Annotation of estrogen-regulated enhancer RNAs [RNA-seq]
Ontology highlight
ABSTRACT: To annotate estrogen-regulated eRNAs in MCF-7 breast cancer cells, we used RNA-sequencing of polyA-depleted and polyA-enriched RNA fractions to assemble the gene bodies of eRNAs.
Project description:To annotate estrogen-regulated eRNAs in MCF-7 breast cancer cells, we used precision nuclear run-on and sequencing of capped RNA (PRO-cap) to determine the transcription start sites of eRNAs.
Project description:A series of MCF-7 variants were previously developed that are either estrogen-dependent for growth (MCF-7:WS8 cells), or resistant to estrogen deprivation and refractory (MCF-7:2A) or sensitive (MCF-7:5C) to E2-induced apoptosis. To identify genes associated with E2-induced apoptosis, estrogen deprivation-resistant/apoptotic-sensitive 5C cells were compared to both estrogen-dependent MCF-7:WS8 and estrogen deprivation/apoptotic-refractory MCF-7:2A cells
Project description:A series of MCF-7 variants were previously developed that are estrogen-dependent for growth (MCF-7:WS8 cells), or resistant to estrogen deprivation/vulnerable to fast (MCF-7:5C) and delayed (MCF-7:2A) E2-inducible apoptosis. To identify miRNAs associated with aromatase inhibitor (AI)-resistance and vulnerability to E2-induced apoptosis, estrogen deprivation-resistant 5C and 2A cells were compared to estrogen-dependent WS8 cells and among each other.
Project 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 ?-estradiol (E2)-bound estrogen receptor alpha (ER?) 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 ER? binding. Cohesin, present on many ER?-regulated enhancers even prior to ligand treatment, apparently contributes to E2-dependent gene activation by stabilizing E2/ER?/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 - ER?, 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.
Project description:We have evaluated the effects of estrogen over SOX2 expression by maintaining MCF-7 under three distinct conditions: estrogen deprivation for a year (LT -E2 MCF-7), treating MCF-7 cells with estrogen for 48 hours (48hr +E2 MCF-7), and maintaining cells under estrogenic conditions for a year (LT +E2 MCF-7).
Project description:Estrogen receptor alpha (ERα) signaling mainly occupies on distal enhancers within genome and plays an essential role in ERα-positive breast cancer. ERα usually requires co-factors to regulate the enhancer activity. By analysis of genome-wide nascent transcript profiling in breast cancer cells, we identified a special group of eRNAs that are functionally important for estrogen-induced transcriptional repression. In addition to stabilizing promoter-enhancer looping structure, these eRNAs recruit ERα to particular enhancers of target genes, facilitate the hierarchical formation of a functional transcriptional complex, and cause gene silencing. Interestingly, we found that ERα directly binds to eRNAs and its DNA-binding domain mediates the interaction with RNA molecules. Our ChIP-seq data in MCF-7 cells indicating that ERαwas not able to bind to E2-activated enhancers, which is consistent with the DNA-binding function reported before; moreover the DBD-truncated ERα was not able to bind to E2-repressed enhancers as well, which further supports our hypothesis that eRNA from E2-repressed enhancers help to recruit ERα to specific enhancers through the interaction with DBD domain. Substitution of ERα-DDBD showed a global effect on both induction and repression of ERα target genes as examined by RNA-seq. Further, these eRNAs help with the formation of a specific ERα-centered transcriptional complex and promote the association of the histone demethylase KDM2A, which dismisses RNA polymerase II from designated enhancers and suppresses transcription of target genes. Our work demonstrated that eRNAs, as a distinctive class of cis-acting molecules besides chromatin regulatory elements, play an important role in modulating and refining locus-specific transcriptional program.
Project description:Estrogen receptor alpha (ERα) signaling mainly occupies on distal enhancers within genome and plays an essential role in ERα-positive breast cancer. ERα usually requires co-factors to regulate the enhancer activity. By analysis of genome-wide nascent transcript profiling in breast cancer cells, we identified a special group of eRNAs that are functionally important for estrogen-induced transcriptional repression. In addition to stabilizing promoter-enhancer looping structure, these eRNAs recruit ERα to particular enhancers of target genes, facilitate the hierarchical formation of a functional transcriptional complex, and cause gene silencing. Interestingly, we found that ERα directly binds to eRNAs and its DNA-binding domain mediates the interaction with RNA molecules. Our ChIP-seq data in MCF-7 cells indicating that ERαwas not able to bind to E2-activated enhancers, which is consistent with the DNA-binding function reported before; moreover the DBD-truncated ERα was not able to bind to E2-repressed enhancers as well, which further supports our hypothesis that eRNA from E2-repressed enhancers help to recruit ERα to specific enhancers through the interaction with DBD domain. Substitution of ERα-DDBD showed a global effect on both induction and repression of ERα target genes as examined by RNA-seq. Further, these eRNAs help with the formation of a specific ERα-centered transcriptional complex and promote the association of the histone demethylase KDM2A, which dismisses RNA polymerase II from designated enhancers and suppresses transcription of target genes. Our work demonstrated that eRNAs, as a distinctive class of cis-acting molecules besides chromatin regulatory elements, play an important role in modulating and refining locus-specific transcriptional program.
Project description:A series of MCF-7 variants were previously developed that are either estrogen-dependent for growth (MCF-7:WS8 cells), or resistant to estrogen deprivation and refractory (MCF-7:2A) or sensitive (MCF-7:5C) to E2-induced apoptosis. To identify genes associated with E2-induced apoptosis, estrogen deprivation-resistant/apoptotic-sensitive 5C cells were compared to both estrogen-dependent MCF-7:WS8 and estrogen deprivation/apoptotic-refractory MCF-7:2A cells Each cell line was treated with 10-9 M E2 or vehicle control over a 96 h time course consisting of 7 time points (2, 6, 12, 24, 48, 72 and 96 h) using 6 biological replicates per condition. cRNA probes from individual E2-treated samples were competitively hybridized against time-matched pooled control probes using 2-color Agilent 4x44k human oligonucleotide microarrays.