Control of VEGF-A transcriptional programs by pausing and genomic compartmentalization.

Vascular endothelial growth factor A (VEGF-A) is a master regulator of angiogenesis, vascular development and function. In this study we investigated the transcriptional regulation of VEGF-A-responsive genes in primary human aortic endothelial cells (HAECs) and human umbilical vein endothelial cells (HUVECs) using genome-wide global run-on sequencing (GRO-Seq). We demonstrate that half of VEGF-A-regulated gene promoters are characterized by a transcriptionally competent paused RNA polymerase II (Pol II). We show that transition into productive elongation is a major mechanism of gene activation of virtually all VEGF-regulated genes, whereas only ∼40% of the genes are induced at the level of initiation. In addition, we report a comprehensive chromatin interaction map generated in HUVECs using tethered conformation capture (TCC) and characterize chromatin interactions in relation to transcriptional activity. We demonstrate that sites of active transcription are more likely to engage in chromatin looping and cell type-specific transcriptional activity reflects the boundaries of chromatin interactions. Furthermore, we identify large chromatin compartments with a tendency to be coordinately transcribed upon VEGF-A stimulation. We provide evidence that these compartments are enriched for clusters of regulatory regions such as super-enhancers and for disease-associated single nucleotide polymorphisms (SNPs). Collectively, these findings provide new insights into mechanisms behind VEGF-A-regulated transcriptional programs in endothelial cells

Nuclear factor E2-related factor 2 deficiency impairs atherosclerotic lesion development but promotes features of plaque instability in hypercholesterolaemic mice

Aims Oxidative stress and inflammation play an important role in the progression of atherosclerosis. Transcription factor NF-E2-related factor 2 (Nrf2) has antioxidant and anti-inflammatory effects in the vessel wall, but paradoxically, global loss of Nrf2 in apoE deficient mice alleviates atherosclerosis. In this study, we investigated the effect of global Nrf2 deficiency on early and advanced atherogenesis in alternative models of atherosclerosis, LDL receptor deficient mice (LDLR-/-), and LDLR-/- mice expressing apoB-100 only (LDLR-/- ApoB(100/100)) having a humanized lipoprotein profile. Methods and results LDLR-/- mice were fed a high-fat diet (HFD) for 6 or 12weeks and LDLR(-/-)ApoB(100/100) mice a regular chow diet for 6 or 12months. Nrf2 deficiency significantly reduced early and more advanced atherosclerosis assessed by lesion size and coverage in the aorta in both models. Nrf2 deficiency in LDLR-/- mice reduced total plasma cholesterol after 6weeks of HFD and triglycerides in LDLR(-/-)ApoB(100/100) mice on a chow diet. Nrf2 deficiency aggravated aortic plaque maturation in aged LDLR(-/-)ApoB(100/100) mice as it increased plaque calcification. Moreover, approximate to 36% of Nrf2(-/-)LDLR(-/-)ApoB(100/100) females developed spontaneous myocardial infarction (MI) or sudden death at 5 to 12months of age. Interestingly, Nrf2 deficiency increased plaque instability index, enhanced plaque inflammation and calcification, and reduced fibrous cap thickness in brachiocephalic arteries of LDLR(-/-)ApoB(100/100) female mice at age of 12months. Conclusions Absence of Nrf2 reduced atherosclerotic lesion size in both atherosclerosis models, likely via systemic effects on lipid metabolism. However, Nrf2 deficiency in aged LDLR(-/-)ApoB(100/100) mice led to an enhanced atherosclerotic plaque instability likely via increased plaque inflammation and oxidative stress, which possibly predisposed to MI and sudden death.Peer reviewe

The Keap1-Nrf2 pathway: Mechanisms of activation and dysregulation in cancer

The Keap1-Nrf2 pathway is the major regulator of cytoprotective responses to oxidative and electrophilic stress. Although cell signaling pathways triggered by the transcription factor Nrf2 prevent cancer initiation and progression in normal and premalignant tissues, in fully malignant cells Nrf2 activity provides growth advantage by increasing cancer chemoresistance and enhancing tumor cell growth. In this graphical review, we provide an overview of the Keap1-Nrf2 pathway and its dysregulation in cancer cells. We also briefly summarize the consequences of constitutive Nrf2 activation in cancer cells and how this can be exploited in cancer gene therapy

Control of VEGF-A transcriptional programs by pausing and genomic compartmentalization.

Vascular endothelial growth factor A (VEGF-A) is a master regulator of angiogenesis, vascular development and function. In this study we investigated the transcriptional regulation of VEGF-A-responsive genes in primary human aortic endothelial cells (HAECs) and human umbilical vein endothelial cells (HUVECs) using genome-wide global run-on sequencing (GRO-Seq). We demonstrate that half of VEGF-A-regulated gene promoters are characterized by a transcriptionally competent paused RNA polymerase II (Pol II). We show that transition into productive elongation is a major mechanism of gene activation of virtually all VEGF-regulated genes, whereas only ∼40% of the genes are induced at the level of initiation. In addition, we report a comprehensive chromatin interaction map generated in HUVECs using tethered conformation capture (TCC) and characterize chromatin interactions in relation to transcriptional activity. We demonstrate that sites of active transcription are more likely to engage in chromatin looping and cell type-specific transcriptional activity reflects the boundaries of chromatin interactions. Furthermore, we identify large chromatin compartments with a tendency to be coordinately transcribed upon VEGF-A stimulation. We provide evidence that these compartments are enriched for clusters of regulatory regions such as super-enhancers and for disease-associated single nucleotide polymorphisms (SNPs). Collectively, these findings provide new insights into mechanisms behind VEGF-A-regulated transcriptional programs in endothelial cells

In Vitro and In Vivo Pipeline for Validation of Disease-Modifying Effects of Systems Biology-Derived Network Treatments for Traumatic Brain Injury—Lessons Learned

We developed a pipeline for the discovery of transcriptomics-derived disease-modifying therapies and used it to validate treatments in vitro and in vivo that could be repurposed for TBI treatment. Desmethylclomipramine, ionomycin, sirolimus and trimipramine, identified by in silico LINCS analysis as candidate treatments modulating the TBI-induced transcriptomics networks, were tested in neuron-BV2 microglial co-cultures, using tumour necrosis factor α as a monitoring biomarker for neuroinflammation, nitrite for nitric oxide-mediated neurotoxicity and microtubule associated protein 2-based immunostaining for neuronal survival. Based on (a) therapeutic time window in silico, (b) blood-brain barrier penetration and water solubility, (c) anti-inflammatory and neuroprotective effects in vitro (p < 0.05) and (d) target engagement of Nrf2 target genes (p < 0.05), desmethylclomipramine was validated in a lateral fluid-percussion model of TBI in rats. Despite the favourable in silico and in vitro outcomes, in vivo assessment of clomipramine, which metabolizes to desmethylclomipramine, failed to demonstrate favourable effects on motor and memory tests. In fact, clomipramine treatment worsened the composite neuroscore (p < 0.05). Weight loss (p < 0.05) and prolonged upregulation of plasma cytokines (p < 0.05) may have contributed to the worsened somatomotor outcome. Our pipeline provides a rational stepwise procedure for evaluating favourable and unfavourable effects of systems-biology discovered compounds that modulate post-TBI transcriptomics

The Effects of Sequence Variation on Genome-wide NRF2 Binding––New Target Genes and Regulatory SNPs

Transcription factor binding specificity is crucial for proper target gene regulation. Motif discovery algorithms identify the main features of the binding patterns, but the accuracy on the lower affinity sites is often poor. Nuclear factor E2-related factor 2 (NRF2) is a ubiquitous redox-activated transcription factor having a key protective role against endogenous and exogenous oxidant and electrophile stress. Herein, we decipher the effects of sequence variation on the DNA binding sequence of NRF2, in order to identify both genome-wide bind- ing sites for NRF2 and disease-associated regulatory SNPs (rSNPs) with drastic effects on NRF2 binding. Interactions between NRF2 and DNA were studied using molecular modelling, and NRF2 chromatin immunoprecipitation-sequence datasets together with protein binding microarray measurements were utilized to study binding sequence variation in detail. The binding model thus generated was used to identify genome-wide binding sites for NRF2, and genomic binding sites with rSNPs that have strong effects on NRF2 binding and reside on active regulatory elements in human cells. As a proof of concept, miR-126–3p and -5p were identified as NRF2 target microRNAs, and a rSNP (rs113067944) residing on NRF2 target gene (Ferritin, light polypeptide, FTL) promoter was experimentally verified to decrease NRF2 binding and result in decreased transcriptional activity