remains unclear. Here we investigated the function of the highly conserved histone variant H2A.Z in pre-mRNA splicing using the intron-rich model yeast Schizosaccharomyces pombe Using epistatic miniarray profiles (EMAPs) to survey the genetic interaction landscape of the Swr1 nucleosome remodeling complex, which deposits H2A.Z, we uncovered evidence for functional interactions with components of the spliceosome. In support of these genetic connections, splicing-specific microarrays show that H2A.Z and the Swr1 ATPase are required during temperature stress for the efficient splicing of a subset of introns. Notably, affected introns are enriched for H2A.Z occupancy and more likely to contain nonconsensus splice sites. To test the significance of the latter correlation, we mutated the splice sites in an affected intron to consensus and found that this suppressed the requirement for H2A.Z in splicing of that intron. These data suggest that H2A.Z occupancy promotes cotranscriptional splicing of suboptimal introns that may otherwise be discarded via proofreading ATPases. Consistent with this model, we show that overexpression of splicing ATPase Prp16 suppresses both the growth and splicing defects seen in the absence of H2A.Z.

Paralog dispensability shapes homozygous deletion patterns in tumor genomes.

Genomic instability is a hallmark of cancer, resulting in tumor genomes having large numbers of genetic aberrations, including homozygous deletions of protein coding genes. That tumor cells remain viable in the presence of such gene loss suggests high robustness to genetic perturbation. In model organisms and cancer cell lines, paralogs have been shown to contribute substantially to genetic robustness-they are generally more dispensable for growth than singletons. Here, by analyzing copy number profiles of > 10,000 tumors, we test the hypothesis that the increased dispensability of paralogs shapes tumor genome evolution. We find that genes with paralogs are more likely to be homozygously deleted and that this cannot be explained by other factors known to influence copy number variation. Furthermore, features that influence paralog dispensability in cancer cell lines correlate with paralog deletion frequency in tumors. Finally, paralogs that are broadly essential in cancer cell lines are less frequently deleted in tumors than non-essential paralogs. Overall, our results suggest that homozygous deletions of paralogs are more frequently observed in tumor genomes because paralogs are more dispensable.

Cross-species identification of PIP5K1-, splicing- and ubiquitin-related pathways as potential targets for RB1-deficient cells.

The RB1 tumor suppressor is recurrently mutated in a variety of cancers including retinoblastomas, small cell lung cancers, triple-negative breast cancers, prostate cancers, and osteosarcomas. Finding new synthetic lethal (SL) interactions with RB1 could lead to new approaches to treating cancers with inactivated RB1. We identified 95 SL partners of RB1 based on a Drosophila screen for genetic modifiers of the eye phenotype caused by defects in the RB1 ortholog, Rbf1. We validated 38 mammalian orthologs of Rbf1 modifiers as RB1 SL partners in human cancer cell lines with defective RB1 alleles. We further show that for many of the RB1 SL genes validated in human cancer cell lines, low activity of the SL gene in human tumors, when concurrent with low levels of RB1 was associated with improved patient survival. We investigated higher order combinatorial gene interactions by creating a novel Drosophila cancer model with co-occurring Rbf1, Pten and Ras mutations, and found that targeting RB1 SL genes in this background suppressed the dramatic tumor growth and rescued fly survival whilst having minimal effects on wild-type cells. Finally, we found that drugs targeting the identified RB1 interacting genes/pathways, such as UNC3230, PYR-41, TAK-243, isoginkgetin, madrasin, and celastrol also elicit SL in human cancer cell lines. In summary, we identified several high confidence, evolutionarily conserved, novel targets for RB1-deficient cells that may be further adapted for the treatment of human cancer.

Exploiting Cancer Synthetic Lethality in Cancer-Lessons Learnt from PARP Inhibitors.

PARP inhibitors now have proven utility in the treatment of homologous recombination (HR) defective cancers. These drugs, and the synthetic lethality effect they exploit, have not only taught us how to approach the treatment of HR defective cancers but have also illuminated how resistance to a synthetic lethal approach can occur, how cancer-associated synthetic lethal effects are perhaps more complex than we imagine, how the better use of biomarkers could improve the success of treatment and even how drug resistance might be targeted. Here, we discuss some of the lessons learnt from the study of PARP inhibitor synthetic lethality and how these lessons might have wider application. Specifically, we discuss the concept of synthetic lethal penetrance, phenocopy effects in cancer such as BRCAness, synthetic lethal resistance, the polygenic and complex nature of synthetic lethal interactions, how evolutionary double binds could be exploited in treatment as well as future horizons for the field.

Paralog buffering contributes to the variable essentiality of genes in cancer cell lines.

What makes a gene essential for cellular survival? In model organisms, such as budding yeast, systematic gene deletion studies have revealed that paralog genes are less likely to be essential than singleton genes and that this can partially be attributed to the ability of paralogs to buffer each other's loss. However, the essentiality of a gene is not a fixed property and can vary significantly across different genetic backgrounds. It is unclear to what extent paralogs contribute to this variation, as most studies have analyzed genes identified as essential in a single genetic background. Here, using gene essentiality profiles of 558 genetically heterogeneous tumor cell lines, we analyze the contribution of paralogy to variable essentiality. We find that, compared to singleton genes, paralogs are less frequently essential and that this is more evident when considering genes with multiple paralogs or with highly sequence-similar paralogs. In addition, we find that paralogs derived from whole genome duplication exhibit more variable essentiality than those derived from small-scale duplications. We provide evidence that in 13-17% of cases the variable essentiality of paralogs can be attributed to buffering relationships between paralog pairs, as evidenced by synthetic lethality. Paralog pairs derived from whole genome duplication and pairs that function in protein complexes are significantly more likely to display such synthetic lethal relationships. Overall we find that many of the observations made using a single strain of budding yeast can be extended to understand patterns of essentiality in genetically heterogeneous cancer cell lines.

Accurate prediction of kinase-substrate networks using knowledge graphs.

Phosphorylation of specific substrates by protein kinases is a key control mechanism for vital cell-fate decisions and other cellular processes. However, discovering specific kinase-substrate relationships is time-consuming and often rather serendipitous. Computational predictions alleviate these challenges, but the current approaches suffer from limitations like restricted kinome coverage and inaccuracy. They also typically utilise only local features without reflecting broader interaction context. To address these limitations, we have developed an alternative predictive model. It uses statistical relational learning on top of phosphorylation networks interpreted as knowledge graphs, a simple yet robust model for representing networked knowledge. Compared to a representative selection of six existing systems, our model has the highest kinome coverage and produces biologically valid high-confidence predictions not possible with the other tools. Specifically, we have experimentally validated predictions of previously unknown phosphorylations by the LATS1, AKT1, PKA and MST2 kinases in human. Thus, our tool is useful for focusing phosphoproteomic experiments, and facilitates the discovery of new phosphorylation reactions. Our model can be accessed publicly via an easy-to-use web interface (LinkPhinder).

Hierarchical modularity and the evolution of genetic interactomes across species.

To date, cross-species comparisons of genetic interactomes have been restricted to small or functionally related gene sets, limiting our ability to infer evolutionary trends. To facilitate a more comprehensive analysis, we constructed a genome-scale epistasis map (E-MAP) for the fission yeast Schizosaccharomyces pombe, providing phenotypic signatures for ~60% of the nonessential genome. Using these signatures, we generated a catalog of 297 functional modules, and we assigned function to 144 previously uncharacterized genes, including mRNA splicing and DNA damage checkpoint factors. Comparison with an integrated genetic interactome from the budding yeast Saccharomyces cerevisiae revealed a hierarchical model for the evolution of genetic interactions, with conservation highest within protein complexes, lower within biological processes, and lowest between distinct biological processes. Despite the large evolutionary distance and extensive rewiring of individual interactions, both networks retain conserved features and display similar levels of functional crosstalk between biological processes, suggesting general design principles of genetic interactomes.

MED12-related XLID disorders are dose-dependent of immediate early genes (IEGs) expression.

Mediator occupies a key role in protein coding genes expression in mediating the contacts between gene specific factors and the basal transcription machinery but little is known regarding the role of each Mediator subunits. Mutations in MED12 are linked with a broad spectrum of genetic disorders with X-linked intellectual disability that are difficult to range as Lujan, Opitz-Kaveggia or Ohdo syndromes. Here, we investigated several MED12 patients mutations (p.R206Q, p.N898D, p.R961W, p.N1007S, p.R1148H, p.S1165P and p.R1295H) and show that each MED12 mutations cause specific expression patterns of JUN, FOS and EGR1 immediate early genes (IEGs), reflected by the presence or absence of MED12 containing complex at their respective promoters. Moreover, the effect of MED12 mutations has cell-type specificity on IEG expression. As a consequence, the expression of late responsive genes such as the matrix metalloproteinase-3 and the RE1 silencing transcription factor implicated respectively in neural plasticity and the specific expression of neuronal genes is disturbed as documented for MED12/p.R1295H mutation. In such case, JUN and FOS failed to be properly recruited at their AP1-binding site. Our results suggest that the differences between MED12-related phenotypes are essentially the result of distinct IEGs expression patterns, the later ones depending on the accurate formation of the transcription initiation complex. This might challenge clinicians to rethink the traditional syndromes boundaries and to include genetic criterion in patients' diagnostic.

High-resolution network biology: connecting sequence with function.

Proteins are not monolithic entities; rather, they can contain multiple domains that mediate distinct interactions, and their functionality can be regulated through post-translational modifications at multiple distinct sites. Traditionally, network biology has ignored such properties of proteins and has instead examined either the physical interactions of whole proteins or the consequences of removing entire genes. In this Review, we discuss experimental and computational methods to increase the resolution of protein-protein, genetic and drug-gene interaction studies to the domain and residue levels. Such work will be crucial for using interaction networks to connect sequence and structural information, and to understand the biological consequences of disease-associated mutations, which will hopefully lead to more effective therapeutic strategies.

Genetic interaction mapping reveals a role for the SWI/SNF nucleosome remodeler in spliceosome activation in fission yeast.

Although numerous regulatory connections between pre-mRNA splicing and chromatin have been demonstrated, the precise mechanisms by which chromatin factors influence spliceosome assembly and/or catalysis remain unclear. To probe the genetic network of pre-mRNA splicing in the fission yeast Schizosaccharomyces pombe, we constructed an epistatic mini-array profile (E-MAP) and discovered many new connections between chromatin and splicing. Notably, the nucleosome remodeler SWI/SNF had strong genetic interactions with components of the U2 snRNP SF3 complex. Overexpression of SF3 components in ΔSWI/SNF cells led to inefficient splicing of many fission yeast introns, predominantly those with non-consensus splice sites. Deletion of SWI/SNF decreased recruitment of the splicing ATPase Prp2, suggesting that SWI/SNF promotes co-transcriptional spliceosome assembly prior to first step catalysis. Importantly, defects in SWI/SNF as well as SF3 overexpression each altered nucleosome occupancy along intron-containing genes, illustrating that the chromatin landscape both affects--and is affected by--co-transcriptional splicing.

Ultrahigh Q whispering gallery mode electro-optic resonators on a silicon photonic chip.

Crystalline whispering gallery mode (WGM) electro-optic resonators made of LiNbO3 and LiTaO3 are critical for a wide range of applications in nonlinear and quantum optics, as well as RF photonics, due to their remarkably ultrahigh Q(>108) and large electro-optic coefficient. Achieving efficient coupling of these resonators to planar on-chip optical waveguides is essential for any high-yield and robust practical applications. However, it has been very challenging to demonstrate such coupling while preserving the ultrahigh Q properties of the resonators. Here, we show how the silicon photonic platform can overcome this long-standing challenge. Silicon waveguides with appropriate designs enable efficient and strong coupling to these WGM electro-optic resonators. We discuss various integration architectures of these resonators onto a silicon chip and experimentally demonstrate critical coupling of a planar Si waveguide and an ultrahigh QLiTaO3 resonator (Q∼108). Our results show a promising path for widespread and practical applications of these resonators on a silicon photonic platform.

Genome-wide epistatic expression quantitative trait loci discovery in four human tissues reveals the importance of local chromosomal interactions governing gene expression.

BACKGROUND: Epistasis (synergistic interaction) among SNPs governing gene expression is likely to arise within transcriptional networks. However, the power to detect it is limited by the large number of combinations to be tested and the modest sample sizes of most datasets. By limiting the interaction search space firstly to cis-trans and then cis-cis SNP pairs where both SNPs had an independent effect on the expression of the most variable transcripts in the liver and brain, we greatly reduced the size of the search space. RESULTS: Within the cis-trans search space we discovered three transcripts with significant epistasis. Surprisingly, all interacting SNP pairs were located nearby each other on the chromosome (within 290 kb-2.16 Mb). Despite their proximity, the interacting SNPs were outside the range of linkage disequilibrium (LD), which was absent between the pairs (r(2) < 0.01). Accordingly, we redefined the search space to detect cis-cis interactions, where a cis-SNP was located within 10 Mb of the target transcript. The results of this show evidence for the epistatic regulation of 50 transcripts across the tissues studied. Three transcripts, namely, HLA-G, PSORS1C1 and HLA-DRB5 share common regulatory SNPs in the pre-frontal cortex and their expression is significantly correlated. This pattern of epistasis is consistent with mediation via long-range chromatin structures rather than the binding of transcription factors in trans. Accordingly, some of the interactions map to regions of the genome known to physically interact in lymphoblastoid cell lines while others map to known promoter and enhancer elements. SNPs involved in interactions appear to be enriched for promoter markers. CONCLUSIONS: In the context of gene expression and its regulation, our analysis indicates that the study of cis-cis or local epistatic interactions may have a more important role than interchromosomal interactions.

The yeast SR-like protein Npl3 links chromatin modification to mRNA processing.

Eukaryotic gene expression involves tight coordination between transcription and pre-mRNA splicing; however, factors responsible for this coordination remain incompletely defined. Here, we explored the genetic, functional, and biochemical interactions of a likely coordinator, Npl3, an SR-like protein in Saccharomyces cerevisiae that we recently showed is required for efficient co-transcriptional recruitment of the splicing machinery. We surveyed the NPL3 genetic interaction space and observed a significant enrichment for genes involved in histone modification and chromatin remodeling. Specifically, we found that Npl3 genetically interacts with both Bre1, which mono-ubiquitinates histone H2B as part of the RAD6 Complex, and Ubp8, the de-ubiquitinase of the SAGA Complex. In support of these genetic data, we show that Bre1 physically interacts with Npl3 in an RNA-independent manner. Furthermore, using a genome-wide splicing microarray, we found that the known splicing defect of a strain lacking Npl3 is exacerbated by deletion of BRE1 or UBP8, a phenomenon phenocopied by a point mutation in H2B that abrogates ubiquitination. Intriguingly, even in the presence of wild-type NPL3, deletion of BRE1 exhibits a mild splicing defect and elicits a growth defect in combination with deletions of early and late splicing factors. Taken together, our data reveal a connection between Npl3 and an extensive array of chromatin factors and describe an unanticipated functional link between histone H2B ubiquitination and pre-mRNA splicing.

Genetic interactions under the microscope.

Traditional genetic interaction screens profile phenotypes at aggregate level, missing interactions that may influence the distribution of single cells in specific states. Here, Heigwer and colleagues use an imaging approach to generate a large-scale high-resolution genetic interaction map in Drosophila cells and demonstrate its utility for understanding gene function.

Antibody reliability influences observed mRNA-protein correlations in tumour samples.

Reverse phase protein arrays (RPPA) have been used to quantify the abundance of hundreds of proteins across thousands of tumour samples in the Cancer Genome Atlas. By number of samples, this is the largest tumour proteomic dataset available and it provides an opportunity to systematically assess the correlation between mRNA and protein abundances. However, the RPPA approach is highly dependent on antibody reliability and approximately one-quarter of the antibodies used in the the Cancer Genome Atlas are deemed to be somewhat less reliable. Here, we assess the impact of antibody reliability on observed mRNA-protein correlations. We find that, in general, proteins measured with less reliable antibodies have lower observed mRNA-protein correlations. This is not true of the same proteins when measured using mass spectrometry. Furthermore, in cell lines, we find that when the same protein is quantified by both mass spectrometry and RPPA, the overall correlation between the two measurements is lower for proteins measured with less reliable antibodies. Overall our results reinforce the need for caution in using RPPA measurements from less reliable antibodies.

Gene essentiality in cancer is better predicted by mRNA abundance than by gene regulatory network-inferred activity.

Gene regulatory networks (GRNs) are often deregulated in tumor cells, resulting in altered transcriptional programs that facilitate tumor growth. These altered networks may make tumor cells vulnerable to the inhibition of specific regulatory proteins. Consequently, the reconstruction of GRNs in tumors is often proposed as a means to identify therapeutic targets. While there are examples of individual targets identified using GRNs, the extent to which GRNs can be used to predict sensitivity to targeted intervention in general remains unknown. Here we use the results of genome-wide CRISPR screens to systematically assess the ability of GRNs to predict sensitivity to gene inhibition in cancer cell lines. Using GRNs derived from multiple sources, including GRNs reconstructed from tumor transcriptomes and from curated databases, we infer regulatory gene activity in cancer cell lines from ten cancer types. We then ask, in each cancer type, if the inferred regulatory activity of each gene is predictive of sensitivity to CRISPR perturbation of that gene. We observe slight variation in the correlation between gene regulatory activity and gene sensitivity depending on the source of the GRN and the activity estimation method used. However, we find that there is consistently a stronger relationship between mRNA abundance and gene sensitivity than there is between regulatory gene activity and gene sensitivity. This is true both when gene sensitivity is treated as a binary and a quantitative property. Overall, our results suggest that gene sensitivity is better predicted by measured expression than by GRN-inferred activity.

Targeting synthetic lethal paralogs in cancer.

Synthetic lethal interactions, where mutation of one gene renders cells sensitive to inhibition of another gene, can be exploited for the development of targeted therapeutics in cancer. Pairs of duplicate genes (paralogs) often share common functionality and hence are a potentially rich source of synthetic lethal interactions. Because the majority of human genes have paralogs, exploiting such interactions could be a widely applicable approach for targeting gene loss in cancer. Moreover, existing small-molecule drugs may exploit synthetic lethal interactions by inhibiting multiple paralogs simultaneously. Consequently, the identification of synthetic lethal interactions between paralogs could be extremely informative for drug development. Here we review approaches to identify such interactions and discuss some of the challenges of exploiting them.

Complex synthetic lethality in cancer.

The concept of synthetic lethality has been widely applied to identify therapeutic targets in cancer, with varying degrees of success. The standard approach normally involves identifying genetic interactions between two genes, a driver and a target. In reality, however, most cancer synthetic lethal effects are likely complex and also polygenic, being influenced by the environment in addition to involving contributions from multiple genes. By acknowledging and delineating this complexity, we describe in this article how the success rate in cancer drug discovery and development could be improved.

Practical experimental certification of computational quantum gates using a twirling procedure.

Because of the technical difficulty of building large quantum computers, it is important to be able to estimate how faithful a given implementation is to an ideal quantum computer. The common approach of completely characterizing the computation process via quantum process tomography requires an exponential amount of resources, and thus is not practical even for relatively small devices. We solve this problem by demonstrating that twirling experiments previously used to characterize the average fidelity of quantum memories efficiently can be easily adapted to estimate the average fidelity of the experimental implementation of important quantum computation processes, such as unitaries in the Clifford group, in a practical and efficient manner with applicability in current quantum devices. Using this procedure, we demonstrate state-of-the-art coherent control of an ensemble of magnetic moments of nuclear spins in a single crystal solid by implementing the encoding operation for a 3-qubit code with only a 1% degradation in average fidelity discounting preparation and measurement errors. We also highlight one of the advances that was instrumental in achieving such high fidelity control.