Resultados de investigación

Expression profiling by high throughput sequencing.--RNA-seq experiments in human RPE cells were generated upon different times of merbarone treatment: 1) 4h merbarone treatment (2 replicates) and 2) 8h merbarone treatment (2 replicates), Resources available on the publisher's site:https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE198093, The inference of Gene Regulatory Networks (GRN) enables in silico generation of biological models, which are capable of integrat- ing large amounts of diverse biological information. These often base on gene expression data. In this project, several GRNs were reconstructed out of RPE-1 cells RNAseq data in order to identify cellular processes affected by antitumoral drug merbarone. Merbarone catalytically inhib- its the enzyme Topoisomerase II. Additionally, it is intended to provide an overview and a general pipeline for GRN inference and reconstruc- tion, from transcription data preprocessing to network validation and analysis., Peer reviewed

Expression profiling by high throughput sequencing.--2 RNA-seq experiments in human RPE cells were generated, each one consisting on 2 conditions (control VS merbarone treatment): 1) Control VS 30m merbarone: 30m vehicle treatment (3 replicates) and 30m merbarone treatment (3 replicates); 2) Control VS 2h merbarone: Untreated (2 replicates) and 2h merbarone treatment (2 replicates)., Resources available on the publisher's site: https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE141799, During transcription, DNA supercoiling generated by the advance of RNA polymerase II (Pol II) is resolved by DNA topoisomerases, enzymes that bind chromatin and produce transient breaks to relax DNA. Recently, this idea of mere facilitators of transcription progression is changing, as topoisomerases are being assigned new functions in regulating the expression of specific genes. In fact, mammalian type II topoisomerases, both the [Symbol] (TOP2A) and [Symbol] (TOP2B) paralogs, are enriched at promoter regions, where they have been proposed to trigger transcription through the generation of DNA double-strand breaks (DSBs). However, this is difficult to reconcile with the intrinsic catalytic properties of TOP2 and the high risk of genome instability that continuous production and repair of DSBs implies. Here, we show that TOP2A enforces promoter-proximal pausing of Pol II by removing transcription-associated negative DNA supercoiling. Interestingly, this topological balance and its disruption is essential for the transcriptional control of Immediate Early Genes (IEGs) and their typical bursting behaviour in response to stimulus. We therefore uncover a novel layer of transcriptional regulation that relies on canonical functions of TOP2A that are independent of aberrant DSB formation, providing a topological framework for the control of promoter-proximal pausing and the tight regulation of IEGs., Peer reviewed

Ghent University European Commission Research Foundation - Flanders Junta de Andalucía, Peer reviewed

Supplementary Figure 1. RfxCas13d protein is less efficient than RfxCas13d mRNA when targeting mid- and late-zygotically expressed mRNAs. Supplementary Figure 2. Increasing the length of gRNA spacers does not improve CRISPR-RfxCas13d activity in zebrafish embryos. Supplementary Figure 3. In vitro transcribed gRNAs can induce toxicity. Supplementary Figure 4. Optimization of NLS versions to increase CRISPRRfxCas13d nuclear RNA-targeting. Supplementary Figure 5. Highly efficient CRISPR-RfxCas13d gRNAs can be identified when injecting up to 25 gRNAs together. Supplementary Figure 6. In vivo activity of CRISPR-RfxCas13d gRNAs activity predicted by ex vivo computational models. Supplementary Figure 7. Highly abundant dsRed mRNA partially buffers the collateral activity triggered by CRISPR-RfxCas13d. Supplementary Figure 8. CRISPR-RfxCas13d does not induce a physiologically relevant collateral activity when targeting highly expressed endogenous mRNAs. Supplementary Figure 9. CRISPR-Cas7-11 RNP complexes do not exhibit collateral activity. Supplementary Figure 10. CRISPR-DjCas13d RNP complexes induce less unspecific RNA degradation and collateral activity than CRISPRRfxCas13d. Supplementary Figure 11. DjCas13d did not lead to massive deregulation upon collateral activity. Supplementary Figure 12. DjCas13d in vitro transcribed gRNAs do not induce toxicity. -- Description of Additional Supplementary Files File Name: Supplementary Data 1 Description: Summary of statistical information of data represented in Figures and Supplementary Figures. File Name: Supplementary Data 2 Description: RfxCas13d, DjCas13d and Cas7-11 gRNAs to KD maternal, zygotically transcribed or reporter mRNAs. File Name: Supplementary Data 3 Description: RfxCas13d gRNAs used in multiplexing experiment. File Name: Supplementary Data 4 Description: RfxCas13d gRNAs employed to interrogate existing computational models to predict gRNAs efficiency in vivo. File Name: Supplementary Data 5 Description: Oligonucleotides employed in this study for fill-in PCR to generate DNA templates for in vitro-transcribed gRNAs of RfxCas13d and DjCas13d. File Name: Supplementary Data 6 Description: Oligonucleotides employed in this study to clone different NLS versions of RfxCas13d into pT3TS plasmid and Cas7-11 ORF into pET28b plasmid for bacterial protein expression, and to generate high-fidelity version of RfxCas13d. File Name: Supplementary Data 7 Description: miR-430 duplexes and oligonucleotides employed in this study for qRT-PCR., Peer reviewed

Additional file 1. Supplementary Figures S1-22 (.pdf)., Ghent University Special Research Fund H2020 Marie Skłodowska-Curie Actions European Joint Programme on Rare Diseases (EJP RD) Research Foundation Flanders (FWO) Foundation John W. Mouton Pro Retina & Marie-Claire Liénaert Ghent University Fund Alzheimer and Neurodegenerative Diseases Junta de Andalucía, Peer reviewed

Additional file 3. BED file with Hi-C boundaries in RPE/choroid (25 kb resolution)., Ghent University European Commission Research Foundation - Flanders Junta de Andalucía, Peer reviewed

Additional file 4. BED file with Hi-C CHESS regions with differential 3D topology between neural retina vs. RPE/choroid., Ghent University European Commission Research Foundation - Flanders Junta de Andalucía, Peer reviewed

Additional file 5. BEDPE file with Hi-C loops in neural retina., Ghent University European Commission Research Foundation - Flanders Junta de Andalucía, Peer reviewed

Additional file 6. BEDPE file with Hi-C loops in RPE/choroid., Ghent University European Commission Research Foundation - Flanders Junta de Andalucía, Peer reviewed

Ghent University European Commission Research Foundation - Flanders Junta de Andalucía, Peer reviewed