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A massively parallel reporter assay dissects the influence of chromatin structure on cis-regulatory activity

Nature Biotechnology | Download Citation

Abstract

A gene's position in the genome can profoundly affect its expression because regional differences in chromatin modulate the activity of locally acting cis-regulatory sequences (CRSs). Here we study how CRSs and regional chromatin act in concert on a genome-wide scale. We present a massively parallel reporter gene assay that measures the activities of hundreds of different CRSs, each integrated at many specific genomic locations. Although genome location strongly affected CRS activity, the relative strengths of CRSs were maintained at all chromosomal locations. The intrinsic activities of CRSs also correlated with their activities in plasmid-based assays. We explain our data with a quantitative model in which expression levels are set by independent contributions from local CRSs and the regional chromatin environment, rather than by more complex sequence- or protein-specific interactions between these two factors. The methods we present will help investigators determine when regulatory information is integrated in a modular fashion and when regulatory sequences interact in more complex ways.

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Acknowledgements

We thank S. Elgin and members of the Cohen laboratory for their critical feedback on the manuscript. We thank J. Hoisington-Lopez for assistance with high-throughput sequencing. We also thank the Alvin J. Siteman Cancer Center at Washington University School of Medicine and Barnes-Jewish Hospital in St. Louis, Missouri, for the use of the Siteman Flow Cytometry Core, which provided single-cell sorting services. The Siteman Cancer Center is supported in part by NCI Cancer Center Support Grant P30-CA91842. This work was also supported by the Hope Center Viral Vectors Core at Washington University School of Medicine and by grants to B.A.C. from the National Institutes of Health, R01-GM092910 and R01-HG008687.

Author information

Author notes

    • Brett B Maricque

    Present address: Department of Biological Sciences, Howard Hughes Medical Institute, Columbia University, New York, New York, USA.

    • Brett B Maricque
    •  & Hemangi G Chaudhari

    These authors contributed equally to this work.

Affiliations

  1. The Edison Family Center for Genome Sciences and Systems Biology, Washington University School of Medicine, Saint Louis, Missouri, USA.

    • Brett B Maricque
    • , Hemangi G Chaudhari
    •  & Barak A Cohen
  2. Department of Genetics, Washington University School of Medicine, Saint Louis, Missouri, USA.

    • Brett B Maricque
    • , Hemangi G Chaudhari
    •  & Barak A Cohen

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Contributions

B.B.M., H.G.C. and B.A.C. conceived the landing pad system. B.B.M. and H.G.C. designed and conducted all the experiments. B.B.M., H.G.C. and B.A.C. wrote the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Barak A Cohen.

Integrated supplementary information

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–7

  2. 2.

    Life Sciences Reporting Summary

Excel files

  1. 1.

    Supplementary Table 1

    Landing pad locations and annotations

  2. 2.

    Supplementary Table 2

    Distance to nearest TAD boundaries

  3. 3.

    Supplementary Table 3

    CRS sequence and expression data for Library 1

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    Supplementary Table 4

    Composition of Library 2

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    Supplementary Table 5

    Expression data for Library 2

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    Supplementary Table 6

    Primers used in this study

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DOI

https://doi.org/10.1038/nbt.4285