DNA damage during S-phase mediates the proliferation-quiescence decision in the subsequent G1 via p21 expression

Following DNA damage caused by exogenous sources, such as ionizing radiation, the tumour suppressor p53 mediates cell cycle arrest via expression of the CDK inhibitor, p21. However, the role of p21 in maintaining genomic stability in the absence of exogenous DNA-damaging agents is unclear. Here, using live single-cell measurements of p21 protein in proliferating cultures, we show that naturally occurring DNA damage incurred over S-phase causes p53-dependent accumulation of p21 during mother G2- and daughter G1-phases. High p21 levels mediate G1 arrest via CDK inhibition, yet lower levels have no impact on G1 progression, and the ubiquitin ligases CRL4Cdt2 and SCFSkp2 couple to degrade p21 prior to the G1/S transition. Mathematical modelling reveals that a bistable switch, created by CRL4Cdt2, promotes irreversible S-phase entry by keeping p21 levels low, preventing premature S-phase exit upon DNA damage. Thus, we characterize how p21 regulates the proliferation-quiescence decision to maintain genomic stability

PhenoM: a database of morphological phenotypes caused by mutation of essential genes in Saccharomyces cerevisiae

About one-fifth of the genes in the budding yeast are essential for haploid viability and cannot be functionally assessed using standard genetic approaches such as gene deletion. To facilitate genetic analysis of essential genes, we and others have assembled collections of yeast strains expressing temperature-sensitive (ts) alleles of essential genes. To explore the phenotypes caused by essential gene mutation we used a panel of genetically engineered fluorescent markers to explore the morphology of cells in the ts strain collection using high-throughput microscopy. Here, we describe the design and implementation of an online database, PhenoM (Phenomics of yeast Mutants), for storing, retrieving, visualizing and data mining the quantitative single-cell measurements extracted from micrographs of the ts mutant cells. PhenoM allows users to rapidly search and retrieve raw images and their quantified morphological data for genes of interest. The database also provides several data-mining tools, including a PhenoBlast module for phenotypic comparison between mutant strains and a Gene Ontology module for functional enrichment analysis of gene sets showing similar morphological alterations. The current PhenoM version 1.0 contains 78 194 morphological images and 1 909 914 cells covering six subcellular compartments or structures for 775 ts alleles spanning 491 essential genes. PhenoM is freely available at http://phenom.ccbr.utoronto.ca/

A Quantitative RNAi Screen for JNK Modifiers Identifies Pvr as a Novel Regulator of Drosophila Immune Signaling

Drosophila melanogaster responds to gram-negative bacterial challenges through the IMD pathway, a signal transduction cassette that is driven by the coordinated activities of JNK, NF-κB and caspase modules. While many modifiers of NF-κB activity were identified in cell culture and in vivo assays, the regulatory apparatus that determines JNK inputs into the IMD pathway is relatively unexplored. In this manuscript, we present the first quantitative screen of the entire genome of Drosophila for novel regulators of JNK activity in the IMD pathway. We identified a large number of gene products that negatively or positively impact on JNK activation in the IMD pathway. In particular, we identified the Pvr receptor tyrosine kinase as a potent inhibitor of JNK activation. In a series of in vivo and cell culture assays, we demonstrated that activation of the IMD pathway drives JNK-dependent expression of the Pvr ligands, Pvf2 and Pvf3, which in turn act through the Pvr/ERK MAP kinase pathway to attenuate the JNK and NF-κB arms of the IMD pathway. Our data illuminate a poorly understood arm of a critical and evolutionarily conserved innate immune response. Furthermore, given the pleiotropic involvement of JNK in eukaryotic cell biology, we believe that many of the novel regulators identified in this screen are of interest beyond immune signaling

Integrin-Dependent Activation of the JNK Signaling Pathway by Mechanical Stress

Mechanical force is known to modulate the activity of the Jun N-terminal kinase (JNK) signaling cascade. However, the effect of mechanical stresses on JNK signaling activation has previously only been analyzed by in vitro detection methods. It still remains unknown how living cells activate the JNK signaling cascade in response to mechanical stress and what its functions are in stretched cells

Posterior Association Networks and Functional Modules Inferred from Rich Phenotypes of Gene Perturbations

Combinatorial gene perturbations provide rich information for a systematic exploration of genetic interactions. Despite successful applications to bacteria and yeast, the scalability of this approach remains a major challenge for higher organisms such as humans. Here, we report a novel experimental and computational framework to efficiently address this challenge by limiting the ‘search space’ for important genetic interactions. We propose to integrate rich phenotypes of multiple single gene perturbations to robustly predict functional modules, which can subsequently be subjected to further experimental investigations such as combinatorial gene silencing. We present posterior association networks (PANs) to predict functional interactions between genes estimated using a Bayesian mixture modelling approach. The major advantage of this approach over conventional hypothesis tests is that prior knowledge can be incorporated to enhance predictive power. We demonstrate in a simulation study and on biological data, that integrating complementary information greatly improves prediction accuracy. To search for significant modules, we perform hierarchical clustering with multiscale bootstrap resampling. We demonstrate the power of the proposed methodologies in applications to Ewing's sarcoma and human adult stem cells using publicly available and custom generated data, respectively. In the former application, we identify a gene module including many confirmed and highly promising therapeutic targets. Genes in the module are also significantly overrepresented in signalling pathways that are known to be critical for proliferation of Ewing's sarcoma cells. In the latter application, we predict a functional network of chromatin factors controlling epidermal stem cell fate. Further examinations using ChIP-seq, ChIP-qPCR and RT-qPCR reveal that the basis of their genetic interactions may arise from transcriptional cross regulation. A Bioconductor package implementing PAN is freely available online at http://bioconductor.org/packages/release/bioc/html/PANR.html

Occupational Radiation Protection in Interventional Radiology: A Joint Guideline of the Cardiovascular and Interventional Radiology Society of Europe and the Society of Interventional Radiology

No Abstract

Data-analysis strategies for image-based cell profiling

Image-based cell profiling is a high-throughput strategy for the quantification of phenotypic differences among a variety of cell populations. It paves the way to studying biological systems on a large scale by using chemical and genetic perturbations. The general workflow for this technology involves image acquisition with high-throughput microscopy systems and subsequent image processing and analysis. Here, we introduce the steps required to create high-quality image-based (i.e., morphological) profiles from a collection of microscopy images. We recommend techniques that have proven useful in each stage of the data analysis process, on the basis of the experience of 20 laboratories worldwide that are refining their image-based cell-profiling methodologies in pursuit of biological discovery. The recommended techniques cover alternatives that may suit various biological goals, experimental designs, and laboratories' preferences.Peer reviewe