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Identification of Transcribed Enhancers by Genome-Wide Chromatin Immunoprecipitation Sequencing

Protocol
First Online:
Part of the Methods in Molecular Biology book series (MIMB, volume 1468)

Abstract

Recent work has shown that RNA polymerase II-mediated transcription at distal cis-regulatory elements serves as a mark of highly active enhancers. Production of noncoding RNAs at enhancers, termed eRNAs, correlates with higher expression of genes that the enhancer interacts with; hence, eRNAs provide a new tool to model gene activity in normal and disease tissues. Moreover, this unique class of noncoding RNA has diverse roles in transcriptional regulation. Transcribed enhancers can be identified by a common signature of epigenetic marks by overlaying a series of genome-wide chromatin immunoprecipitation and RNA sequencing datasets. A computational approach to filter non-enhancer elements and other classes of noncoding RNAs is essential to not cloud downstream analysis. Here we present a protocol that combines wet and dry bench methods to accurately identify transcribed enhancers genome-wide as well as an experimental procedure to validate these datasets.

Key words

eRNA Chromatin immunoprecipitation sequencing Global run on sequencing Noncoding RNA Transcribed enhancer ENCODE 
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References

  1. 1.
    Heintzman ND, Stuart RK, Hon G et al (2007) Distinct and predictive chromatin signatures of transcriptional promoters and enhancers in the human genome. Nat Genet 39:311–318CrossRefPubMedGoogle Scholar
  2. 2.
    Heintzman ND, Hon GC, Hawkins RD et al (2009) Histone modifications at human enhancers reflect global cell-type-specific gene expression. Nature 459:108–112CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Visel A, Blow MJ, Li Z et al (2009) ChIP-seq accurately predicts tissue-specific activity of enhancers. Nature 457:854–858CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Zentner GE, Tesar PJ, Scacheri PC (2011) Epigenetic signatures distinguish multiple classes of enhancers with distinct cellular functions. Genome Res 21:1273–1283CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Rada-Iglesias A, Bajpai R, Swigut T et al (2011) A unique chromatin signature uncovers early developmental enhancers in humans. Nature 470:279–283CrossRefPubMedGoogle Scholar
  6. 6.
    Creyghton MP, Cheng AW, Welstead GG et al (2010) Histone H3K27ac separates active from poised enhancers and predicts developmental state. Proc Natl Acad Sci U S A 107:21931–21936CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Stadler MB, Murr R, Burger L et al (2011) DNA-binding factors shape the mouse methylome at distal regulatory regions. Nature 480:490–495PubMedGoogle Scholar
  8. 8.
    Kim T-K, Hemberg M, Gray JM et al (2010) Widespread transcription at neuronal activity-regulated enhancers. Nature 465:182–187CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    De Santa F, Barozzi I, Mietton F et al (2010) A large fraction of extragenic RNA pol II transcription sites overlap enhancers. PLoS Biol 8, e1000384CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Koch F, Fenouil R, Gut M et al (2011) Transcription initiation platforms and GTF recruitment at tissue specific enhancers and promoters. Nat Struct Mol Biol 18:956–963CrossRefPubMedGoogle Scholar
  11. 11.
    Wang D, Garcia-Bassets I, Benner C et al (2011) Reprogramming transcription by distinct classes of enhancers functionally defined by eRNA. Nature 474:390–394CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Orom UA, Derrien T, Beringer M et al (2010) Long noncoding RNAs with enhancer-like function in human cells. Cell 143:46–58CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Lai F, Orom UA, Cesaroni M et al (2013) Activating RNAs associate with mediator to enhance chromatin architecture and transcription. Nature 494:497–501CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Pulakanti K, Pienello L, Stelloh C et al (2013) Enhancer transcribed RNAs are produced from hypomethylated genomic regions in a Tet-dependent manner. Epigenetics 8:1303–1320CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Schaukowitch K, Joo JY, Liu X et al (2014) Enhancer RNA facilitates NELF release from immediate early genes. Mol Cell 56:29–42CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Maruyama A, Mimura J, Itoh K (2014) Noncoding RNA derived from the region adjacent to the human HO-1 E2 enhancer selectively regulates HO-1 gene induction by modulating Pol II binding. Nucleic Acids Res 42:13599–13614CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Whyte WA, Orlando DA, Hnisz D et al (2013) Master transcription factors and mediator establish super-enhancers at key cell identity genes. Cell 153:307–319CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Rao S, Zhen S, Roumiantsev S et al (2010) Differential roles of Sall4 isoforms in embryonic stem cell pluripotency. Mol Cell Biol 30:5364–5380CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Kim J, Cantor AB, Orkin SH, Wang J (2009) Use of in vivo biotinylation to study protein-protein and protein-DNA interactions in mouse embryonic stem cells. Nat Protoc 4:506–517CrossRefPubMedGoogle Scholar
  20. 20.
    Nelson JD, Denisenko O, Bomsztyk K (2006) Protocol for the fast chromatin immunoprecipitation (ChIP) method. Nat Protoc 1:179–185CrossRefPubMedGoogle Scholar
  21. 21.
    Broad (2010) Broad ChIP protocol for full REMC (6 marks). http://www.roadmapepigenomics.org/protocols/type/experimental/. Accessed 19 July 2015
  22. 22.
    Das PP, Shao Z, Beyaz S et al (2014) Distinct and combinatorial functions of Jmjd2b/Kdm4b and Jmjd2c/Kdm4c in mouse embryonic stem cell identity. Mol Cell 53:32–48CrossRefPubMedGoogle Scholar
  23. 23.
    Barrett T, Wilhite SE, Ledoux P et al (2013) NCBI GEO: archive for functional genomics data sets—update. Nucleic Acids Res 41:D991–D995CrossRefPubMedGoogle Scholar
  24. 24.
    Babraham Bioinformatics (2015) FastQC. http://www.bioinformatics.bbsrc.ac.uk/projects/fastqc. Accessed 19 July 2015
  25. 25.
    Bolger AM, Lohse M, Usadel B (2014) Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics 30:2114–2120CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Hannon Lab Cold Spring Harbor (2015) FASTX-Toolkit. http://hannonlab.cshl.edu/fastx_toolkit/index.html. Accessed 19 July 2015
  27. 27.
    Langmead B, Trapnell C, Pop M, Salzberg SL (2009) Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol 10:R25CrossRefPubMedPubMedCentralGoogle Scholar
  28. 28.
    Li H, Handsaker B, Wysoker A et al (2009) The sequence alignment/map (SAM) format and SAMtools. Bioinformatics 25:2078–2079CrossRefPubMedPubMedCentralGoogle Scholar
  29. 29.
    Li H (2011) Statistical framework for SNP calling, mutation discovery, association mapping and population genetical parameter estimation from sequencing data. Bioinformatics 27:2987–2993CrossRefPubMedPubMedCentralGoogle Scholar
  30. 30.
    Zhang Y, Liu T, Meyer CA et al (2008) Model-based analysis of ChIP-Seq (MACS). Genome Biol 9:R137CrossRefPubMedPubMedCentralGoogle Scholar
  31. 31.
    Ji H, Jiang H, Ma W et al (2008) An integrated software system for analyzing ChIP-chip and ChIP-seq data. Nat Biotechnol 26:1293–1300CrossRefPubMedPubMedCentralGoogle Scholar
  32. 32.
    Quinlan AR, Hall IM (2010) BEDTools: a flexible suite of utilities for comparing genomic features. Bioinformatics 26:841–842CrossRefPubMedPubMedCentralGoogle Scholar
  33. 33.
    Landt S, Marinov GK, Kundaje A et al (2012) ChIP-seq guidelines and practices of the ENCODE and modENCODE consortia. Genome Res 22:1813–1831CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2017

Authors and Affiliations

  1. 1.Department of Cell Biology, Neurobiology, and AnatomyMedical College of WisconsinMilwaukeeUSA
  2. 2.BloodCenter of WisconsinBlood Research InstituteMilwaukeeUSA
  3. 3.Department of Biostatistics and Computational BiologyHarvard TH Chan School of Public Health, Dana-Farber Cancer InstituteBostonUSA
  4. 4.Department of PediatricsMedical College of WisconsinMilwaukeeUSA

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