TNFM-NM-1 Signaling Exposes Latent Estrogen Receptor Binding Sites in Breast Cancer Cells
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ABSTRACT:
ORGANISM(S): Homo sapiens
SUBMITTER: W. Lee Kraus
PROVIDER: E-GEOD-59532 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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Project description:The interplay between mitogenic and proinflammatory signaling pathways play key roles in determining the phenotypes and clinical outcomes of breast cancers. We have used global nuclear run-on coupled with deep sequencing to characterize the immediate transcriptional responses of MCF-7 breast cancer cells treated with estradiol, TNFM-NM-1, or both. In addition, we have integrated these data with chromatin immunoprecipitation coupled with deep sequencing for estrogen receptor alpha (ERM-NM-1), the pioneer factor FoxA1 and the p65 subunit of the NF-M-NM-:B transcription factor. Our results indicate extensive transcriptional interplay between these two signaling pathways, which is observed for a number of classical mitogenic and proinflammatory protein-coding genes. In addition, GRO-seq has allowed us to capture the transcriptional crosstalk at the genomic locations encoding for long non-coding RNAs, a poorly characterized class of RNAs which have been shown to play important roles in cancer outcomes. The synergistic and antagonistic interplay between estrogen and TNFM-NM-1 signaling at the gene level is also evident in the patterns of ERM-NM-1 and NF-M-NM-:B binding, which relocalize to new binding sites that are not occupied by either treatment alone. Interestingly, the chromatin accessibility of classical ERM-NM-1 binding sites is predetermined prior to estrogen treatment, whereas ERM-NM-1 binding sites gained upon co-treatment with TNFM-NM-1 require NF-M-NM-:B and FoxA1 to promote chromatin accessibility de novo. Our data suggest that TNFM-NM-1 signaling recruits FoxA1 and NF-M-NM-:B to latent ERM-NM-1 enhancer locations and directly impact ERM-NM-1 enhancer accessibility. Binding of ERM-NM-1 to latent enhancers upon co-treatment, results in increased enhancer transcription, target gene expression and altered cellular response. This provides a mechanistic framework for understanding the molecular basis for integration of mitogenic and proinflammatory signaling in breast cancer. Using GRO-seq and ChIP-seq (ER, FoxA1 and p65) to assay the molecular crosstalk of MCF-7 cells treated with E2, TNFM-NM-1 or both E2+TNFM-NM-1.
2015-03-05 | E-GEOD-59531 | biostudies-arrayexpress
Project description:Estrogen receptor M-NM-1 (ER), a member of the nuclear hormone receptor superfamily, regulates transcriptional activity by ligand-dependent recruitment of cofactors which, in turn, locally alter chromatin structure. It is generally believed that co-factor activity at target promoters leads to a more open, transcriptionally permissive chromatin structure, however, these mechanisms remain to be fully established. Peptidylarginine deiminases (PADIs) catalyze the conversion of positively charged arginine and methylarginine residues to neutrally charged citrulline and this activity has been linked to the gene regulation. Here, we found that PADI2 citrullinate H3 Arginine 26 (H3R26) in vitro and, using a specific H3R26 citrulline (H3Cit26) antibody, we demonstrate that H3Cit26 occurs in vivo following 17M-NM-2-estradiol (E2) stimulation and this unique and pronounced global activation of H3Cit26 is ER-dependent. Using a mammalian-based promoter chromosomal array system, we observed that citrullination at H3R26 is robust and co-localizes with ER at decondensed chromatin loci. Additionally, this histone modification is specifically enriched at ER bound regions of target promoters, forming a permissive chromatin environment for gene transactivation. Interestingly, we have shown in a reciprocal way, that either depletion of PADI2 or inhibition of ER not only dramatically abolished E2-induced activation of H3Cit26 on gene promoters but also affect ER recruitment. Collectively, our results demonstrate that citrullination of H3R26 by PADI2 following estrogen stimulation plays a role in ER target gene activation, likely via decondensation of the local chromatin architecture. Two H3Cit26 ChIP-chip biological replicates under vehicle treatment and two H3Cit26 ChIP-chip biological replicates under E2 stimulation from MCF-7 human breast cancer cells are included.
2012-09-04 | E-GEOD-32599 | biostudies-arrayexpress
Project description:This SuperSeries is composed of the SubSeries listed below. Refer to individual Series
2014-02-27 | E-GEOD-51225 | biostudies-arrayexpress
Project description:We descrive a joint model of transcriptional activation and mRNA accumulation, using estrogen receptor ERM-NM-1 activation in MCF-7 breast cancer cell line, which can be used for inference of transcription rate, RNA processing delay and degradation rate given data from high-throughput sequencing time course experiments. MCF-7 cells were mock treated or with 10nM 17b-E2 to nine time points (5', 10', 20', 40', 80', 160', 320', 640' and 1280'). Genome-wide identification of RNA polymerase II (RNAPII) occupancy and transcriptome profiling (RNA-seq) following E2 induction of MCF-7 cells Please note that the information in the wig.txt files is in gene-specific coordinates, not chromosomic coordinates, as this is the most sensible format for the associated project/paper.
2015-03-04 | E-GEOD-62789 | biostudies-arrayexpress
Project description:Metabolic homeostasis in mammals critically depends on the regulation of fasting-induced genes by CREB in the liver. Previous genome-wide analysis has shown that only a small percentage of CREB target genes are induced in response to fasting-associated signaling pathways. The precise molecular mechanisms by which CREB specifically targets these genes in response to alternating hormonal cues remain to be elucidated. We performed chromatin immunoprecipitation coupled to high-throughput sequencing of CREB in livers from both fasted and re-fed mice. In order to quantitatively compare the extent of CREB-DNA interactions genome-wide between these two physiological conditions we developed a novel, robust analysis method, termed the M-bM-^@M-^Xsingle sample independenceM-bM-^@M-^Y (SSI) test that greatly reduced the number of false positive peaks. We found that CREB remains constitutively bound to its target genes in the liver regardless of the metabolic state. Integration of the CREB cistrome with expression microarrays of fasted and re-fed mouse livers and ChIP-seq data for additional transcription factors revealed that the gene expression switches between the fasted and fed states are associated with co-localization of additional transcription factors at CREB sites. Our results support a model in which CREB is constitutively bound to thousands of potential target genes and combinatorial interactions between DNA-binding factors are necessary to achieve the specific transcriptional response of the liver to fasting. Furthermore, our genome-wide analysis identifies thousands of novel CREB target genes in liver, including a previously unknown role for CREB in regulating ER stress genes in response to nutrient influx. CREB ChIP-seq was performed on mouse liver from fasted and re-fed mice, using 5 separate biological replicates for each condition. GR and C/EBP(beta) ChIP-seq were performed as single replicates on ad-lib fed mice.
2013-05-01 | E-GEOD-45674 | biostudies-arrayexpress
Project description:During differentiation of embryonic stem cells, chromatin reorganizes to establish cell type specific expression programs. Here, we have dissected the linkages between DNA methylation (5mC), hydroxymethylation (5hmC), nucleosome repositioning and binding of the transcription factor CTCF during this process. By integrating MNase-seq and ChIP-seq experiments in mouse embryonic stem cells (ESC) and their differentiated counterparts with biophysical modeling, we find that the interplay between these factors depends on their large-scale genomic context. The mostly unmethylated CpG islands have a reduced nucleosome occupancy and are enriched in cell type-independent binding sites for CTCF. The few remaining methylated CpG dinucleotides are preferentially associated with nucleosomes. In contrast, outside of CpG islands most CpGs are methylated and their average density oscillates so that it is highest in the linker region between nucleosomes. Outside CpG islands binding of TET1, an enzyme that converts 5mC to 5hmC, is associated with labile, MNase-sensitive nucleosomes. Such nucleosomes are poised for eviction in ESCs and become stably bound in differentiated cells where the level of TET1 and 5hmCs goes down. In particular, this has a dramatic effect at variable CTCF sites enriched outside CpG islands, that are occupied by CTCF in ESCs but loose CTCF during differentiation. To rationalize this cell type dependent targeting of CTCF binding in competition with the histone octamer, we have developed a quantitative biophysical model, which allowed predicting differences in CTCF binding due to TET1/5hmC/5mC-dependent nucleosome repositioning. MNase-seq experiments for analysis of mononucleosomes were conducted as described previously (Teif et al. Nature Struct. Mol. Biol., 2012). In addition, a dinucleosome fraction also was gel purified and sequenced. Briefly, embryonic stem cells from 129P2/Ola mice were cultured in ESGRO complete medium (Millipore), harvested and resuspended in low salt buffer (10 mM Hepes, pH 8, 10 mM KCl, 0.5 mM DTT) at 4 M-BM-0C. After disruption of the cells with a douncer the nuclei were collected by centrifugation and washed once with the MNase Buffer (10 mM TrisM-bM-^@M-^SHCl, pH 7.5, 10 mM CaCl2), resuspended in the MNase Buffer and digested with 0.5 units MNase per microliter (Fermentas) and incubation for 6M-bM-^@M-^S11 minutes at 37 M-BM-0C. The MNase digestion was stopped by putting the samples on ice and adding EDTA to a concentration of 10 mM. After digestion with 0.1 M-BM-5g M-BM-5lM-bM-^@M-^S1 RNase A (Fermentas) and removal of protein by phenol and chloroform extraction, the DNA was ethanol precipitated and the resulting DNA pellet was dissolved in H2O. DNA fragments corresponding to mononucleosomes or dinucleosomes were separated on a 2 % agarose gel using an E-Gel electrophoresis system (Life Technologies). The libraries for sequencing were prepared according to the standard Illumina protocol. High-throughput paired-end sequencing of at least 50 bp read length was performed on the Illumina HiSeq 2000 platform at the DKFZ sequencing core facility in Heidelberg, Germany. We obtained about 150 million nucleosome positions per sequencing reaction. ChIP-seq experiments were conducted as described previously (Teif et al. 2012). For each sample, 1 x 106 cells were cross-linked with 1% PFA and cell nuclei were prepared using a swelling buffer (25 mM Hepes, pH 7.8, 1 mM MgCl2, 10 mM KCl, 0.1% NP-40, 1 mM DTT). Chromatin was sheared to mononucleosomal fragments. After IgG preclearance the sheared chromatin was incubated with 4 M-BM-5g of either H3K9me3 (Abcam ab8898) or H3K4me3 (Abcam ab8580) antibody over night. After washes with sonication (10 mM TrisM-bM-^@M-^SHCl, pH 8.0, 200 mM NaCl, 1 mM EDTA, 0.5% N-lauroylsarcosine, 0.1% NaM-bM-^@M-^Sdeoxycholate), high-salt-buffer (50 mM Hepes pH 7.9, 500 mM NaCl, 1mM EDTA, 1% Triton X-100, 0.1% NaM-bM-^@M-^Sdeoxycholate, 0.1% SDS), lithium buffer (20 mM TrisM-bM-^@M-^SHCl pH 8.0, 1 mM EDTA, 250 mM LiCl, 0.5% NP-40, 0.5% NaM-bM-^@M-^Sdeoxycholate) and 10 mM TrisM-bM-^@M-^SHCl, chromatin was eluted from the protein G magnetic beads and the crosslink was reversed over night. After RNase A and proteinase K digestion, DNA was purified and cloned in a barcoded sequencing library for the Illumina HiSeq 2000 sequencing platform (single reads of 50 bp length).
2014-05-07 | E-GEOD-56938 | biostudies-arrayexpress
Project description:Inflammation is associated with many cardiovascular pathologies, but the underlying mechanisms remain unclear. To explore this in more detail, we characterized the transcriptome of an immortalized adult human ventricular cardiomyocyte cell line (AC16) in response to tumor necrosis factor (TNFa). Using a combination of genomic approaches, including global nuclear run-on sequencing (GRO-seq) and chromatin immunoprecipitation coupled with sequencing (ChIP-seq), we identified ~30,000 transcribed regions in AC16 cells, which includes a set of RNA polymerases I and III (Pol I and Pol III) transcribed regions revealed in the presence of M-NM-1-amanitin. The set of transcribed regions produces both protein-coding and non-coding RNAs, many of which have not been annotated previously, including enhancer RNAs originating from NF-M-NM-:B binding sites. In addition, we observed that AC16 cells rapidly and dynamically reorganize their transcriptomes in response to TNFa stimulation in an NF-M-NM-:B-dependent manner, switching from a basal state to a proinflammatory state affecting a spectrum of cardiac-associated protein-coding and non-coding genes. Moreover, we observed distinct Pol II dynamics for up- and downregulated genes, with a rapid release of Pol II into productive elongation for TNFa-stimulated genes. Our studies shed new light on the regulation of the cardiomyocyte transcriptome in response to a proinflammatory signal and help to clarify the link between inflammation and cardiomyocyte function at the transcriptional level. Using GRO-seq and ChIP-seq (p65 and RNA Pol II) over a time course of TNFM-NM-1 signaling in AC16 human cardiomyocytes.
2014-02-27 | E-GEOD-51224 | biostudies-arrayexpress
Project description:JNK1 ChIP-chip and ER-alpha ChIP-chip were performed on NimbleGen genomic tiling arrays to understand the genomic binding patterns of this MAP kinase and receptor and their relationship to gene expression. ChIP-chip analysis of JNK1 occupancy at promoters before and after estrogen treatment. 2 biological replicates (rep1 and rep2, rep3A and rep3B), 2 technical replicates each. ChIP-chip analysis of ER-alpha occupancy at promoters before and after estrogen treatment. 2 biological replicates, 2 technical replicates for one of the replicates (1 and 2).
2012-03-31 | E-GEOD-13200 | biostudies-arrayexpress
Project description:In this study, we examined the effect of inorganic arsenic (iAs) on the estrogen response (E2) in MCF-7 cells that are estrogen receptor positive/progesterone receptor positive (ER+/PR+). Understanding the changes in the transcriptomic response after iAs exposure and the retention of such responses during recovery from iAs exposure can identify driver mechanisms of breast cancer progression and improve therapies for patients with ER+ cancers exposed to iAs.
2020-10-21 | E-MTAB-9498 | biostudies-arrayexpress
Project description:The winged helix protein FOXA2 and the nuclear receptor PPARg are highly conserved, regionally-expressed transcription factors that regulate networks of genes controlling complex metabolic functions. Cistrome analysis for FOXA2 in mouse liver and PPARg in mouse adipocytes has previously produced consensus binding sites that are nearly identical to those used by the factors in human cells. Despite this conservation of the canonical binding motif, we report here that the great majority of specific binding regions for FOXA2 in human liver and for PPARg in human adipocytes are not in the orthologous locations to the mouse genome. Nevertheless, gene-centric analysis reveals strong shared transcription factor occupancy near genes in tissue-specific metabolic pathways that are functionally conserved across species. Genes with only species-specific binding sites fail to show enrichment for these pathways. Thus, the biological functions of transcription factors that control specific metabolic functions are highly shared across species. Two TFs, FOXA2 and PPARg, were studied for genome-wide conservation of binding between mouse and human in specific tissues/cell-types (liver for FOXA2, adipocytes for PPARg). The number of replicates for each TF was chosen to obtain a comparable number of reads between the TFs and species. Human FOXA2 ChIP-seq was performed on two biological replicates of human liver samples, in three technical replicates each. Input DNA was also collected and sequenced from both biological samples. Mouse FOXA2 ChIP-seq was performed on four biological replicates of mouse liver samples. The ChIP and sequencing were repeated on two of these biological replicates to create technical replicates for additional sequence reads. Input DNA was sequenced from three additional mouse livers. Human PPARg ChIP-seq was performed on a human adipocyte cell-line (SGBS) differentiated in two replicate cultures. Input DNA was also collected and sequenced from one culture. Mouse PPARg ChIP-seq was performed on 3T3-L1 cells differentiated into adipocytes in culture in a single replicate, and this sequence data was pooled with existing data previously generated by the same lab, already available in GEO (GSE21314). A standard pool of input DNA sample sequence from multiple mouse tissue was used for analyzing the Mouse PPARg ChIP-seq data.
2011-04-06 | E-GEOD-25836 | biostudies-arrayexpress