Abstract
Enhancers are principal regulators that allow spatiotemporal tissue-specific control of gene expression. While mounting evidence suggests that enhancer-derived long noncoding RNAs (long ncRNAs), including enhancer RNAs (eRNAs), are an important component of enhancer function, their expression has not been broadly analyzed at a single cell level via imaging techniques. This protocol describes a method to image eRNA in single cells by in situ hybridization followed by tyramide signal amplification (TSA). The procedure can be multiplexed to simultaneously visualize both eRNA and protein-coding transcript at the site of transcriptional elongation, thereby permitting analysis of dynamics between the two transcript species in single cells. Our approach is not limited to eRNAs, but can be implemented on other transcripts.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Banerji J, Rusconi S, Schaffner W (1981) Expression of beta-globin gene is enhanced by remote SV40 DNA sequences. Cell 27:299–308
Levine M, Cattoglio C, Tjian R (2014) Looping back to leap forward: transcription enters a new era. Cell 157:13–25
Collins P, Antoniou M, Grosveld F (1990) Definition of the minimal requirements within the human beta-globin gene and the dominant control region for high level expression. EMBO J 9:233–240
Tuan D, Kong S, Hu K (1992) Transcription of the hypersensitive site HS2 enhancer in erythroid cells. Proc Natl Acad Sci U S A 89:11219–11223
Kim TK, Hemberg M, Gray JM et al (2010) Widespread transcription at neuronal activity-regulated enhancers. Nature 465:182–187
Andersson R, Gebhard C, Miguel-Escalada I et al (2014) An atlas of active enhancers across human cell types and tissues. Nature 507:455–461
Li W, Notani D, Ma Q et al (2013) Functional roles of enhancer RNAs for oestrogen-dependent transcriptional activation. Nature 498:516–520
Lam MTY, Cho H, Lesch HP et al (2013) Rev-Erbs repress macrophage gene expression by inhibiting enhancer-directed transcription. Nature 498:511–515
Melo CA, Drost J, Wijchers PJ (2013) eRNAs are required for p53-dependent enhancer activity and gene transcription. Mol Cell 49:524–535
Mousavi K, Zare H, Dell’orso S et al (2013) eRNAs promote transcription by establishing chromatin accessibility at defined genomic loci. Mol Cell 51:606–617
Parker SC, Stitzel ML, Taylor DL et al (2013) Chromatin stretch enhancer states drive cell-specific gene regulation and harbor human disease risk variants. Proc Natl Acad Sci U S A 110:17921–17926
Whyte WA, Orlando DA, Hnisz D et al (2013) Master transcription factors and mediator establish super-enhancers at key cell identity genes. Cell 153:307–319
Hah N, Benner C, Chong LW et al (2015) Inflammation-sensitive super enhancers form domains of coordinately regulated enhancer RNAs. Proc Natl Acad Sci U S A 112:E297–E302
Raj A, van den Bogaard P, Rifkin SA et al (2008) Imaging individual mRNA molecules using multiple singly labeled probes. Nat Methods 5:877–879
Batish M, Raj A, Tyagi S (2011) Single molecule imaging of RNA in situ. In: Gerst JE (ed) RNA detection and visualization: methods and protocols, vol 714, Methods in molecular biology. Springer, Heidelberg, pp 3–13
Bobrow MN, Harris TD, Krista H et al (1989) Catalyzed reporter deposition, a novel method of signal amplification application to immunoassays. J Immunol Methods 125:279–285
van Gijlswijk RPM, Zijlmans HJMAA, Wiegant J et al (1997) Fluorochrome-labeled tyramides: use in immunocytochemistry and fluorescence in situ hybridization. J Histochem Cytochem 45:375–382
Wang KC, Yang YW, Liu B et al (2011) A long noncoding RNA maintains active chromatin to coordinate homeotic gene expression. Nature 472:120–124
Levesque MJ, Raj A (2013) Single-chromosome transcriptional profiling reveals chromosomal gene expression regulation. Nat Methods 10:246–248
Fanucchi S, Shibayama Y, Burd S et al (2013) Chromosomal contact permits transcription between coregulated genes. Cell 155:606–620
Yildiz A, Forkey JN, McKinney SA et al (2003) Myosin V walks hand-over-hand: single fluorophore imaging with 1.5-nm localization. Science 300:2061–2065
Acknowledgments
We acknowledge contributions from Robyn Brackin who helped develop the TSA technique in our lab. This work was supported by grant PG-V2KYPO7 from the Council for Industrial and Scientific Research (CSIR, South Africa) and by a grant from the Emerging Research Area Program of The Department of Science and Technology (DST, South Africa) and grant PTDC/SAU-GMG/115652/2009 from the Fundação para a Ciência e a Tecnologia (FCT, Portugal), all to Musa M. Mhlanga.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2017 Springer Science+Business Media New York
About this protocol
Cite this protocol
Shibayama, Y., Fanucchi, S., Mhlanga, M.M. (2017). Visualization of Enhancer-Derived Noncoding RNA. In: Ørom, U. (eds) Enhancer RNAs. Methods in Molecular Biology, vol 1468. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-4035-6_3
Download citation
DOI: https://doi.org/10.1007/978-1-4939-4035-6_3
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-4033-2
Online ISBN: 978-1-4939-4035-6
eBook Packages: Springer Protocols