Resultados de investigación

Expression profiling by array, Resources available on the publisher's site: https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE29420, The yeast PMR1 (ATP2C1) gene codes for the eukaryotic prototype of a high affinity P-type ATPase required for Ca2+/Mn2+ transport into the Golgi. Cells lacking PMR1 exhibit multiple genetic interactions with genes involved in DNA recombination and replication, a fact that is not yet understood. We find that deletion of PMR1 causes a delay in DNA replication initiation, progression and G2/M transition and induces the transcriptional up-regulation of genes involved in cell cycle regulation, including CLB5 and SWE1. Interestingly, pmr1∆ clb5∆ double mutants exhibit a dramatic delay in DNA replication and increased DNA breakage, while endoreplication and the formation of multi-nucleated, giant yeast is observed in pmr1∆ swe1∆ cells. Because these phenotypes can be attributed to impeded Mn2+-pump function, we provide a model in which Mn2+ interferes with Mg2+ in the nucleus, and vice versa, Mg2+ interferes with Mn2+ in the Golgi. Consequently, cell cycle progression is challenged by aberrant catalytic activities of enzymes involved in replication and protein glycosylation., Peer reviewed

Genome binding/occupancy profiling by high throughput sequencing Expression profiling by high throughput sequencing, Resources available on the publisher's site: https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE140552, This SuperSeries is composed of the SubSeries listed below.--Organism: Mus musculus, Peer reviewed

This PDF file includes: Extended Methods Figures S1 to S11, Extended Methods; Figures S1 to S11, Peer reviewed

Expression profiling by high throughput sequencing Genome binding/occupancy profiling by high throughput sequencing, Resources available on the publisher's site: https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE207917, Peer reviewed

RNA-seq profiles of immortalized human fibroblasts (FSE-hTert) infected with ER:HRASG12V with or without 4-OHT treatment for 2 or 4 days (3 biological replicaes per condition), Resources available on the publisher's site:https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE168738, DNA replication is a complex process tightly regulated to ensure faithful genome duplication, and its perturbation leads to DNA damage and genomic instability. Replication stress is commonly associated with slow and stalled replication forks. Recently, accelerated replication has emerged as a non-canonical form of replication stress. However, the molecular basis underlying fork acceleration is largely unknown. Here we show that mutated HRAS activation leads to increased topoisomerase 1 (TOP1) expression leading to aberrant replication fork acceleration and DNA damage by decreasing RNA-DNA hybrids or R-loops. In these cells, restoration of TOP1 expression or mild replication inhibition rescues the perturbed replication and reduces DNA damage. Furthermore, TOP1 or RNaseH1 overexpression induces accelerated replication and DNA damage, highlighting the importance of TOP1 equilibrium in regulating R-loop homeostasis to ensure faithful DNA replication and genome integrity. Altogether, our results reveal a novel mechanism of oncogene-induced DNA damage induced by aberrant replication fork acceleration., Peer reviewed

Sample Processing Protocol Cells were mock treated or UV-C irradiated (20 J/m2) and harvested after 1 h. Cell pellets were lysed for 20 min on ice in EBC-150 buffer (50 mM Tris pH 7.5, 150 mM NaCl, 0.5 % NP-40, 2 mM MgCl2, protease inhibitor cocktail (Roche)) supplemented with 500 U/mL Benzonase® Nuclease (Novagen). Cell lysates were incubated for 1.5 h at 4°C with GFP-Trap®_A beads (Chromotek). The beads were then washed six times with EBC-150 buffer (50 mM Tris pH 7.5, 150 mM NaCl, 0.5 % NP-40, 1 mM EDTA, protease inhibitor cocktail (Roche), 5 time ammonium bicarbonate 50mM. and Trypsinized overnight. Peptides were later analysed by mass spectrometry. Data Processing Protocol Mass spectrometry was performed essentially as previously described . Samples were analyzed on a Q-Exactive Orbitrap mass spectrometer (Thermo Scientific, Germany) coupled to an EASY-nanoLC 1000 system (Proxeon, Odense, Denmark). Digested peptides were separated using a 20 cm fused silica capillary (ID: 75 µm, OD: 375 µm, Polymicro Technologies, California, US) in-house packed with 1.9 µm C18-AQ beads (Reprospher-DE, Pur, Dr. Maisch, Ammerburch-Entringen, Germany). Peptides were separated by liquid chromatography using a gradient from 2 % to 30 % acetonitrile in 0.1% formic acid. For the ALC1 samples and related GFP controls the gradient length was 40 min. For the XPC,PARP1 and PARP2 samples grandient length was 100 min. Every gradient was followed by an increase to 95% acetonitrile and back to 2% acetonitrile in 0.1% formic acid for chromatography column re-conditioning. Flow rate of was set 200 nl/min for 2 h. The mass spectrometer was operated in positive-ion mode at 2.8 kV with the capillary heated to 250°C. Data-dependent acquisition mode was used to automatically switch between full scan MS and MS/MS scans, employing a top 7 method. Full scan MS spectra were obtained with a resolution of 70.000, a target value of 3x106 and a scan range of 400-2.000 m/z (XPC samples) or 300-1600 m/z (ALC1, PARP1 and PARP2 samples). Higher-Collisional Dissociation (HCD) tandem mass spectra (MS/MS) were recorded with a resolution of 35,000, a target value of 1x105 and a normalized collision energy of 25%. The precursor ion masses selected for MS/MS analysis were subsequently dynamically excluded from MS/MS analysis for 60s and Precursor ions with a charge state of 1 and greater than 6 were excluded from triggering MS/MS events. Maximum injection times for MS and MS/MS were 20 and 120 ms (XPC) or 250 and 120 ms (ALC1, PARP1 and PARP2), respectively. Mass spectrometry data analysis. All raw data were analyzed using MaxQuant (version 1.6.6.0) as described previously. We performed the search against an in silico digested UniProt reference proteome for Homo sapiens including canonical and isoform sequences (27th May 2019). Database searches were performed according to standard settings with the following modifications. Digestion with Trypsin/P was used, allowing 4 missed cleavages. Oxidation (M), Acetyl (Protein N-term) were allowed as variable modifications with a maximum number of 3. Carbamidomethyl (C) was disabled as a fixed modification. Label-Free Quantification was enabled, not allowing Fast LFQ. iBAQ was calculated., Cells employ global genome DNA damage repair (GGR) to eliminate a broad spectrum of DNA lesions, including those induced by UV light. The lesion-recognition factor XPC initiates repair of helix-distorting DNA lesions, but binds inefficiently to lesions that cause poor helix distortion. How such difficult-to-repair lesions are detected in chromatin is unknown. Here, we identify the poly-(ADP-ribose) polymerases PARP1 and PARP2 as constitutive interactors of XPC. The close interaction between these proteins results in the PARylation of XPC at UV lesions, and an XPC-dependent stimulation of the poly-(ADP-ribose) response, which facilitates the recruitment of the poly-(ADP-ribose)-dependent chromatin remodeler ALC1. Both ALC1 and in particular PARP2 are required for the efficient clearing of difficult-to-repair DNA lesions. Our study offers key insights into the molecular mechanisms of GGR by revealing a molecular bookmarking system, which primes chromatin containing difficult-to-repair DNA lesions for efficient repair., Peer reviewed

Sample Processing Protocol Sample preparation Samples were dissolved in Urea 6M, 200mM ammonium bicarbonate, reduced with dithiothreitol (10 mM, 1 h, 37 °C) and alkylated in the dark with iodoacetamide (20 mM, 30 min, 25 °C). The protein mixture was then diluted 10 times with 200mM ammonium bicarbonate before being digested at 37 ºC overnight with trypsin (ration protein:enzyme 10:1). Peptides generated in the digestion were desalted with C18 column (Empore 3M, St. Paul, MN, USA), evaporated to dryness and resuspended in 0.1% formic acid. Chromatographic and mass spectrometric analysis Samples were analyzed using an LTQ-Orbitrap XL mass spectrometer (Thermo Fisher Scientific) coupled to an Agilent Technologies 1200 Series. Peptides were loaded onto C18 Zorbax precolumn (Agilent Technologies) and were separated by reversed-phase chromatography using a 12-cm column with an inner diameter of 75 μm, packed with 5 μm C18 particles (Nikkyo Technos Co.) using a 60 minute chromatographic gradients (3% to 35% acetonitrile, 0.1% formic acid). The mass spectrometer was operated in positive ionization mode with nanospray voltage set at 2.5 kV and source temperature at 200 °C. Ultramark 1621 for the FT mass analyzer was used for external calibration prior the analyses. Moreover, an internal calibration was also performed using the background polysiloxane ion signal at m/z 445.1200. The instrument was operated in data dependent acquisition (DDA) mode and full MS scans with 1 micro scans at resolution of 60.000 were used over a mass range of m/z 350-2000 with detection in the Orbitrap. Auto gain control (AGC) was set to 1E6, dynamic exclusion (60 seconds) and charge state filtering disqualifying singly charged peptides were activated. In each cycle of DDA analysis, following each survey scan the top ten most intense ions with multiple charged ions above a threshold ion count of 5000 were selected for fragmentation at normalized collision energy of 35%. Fragment ion spectra produced via collision-induced dissociation (CID) were acquired in the Ion Trap, AGC was set to 5e4 and isolation window to 2.0 m/z. All data were acquired with Xcalibur software. Data Processing Protocol Proteome Discoverer software suite (Thermo Fisher Scientific) and Mascot search engine Matrix Science) were used for peptide identification and quantitation. The data were searched against Swiss-Prot Human database. A precursor ion mass tolerance of 7 ppm at the MS1 level was used, and up to three miscleavages for trypsin were allowed. The fragment ion mass tolerance was set to 0.5 Da. Oxidation of methionine and N-terminal protein acetylation were used as variable modifications whereas carbamidomethylation on cysteines was set as a fixed modification, High mobility group proteins are chromatin regulators with essential functions in development, cell differentiation and cell proliferation. The protein HMG20A contains three well defined domains: an amino terminal intrinsically distorted domain, a high mobility group box with higher affinity for double stranded four-way-junction DNA than for lineal DNA and a long coiled-coil domain. Here we have found by a proteomic study that HMG20A copurify with the histone reader protein PHF14. Deletion analysis of the interaction domains and structural modeling by using AlphaFold2 software indicated that HMG20A forms a complex with PHF14 through the establishment of a two-stranded alpha-helical coiled-coil. Furthermore, PHF14 was essential for the stability and the chromatin localization of HMG20A. Transcriptomic analysis of triple negative human breast cancer cells demonstrated that PHF14 and HMG20A share a large subset of targets. PHF14 or HMG20A deficiency increased epithelial markers such as E-cadherin or p63 and components of the Hippo pathway and impaired mesenchymal phenotypes including cell migration, and invasion. Expression of mesenchymal adhesion molecules involved in metastasis such as L1CAM and LRRC15 were downregulated in PHF14 or HMG20A depleted cells in agreement with a decreased homotypic cell-cell adhesion ability. Inducible CRISPR inactivation of PHF14 or HMG20A genes impaired normal TGFβ-trigged epithelial to mesenchymal transition in mouse normal breast epithelial cells. Finally, we performed a meta-analysis of PHF14 and HMG20A genes in cancer databases. PHF14 gene is often amplified in different cancers which is associated to poor prognosis. In fact, high levels of PHF14 mRNA are associated to poor prognosis in many types of cancers. High expression of HMG20A and PHF14 are commonly associated to poor prognosis in sarcoma, colon and pancreas adenocarcinomas. In these tumors we found a clear correlation between the mRNA levels of both factors and also similar patterns of co-expressed genes with HMG20A and PHF14, indicating similar functions., Peer reviewed

Expression profiling by high throughput sequencing.--mRNA profiles of the triple-negative breast cancer cell line MDA-MB-231 treated with siControl, siPHF14 or siHMG20A were generated by sequencing, in duplicates, using Illumina Nexseq., Resources available on the publisher's site: https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE193476, High mobility group proteins are chromatin regulators with essential functions in development, cell differentiation and cell proliferation. We have shown that the high mobility group protein HMG20A copurifies with the histone reader PHF14. To understand the common functions of HMG20A and PHF14 proteins in regulating the transcriptome, we have performed RNA-Seq expression analysis following depletion of each protein in the triple-negative breast cancer cell line MDA-MB-231. Results indicate that genes regulated by PHF14 largely overlap with those regulated by HMG20A., Peer reviewed

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