1. Chromosomes and Gene Expression
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Chromatin-associated RNA sequencing (ChAR-seq) maps genome-wide RNA-to-DNA contacts

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Cite this article as: eLife 2018;7:e27024 doi: 10.7554/eLife.27024

Abstract

RNA is a critical component of chromatin in eukaryotes, both as a product of transcription, and as an essential constituent of ribonucleoprotein complexes that regulate both local and global chromatin states. Here we present a proximity ligation and sequencing method called Chromatin-Associated RNA sequencing (ChAR-seq) that maps all RNA-to-DNA contacts across the genome. Using Drosophila cells we show that ChAR-seq provides unbiased, de novo identification of targets of chromatin-bound RNAs including nascent transcripts, chromosome-specific dosage compensation ncRNAs, and genome-wide trans-associated RNAs involved in co-transcriptional RNA processing.

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Author details

  1. Jason C Bell

    Department of Biochemistry, Stanford University, Stanford, United States
    For correspondence
    jcbell@stanford.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-5480-7975
  2. David Jukam

    Department of Biology, Stanford University, Stanford, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-4167-2754
  3. Nicole A Teran

    Department of Biochemistry, Stanford University, Stanford, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-9625-5010
  4. Viviana I Risca

    Department of Genetics, Stanford University, Stanford, United States
    Competing interests
    The authors declare that no competing interests exist.
  5. Owen K Smith

    Department of Biochemistry, Stanford University, Stanford, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-0880-2801
  6. Whitney L Johnson

    Department of Biochemistry, Stanford University, Stanford, United States
    Competing interests
    The authors declare that no competing interests exist.
  7. Jan M Skotheim

    Department of Biology, Stanford University, Stanford, United States
    Competing interests
    The authors declare that no competing interests exist.
  8. William James Greenleaf

    Department of Genetics, Stanford University, Stanford, United States
    Competing interests
    The authors declare that no competing interests exist.
  9. Aaron F Straight

    Department of Biochemistry, Stanford University, Stanford, United States
    For correspondence
    astraigh@stanford.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-5885-7881

Funding

National Institutes of Health (Stanford Center for Systems Biology (NIH P50 GM107615) Seed Grant)

  • Jason C Bell
  • David Jukam
  • Viviana I Risca
  • Whitney L Johnson

Howard Hughes Medical Institute (HHMI-Simons Faculty Scholar Award)

  • Jan M Skotheim

National Institutes of Health (P50HG00773501)

  • William James Greenleaf

National Institutes of Health (R01GM106005)

  • Aaron F Straight

Stanford University School of Medicine (Dean's Fellowship)

  • Jason C Bell

National Institutes of Health (R01HG009909)

  • William James Greenleaf
  • Aaron F Straight

National Institutes of Health (R21HG007726)

  • William James Greenleaf

National Institutes of Health (NIH Ruth Kirchstein National Research Service Award (F32GM116338))

  • Jason C Bell

National Institutes of Health (NIH Ruth Kirchstein National Research Service Award (F32GM108295 ))

  • David Jukam

Stanford University (Walter V. and Idun Berry Fellowship)

  • Viviana I Risca

National Institutes of Health (Stanford Genetics Training Program (5T32HG000044-19))

  • Nicole A Teran

National Institutes of Health (Molecular Pharmacology Training Grant (NIH T32-GM113854-02))

  • Owen K Smith

National Institutes of Health (NIH T32 Training Fellowship (GM007276))

  • Whitney L Johnson

National Science Foundation (DGE-114747)

  • Whitney L Johnson

National Institutes of Health (RO1 HD085135)

  • Jan M Skotheim
  • Aaron F Straight

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Reviewing Editor

  1. Job Dekker, University of Massachusetts Medical School, United States

Publication history

  1. Received: March 21, 2017
  2. Accepted: April 11, 2018
  3. Accepted Manuscript published: April 12, 2018 (version 1)
  4. Version of Record published: May 21, 2018 (version 2)

Copyright

© 2018, Bell et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

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Further reading

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    During mitosis chromosomes reorganise into highly compact, rod-shaped forms, thought to consist of consecutive chromatin loops around a central protein scaffold. Condensin complexes are involved in chromatin compaction, but the contribution of other chromatin proteins, DNA sequence and histone modifications is less understood. A large region of fission yeast DNA inserted into a mouse chromosome was previously observed to adopt a mitotic organisation distinct from that of surrounding mouse DNA. Here we show that a similar distinct structure is common to a large subset of insertion events in both mouse and human cells and is coincident with the presence of high levels of heterochromatic H3 lysine 9 trimethylation (H3K9me3). Hi-C and microscopy indicate that the heterochromatinised fission yeast DNA is organised into smaller chromatin loops than flanking euchromatic mouse chromatin. We conclude that heterochromatin alters chromatin loop size, thus contributing to the distinct appearance of heterochromatin on mitotic chromosomes.

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    Shadow enhancers, groups of seemingly redundant enhancers, are found in a wide range of organisms and are critical for robust developmental patterning. However, their mechanism of action is unknown. We hypothesized that shadow enhancers drive consistent expression levels by buffering upstream noise through a separation of transcription factor (TF) inputs at the individual enhancers. By measuring the transcriptional dynamics of several Kruppel shadow enhancer configurations in live Drosophila embryos, we showed that individual member enhancers act largely independently. We found that TF fluctuations are an appreciable source of noise that the shadow enhancer pair can better buffer than duplicated enhancers. The shadow enhancer pair is also uniquely able to maintain low levels of expression noise across a wide range of temperatures. A stochastic model demonstrated the separation of TF inputs is sufficient to explain these findings. Our results suggest the widespread use of shadow enhancers is partially due to their noise suppressing ability.