Project description:Gut microbiome research is rapidly moving towards the functional characterization of the microbiota by means of shotgun meta-omics. Here, we selected a cohort of healthy subjects from an indigenous and monitored Sardinian population to analyze their gut microbiota using both shotgun metagenomics and shotgun metaproteomics. We found a considerable divergence between genetic potential and functional activity of the human healthy gut microbiota, in spite of a quite comparable taxonomic structure revealed by the two approaches. Investigation of inter-individual variability of taxonomic features revealed Bacteroides and Akkermansia as remarkably conserved and variable in abundance within the population, respectively. Firmicutes-driven butyrogenesis (mainly due to Faecalibacterium spp.) was shown to be the functional activity with the higher expression rate and the lower inter-individual variability in the study cohort, highlighting the key importance of the biosynthesis of this microbial by-product for the gut homeostasis. The taxon-specific contribution to functional activities and metabolic tasks was also examined, giving insights into the peculiar role of several gut microbiota members in carbohydrate metabolism (including polysaccharide degradation, glycan transport, glycolysis and short-chain fatty acid production). In conclusion, our results provide useful indications regarding the main functions actively exerted by the gut microbiota members of a healthy human cohort, and support metaproteomics as a valuable approach to investigate the functional role of the gut microbiota in health and disease.
Project description:Evaluation of the genome wide impact on gene expression of DNA-PK knockdown or enzymatic inhibition. DNA-PK expression is elevated in multiple tumor types. DNA-PK has recently been implicated in transcriptional regulation, so understanding genes modulated by DNA-PK may provide insight into disease progression 6 samples were analyzed. A genome-wide expression array was performed on a GeneChip Human Gene 2.0ST Array (Affymetrix, 902112) with C4-2 cells depleted of DNA-PK or treated with DNA-PK inhibitor for 24 hours
Project description:This project contains raw data, intermediate files and results is a re-analysis of the publicly available dataset from the PRIDE dataset PXD005780. The RAW files were processed using ThermoRawFileParser, SearchGUI and PeptideShaker through standard settings (see ‘Data Processing Protocol’). This reanalysis work is part of the MetaPUF (MetaProteomics with Unknown Function) project, which is a collaboration between EMBL-EBI and the University of Luxembourg. The dataset was selected with the following conditions: 1. It has been made publicly available in PRIDE and focuses on metaproteomics of the human gut; 2. The corresponding metagenomics assemblies were also available from ENA (European Nucleotide Archive) or MGnify. The processed peptide reports for each sample are available to view at the contig level on the MGnify website. In total, the reanalysis identified 15,417 unique proteins from 15 samples.
Project description:The Mre11-Rad50-Nbs1 (MRN) complex recognizes and processes DNA double-strand breaks for homologous recombination by performing short-range removal of 5ʹ strands. Endonucleolytic processing by MRN requires a stably bound protein at the break site—a role we postulate is played by DNA-dependent protein kinase (DNA-PK) in mammals. Here we interrogate the sites of MRN-dependent processing by isolating and sequencing DNA-PK-bound DNA fragments that are products of MRN cleavage. These intermediates are generated with highest efficiency when DNA-PK is catalytically blocked, yielding products within 200 bp of the break site, whereas DNA-PK products in the absence of kinase inhibition show much greater dispersal. Use of light-activated Cas9 to induce breaks facilitates temporal resolution of DNA-PK and Mre11 binding, showing that Mre11 and DNA-PK both bind to DNA ends before release of DNA-PK-bound products. These results support a sequential model of double-strand break repair involving collaborative interactions between homologous and non-homologous repair complexes.
Project description:We have previously shown that RNA polymerase II (Pol II) pause release and transcriptional elongation involve phosphorylation of the factor TRIM28 by the DNA damage response (DDR) kinases ATM and DNA-PK. Here, we report a significant role for DNA breaks and DDR signaling in the mechanisms of transcriptional elongation in stimulus-inducible genes in humans. Our data show the enrichment of TRIM28 and γH2AX on serum-induced genes and the important function of DNA-PK for Pol II pause release and transcriptional activation-coupled DDR signaling on these genes. γH2AX accumulation decreases when P-TEFb is inhibited, confirming that DDR signaling results from transcriptional elongation. In addition, transcriptional elongation-coupled DDR signaling involves topoisomerase II because inhibiting this enzyme interferes with Pol II pause release and γH2AX accumulation. Our findings propose that DDR signaling is required for effective Pol II pause release and transcriptional elongation through a novel mechanism involving TRIM28, DNA-PK, and topoisomerase II 42 samples in total. IP targets were gammaH2ax, s2-pol-II, pol-II, pTRIM28, DNA-pk, topo-IIB. Experimental conditions included DMSO treatment (control), pTEFb, topoII-i, dnapk-i. Matched non-specific IP samples used for control in peak calling.