Expanding the repertoire of glucocorticoid receptor target genes by engineering genomic response elements

The glucocorticoid receptor (GR), a hormone-activated transcription factor, binds to a myriad of genomic binding sites yet seems to regulate a much smaller number of genes. Genome-wide analysis of GR binding and gene regulation has shown that the likelihood of GR-dependent regulation increases with decreased distance of its binding to the transcriptional start site of a gene. To test if we can adopt this knowledge to expand the repertoire of GR target genes, we used CRISPR/Cas-mediated homology-directed repair to add a single GR-binding site directly upstream of the transcriptional start site of each of four genes. To our surprise, we found that the addition of a single GR-binding site can be enough to convert a gene into a GR target. The gain of GR-dependent regulation was observed for two of four genes analyzed and coincided with acquired GR binding at the introduced binding site. However, the gene-specific gain of GR-dependent regulation could not be explained by obvious differences in chromatin accessibility between converted genes and their non-converted counterparts. Furthermore, by introducing GR-binding sequences with different nucleotide compositions, we show that activation can be facilitated by distinct sequences without obvious differences in activity between the GR-binding sequence variants we tested. The approach to use genome engineering to build genomic response elements facilitates the generation of cell lines with tailored repertoires of GR-responsive genes and a framework to test and refine our understanding of the cis-regulatory logic of gene regulation by testing if engineered response elements behave as predicted

Dormant but Active: Chilling Accumulation Modulates the Epigenome and Transcriptome of Prunus avium During Bud Dormancy

Indexación: Scopus.Temperate deciduous fruit tree species like sweet cherry (Prunus avium) require long periods of low temperatures to trigger dormancy release and flowering. In addition to sequence-based genetic diversity, epigenetic variation may contribute to different chilling requirements among varieties. For the low chill variety ‘Royal Dawn’ and high chill variety ‘Kordia’, we studied the methylome of floral buds during chilling accumulation using MethylC-seq to identify differentially methylated regions (DMRs) during chilling hours (CH) accumulation, followed by transcriptome analysis to correlate changes in gene expression with DNA methylation. We found that during chilling accumulation, DNA methylation increased from 173 CH in ‘Royal Dawn’ and 443 CH in ‘Kordia’ and was mostly associated with the CHH context. In addition, transcriptional changes were observed from 443 CH in ‘Kordia’ with 1,210 differentially expressed genes, increasing to 4,292 genes at 1,295 CH. While ‘Royal Dawn’ showed approximately 5,000 genes differentially expressed at 348 CH and 516 CH, showing a reprogramming that was specific for each genotype. From conserved upregulated genes that overlapped with hypomethylated regions and downregulated genes that overlapped with hypermethylated regions in both varieties, we identified genes related to cold-sensing, cold-signaling, oxidation-reduction process, metabolism of phenylpropanoids and lipids, and a MADS-box SVP-like gene. As a complementary analysis, we used conserved and non-conserved DEGs that presented a negative correlation between DNA methylations and mRNA levels across all chilling conditions, obtaining Gene Ontology (GO) categories related to abiotic stress, metabolism, and oxidative stress. Altogether, this data indicates that changes in DNA methylation precedes transcript changes and may occur as an early response to low temperatures to increase the cold tolerance in the endodormancy period, contributing with the first methylome information about the effect of environmental cues over two different genotypes of sweet cherry. © Copyright © 2020 Rothkegel, Sandoval, Soto, Ulloa, Riveros, Lillo-Carmona, Cáceres-Molina, Almeida and Meneses.https://www.frontiersin.org/articles/10.3389/fpls.2020.01115/ful

How to look next? A data-driven approach for scanpath prediction

By and large, current visual attention models mostly rely, when considering static stimuli, on the following procedure. Given an image, a saliency map is computed, which, in turn, might serve the purpose of predicting a sequence of gaze shifts, namely a scanpath instantiating the dynamics of visual attention deployment. The temporal pattern of attention unfolding is thus confined to the scanpath generation stage, whilst salience is conceived as a static map, at best conflating a number of factors (bottom-up information, top-down, spatial biases, etc.). In this note we propose a novel sequential scheme that consists of a three-stage processing relying on a center-bias model, a context/layout model, and an object-based model, respectively. Each stage contributes, at different times, to the sequential sampling of the final scanpath. We compare the method against classic scanpath generation that exploits state-of-the-art static saliency model. Results show that accounting for the structure of the temporal unfolding leads to gaze dynamics close to human gaze behaviour

Fluorescence Dequenching Makes Haem-Free Soluble Guanylate Cyclase Detectable in Living Cells

In cardiovascular disease, the protective NO/sGC/cGMP signalling-pathway is impaired due to a decreased pool of NO-sensitive haem-containing sGC accompanied by a reciprocal increase in NO-insensitive haem-free sGC. However, no direct method to detect cellular haem-free sGC other than its activation by the new therapeutic class of haem mimetics, such as BAY 58-2667, is available. Here we show that fluorescence dequenching, based on the interaction of the optical active prosthetic haem group and the attached biarsenical fluorophor FlAsH can be used to detect changes in cellular sGC haem status. The partly overlap of the emission spectrum of haem and FlAsH allows energy transfer from the fluorophore to the haem which reduces the intensity of FlAsH fluorescence. Loss of the prosthetic group, e.g. by oxidative stress or by replacement with the haem mimetic BAY 58-2667, prevented the energy transfer resulting in increased fluorescence. Haem loss was corroborated by an observed decrease in NO-induced sGC activity, reduced sGC protein levels, and an increased effect of BAY 58-2667. The use of a haem-free sGC mutant and a biarsenical dye that was not quenched by haem as controls further validated that the increase in fluorescence was due to the loss of the prosthetic haem group. The present approach is based on the cellular expression of an engineered sGC variant limiting is applicability to recombinant expression systems. Nevertheless, it allows to monitor sGC's redox regulation in living cells and future enhancements might be able to extend this approach to in vivo conditions

Perturbation Centrality and Turbine: A Novel Centrality Measure Obtained Using a Versatile Network Dynamics Tool

Analysis of network dynamics became a focal point to understand and predict changes of complex systems. Here we introduce Turbine, a generic framework enabling fast simulation of any algorithmically definable dynamics on very large networks. Using a perturbation transmission model inspired by communicating vessels, we define a novel centrality measure: perturbation centrality. Hubs and inter-modular nodes proved to be highly efficient in perturbation propagation. High perturbation centrality nodes of the Met-tRNA synthetase protein structure network were identified as amino acids involved in intra-protein communication by earlier studies. Changes in perturbation centralities of yeast interactome nodes upon various stresses well recapitulated the functional changes of stressed yeast cells. The novelty and usefulness of perturbation centrality was validated in several other model, biological and social networks. The Turbine software and the perturbation centrality measure may provide a large variety of novel options to assess signaling, drug action, environmental and social interventions. The Turbine algorithm is available at: http://www.turbine.linkgroup.huComment: 21 pages, 4 figues, 1 table, 58 references + a Supplement of 52 pages, 10 figures, 9 tables and 39 references; Turbine algorithm is available at: http://www.turbine.linkgroup.h