Kidney tumours are among the most common solid tumours in children, comprising distinct subtypes differing in many aspects, including cell-of-origin, genetics, and pathology. Pre-clinical cell models ...capturing the disease heterogeneity are currently lacking. Here, we describe the first paediatric cancer organoid biobank. It contains tumour and matching normal kidney organoids from over 50 children with different subtypes of kidney cancer, including Wilms tumours, malignant rhabdoid tumours, renal cell carcinomas, and congenital mesoblastic nephromas. Paediatric kidney tumour organoids retain key properties of native tumours, useful for revealing patient-specific drug sensitivities. Using single cell RNA-sequencing and high resolution 3D imaging, we further demonstrate that organoid cultures derived from Wilms tumours consist of multiple different cell types, including epithelial, stromal and blastemal-like cells. Our organoid biobank captures the heterogeneity of paediatric kidney tumours, providing a representative collection of well-characterised models for basic cancer research, drug-screening and personalised medicine.
De-novo reverse-engineering of genome-scale regulatory networks is a fundamental problem of biological and translational research. One of the major obstacles in developing and evaluating approaches ...for de-novo gene network reconstruction is the absence of high-quality genome-scale gold-standard networks of direct regulatory interactions. To establish a foundation for assessing the accuracy of de-novo gene network reverse-engineering, we constructed high-quality genome-scale gold-standard networks of direct regulatory interactions in Saccharomyces cerevisiae that incorporate binding and gene knockout data. Then we used 7 performance metrics to assess accuracy of 18 statistical association-based approaches for de-novo network reverse-engineering in 13 different datasets spanning over 4 data types. We found that most reconstructed networks had statistically significant accuracies. We also determined which statistical approaches and datasets/data types lead to networks with better reconstruction accuracies. While we found that de-novo reverse-engineering of the entire network is a challenging problem, it is possible to reconstruct sub-networks around some transcription factors with good accuracy. The latter transcription factors can be identified by assessing their connectivity in the inferred networks. Overall, this study provides the gene network reverse-engineering community with a rigorous assessment of the accuracy of S. cerevisiae gene network reconstruction and variability in performance of various approaches for learning both the entire network and sub-networks around transcription factors.
To understand regulatory systems, it would be useful to uniformly determine how different components contribute to the expression of all other genes. We therefore monitored mRNA expression ...genome-wide, for individual deletions of one-quarter of yeast genes, focusing on (putative) regulators. The resulting genetic perturbation signatures reflect many different properties. These include the architecture of protein complexes and pathways, identification of expression changes compatible with viability, and the varying responsiveness to genetic perturbation. The data are assembled into a genetic perturbation network that shows different connectivities for different classes of regulators. Four feed-forward loop (FFL) types are overrepresented, including incoherent type 2 FFLs that likely represent feedback. Systematic transcription factor classification shows a surprisingly high abundance of gene-specific repressors, suggesting that yeast chromatin is not as generally restrictive to transcription as is often assumed. The data set is useful for studying individual genes and for discovering properties of an entire regulatory system.
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•A systems analyses resource reports expression signatures for 1,484 yeast gene knockouts•Analysis reveals pathway branching, connectivity, and responsiveness to perturbations•Four feed-forward loop types are overrepresented in the genetic perturbation network•Transcription factor classification shows an abundance of gene-specific repressors
A comprehensive mRNA expression profiling study reports the effects of over 1,400 individual gene deletions in yeast, focusing on nonessential regulators of gene expression. Analysis of the data indicates a surprisingly high abundance of repressors, suggesting that chromatin itself may not be generally restrictive to transcription, as previously supposed.
Cancer is generally characterized by acquired genomic aberrations in a broad spectrum of types and sizes, ranging from single nucleotide variants to structural variants (SVs). At least 30% of cancers ...have a known pathogenic SV used in diagnosis or treatment stratification. However, research into the role of SVs in cancer has been limited due to difficulties in detection. Biological and computational challenges confound SV detection in cancer samples, including intratumor heterogeneity, polyploidy, and distinguishing tumor-specific SVs from germline and somatic variants present in healthy cells. Classification of tumor-specific SVs is challenging due to inconsistencies in detected breakpoints, derived variant types and biological complexity of some rearrangements. Full-spectrum SV detection with high recall and precision requires integration of multiple algorithms and sequencing technologies to rescue variants that are difficult to resolve through individual methods. Here, we explore current strategies for integrating SV callsets and to enable the use of tumor-specific SVs in precision oncology.
Defining protein complexes is critical to virtually all aspects of cell biology. Two recent affinity purification/mass spectrometry studies in Saccharomyces cerevisiae have vastly increased the ...available protein interaction data. The practical utility of such high throughput interaction sets, however, is substantially decreased by the presence of false positives. Here we created a novel probabilistic metric that takes advantage of the high density of these data, including both the presence and absence of individual associations, to provide a measure of the relative confidence of each potential protein-protein interaction. This analysis largely overcomes the noise inherent in high throughput immunoprecipitation experiments. For example, of the 12,122 binary interactions in the general repository of interaction data (BioGRID) derived from these two studies, we marked 7504 as being of substantially lower confidence. Additionally, applying our metric and a stringent cutoff we identified a set of 9074 interactions (including 4456 that were not among the 12,122 interactions) with accuracy comparable to that of conventional small scale methodologies. Finally we organized proteins into coherent multisubunit complexes using hierarchical clustering. This work thus provides a highly accurate physical interaction map of yeast in a format that is readily accessible to the biological community.
Adaptive evolution is generally assumed to progress through the accumulation of beneficial mutations. However, as deleterious mutations are common in natural populations, they generate a strong ...selection pressure to mitigate their detrimental effects through compensatory genetic changes. This process can potentially influence directions of adaptive evolution by enabling evolutionary routes that are otherwise inaccessible. Therefore, the extent to which compensatory mutations shape genomic evolution is of central importance. Here, we studied the capacity of the baker's yeast genome to compensate the complete loss of genes during evolution, and explored the long-term consequences of this process. We initiated laboratory evolutionary experiments with over 180 haploid baker's yeast genotypes, all of which initially displayed slow growth owing to the deletion of a single gene. Compensatory evolution following gene loss was rapid and pervasive: 68% of the genotypes reached near wild-type fitness through accumulation of adaptive mutations elsewhere in the genome. As compensatory mutations have associated fitness costs, genotypes with especially low fitnesses were more likely to be subjects of compensatory evolution. Genomic analysis revealed that as compensatory mutations were generally specific to the functional defect incurred, convergent evolution at the molecular level was extremely rare. Moreover, the majority of the gene expression changes due to gene deletion remained unrestored. Accordingly, compensatory evolution promoted genomic divergence of parallel evolving populations. However, these different evolutionary outcomes are not phenotypically equivalent, as they generated diverse growth phenotypes across environments. Taken together, these results indicate that gene loss initiates adaptive genomic changes that rapidly restores fitness, but this process has substantial pleiotropic effects on cellular physiology and evolvability upon environmental change. Our work also implies that gene content variation across species could be partly due to the action of compensatory evolution rather than the passive loss of genes.
Protein–DNA interactions are dynamic, and these dynamics are an important aspect of chromatin‐associated processes such as transcription or replication. Due to a lack of methods to study on‐ and ...off‐rates across entire genomes, protein–DNA interaction dynamics have not been studied extensively. Here, we determine in vivo off‐rates for the Saccharomyces cerevisiae chromatin organizing factor Abf1, at 191 sites simultaneously across the yeast genome. Average Abf1 residence times span a wide range, varying between 4.2 and 33 min. Sites with different off‐rates are associated with different functional characteristics. This includes their transcriptional dependency on Abf1, nucleosome positioning and the size of the nucleosome‐free region, as well as the ability to roadblock RNA polymerase II for termination. The results show how off‐rates contribute to transcription factor function and that DIVORSEQ (Determining In Vivo Off‐Rates by SEQuencing) is a meaningful way of investigating protein–DNA binding dynamics genome‐wide.
Synopsis
Using a new method (DIVORSEQ), genome‐wide off‐rates are determined for a DNA binding transcription factor. Abf1 exhibits site‐specific residence times that range from 4 to 33 min and which are associated with different functional characteristics.
A method to determine in vivo off‐rates across the genome.
Long Abf1 residence times are associated with large nucleosome free regions.
Changes in promoter activity closely follow Abf1 off‐rates.
Stable Abf1 binding is associated with efficient roadblock termination.
Using a new method (DIVORSEQ), genome‐wide off‐rates are determined for a DNA binding transcription factor. Abf1 exhibits site‐specific residence times that range from 4 to 33 min and which are associated with different functional characteristics.
Packaging of DNA into chromatin has a profound impact on gene expression. To understand how changes in chromatin influence transcription, we analyzed 165 mutants of chromatin machinery components in
...Saccharomyces cerevisiae. mRNA expression patterns change in 80% of mutants, always with specific effects, even for loss of widespread histone marks. The data are assembled into a network of chromatin interaction pathways. The network is function based, has a branched, interconnected topology, and lacks strict one-to-one relationships between complexes. Chromatin pathways are not separate entities for different gene sets, but share many components. The study evaluates which interactions are important for which genes and predicts additional interactions, for example between Paf1C and Set3C, as well as a role for Mediator in subtelomeric silencing. The results indicate the presence of gene-dependent effects that go beyond context-dependent binding of chromatin factors and provide a framework for understanding how specificity is achieved through regulating chromatin.
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► Loss of chromatin regulators results in highly specific effects on mRNA expression ► These effects are much more specific than the location of regulators or of marks ► The perturbation signatures identify a network of interactions between regulators ► Chromatin interaction pathways are highly branched and interconnected
Rhabdomyosarcomas (RMS) are mesenchyme‐derived tumors and the most common childhood soft tissue sarcomas. Treatment is intense, with a nevertheless poor prognosis for high‐risk patients. Discovery of ...new therapies would benefit from additional preclinical models. Here, we describe the generation of a collection of 19 pediatric RMS tumor organoid (tumoroid) models (success rate of 41%) comprising all major subtypes. For aggressive tumors, tumoroid models can often be established within 4–8 weeks, indicating the feasibility of personalized drug screening. Molecular, genetic, and histological characterization show that the models closely resemble the original tumors, with genetic stability over extended culture periods of up to 6 months. Importantly, drug screening reflects established sensitivities and the models can be modified by CRISPR/Cas9 with TP53 knockout in an embryonal RMS model resulting in replicative stress drug sensitivity. Tumors of mesenchymal origin can therefore be used to generate organoid models, relevant for a variety of preclinical and clinical research questions.
Synopsis
The first collection of comprehensively characterized tumor organoid (tumoroid) models of pediatric rhabdomyosarcomas (RMS) as novel preclinical models for this highly aggressive pediatric cancer entity.
This is the first collection of purely mesenchymal tumoroid models and only the second tumoroid collection of pediatric cancers.
RMS tumoroid models faithfully recapitulate molecular alterations of the parent tumor with retained transcriptional and clonal heterogeneity.
They are rapidly established and expanded with the ability to perform drug screening as fast as 27 days after sample acquisition (median 81 days).
Lastly, they are amenable to CRISPR/Cas9 editing to recapitulate mutations conferring poor prognosis in RMS (e.g., in TP53).
The first collection of comprehensively characterized tumor organoid (tumoroid) models of pediatric rhabdomyosarcomas (RMS) as novel preclinical models for this highly aggressive pediatric cancer entity.
The interpretation of genetic variants after genome-wide analysis is complex in heterogeneous disorders such as intellectual disability (ID). We investigate whether algorithms can be used to detect ...if a facial gestalt is present for three novel ID syndromes and if these techniques can help interpret variants of uncertain significance.
Facial features were extracted from photos of ID patients harboring a pathogenic variant in three novel ID genes (PACS1, PPM1D, and PHIP) using algorithms that model human facial dysmorphism, and facial recognition. The resulting features were combined into a hybrid model to compare the three cohorts against a background ID population.
We validated our model using images from 71 individuals with Koolen-de Vries syndrome, and then show that facial gestalts are present for individuals with a pathogenic variant in PACS1 (p = 8 × 10
), PPM1D (p = 4.65 × 10
), and PHIP (p = 6.3 × 10
). Moreover, two individuals with a de novo missense variant of uncertain significance in PHIP have significant similarity to the expected facial phenotype of PHIP patients (p < 1.52 × 10
).
Our results show that analysis of facial photos can be used to detect previously unknown facial gestalts for novel ID syndromes, which will facilitate both clinical and molecular diagnosis of rare and novel syndromes.
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