Effect of JMJD2B depletion on the ER signaling pathway
Ontology highlight
ABSTRACT: JMJD2B is expressed in a high proportion of human breast tumors, and the expression levels significantly correlate with estrogen receptor (ER) positivity. To assess the effect of JMJD2B depletion on the ER signaling pathway, we performed genome-wide gene expression analysis using the Affymetrix Human Gene 1.0 ST array. RNA was extracted from steroid-depleted control MCF-7 cells (control E2(-)), control MCF-7 cells treated with E2 (control E2(+)), steroid-depleted JMJD2B-depleted MCF-7 cells (siJ2B E2(-)), and JMJD2B-depleted MCF-7 cells treated with E2 (siJ2B E2(+)).
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.
Project description:The human steroid receptor RNA activator (SRA) gene encodes both non-coding RNAs (ncRNAs) and protein-generating isoforms. However, the breadth of endogenous target genes that might be regulated by SRA RNAs remains largely unknown. To address this, we depleted SRA RNA in two human cancer cell lines (HeLa and MCF-7) with small interfering RNAs, then assayed for changes in gene expression by microarray analyses using Affymetrix HGU133+2 arrays. We also tested if SRA depletion affects estradiol-regulated genes in MCF-7 breast cancer cells. We transiently transfected HeLa or MCF-7 human cancer cell lines with with non-targeting (NT) or SRA-targeting siRNAs. Six total HeLa RNA samples were analyzed on an Affymetrix HGU133+2 array, representing three biological triplicates. Likewise, 12 total MCF-7 RNA samples were analyzed on Affymetrix HGU133+2 arrays, representing biological trilplicates for both NT and siSRA transfected cells and with/without 10 nM estradiol (E2) for 6 hours.
Project description:Long noncoding RNAs (lncRNAs) are important regulators of chromatin; however, the mechanistic roles for many lncRNAs are poorly understood in part because their direct interactions with genomic loci and proteins are difficult to assess. We used CHART-seq to map the genomic binding sites for two highly expressed human lncRNAs, NEAT1 and MALAT1, which localize within the nucleus to paraspeckles and nuclear speckles, respectively. We show that NEAT1 and MALAT1 localize to hundreds of genomic sites in human cells, primarily over active genes. NEAT1 and MALAT1 exhibit colocalization to many of these loci, but display distinct gene body binding patterns at these sites, suggesting independent but complementary functions for these RNAs. Protein mass spectrometry analysis of CHART-enriched material (CHART-MS) identified numerous proteins enriched by both lncRNAs, supporting complementary binding and function, in addition to unique associated proteins. Transcriptional inhibition or stimulation affects the localization of NEAT1 to active chromatin sites, implying that DNA sequence itself does not target NEAT1 to chromatin and that localization responds to cues involved in the transcription process. Paired-end CHART-seq was performed for a single replicate of each capture oligonucleotide in untreated MCF-7 cells to establish binding sites of these RNAs, for a total of 6 samples. To investigate the effects of transcriptional inhibition and E2 stimulation on the localization of these RNAs, we performed paired-end CHART-seq with each capture oligonucleotide for two biological replicates of flavopiridol- and vehicle (DMSO)-treated MCF-7 cells and for two biological replicates of E2- and vehicle (ethanol)-treated MCF-7 cells. To establish the overlap of NEAT1 and MALAT1 binding sites with a known component of paraspeckles (NEAT1-containing subnuclear body), we performed paired-end ChIP-seq for the paraspeckle component PSF in MCF-7 cells, as well as a single-end biological replicate.
Project description:To examine the effect of E2 treatment for the miRNA expression in human MCF-7 cells, MCF-7 cells were treated with or without E2 (100 nM) for 4 hr or 24 hr. Four group experiments; E2 (100 nM) treatment for 4 hr (MCF-7 E2 4h_1, MCF-7 E2 4h_2), vehicle (ethanol) treatment for 4 hr as Mock control (MCF-7 Mock1, MCF-7 Mock2), E2 (100 nM) treatment for 24 hr (MCF-7 E2 24h_1, MCF-7 E2 24h_2, MCF-7 E2 24h_3), vehicle treatment for 24 hr as Mock control (MCF-7 Mock_3, MCF-7 Mock_4, MCF-7 Mock_5).
Project description:MCF-7 is an estrogen receptor-positive breast cancer cell line. This experiment is designed to study (1) the effect of estradiol (E2) exposure and (2) lysine methyltransferase 2B (KMT2B) knockdown in MCF-7 cells. Cells were grown for 72 hours prior to treatment with vehicle or 10 nM E2 for 4 and 24 hours. Additionally, to assess the effect of KMT2B knockdown, MCF-7 cells were transfected with KMT2B targeting siRNA or scrambled control siRNA in the absence or presence of E2. RNA were isolated using Trizol and hybridized to Affymetrix GeneChip Human Genome U133 Plus 2.0 array.
Project description:Estrogens(E2) are important steroid hormones that regulate differentiation, proliferation, and apoptosis in hormone-dependent breast cancer.In order to detect the E2-dependent transcription program associated with the observed cell cycle response, we analyzed the effect of H2ac knockdown on MCF-7 gene expression using microarray. Interestingly, we noticed that 51% of the E2-upregulated genes are down-regulated by depletion of H2ac. The data also show that H2ac regulated E2-dependent genes through E2-induction signaling pathway. MCF-7 cell line was transfected with scrambled or H2ac siRNA in the absence or persence of E2.
Project description:Estrogen receptor alpha plays a critical role in breast cancer and is a major target in endocrine therapy. HIF-1 alpha have been associated with ER alpha and predict a worse outcome. Recent studies indicate that histone demethylase JMJD2B is a HIF-1 alpha target. However, little is known about the biological functions of JMJD2B, especially in breast cancer. To elucidate the mechanism by which JMJD2B reguates gene expression in normoxia and hypoxia, MCF-7 breast cancer cells were depleted forJMJD2B in normoxia and hypoxia. Our results provide insight into JMJD2B regulation of gene expression in breast cancer cells in normoxia and hypoxia.
Project description:Estrogen receptor alpha plays a critical role in breast cancer and is a major target in endocrine therapy. HIF-1 alpha have been associated with ER alpha and predict a worse outcome. Recent studies indicate that histone demethylase JMJD2B is a HIF-1 alpha target. However, little is known about the biological functions of JMJD2B, especially in breast cancer. To elucidate the mechanism by which JMJD2B reguates gene expression in normoxia and hypoxia, MCF-7 breast cancer cells were depleted forJMJD2B in normoxia and hypoxia. Our results provide insight into JMJD2B regulation of gene expression in breast cancer cells in normoxia and hypoxia. MCF7 cells were subjected to transfection with siRNA controls and two different siRNA oligos against JMJD2B for 24 hours. Cells were treated in normoxia and hypoxia for another 16 hours.
Project description:Estrogen receptor-α (ERα) is an important driver of breast cancer and is the target for hormonal therapies, anti-estrogens and drugs that limit estrogen biosynthesis (aromatase inhibitors). Mutations in the ESR1 gene identified in metastatic breast cancer provide a potential mechanism for acquired resistance to hormone therapies. We have used CRISPR-Cas9 mediated genome editing in the MCF-7 breast cancer cell line, generating MCF-7-Y537S. MCF-7-Y537S cells encode a wild-type (tyrosine 537) and a mutant (serine 537) allele. Growth of the line is estrogen-independent and expression of ERα target genes is elevated in the absence of estrogen. ER ChIP-seq was carried out to map global ERα binding sites in the presence and absence of estrogen. RNA-seq following estrogen treatment was used for gene expression analysis. We show that expression of ER target genes and ER recruitment to ER binding regions is similar in MCF-7 and MCF-7-Y537S cells, except that ER recruitment to DNA and expression of ER target genes is frequently elevated in the absence of estrogen. Hormone depleted MCF7 Luc or Y537S cells were treated with 10nM E2 or ethanol, as vehicle control, for 8 hours, with 3 replicates (2 replicates for Y537S + E2). RNA-seq was carried out using Illumina Hiseq 2500.
Project description:We used ChIP-Seq to map ERalpha binding sites and to profile changes in RNA polymerase II (RNAPII) occupancy in MCF-7 cells in response to estradiol (E2), tamoxifen or fulvestrant. We identified 10,205 high confidence ERalpha binding sites in response to E2 of which 68% contained an estrogen response element (ERE) and only 7% contained a FOXA1 motif. Remarkably, 596 genes already change significantly in RNAPII occupancy (59% up and 41% down) following one hour of E2 exposure. Although pausing of RNA polymerase II occurs frequently in MCF-7 cells (17%) it is only observed on a minority of E2-regulated genes (4%). Tamoxifen and fulvestrant partially reduce ERalpha DNA binding and prevent RNAPII loading on the promoter and coding body on E2-upregulated genes. Both antagonists act differently on E2-downregulated genes. Tamoxifen acts as an agonist, also downregulating these genes while fulvestrant antagonizes E2 induced repression and often increases RNAPII occupancy. Furthermore our data identified genes preferentially regulated by tamoxifen but not by E2 or fulvestrant. Thus, antagonist loaded ERalpha acts mechanistically different on E2-activated and E2-repressed genes. Examination of ERalpha binding sites upon the binding of different ligands and association with transcription via RNAPII occupancy.