Antibody validation in estrogen receptor beta research
Ontology highlight
ABSTRACT: Three antibodies targeting estrogen receptor beta were evaluated with respect to their specificity towards the target protein, by immunoprecipitation followed by LC-Orbitrap MS.
Project description:Estrogen Receptor subtypes (ERα and ERβ) are transcription factors sharing similar structure, however, they often perform opposite roles in breast cancer’s cell proliferation and tumor progression. Besides the well-characterized genomic actions of ERs upon ligand binding, rapid non-genomic cytoplasmic changes together with the recently discovered ligand-free action of ERs are emerging as key regulators of tumorigenesis. The identification of cytoplasmic interaction partners of unliganded ERα and ERβ may help characterize the molecular basis of the extra-nuclear mechanism of action of these receptors, revealing novel mechanisms to explain their role in breast cancer response or resistance to endocrine therapy. To this aim, in this study, cytoplasmic extracts from stably expressing TAP-ERα and -ERβ MCF-7 cell clones were subjected to interaction proteomics in the absence of estrogen stimulation, leading to the identification of 84 and 142 proteins associated with unliganded ERα and ERβ, respectively. Functional analyses of ER subtype-specific interactomes revealed significant differences in the molecular pathways associated to each receptor in the cytoplasm. This work reports the first identification of the unliganded ERα and ERβ cytoplasmic interactomes in breast cancer cells, providing novel experimental evidence on the non-genomic effects of ERs in the absence of hormonal stimulus.
Project description:The closely related transcription factors (TFs), estrogen receptors ERα and ERβ, regulate divergent gene expression programs and proliferative outcomes in breast cancer. Utilizing MCF-7 breast cancer cells with ERα, ERβ, or both receptors as a model system to define the basis of differing response specification by related TFs, we show that these TFs and their key coregulators, SRC3 and RIP140, generate overlapping as well as unique chromatin-binding and transcription-regulating modules. Cistrome and transcriptome analyses and use of clustering algorithms delineated 11 clusters representing different chromatin-bound receptor and coregulator assemblies that could be functionally associated through enrichment analysis with distinct patterns of gene regulation and preferential coregulator usage, RIP140 with ERβ and SRC3 with ERα. The receptors modified each other’s transcriptional effect, and ERβ countered the proliferative drive of ERα through several novel mechanisms associated with specific binding site clusters. Our findings delineate distinct TF-coregulator assemblies that function as control nodes specifying precise patterns of gene regulation, proliferation, and metabolism, as exemplified by two of the most important nuclear hormone receptors in human breast cancer. MCF-7 cells expressing endogenous ERalpha were infected with adenovirus carrying either estrogen receptor beta (AdERb) or no insert (Ad) at multiplicity of infection (moi) of 50. ERβ only cells were generated from these cells by knockdown of ERα in parental cells using the following siERα sequences from Dharmacon: forward, 5’-UCAUCGCAUUCCUUGCAAAdTdT-3’, and reverse, 5’-UUUGCAAGGAAUGCGAUGAdTdT-3’. siRNA experiments were performed as previously described, and resulted in knocknown of ERα mRNA and protein by greater than 95% (GSE4006, PMID 16809442). Briefly, cells were transfected with 20 nM siCtrl [GSE4006] or siERα for 48 h after infection. Then cells were treated with 0.1% EtOH (Veh) or 10 nM E2 (Sigma-Aldrich) for 24h. All experiments were conducted with two replicates. key words; Adenovirus infection,siRNA knock-down, ligand treatment
Project description:Cocksfoot grass (Dactylis glomerata) collected from Wytham, Oxford, UK, was tested for Cocksfoot streak virus infection. Small RNA of the grass was extracted and converted to DNA according to Ho, T., et al. (2008) Biochem Biophys Res Commun. 368:433-7, with primers modified to contain 454 adapter nucleotide sequences. The DNA then passed quality control through Bioanalyzer and Nanodrop before sequenced by 454 Life Sciences. Keywords: siRNA One sample analyzed by 454 high-throughput sequencing technology
Project description:Breast cancer (BC) is the most common cancer in women worldwide, and is classified in multiple subtypes, including the so called triple-negative BC (TNBC). This is characterized by lack of estrogen receptor alpha (ERα), progesterone receptor (PR) and epidermal growth factor receptor 2 (HER2/neu), that represent common targets for BC treatment. Their absence limits the number of therapies that may be applied for TNBC treatment, suggesting the need to identify novel therapeutic targets against this disease. Several studies reported that the beta ER subtype (ERβ) is expressed in a sizeable fraction of TNBCs where its presence correlates with improved patient outcome. We evaluated ERβ expression in TNBC tissues by immunohistochemistry using two validated antibodies, demonstrating presence of this protein in 28% of samples. To investigate, in this context, the role of this estrogen receptor in TNBC biology, ERβ-expressing cell lines, representing different TNBC subtypes, were generated. Cellular and functional assays confirmed the antiproliferative activity of ERβ in TNBCs. Interaction proteomics revealed in BC nuclei the presence of several protein complexes associated with this receptor involved in chromatin remodeling, miRNA maturation and mRNA transcription. Transcriptome analyses pointed out tumor subtype-specific signaling pathways deregulation. Interestingly, among these the cholesterol biosynthesis pathway was commonly downregulated in all cell lines tested. Global analyses of ERβ binding to the genome showed its recruitment to regulatory sites of Sterol Regulatory Element-Binding Protein 1 (SREBP1), indicating a direct regulation of this pathway by the receptor. These findings suggest that drugs targeting components of cholesterol biosynthesis pathway may be new potential therapeutic options for TNBC treatment.
Project description:Quantitative mass spectrometry analysis using tandem mass tags (TMT) labelling of estrogen receptor α (ERα) pull downs. Co-immunoprecipitated proteins using a anti Erα antibody were compared quantitatively with material recovered in a mock to identify ERα-interacting proteins.
Project description:The Estrogen Receptor alpha (ERα) controls key cellular functions in hormone responsive breast cancer by assembling in large functional multiprotein complexes. ERα ligands are classified as agonists and antagonist, according to the response they elicit, thus the molecular characterization of the of ERα nuclear iteractome composition following estrogen and antiestrogen stimulation whose is needed to understand their effects on estrogen target tissues, in particular breast cancer. To this aim interaction proteomics coupled to mass spectrometry (MS) was applied to map the ERα nuclear interacting partners in MCF7 breast cancer cell nuclei following estrogen and antiestrogen stimuli.
Project description:Breast cancer (BC) is the most common cancer in women worldwide, and is classified in multiple subtypes, including the so called triple-negative BC (TNBC). This is characterized by lack of estrogen receptor alpha (ERα), progesterone receptor (PR) and epidermal growth factor receptor 2 (HER2/neu), that represent common targets for BC treatment. Their absence limits the number of therapies that may be applied for TNBC treatment, suggesting the need to identify novel therapeutic targets against this disease. Several studies reported that the beta ER subtype (ERβ) is expressed in a sizeable fraction of TNBCs where its presence correlates with improved patient outcome. We evaluated ERβ expression in TNBC tissues by immunohistochemistry using two validated antibodies, demonstrating presence of this protein in 28% of samples. To investigate, in this context, the role of this estrogen receptor in TNBC biology, ERβ-expressing cell lines, representing different TNBC subtypes, were generated. Cellular and functional assays confirmed the antiproliferative activity of ERβ in TNBCs. Interaction proteomics revealed in BC nuclei the presence of several protein complexes associated with this receptor involved in chromatin remodeling, miRNA maturation and mRNA transcription. Transcriptome analyses pointed out tumor subtype-specific signaling pathways deregulation. Interestingly, among these the cholesterol biosynthesis pathway was commonly downregulated in all cell lines tested. Global analyses of ERβ binding to the genome showed its recruitment to regulatory sites of Sterol Regulatory Element-Binding Protein 1 (SREBP1), indicating a direct regulation of this pathway by the receptor. These findings suggest that drugs targeting components of cholesterol biosynthesis pathway may be new potential therapeutic options for TNBC treatment.
Project description:Expression level, control and intercoordination of 66 selected heart rhythm determinant (HRD) genes were compared in atria and ventricles of 4 male and 4 female adult mice. We found that genes encoding various adrenergic receptors, ankyrins, ion channels and transporters, connexins and other components of the intercalated discs form a complex network that is chamber dependent and differs between the two sexes. In addition, most HRD genes in atria had higher expression in males than in females, while in ventricles expression levels were mostly higher in females than in males. Moreover, significant chamber-differences were observed between the sexes, with higher expression in atria than ventricles for males and higher expression in ventricles than atria for females. We have ranked the selected genes according to their prominence in controlling the HRD gene web through expression coordination with the other web genes and protecting the web though their own expression stability. Interestingly, the prominence hierarchy was substantially different between the two sexes. Taken together these findings indicate that the organizational principles of the heart rhythm transcriptome are sex-dependent, with the newly introduced prominence analysis allowing identification of genes that are pivotal for the sexual dichotomy. Four adult male (M) and 4 adult female (F) mice were decapitated, the hearts removed and atria (A) and ventricles (V) collected in separate tubes. 20 µg total RNA extracted in Trizol from each of the 16 samples was reverse transcribed in the presence of fluorescent Alexa Fluor®_647 and Alexa Fluor®_555-aha-dUTPs (Invitrogen, CA) to obtain labeled cDNAs Red and green-labeled samples of biological replicas were then co-hybridized (âmultiple yellowâ strategy) overnight at 50°C with mouse MO36k oligonucleotide arrays printed by Duke University (http://microarray.genome.duke.edu/spotted-arrays) with Operon Mouse Oligo Set, version 4.0. After washing (0.1% SDS and 1% SSC) to remove the non-hybridized cDNA, each array was scanned with GenePix 4000B scanner (MDS, Toronto, Canada) and images were primarily analyzed with GenePixPro 6.0 (Axon Instruments, CA).
Project description:Use of null mutant mice is a powerful way to evaluate the role of specific proteins in brain function. Studies performed on knockout mice have revealed some unexpected roles of the gap junction proteins (the connexins). Thus, analyses of gene expression in connexin43 (Cx43) null brains indicated that deletion of a single gene (Gja1) induced expression level change of numerous other genes located on all chromosomes and involved in a wide diversity of functional pathways. The significant overlap between alterations in gene expression level, control and coordination in Cx43 knockout and knockdown astrocytes raised the possibility that Gja1 represents a transcriptomic node of gene regulatory networks. However, conditional deletion of Gja1 in astrocytes of two mouse strains resulted in remarkably different phenotypes. In order to evaluate the influence of the genetic background on the transcriptome, we performed microarray studies on brains of GFAP-Cre:Cx43f/f C57Bl/6 and 129/SVEV mice. The surprisingly low number of Cx43 core genes (regulated in all Cx43 nulls regardless of strain) and the high number of differently regulated genes in the two Cx43 CKOs indicate high influence of mouse strain on brain transcriptome. The transcriptomes of WT and Cx43 null brains from both C57Bl/6 and SVEV strains were profiled and compared at perinatal and adult time points to learn more about the strain dependence of the Cx43-null phenotype. For this purpose, differently labeled cDNAs from biological replicas (4 of each genotype) were co-hybridized with Duke MO36K mouse oligonucleotide array spotted with 36k Operon oligonucleotides V4.0.