Comment[ArrayExpressAccession] E-GEOD-45822 MAGE-TAB Version 1.1 Public Release Date 2013-06-04 Investigation Title Functional Importance of eRNAs for Estrogen-dependent Gene Transcriptional Activation Comment[Submitted Name] Functional Importance of eRNAs for Estrogen-dependent Gene Transcriptional Activation Experiment Description The functional importance of gene enhancers in regulated gene expression is well established. In addition to widespread transcription of long non-coding RNA (ncRNA) transcripts in mammalian cells, bidirectional ncRNAs referred to as eRNAs are present on enhancers. However, it has remained unclear whether these eRNAs are functional, or merely a reflection of enhancer activation. Here, we report that 17 β-estradiol (E2)-bound estrogen receptor alpha (ERα) on enhancers causes a global increase in eRNA transcription on enhancers adjacent to E2 upregulated coding genes. These induced eRNAs, as functional transcripts, appear to exert important roles for the observed ligand-dependent induction of target coding genes, causing an increased strength of specific enhancer:promoter looping initiated by ERα binding. Cohesin, present on many ERα-regulated enhancers even prior to ligand treatment, apparently contributes to E2-dependent gene activation by stabilizing E2/ERα/eRNA-induced enhancer:promoter looping. Our data indicate that eRNAs are likely to exert important functions in many regulated programs of gene transcription. The ChIP-seqs in this study measure the binding landscape of master transcription regulator of estrogen signaling - ERα, together with common histone marks including H3K27ac and H3K4me1 in MCF7 cells. These data serve as the basis to understand the enhancer map and subsequent analysis of eRNA expression using GRO-seq. The GRO-seq measures the trancription of nascent RNAs in the genome. From MCF7 cells treated with veichle or estrodial, we could identify estrogen-regulated eRNAs and subsequently could study their functions. Term Source Name ArrayExpress EFO Term Source File http://www.ebi.ac.uk/arrayexpress/ http://www.ebi.ac.uk/efo/efo.owl Person Last Name Ma Li Ma Rosenfeld Person First Name Qi W Q M Person Mid Initials G Person Email q1ma@ucsd.edu Person Affiliation UCSD Person Address UCSD, 9500 Gilman Dr,, San Diego, USA Person Roles submitter Protocol Name P-GSE45822-6 P-GSE45822-4 P-GSE45822-1 P-GSE45822-5 P-GSE45822-3 P-GSE45822-2 Protocol Description Basecalls performed using CASAVA version 1.4 Gro-seq reads were aligned to the hg18 genome assembly using Bowtie version 2.0.1. The bigwig files were generated by using Homer v3.9, which the total tags are normalized to 1.00e+07. Genome_build: hg18 Basecalls performed using CASAVA version 1.4 ChIP-seq reads were aligned to the hg18 genome assembly using Bowtie version 2.0.1. The bigwig files were generated by using Homer v3.9, which the total tags are normalized to 1.00e+07. Genome_build: hg18 Before experiment, the cells were changed to deficient MEM plus 5% charcoal treated phenol red free medium to culture for 3 days, followed by treatemnt of either 100nM 17-β-estrodial or ethanol for 1hr. MCF7 cells were subjected to nuclear run-on for 5minutes at 30 degree with BrU labeling. The run-on RNAs were pull down by BrU beads and subjected to library preparation for deep sequencing. We followed a previous published protocol (Ingolia et al., 2009 Science; Wang et al., 2011 Nature). Briefly, the run-on RNA were first added with a polyA tracts by RNA polyA polymerase. This polyA tail enables the run-on RNA to be reverse transcribed into single strand cDNA by Reverse Transcriptase. The cDNA was circularized by CircLigase (Epicentre) and re-linearized by Ape1, subsequently subjected to PCR amplification and deep sequencing. Lysates were clarified from sonicated nuclei and histone-DNA complexes were isolated with antibody. The extracted ChIP DNA was ligated to specific adaptors of Truseq system from Illumina, according to the manufacturer’s instructions. MCF7 obtained from ATCC were cultured in α-MEM media supplemented with 10% FBS in a 5% CO2 humidified incubator. Protocol Type normalization data transformation protocol normalization data transformation protocol sample treatment protocol nucleic acid library construction protocol nucleic acid library construction protocol growth protocol Experimental Factor Name TREATMENT CHIP VENDOR/CATALOG CHIP ANTIBODY Experimental Factor Type treatment chip vendor/catalog chip antibody Publication Title Functional roles of enhancer RNAs for oestrogen-dependent transcriptional activation. Publication Author List Li W, Notani D, Ma Q, Tanasa B, Nunez E, Chen AY, Merkurjev D, Zhang J, Ohgi K, Song X, Oh S, Kim HS, Glass CK, Rosenfeld MG PubMed ID 23728302 Publication DOI 10.1038/nature12210 Comment[SecondaryAccession] GSE45822 Comment[GEOReleaseDate] 2013-06-04 Comment[ArrayExpressSubmissionDate] 2013-04-05 Comment[GEOLastUpdateDate] 2013-06-06 Comment[AEExperimentType] ChIP-seq Comment[AEExperimentType] RNA-seq of coding RNA Comment[SecondaryAccession] SRP020561 Comment[SequenceDataURI] http://www.ebi.ac.uk/ena/data/view/SRR816993-SRR817003 SDRF File E-GEOD-45822.sdrf.txt