title: Estradiol-Induced Transcriptional Regulation of Long Non-Coding RNA, HOTAIR.
abstract: HOTAIR (HOX antisense intergenic RNA) is a 2.2 kb long non-coding RNA (lncRNA), transcribed from the antisense strand of homeobox C (HOXC) gene locus in chromosome 12. HOTAIR acts as a scaffolding lncRNA. It interacts and guides various chromatin-modifying complexes such as PRC2 (polycomb-repressive complex 2) and LSD1 (lysine-specific demethylase 1) to the target gene promoters leading to their gene silencing. Various studies have demonstrated that HOTAIR overexpression is associated with breast cancer. Recent studies from our laboratory demonstrate that HOTAIR is required for viability of breast cancer cells and is transcriptionally regulated by estradiol (E2) in vitro and in vivo. This chapter describes protocols for analysis of the HOTAIR promoter, cloning, transfection and dual luciferase assays, knockdown of protein synthesis by antisense oligonucleotides, and chromatin immunoprecipitation (ChIP) assay. These protocols are useful for studying the estrogen-mediated transcriptional regulation of lncRNA HOTAIR, as well as other protein coding genes and non-coding RNAs.
authors.affiliation: Department of Chemistry and Biochemistry, The University of Texas at Arlington, Arlington, TX, 76019, USA.
authors.forename: Arunoday
authors.initials: A
authors.lastname: Bhan
authors.affiliation: Department of Chemistry and Biochemistry, The University of Texas at Arlington, Arlington, TX, 76019, USA. smandal@uta.edu.
authors.forename: Subhrangsu S
authors.initials: SS
authors.lastname: Mandal
mesh_terms: D001943:Breast Neoplasms / Q000235:genetics / Q000378:metabolism*; D047369:Chromatin Immunoprecipitation; D003001:Cloning, Molecular; D004305:Dose-Response Relationship, Drug; D004958:Estradiol / Q000494:pharmacology*; D004967:Estrogens / Q000494:pharmacology*; D005260:Female; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects*; D017930:Genes, Reporter; D006801:Humans; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D061986:MCF-7 Cells; D016376:Oligonucleotides, Antisense / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic; D062085:RNA, Long Noncoding / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic / Q000187:drug effects*; D015533:Transcriptional Activation / Q000187:drug effects; D014162:Transfection; D057188:Workflow
chemical_list: D004967:Estrogens; C561566:HOTAIR long untranslated RNA, human; D016376:Oligonucleotides, Antisense; D062085:RNA, Long Noncoding; D004958:Estradiol; D008156:Luciferases
keywords: Epigenetics; Estradiol; Estrogen receptor s; Gene expression; HOTAIR; Long non-coding RNA; Mixed lineage leukemia; Polycomb-repressive complex 2