Project description:Long non coding RNAs are implemented in epigenetic changes and regulation of gene expression. HOTAIR is a promising lncRNA concerning epigenetic regulation. We performed HOTAIR overexpression and knockdown experiments in mesenchymal stromal cells derived from bone marrow. After two weeks cells were harvested and RNA and DNA were isolated. Analysis of gene expression was performed with Human Gene 2.0 ST Array (Affymetrix, Santa Clara, USA). Analysis of DNA methylation was performed with Infinium HumanMethylation450 BeadChips (Illumina, San Diego, USA)
Project description:Long non coding RNAs are implemented in epigenetic changes and regulation of gene expression. HOTAIR is a promising lncRNA concerning epigenetic regulation. We performed HOTAIR overexpression and knockdown experiments in mesenchymal stromal cells derived from bone marrow. After two weeks cells were harvested and RNA and DNA were isolated. Analysis of gene expression was performed with Human Gene 2.0 ST Array (Affymetrix, Santa Clara, USA). Analysis of DNA methylation was performed with Infinium HumanMethylation450 BeadChips (Illumina, San Diego, USA)
Project description:Accumulating evidence highlights the role of long non-coding RNAs (lncRNA) in cellular homeostasis, and their dysregulation in disease settings. Most lncRNAs function by interacting with proteins or protein complexes. While several orthogonal methods have been developed to identify these proteins, each method has its inherent strengths and limitations. Here, we combine two RNA-centric methods ChIRP-MS and RNA-BioID to obtain a comprehensive list of proteins that interact with the well-known lncRNA HOTAIR. Overexpression of HOTAIR has been associated with a metastasis-promoting phenotype in various cancers. Although HOTAIR is known to bind with PRC2 and LSD1 protein complexes, an unbiased and comprehensive method to map its interactome has not yet been performed. Both ChIRP-MS and RNA-BioID data sets show an association of HOTAIR with mitoribosomes, suggesting HOTAIR has functions independent of its (post-)transcriptional mode-of-action.
Project description:HOTAIR is a scafold long non-coding RNA tethering PRC2 and Lsd1/REST/coREST complexes to gene promoters to repress transcription of genes involved in epithelial to mesenchymal transition (EMT). To decipher a role of the Lsd1 in HOTAIR function we generated epithelial cell lines expressing full length and truncated versions of HOTAIR missing first 5'-300 (Epi-HOTΔP) and last 3'-500 bp (Epi-HOTΔL) interacting with HOTAIR partners. These cell lines were further used to determine Lsd1 binding sites using chromatin immunoprecipitation approach.
Project description:HOTAIR is a 2.2 kb long noncoding RNA (lncRNA) whose dysregulation has been linked to oncogenesis, defects in pattern formation during early development, and irregularities during the process of epithelial-to-mesenchymal transition (EMT). However, the oncogenic transformation determined by HOTAIR in vivo and its impact on chromatin dynamics are incompletely understood. Here we generate a transgenic mouse model with doxycycline-inducible expression of human HOTAIR in the context of the MMTV-PyMT breast cancer-prone background (iHOT-PyMT mice) to systematically interrogate the cellular mechanisms by which human HOTAIR lncRNA acts to promote breast cancer progression. We isolated breast cancer cells from the primary tumors of iHOT-PyMT mice (named iHOT+ cells) and performed RNA-seq and ATAC-seq of iHOT+ cells treated with 3 conditions: Dox+, Dox- and DoxWD. We showed that HOTAIR overexpression altered both the cellular transcriptome and chromatin accessibility landscape of multiple metastasis-associated genes and promoted epithelial to mesenchymal transition. These alterations are abrogated within several cell cycles after HOTAIR expression is reverted to basal levels, indicating an erasable lncRNA-associated epigenetic memory. These results suggest that a continual role for HOTAIR in programming a metastatic gene regulatory program.
Project description:HOTAIR is a 2.2 kb long noncoding RNA (lncRNA) whose dysregulation has been linked to oncogenesis, defects in pattern formation during early development, and irregularities during the process of epithelial-to-mesenchymal transition (EMT). However, the oncogenic transformation determined by HOTAIR in vivo and its impact on chromatin dynamics are incompletely understood. Here we generate a transgenic mouse model with doxycycline-inducible expression of human HOTAIR in the context of the MMTV-PyMT breast cancer-prone background (iHOT-PyMT mice) to systematically interrogate the cellular mechanisms by which human HOTAIR lncRNA acts to promote breast cancer progression. We isolated breast cancer cells from the primary tumors of iHOT-PyMT mice (named iHOT+ cells) and performed RNA-seq and ATAC-seq of iHOT+ cells treated with 3 conditions: Dox+, Dox- and DoxWD. We showed that HOTAIR overexpression altered both the cellular transcriptome and chromatin accessibility landscape of multiple metastasis-associated genes and promoted epithelial to mesenchymal transition. These alterations are abrogated within several cell cycles after HOTAIR expression is reverted to basal levels, indicating an erasable lncRNA-associated epigenetic memory. These results suggest that a continual role for HOTAIR in programming a metastatic gene regulatory program.
Project description:Purpose: Our experimental results demonstrate an essential role for the lncRNA HOTAIR in Ewing sarcoma. We have repressed HOTAIR expression in three Ewing sarcoma cell lines and overexpressed HOTAIR, alone and with EWS-FLI1, in htert-immortalized human mesenchymal stem cells to evaluate its effects on gene expression in this cancer. Methods: RNA-Seq of Ewing sarcoma cell lines with HOTAIR represssed by GapmeR or treated with nonsilencing control, and hTERT-immortalized hMSCs with expression of control GFP, HOTAIR, or HOTAIR and EWS-FLI1, was used for gene expression analysis. Results: Defined gene expression signatures were defined as driven by HOTAIR in each cell line model, with a consensus set of gene identified in the Ewing sarcoma cell lines. Additionally, a set of genes regulated by HOTAIR independently of EWS-FLI1 was identified in the hTERT-hMSC models. Conclusions: HOTAIR expression regulates a set of genes critical to viability, cell adhesion, cell motility, and other markers of EMT and metastasis in Ewing sarcoma, independently of EWS-FLI1.
Project description:Accumulating evidence highlights the role of long non-coding RNAs (lncRNA) in cellular homeostasis, and their dysregulation in disease settings. Most lncRNAs function by interacting with proteins or protein complexes. While several orthogonal methods have been developed to identify these proteins, each method has its inherent strengths and limitations. Here, we combine two RNA-centric methods ChIRP-MS and RNA-BioID to obtain a comprehensive list of proteins that interact with the well-known lncRNA HOTAIR. Overexpression of HOTAIR has been associated with a metastasis-promoting phenotype in various cancers. Although HOTAIR is known to bind with PRC2 and LSD1 protein complexes, an unbiased and comprehensive method to map its interactome has not yet been performed. Both ChIRP-MS and RNA-BioID data sets show an association of HOTAIR with mitoribosomes, suggesting HOTAIR has functions independent of its (post-)transcriptional mode-of-action.