Ten simple rules for computational biologists collaborating with wet lab researchers.

Computational biologists are frequently engaged in collaborative data analysis with wet lab researchers. These interdisciplinary projects, as necessary as they are to the scientific endeavor, can be surprisingly challenging due to cultural differences in operations and values. In this Ten Simple Rules guide, we aim to help dry lab researchers identify sources of friction and provide actionable tools to facilitate respectful, open, transparent, and rewarding collaborations.

Involvement of circulating factors in the transmission of paternal experiences through the germline.

Environmental factors can change phenotypes in exposed individuals and offspring and involve the germline, likely via biological signals in the periphery that communicate with germ cells. Here, using a mouse model of paternal exposure to traumatic stress, we identify circulating factors involving peroxisome proliferator-activated receptor (PPAR) pathways in the effects of exposure to the germline. We show that exposure alters metabolic functions and pathways, particularly lipid-derived metabolites, in exposed fathers and their offspring. We collected data in a human cohort exposed to childhood trauma and observed similar metabolic alterations in circulation, suggesting conserved effects. Chronic injection of serum from trauma-exposed males into controls recapitulates metabolic phenotypes in the offspring. We identify lipid-activated nuclear receptors PPARs as potential mediators of the effects from father to offspring. Pharmacological PPAR activation in vivo reproduces metabolic dysfunctions in the offspring and grand-offspring of injected males and affects the sperm transcriptome in fathers and sons. In germ-like cells in vitro, both serum and PPAR agonist induce PPAR activation. Together, these results highlight the role of circulating factors as potential communication vectors between the periphery and the germline.

enrichMiR predicts functionally relevant microRNAs based on target collections.

MicroRNAs (miRNAs) are small non-coding RNAs that are among the main post-transcriptional regulators of gene expression. A number of data collections and prediction tools have gathered putative or confirmed targets of these regulators. It is often useful, for discovery and validation, to harness such collections to perform target enrichment analysis in given transcriptional signatures or gene-sets in order to predict involved miRNAs. While several methods have been proposed to this end, a flexible and user-friendly interface for such analyses using various approaches and collections is lacking. enrichMiR (https://ethz-ins.org/enrichMiR/) addresses this gap by enabling users to perform a series of enrichment tests, based on several target collections, to rank miRNAs according to their likely involvement in the control of a given transcriptional signature or gene-set. enrichMiR results can furthermore be visualised through interactive and publication-ready plots. To guide the choice of the appropriate analysis method, we benchmarked various tests across a panel of experiments involving the perturbation of known miRNAs. Finally, we showcase enrichMiR functionalities in a pair of use cases.

The vascular gene Apold1 is dispensable for normal development but controls angiogenesis under pathological conditions.

The molecular mechanisms of angiogenesis have been intensely studied, but many genes that control endothelial behavior and fate still need to be described. Here, we characterize the role of Apold1 (Apolipoprotein L domain containing 1) in angiogenesis in vivo and in vitro. Single-cell analyses reveal that - across tissues - the expression of Apold1 is restricted to the vasculature and that Apold1 expression in endothelial cells (ECs) is highly sensitive to environmental factors. Using Apold1-/- mice, we find that Apold1 is dispensable for development and does not affect postnatal retinal angiogenesis nor alters the vascular network in adult brain and muscle. However, when exposed to ischemic conditions following photothrombotic stroke as well as femoral artery ligation, Apold1-/- mice display dramatic impairments in recovery and revascularization. We also find that human tumor endothelial cells express strikingly higher levels of Apold1 and that Apold1 deletion in mice stunts the growth of subcutaneous B16 melanoma tumors, which have smaller and poorly perfused vessels. Mechanistically, Apold1 is activated in ECs upon growth factor stimulation as well as in hypoxia, and Apold1 intrinsically controls EC proliferation but not migration. Our data demonstrate that Apold1 is a key regulator of angiogenesis in pathological settings, whereas it does not affect developmental angiogenesis, thus making it a promising candidate for clinical investigation.

scanMiR: a biochemically based toolkit for versatile and efficient microRNA target prediction.

MOTIVATION: microRNAs are important post-transcriptional regulators of gene expression, but the identification of functionally relevant targets is still challenging. Recent research has shown improved prediction of microRNA-mediated repression using a biochemical model combined with empirically-derived k-mer affinity predictions; however, these findings are not easily applicable. RESULTS: We translate this approach into a flexible and user-friendly bioconductor package, scanMiR, also available through a web interface. Using lightweight linear models, scanMiR efficiently scans for binding sites, estimates their affinity and predicts aggregated transcript repression. Moreover, flexible 3'-supplementary alignment enables the prediction of unconventional interactions, such as bindings potentially leading to target-directed microRNA degradation or slicing. We showcase scanMiR through a systematic scan for such unconventional sites on neuronal transcripts, including lncRNAs and circRNAs. Finally, in addition to the main bioconductor package implementing these functions, we provide a user-friendly web application enabling the scanning of sequences, the visualization of predicted bindings and the browsing of predicted target repression. AVAILABILITY AND IMPLEMENTATION: scanMiR and companion packages are implemented in R, released under the GPL-3 and accessible on Bioconductor (https://bioconductor.org/packages/release/bioc/html/scanMiR.html) as well as through a shiny web server (https://ethz-ins.org/scanMiR/). SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Pervasive compartment-specific regulation of gene expression during homeostatic synaptic scaling.

Synaptic scaling is a form of homeostatic plasticity which allows neurons to adjust their action potential firing rate in response to chronic alterations in neural activity. Synaptic scaling requires profound changes in gene expression, but the relative contribution of local and cell-wide mechanisms is controversial. Here we perform a comprehensive multi-omics characterization of the somatic and process compartments of primary rat hippocampal neurons during synaptic scaling. We uncover both highly compartment-specific and correlating changes in the neuronal transcriptome and proteome. Whereas downregulation of crucial regulators of neuronal excitability occurs primarily in the somatic compartment, structural components of excitatory postsynapses are mostly downregulated in processes. Local inhibition of protein synthesis in processes during scaling is confirmed for candidate synaptic proteins. Motif analysis further suggests an important role for trans-acting post-transcriptional regulators, including RNA-binding proteins and microRNAs, in the local regulation of the corresponding mRNAs. Altogether, our study indicates that, during synaptic scaling, compartmentalized gene expression changes might co-exist with neuron-wide mechanisms to allow synaptic computation and homeostasis.

Streamlining differential exon and 3' UTR usage with diffUTR.

BACKGROUND: Despite the importance of alternative poly-adenylation and 3' UTR length for a variety of biological phenomena, there are limited means of detecting UTR changes from standard transcriptomic data. RESULTS: We present the diffUTR Bioconductor package which streamlines and improves upon differential exon usage (DEU) analyses, and leverages existing DEU tools and alternative poly-adenylation site databases to enable differential 3' UTR usage analysis. We demonstrate the diffUTR features and show that it is more flexible and more accurate than state-of-the-art alternatives, both in simulations and in real data. CONCLUSIONS: diffUTR enables differential 3' UTR analysis and more generally facilitates DEU and the exploration of their results.

Isoform-resolved correlation analysis between mRNA abundance regulation and protein level degradation.

Profiling of biological relationships between different molecular layers dissects regulatory mechanisms that ultimately determine cellular function. To thoroughly assess the role of protein post-translational turnover, we devised a strategy combining pulse stable isotope-labeled amino acids in cells (pSILAC), data-independent acquisition mass spectrometry (DIA-MS), and a novel data analysis framework that resolves protein degradation rate on the level of mRNA alternative splicing isoforms and isoform groups. We demonstrated our approach by the genome-wide correlation analysis between mRNA amounts and protein degradation across different strains of HeLa cells that harbor a high grade of gene dosage variation. The dataset revealed that specific biological processes, cellular organelles, spatial compartments of organelles, and individual protein isoforms of the same genes could have distinctive degradation rate. The protein degradation diversity thus dissects the corresponding buffering or concerting protein turnover control across cancer cell lines. The data further indicate that specific mRNA splicing events such as intron retention significantly impact the protein abundance levels. Our findings support the tight association between transcriptome variability and proteostasis and provide a methodological foundation for studying functional protein degradation.

YY1 Haploinsufficiency Causes an Intellectual Disability Syndrome Featuring Transcriptional and Chromatin Dysfunction.

Yin and yang 1 (YY1) is a well-known zinc-finger transcription factor with crucial roles in normal development and malignancy. YY1 acts both as a repressor and as an activator of gene expression. We have identified 23 individuals with de novo mutations or deletions of YY1 and phenotypic features that define a syndrome of cognitive impairment, behavioral alterations, intrauterine growth restriction, feeding problems, and various congenital malformations. Our combined clinical and molecular data define "YY1 syndrome" as a haploinsufficiency syndrome. Through immunoprecipitation of YY1-bound chromatin from affected individuals' cells with antibodies recognizing both ends of the protein, we show that YY1 deletions and missense mutations lead to a global loss of YY1 binding with a preferential retention at high-occupancy sites. Finally, we uncover a widespread loss of H3K27 acetylation in particular on the YY1-bound enhancers, underscoring a crucial role for YY1 in enhancer regulation. Collectively, these results define a clinical syndrome caused by haploinsufficiency of YY1 through dysregulation of key transcriptional regulators.

Exploratory rearing: a context- and stress-sensitive behavior recorded in the open-field test.

Stressful experiences are linked to anxiety disorders in humans. Similar effects are observed in rodent models, where anxiety is often measured in classic conflict tests such as the open-field test. Spontaneous rearing behavior, in which rodents stand on their hind legs to explore, can also be observed in this test yet is often ignored. We define two forms of rearing, supported rearing (in which the animal rears against the walls of the arena) and unsupported rearing (in which the animal rears without contacting the walls of the arena). Using an automated open-field test, we show that both rearing behaviors appear to be strongly context dependent and show clear sex differences, with females rearing less than males. We show that unsupported rearing is sensitive to acute stress, and is reduced under more averse testing conditions. Repeated testing and handling procedures lead to changes in several parameters over varying test sessions, yet unsupported rearing appears to be rather stable within a given animal. Rearing behaviors could therefore provide an additional measure of anxiety in rodents relevant for behavioral studies, as they appear to be highly sensitive to context and may be used in repeated testing designs.

RNAontheBENCH: computational and empirical resources for benchmarking RNAseq quantification and differential expression methods.

RNA sequencing (RNAseq) has become the method of choice for transcriptome analysis, yet no consensus exists as to the most appropriate pipeline for its analysis, with current benchmarks suffering important limitations. Here, we address these challenges through a rich benchmarking resource harnessing (i) two RNAseq datasets including ERCC ExFold spike-ins; (ii) Nanostring measurements of a panel of 150 genes on the same samples; (iii) a set of internal, genetically-determined controls; (iv) a reanalysis of the SEQC dataset; and (v) a focus on relative quantification (i.e. across-samples). We use this resource to compare different approaches to each step of RNAseq analysis, from alignment to differential expression testing. We show that methods providing the best absolute quantification do not necessarily provide good relative quantification across samples, that count-based methods are superior for gene-level relative quantification, and that the new generation of pseudo-alignment-based software performs as well as established methods, at a fraction of the computing time. We also assess the impact of library type and size on quantification and differential expression analysis. Finally, we have created a R package and a web platform to enable the simple and streamlined application of this resource to the benchmarking of future methods.

Cell reprogramming requires silencing of a core subset of polycomb targets.

Transcription factor (TF)-induced reprogramming of somatic cells into induced pluripotent stem cells (iPSC) is associated with genome-wide changes in chromatin modifications. Polycomb-mediated histone H3 lysine-27 trimethylation (H3K27me3) has been proposed as a defining mark that distinguishes the somatic from the iPSC epigenome. Here, we dissected the functional role of H3K27me3 in TF-induced reprogramming through the inactivation of the H3K27 methylase EZH2 at the onset of reprogramming. Our results demonstrate that surprisingly the establishment of functional iPSC proceeds despite global loss of H3K27me3. iPSC lacking EZH2 efficiently silenced the somatic transcriptome and differentiated into tissues derived from the three germ layers. Remarkably, the genome-wide analysis of H3K27me3 in Ezh2 mutant iPSC cells revealed the retention of this mark on a highly selected group of Polycomb targets enriched for developmental regulators controlling the expression of lineage specific genes. Erasure of H3K27me3 from these targets led to a striking impairment in TF-induced reprogramming. These results indicate that PRC2-mediated H3K27 trimethylation is required on a highly selective core of Polycomb targets whose repression enables TF-dependent cell reprogramming.

miRNA-mediated inhibition of an actomyosin network in hippocampal pyramidal neurons restricts sociability in adult male mice.

Social deficits are frequently observed in patients suffering from neurodevelopmental disorders, but the molecular mechanisms regulating sociability are still poorly understood. We recently reported that the loss of the microRNA (miRNA) cluster miR-379-410 leads to hypersocial behavior and anxiety in mice. Here, we show that ablating miR-379-410 in excitatory neurons of the postnatal mouse hippocampus recapitulates hypersociability, but not anxiety. At the cellular level, miR-379-410 loss in excitatory neurons leads to larger dendritic spines, increased excitatory synaptic transmission, and upregulation of an actomyosin gene network. Re-expression of three cluster miRNAs, as well as pharmacological inhibition of the actomyosin activator ROCK, is sufficient to reinstate normal sociability in miR-379-410 knockout mice. Several actomyosin genes and miR-379-410 family members are reciprocally dysregulated in isogenic human induced pluripotent stem cell (iPSC)-derived neurons harboring a deletion present in patients with Williams-Beuren syndrome, characterized by hypersocial behavior. Together, our results show an miRNA-actomyosin pathway involved in social behavior regulation.

On the identification of differentially-active transcription factors from ATAC-seq data.

ATAC-seq has emerged as a rich epigenome profiling technique, and is commonly used to identify Transcription Factors (TFs) underlying given phenomena. A number of methods can be used to identify differentially-active TFs through the accessibility of their DNA-binding motif, however little is known on the best approaches for doing so. Here we benchmark several such methods using a combination of curated datasets with various forms of short-term perturbations on known TFs, as well as semi-simulations. We include both methods specifically designed for this type of data as well as some that can be repurposed for it. We also investigate variations to these methods, and identify three particularly promising approaches (chromVAR-limma with critical adjustments, monaLisa and a combination of GC smooth quantile normalization and multivariate modeling). We further investigate the specific use of nucleosome-free fragments, the combination of top methods, and the impact of technical variation. Finally, we illustrate the use of the top methods on a novel dataset to characterize the impact on DNA accessibility of TRAnscription Factor TArgeting Chimeras (TRAFTAC), which can deplete TFs - in our case NFkB - at the protein level.

Noradrenaline release from the locus coeruleus shapes stress-induced hippocampal gene expression.

Exposure to an acute stressor triggers a complex cascade of neurochemical events in the brain. However, deciphering their individual impact on stress-induced molecular changes remains a major challenge. Here, we combine RNA sequencing with selective pharmacological, chemogenetic, and optogenetic manipulations to isolate the contribution of the locus coeruleus-noradrenaline (LC-NA) system to the acute stress response in mice. We reveal that NA release during stress exposure regulates a large and reproducible set of genes in the dorsal and ventral hippocampus via ß-adrenergic receptors. For a smaller subset of these genes, we show that NA release triggered by LC stimulation is sufficient to mimic the stress-induced transcriptional response. We observe these effects in both sexes, and independent of the pattern and frequency of LC activation. Using a retrograde optogenetic approach, we demonstrate that hippocampus-projecting LC neurons directly regulate hippocampal gene expression. Overall, a highly selective set of astrocyte-enriched genes emerges as key targets of LC-NA activation, most prominently several subunits of protein phosphatase 1 (Ppp1r3c, Ppp1r3d, Ppp1r3g) and type II iodothyronine deiodinase (Dio2). These results highlight the importance of astrocytic energy metabolism and thyroid hormone signaling in LC-mediated hippocampal function and offer new molecular targets for understanding how NA impacts brain function in health and disease.

Sperm chromatin accessibility's involvement in the intergenerational effects of stress hormone receptor activation.

Dexamethasone is a stress hormone receptor agonist used widely in clinics. We and others previously showed that paternal administration of dexamethasone in mice affects the phenotype of their offspring. The substrate of intergenerational transmission of environmentally induced effects often involves changes in sperm RNA, yet other epigenetic modifications in the germline can be affected and are also plausible candidates. First, we tested the involvement of altered sperm RNAs in the transmission of dexamethasone induced phenotypes across generations. We did this by injecting sperm RNA into naïve fertilized oocytes, before performing metabolic and behavioral phenotyping of the offspring. We observed phenotypic changes in discordance with those found in offspring generated by in vitro fertilization using sperm from dexamethasone exposed males. Second, we investigated the effect of dexamethasone on chromatin accessibility using ATAC sequencing and found significant changes at specific genomic features and gene regulatory loci. Employing q-RT-PCR, we show altered expression of a gene in the tissue of offspring affected by accessibility changes in sperm. Third, we establish a correlation between specific DNA modifications and stress hormone receptor activity as a likely contributing factor influencing sperm accessibility. Finally, we independently investigated this dependency by genetically reducing thymine-DNA glycosylase levels and observing concomitant changes at the level of chromatin accessibility and stress hormone receptor activity.

Meta-analysis of (single-cell method) benchmarks reveals the need for extensibility and interoperability.

Computational methods represent the lifeblood of modern molecular biology. Benchmarking is important for all methods, but with a focus here on computational methods, benchmarking is critical to dissect important steps of analysis pipelines, formally assess performance across common situations as well as edge cases, and ultimately guide users on what tools to use. Benchmarking can also be important for community building and advancing methods in a principled way. We conducted a meta-analysis of recent single-cell benchmarks to summarize the scope, extensibility, and neutrality, as well as technical features and whether best practices in open data and reproducible research were followed. The results highlight that while benchmarks often make code available and are in principle reproducible, they remain difficult to extend, for example, as new methods and new ways to assess methods emerge. In addition, embracing containerization and workflow systems would enhance reusability of intermediate benchmarking results, thus also driving wider adoption.

Harnessing PROTAC technology to combat stress hormone receptor activation.

Counteracting the overactivation of glucocorticoid receptors (GR) is an important therapeutic goal in stress-related psychiatry and beyond. The only clinically approved GR antagonist lacks selectivity and induces unwanted side effects. To complement existing tools of small-molecule-based inhibitors, we present a highly potent, catalytically-driven GR degrader, KH-103, based on proteolysis-targeting chimera technology. This selective degrader enables immediate and reversible GR depletion that is independent of genetic manipulation and circumvents transcriptional adaptations to inhibition. KH-103 achieves passive inhibition, preventing agonistic induction of gene expression, and significantly averts the GR's genomic effects compared to two currently available inhibitors. Application in primary-neuron cultures revealed the dependency of a glucocorticoid-induced increase in spontaneous calcium activity on GR. Finally, we present a proof of concept for application in vivo. KH-103 opens opportunities for a more lucid interpretation of GR functions with translational potential.

Reuniting philosophy and science to advance cancer research.

Cancers rely on multiple, heterogeneous processes at different scales, pertaining to many biomedical fields. Therefore, understanding cancer is necessarily an interdisciplinary task that requires placing specialised experimental and clinical research into a broader conceptual, theoretical, and methodological framework. Without such a framework, oncology will collect piecemeal results, with scant dialogue between the different scientific communities studying cancer. We argue that one important way forward in service of a more successful dialogue is through greater integration of applied sciences (experimental and clinical) with conceptual and theoretical approaches, informed by philosophical methods. By way of illustration, we explore six central themes: (i) the role of mutations in cancer; (ii) the clonal evolution of cancer cells; (iii) the relationship between cancer and multicellularity; (iv) the tumour microenvironment; (v) the immune system; and (vi) stem cells. In each case, we examine open questions in the scientific literature through a philosophical methodology and show the benefit of such a synergy for the scientific and medical understanding of cancer.

MicroRNA-138 controls hippocampal interneuron function and short-term memory in mice.

The proper development and function of neuronal circuits rely on a tightly regulated balance between excitatory and inhibitory (E/I) synaptic transmission, and disrupting this balance can cause neurodevelopmental disorders, for example, schizophrenia. MicroRNA-dependent gene regulation in pyramidal neurons is important for excitatory synaptic function and cognition, but its role in inhibitory interneurons is poorly understood. Here, we identify miR138-5p as a regulator of short-term memory and inhibitory synaptic transmission in the mouse hippocampus. Sponge-mediated miR138-5p inactivation specifically in mouse parvalbumin (PV)-expressing interneurons impairs spatial recognition memory and enhances GABAergic synaptic input onto pyramidal neurons. Cellular and behavioral phenotypes associated with miR138-5p inactivation are paralleled by an upregulation of the schizophrenia (SCZ)-associated Erbb4, which we validated as a direct miR138-5p target gene. Our findings suggest that miR138-5p is a critical regulator of PV interneuron function in mice, with implications for cognition and SCZ. More generally, they provide evidence that microRNAs orchestrate neural circuit development by fine-tuning both excitatory and inhibitory synaptic transmission.