Shift in MSL1 alternative polyadenylation in response to DNA damage protects cancer cells from chemotherapeutic agent-induced apoptosis
DNA damage reshapes the cellular transcriptome by modulating RNA transcription and processing. In cancer cells, these changes can alter the expression of genes in the immune surveillance and cell death pathways. Here, we investigate how DNA damage impacts alternative polyadenylation (APA) using the PAPERCLIP technique. We find that APA shifts are a coordinated response for hundreds of genes to DNA damage, and we identify PCF11 as an important contributor of DNA damage-induced APA shifts. One of these APA shifts results in upregulation of the full-length MSL1 mRNA isoform, which protects cells from DNA damage-induced apoptosis and promotes cell survival from DNA-damaging agents. Importantly, blocking MSL1 upregulation enhances cytotoxicity of chemotherapeutic agents even in the absence of p53 and overcomes chemoresistance. Our study demonstrates that characterizing adaptive APA shifts to DNA damage has therapeutic implications and reveals a link between PCF11, the MSL complex, and DNA damage-induced apoptosis.
Attenuated activation of pulmonary immune cells in mRNA-1273–vaccinated hamsters after SARS-CoV-2 infection
The mRNA-1273 vaccine is effective against SARS-CoV-2 and was granted emergency use authorization by the FDA. Clinical studies, however, cannot provide the controlled response to infection and complex immunological insight that are only possible with preclinical studies. Hamsters are the only model that reliably exhibits severe SARS-CoV-2 disease similar to that in hospitalized patients, making them pertinent for vaccine evaluation. We demonstrate that prime or prime-boost administration of mRNA-1273 in hamsters elicited robust neutralizing antibodies, ameliorated weight loss, suppressed SARS-CoV-2 replication in the airways, and better protected against disease at the highest prime-boost dose. Unlike in mice and nonhuman primates, low-level virus replication in mRNA-1273–vaccinated hamsters coincided with an anamnestic response. Single-cell RNA sequencing of lung tissue permitted high-resolution analysis that is not possible in vaccinated humans. mRNA-1273 prevented inflammatory cell infiltration and the reduction of lymphocyte proportions, but enabled antiviral responses conducive to lung homeostasis. Surprisingly, infection triggered transcriptome programs in some types of immune cells from vaccinated hamsters that were shared, albeit attenuated, with mock-vaccinated hamsters. Our results support the use of mRNA-1273 in a 2-dose schedule and provide insight into the potential responses within the lungs of vaccinated humans who are exposed to SARS-CoV-2.
Interpreting the effects of genetic variants is key to understanding individual susceptibility to disease and designing personalized therapeutic approaches. Modern experimental technologies are enabling the generation of massive compendia of human genome sequence data and associated molecular and phenotypic traits, together with genome-scale expression, epigenomics and other functional genomic data. Integrative computational models can leverage these data to understand variant impact, elucidate the effect of dysregulated genes on biological pathways in specific disease and tissue contexts, and interpret disease risk beyond what is feasible with experiments alone. In this Review, we discuss recent developments in machine learning algorithms for genome interpretation and for integrative molecular-level modelling of cells, tissues and organs relevant to disease. More specifically, we highlight existing methods and key challenges and opportunities in identifying specific disease-causing genetic variants and linking them to molecular pathways and, ultimately, to disease phenotypes.
Transcription factors (TFs) often function as a module including both master factors and mediators binding at cis-regulatory regions to modulate nearby gene transcription. ChIP-seq profiling of multiple TFs makes it feasible to infer functional TF modules. However, when inferring TF modules based on co-localization of ChIP-seq peaks, often many weak binding events are missed, especially for mediators, resulting in incomplete identification of modules. To address this problem, we develop a ChIP-seq data-driven Gibbs Sampler to infer Modules (ChIP-GSM) using a Bayesian framework that integrates ChIP-seq profiles of multiple TFs. ChIP-GSM samples read counts of module TFs iteratively to estimate the binding potential of a module to each region and, across all regions, estimates the module abundance. Using inferred module-region probabilistic bindings as feature units, ChIP-GSM then employs logistic regression to predict active regulatory elements. Validation of ChIP-GSM predicted regulatory regions on multiple independent datasets sharing the same context confirms the advantage of using TF modules for predicting regulatory activity. In a case study of K562 cells, we demonstrate that the ChIP-GSM inferred modules form as groups, activate gene expression at different time points, and mediate diverse functional cellular processes. Hence, ChIP-GSM infers biologically meaningful TF modules and improves the prediction accuracy of regulatory region activities.
Experimental approaches to study tissue specificity enable insight into the nature and organization of the cell types and tissues that constitute complex multicellular organisms. Machine learning provides a powerful tool to investigate and interpret tissue-specific experimental data. In this Review, we first provide a brief introduction to key single-cell and whole-tissue approaches that allow investigation of tissue specificity and then highlight two classes of machine-learning-based methods, which can be applied to analyse, model and interpret these experimental data. Deep learning methods can predict tissue-dependent effects of individual mutations on gene expression, alternative splicing and disease phenotypes. Network-based approaches can capture relationships between biomolecules, integrate large heterogeneous data compendia to model molecular circuits and identify tissue-specific functional relationships and regulatory connections. We conclude with an outlook to future possibilities in examining multicellular complexity by combining high-resolution, large-scale multiomics data sets and interpretable machine learning models.
CROTON: an automated and variant-aware deep learning framework for predicting CRISPR/Cas9 editing outcomes
CRISPR/Cas9 is a revolutionary gene-editing technology that has been widely utilized in biology, biotechnology and medicine. CRISPR/Cas9 editing outcomes depend on local DNA sequences at the target site and are thus predictable. However, existing prediction methods are dependent on both feature and model engineering, which restricts their performance to existing knowledge about CRISPR/Cas9 editing
We use theory and numerical computation to determine the shape of an axisymmetric fluid membrane with a resistance to bending and constant area. The membrane connects two rings in the classic geometry that produces a catenoidal shape in a soap film. In our problem, we find infinitely many branches of solutions for the shape and external force as functions of the separation of the rings, analogous to the infinite family of eigenmodes for the Euler buckling of a slender rod. Special attention is paid to the catenoid, which emerges as the shape of maximal allowable separation when the area is less than a critical area equal to the planar area enclosed by the two rings. A perturbation theory argument directly relates the tension of catenoidal membranes to the stability of catenoidal soap films in this regime. When the membrane area is larger than the critical area, we find additional cylindrical tether solutions to the shape equations at large ring separation, and that arbitrarily large ring separations are possible. These results apply for the case of vanishing Gaussian curvature modulus; when the Gaussian curvature modulus is nonzero and the area is below the critical area, the force and the membrane tension diverge as the ring separation approaches its maximum value. We also examine the stability of our shapes and analytically show that catenoidal membranes have markedly different stability properties than their soap film counterparts.
To provide a multi-omics resource and investigate transcriptional regulatory mechanisms, we profile the transcriptome, chromatin accessibility, and methylation status of over 70,000 single nuclei (sn) from adult mouse pituitaries. Paired snRNAseq and snATACseq datasets from individual animals highlight a continuum between developmental epigenetically-encoded cell types and transcriptionally-determined transient cell states. Co-accessibility analysis-based identification of a putative Fshb cis-regulatory domain that overlaps the fertility-linked rs11031006 human polymorphism, followed by experimental validation illustrate the use of this resource for hypothesis generation. We also identify transcriptional and chromatin accessibility programs distinguishing each major cell type. Regulons, which are co-regulated gene sets sharing binding sites for a common transcription factor driver, recapitulate cell type clustering. We identify both cell type-specific and sex-specific regulons that are highly correlated with promoter accessibility, but not with methylation state, supporting the centrality of chromatin accessibility in shaping cell-defining transcriptional programs. The sn multi-omics atlas is accessible at snpituitaryatlas.princeton.edu.
SynNotch-CAR T cells overcome challenges of specificity, heterogeneity, and persistence in treating glioblastoma
Two major hurdles in chimeric antigen receptor (CAR) T cell therapy for solid tumors are ensuring specificity to tumor cells without affecting healthy cells and avoiding tumor escape due to antigen loss. To address these challenges, Hyrenius-Wittsten et al. and Choe et al. developed synthetic notch (synNotch)–CAR T cells targeting solid tumor antigens and used them to treat mouse models of mesothelioma, ovarian cancer, and glioblastoma. In both studies, the authors demonstrated that synNotch-CAR T cells were better at controlling tumors than traditional CAR T cells and did not result in toxicity or damage to healthy tissue. These results suggest that synNotch-CAR T cells may be an effective treatment strategy for solid tumors.
Systematic study of tissue-specific function of enhancers and their disease associations is a major challenge. We present an integrative machine-learning framework, FENRIR, that integrates thousands of disparate epigenetic and functional genomics datasets to infer tissue-specific functional relationships between enhancers for 140 diverse human tissues and cell types, providing a regulatory-region-centric approach to systematically identify disease-associated enhancers. We demonstrated its power to accurately prioritize enhancers associated with 25 complex diseases. In a case study on autism, FENRIR-prioritized enhancers showed a significant proband-specific de novo mutation enrichment in a large, sibling-controlled cohort, indicating pathogenic signal. We experimentally validated transcriptional regulatory activities of eight enhancers, including enhancers not previously reported with autism, and demonstrated their differential regulatory potential between proband and sibling alleles. Thus, FENRIR is an accurate and effective framework for the study of tissue-specific enhancers and their role in disease. FENRIR can be accessed at fenrir.flatironinstitute.org/.
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