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

Constructing endothelial-specific hypoxia-regulated vectors

Soluspesifinen geenien ilmentyminen on molekyylibiologian yksi suurista tutkimusalueista. Geenit aktivoituvat eri solutyypeissä eri tavoin, vaikka soluissa on sama geneettinen perimä. Tutkimuksissa on löydetty useita tekijöitä mm. enhanserit ja enhanseri-RNA molekyylit, jotka vaikuttavat mm. solukohtaiseen geenin ilmentymiseen. Enhanserit ovat alueita genomissa, jotka sitovat proteiineja ja vuorovaikuttavat transkriptiokompleksin kanssa lisäten geenin transkriptiota. Enhanseri-RNA:t ovat enhaserialueilta tuottettuja RNA-molekyylejä. Geenin ilmentymiseen vaikuttaa todella moniulotteinen säätelyjärjestelmä, kun mukaan lasketaan kaikki transkriptioon ja translaatioon vaikuttavat tekijät. Endoteelisolut ovat veri- ja imusuonten seinämien soluja, jotka muodostavat ohuen kerroksen suonten sisäpinnalle. Solukerroksen tehtävä on säädellä mm. nesteiden ja ravinteiden pääsyä suonesta ympäröivään kudokseen. Endoteelisolut ovat myös suuressa roolissa uusien verisuonten muodostumisessa niin normaalioloissa kuin syövissäkin. Hypoksia on eräs tekijä, joka aktivoi verisuonten muodostumista endoteelisoluista. Hypoksia on fysiologinen tila, jossa solu, kudos tai koko keho kärsii hapen puutteesta. Hypoksia johtaa vakavimmillaan hapenpuutteesta kärsivän kudoksen kuolemaan eli nekroosiin. Hypoksia voi muodostua, mikäli verenkierto häiriintyy esim. verisuonen tukkeuman tms. takia eikä veri kulkeudu normaalisti kudokseen. Työn tarkoitus oli luoda endoteelispesifisiä hypoksia-aktivoituvia vektoreita. Genomista valittiin yksi promoottori, kolme enhanseria ja kolme 3’-UTR-aluetta. Alueet kloonattiin napanuoran verisuonten endoteelisoluista (HUVEC) eristetystä genomista ja liitettiin pGL4.10 pohjaisiin lusiferaasi-vektoreihin. Tuotetut konstruktit transfektoitiin viiteen solulinjaan, joista kaksi oli endoteelisolulinjoja. Transfektion jälkeen soluja kasvatettiin 24 tuntia normaalissa happipitoisuudessa tai 1%-hypoksiassa. Käsittelyiden jälkeen konstruktien lusiferaasi-aktiivisuus mitattiin soluista ja aktiivisuuksia vertailtiin keskenään. Tulokset osoittavat soluspesifisyyden ja hypoksia-aktivoitumisen olevan monen eri tekijän summa. Geenin ilmentyminen oli suurempaa kahdella konstruktilla kahdessa solulinjassa verrattuna kontrolleihin. Konstruktien luciferaasi-geeni aktivoitui endoteelisoluissa, mutta myös muissa solulinjoissa. Hypoksia-aktivoitumisesta oli tilastollisesti merkittäviä tuloksia yhden konstruktin kohdalla, joskin rohkaisevia tuloksia saatiin myös muilla konstrukteilla.Cell specific gene expression is one of the most interesting fields in molecular biology. Genes are expressed differently in different cell types even the cells have same genetic material. Studies have found multiple factors affecting cell specific gene expression, enhancer regions and enhancer-RNA (eRNA) molecules among other things. Enhancers are DNA regions that can bind transcription factors and then interact with transcription complexes enhancing the expression. eRNAs are RNA-molecules transcribed from the enhancer regions. Cell specific gene expression is a complex system when all factors affecting transcription and translation are counted in. Endothelial cells are a thin layer of cells in blood- and lymphatic vessel walls. Main functions of endothelial cells are to regulate access of fluids and nutrients from vessels to the surrounding tissues. Endothelial cells play also a critical role in angiogenesis in normal situations and also in cancers and in cardiovascular diseases. Hypoxia is one factor that can activate angiogenesis. Hypoxia is a physiological condition where a cell, a tissue or the whole body is lacking oxygen. It can lead to cell deaths or even tissue necrosis. Hypoxia can be formed when blood circulation is disturbed for example by partial blockage of a blood vessel so that blood cannot flow freely. The aim of this study was to produce endothelial-specific hypoxia-regulated vectors. One promoter-, three enhancer- and three 3’UTR-regions were selected to be cloned from the genome of human umbilical vein endothelial cells (HUVEC). Cloned regions were inserted into pGL4.10 based vectors containing luciferase gene. The constructs were transfected into five cell lines where two of them were endothelial cells. After transfection cells were subjected to normoxia or 1%-hypoxia. After the treatments luciferase activities were measured and compared. Results indicate that cell specific gene expression and enhancing is sum of a multiple factors. Gene expressions were higher with two of the constructs compared to the controls. All constructs expressed luciferase in endothelial cells but also in other cell types. One construct showed statistically significant hypoxia activation and the other constructs gave promising results of hypoxia activation

Data from: Fight evolution with evolution: plasmid-dependent phages with a wide host range prevent the spread of antibiotic resistances

The emergence of pathogenic bacteria resistant to multiple antibiotics is a serious worldwide public health concern. Whenever antibiotics are applied, the genes encoding for antibiotic resistance are selected for within bacterial populations. This has led to the prevalence of conjugative plasmids that carry resistance genes and can transfer themselves between diverse bacterial groups. In this study, we investigated whether it is feasible to attempt to prevent the spread of antibiotic resistances with a lytic bacteriophage, which can replicate in a wide range of gram-negative bacteria harboring conjugative drug-resistance conferring plasmids. The counter-selection against the plasmid was shown to be effective, reducing the frequency of multi-resistant bacteria that formed via horizontal transfer by several orders of magnitude. This was true also in the presence of an antibiotic against which the plasmid provided resistance. Majority of the multi-resistant bacteria subjected to phage selection also lost their conjugation capability. Overall this study suggests that, while we are obligated to maintain the selection for the spread of the drug resistances, the ‘fight evolution with evolution’ approach could help us even out the outcome to our favor

Fight evolution with evolution: Plasmid-dependent phages with a wide host range prevent the spread of antibiotic resistance

The emergence of pathogenic bacteria resistant to multiple antibiotics is a serious worldwide public health concern. Whenever antibiotics are applied, the genes encoding for antibiotic resistance are selected for within bacterial populations. This has le

Ojala et al. (2013) - raw data

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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

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