Investigation Title Transcription Initiation Activity Sets Replication Origin Efficiency in Mammalian Cells Comment[Submitted Name] Transcription Initiation Activity Sets Replication Origin Efficiency in Mammalian Cells Experimental Design other Experimental Design Term Source REF Comment[SecondaryAccession] GSE15082 Comment[ArrayExpressReleaseDate] 2010-05-22 Comment[AEMIAMESCORE] 3 Comment[Publication DOI] 10.1371/journal.pgen.1000446 Comment[ArrayExpressAccession] E-GEOD-15082 Comment[MAGETAB TimeStamp_Version] 2010-08-01 21:23:58 Last Changed Rev: 13058 Experimental Factor Name DNA TYPE Experimental Factor Type dna type Experimental Factor Term Source REF Person Last Name Diaz-Uriarte Brockdorff Gómez Sequeira-Mendes Apedaile Huntley Díaz-Uriarte Person First Name Ramon Neil María Joana Anwyn Derek Ramón Person Mid Initials Person Email rdiaz02@gmail.com Person Phone +34-91-732-8000 Person Fax Person Address Structural Biology and Biocomputing Programme, Spanish National Cancer Research Centre (CNIO), Melchor Fernandez Almagro 3, Madrid, Madrid, Spain Person Affiliation Spanish National Cancer Research Centre (CNIO) Person Roles submitter Person Roles Term Source REF The MGED Ontology Quality Control Type Quality Control Term Source REF Replicate Type Replicate Term Source REF Normalization Type Normalization Term Source REF Date of Experiment Public Release Date 2010-05-22 PubMed ID 19360092 Publication DOI 19360092 Publication Author List Sequeira-Mendes J, Díaz-Uriarte R, Apedaile A, Huntley D, Brockdorff N, Gómez M Publication Title Transcription initiation activity sets replication origin efficiency in mammalian cells. Publication Status journal_article Publication Status Term Source REF The MGED Ontology Experiment Description Genomic mapping of DNA replication origins (ORIs) in mammals provides a powerful means for understanding the regulatory complexity of our genome. Here we combine a genome-wide approach to identify preferential sites of DNA replication initiation at 0.4% of the mouse genome with detailed molecular analysis at distinct classes of ORIs according to their location relative to the genes. Our study reveals that 85% of the replication initiation sites in mouse embryonic stem (ES) cells are associated with transcriptional units. Nearly half of the identified ORIs map at promoter regions and, interestingly, ORI density strongly correlates with promoter density, reflecting the coordinated organisation of replication and transcription in the mouse genome. Detailed analysis of ORI activity showed that CpG island promoter-ORIs are the most efficient ORIs in ES cells and both ORI specification and firing efficiency are maintained across cell types. Remarkably, the distribution of replication initiation sites at promoter-ORIs exactly parallels that of transcription start sites (TSS) suggesting a co-evolution of the regulatory regions driving replication and transcription. Moreover, we found that promoter-ORIs are significantly enriched in CAGE tags derived from early embryos relative to all promoters. This association implies that transcription initiation early in development sets the probability of ORI activation unveiling a new hallmark in ORI efficiency regulation in mammalian cells. Two biological replicates of lambda-exonuclease treated short nascent strands (100-600 or 300-800 nt in length) were co-hybridised with genomic DNA from the same cells to tiled genomic array covering 10.1 Mb of the mouse genome (Agilent Technologies) Protocol Name P-GSE15082-3 P-GSE15082-2 P-GSE15082-4 P-GSE15082-5 P-GSE15082-6 P-GSE15082-7 P-GSE15082-8 P-GSE15082-9 P-GSE15082-1 Protocol Type grow specified_biomaterial_action nucleic_acid_extraction labeling hybridization image_aquisition image_aquisition feature_extraction bioassay_data_transformation Protocol Description The mouse embryonic stem cell line PGK12.1 was grown as described in Penny GD, Kay GF, Sheardown SA, Rastan S, Brockdorff N (1996; Requirement for Xist in X chromosome inactivation. Nature 379: 131-137). Mouse embryonic fibroblasts (MEFs) were derived from 12.5 dpc CD1 embryos and grown in F12 Nutrient Mixture (Ham) medium supplemented with 10% FCS, 1x105 U/ml penicillin, 100 mg/ml streptomycin, 2 mM L- lutamine, 1x non-essential amino acids, and 50 M beta-mercaptoethanol (Invitrogen). NIH/3T3 cells were cultivated as recommended in the ATCC. No treatment was applied Genomic DNA isolation and nascent strands fractionation was performed as described in Gómez M, Antequera F (2008; Overreplication of short DNA regions during S phase in human cells. Genes & Development 22: 375-385) The DNA samples hybridised in the arrays were labelled as described on the protocol published by Agilent (Agilent_Technologies, 2005). Briefly, each sample (nascent strands or control) resuspended in 21 μl H2O was mixed with 20 μl of 2.5x Random primer buffer (BioPrime DNA Labeling System, Invitrogen) and incubated at 95º C for 5 minutes. This mix was transferred immediately to ice and the following reagents added: dNTPs (2 mM each of dATP, dGTP and dTTP and 0.5 mM dCTP), 3.75 μl Cy 3-dCTP or Cy 5-dCTP (Amersham), and 20 U Klenow enzyme. The reactions were incubated at 37ºC for 3 hours, an extra 10 U Klenow enzyme added and then incubated for further 2 hours. Corresponding nascent strands and control samples were pooled, diluted in 400 μl of TE pH 8.0 and purified on a MicroCon YM-30 filter column (Millipore) to remove the unincorporated label, following manufacturer’s instructions. The labelled DNA was transferred to a fresh 1.5 ml tube and brought to a final volume of 150 μl with nuclease-free water and stored in the dark at -20ºC until hybridisation. The labelled DNA was denatured at 95º C for 3 minutes, transferred to ice, mixed with 100 μl H20 and 250 μl 2x Hybridisation buffer (Agilent) and loaded onto a fresh array slide placed into a Agilent SureHyb chamber base. The chamber was sealed and slides hybridised at 65º C for 40 h at 10 rpm. Prior to scanning the slides were washed 5 minutes at room temperature in Wash Buffer 1 and then 5 minutes at room temperature in Wash Buffer 2 (OGT). All labelled samples were sent to Oxford Gene Technology facilities (OGT, Oxford, UK), using the Agilent platform and protocols for the hybridisation and scanning of the arrays. Data was scanned at 100% and 50% PMT and the 100% PMT data feature extracted using local background and spatial detrending to account for differences in hybridisation intensities across the array. Poor quality features were discarded, as were those with very high signal intensities, following standard Agilent protocols. Raw datasets from each experiment were loess normalised to remove signal intensity-dependent bias using GeneSpringX software (Agilent). ID_REF =
VALUE = normalized log2 ratio (Cy5/Cy3 or Cy5/Cy3) representing test/reference Protocol Parameters Protocol Hardware Protocol Software Protocol Contact Protocol Term Source REF The MGED Ontology The MGED Ontology SDRF File E-GEOD-15082.sdrf.txt Term Source Name NCBI Taxonomy ArrayExpress The MGED Ontology EFO Term Source File http://www.ncbi.nlm.nih.gov/Taxonomy/ http://www.ebi.ac.uk/arrayexpress http://mged.sourceforge.net/ontologies/MGEDontology.php http://efo.svn.sourceforge.net/viewvc/efo/trunk/src/efoinowl/efo.owl Term Source Version