Estradiol-Induced Transcriptional Regulation of Long Non-Coding RNA, HOTAIR

Part of the Methods in Molecular Biology book series (MIMB, volume 1366)

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.

Key words

Long non-coding RNA HOTAIR Estradiol Estrogenreceptors Gene expression Epigenetics Mixed lineage leukemia Polycomb-repressive complex 2 

Notes

Acknowledgements

We thank all the Mandal lab members for helpful discussions. Research in Mandal laboratory is supported by a grant from NIH (1R15 ES019129-01).

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Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  1. 1.Department of Chemistry and BiochemistryThe University of Texas at ArlingtonArlingtonUSA

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