Systematic dissection of regulatory motifs in 2000 predicted human enhancers using a massively parallel reporter assay (original) (raw)

1. Jason Ernst1,2,5,
2. Alexandre Melnikov2,
3. Peter Rogov2,
4. Li Wang2,
5. Xiaolan Zhang2,
6. Jessica Alston2,3,
7. Tarjei S. Mikkelsen2,4 and
8. Manolis Kellis1,2,6
9. 1Computer Science and Artificial Intelligence Laboratory, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA;
10. 2Broad Institute, Cambridge, Massachusetts 02142, USA;
11. 3Program in Biological and Biomedical Sciences and Department of Genetics, Harvard Medical School, Boston, Massachusetts 02115, USA;
12. 4Harvard Stem Cell Institute and Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, Massachusetts 02138, USA

• ↵5 Present address: Department of Biological Chemistry, University of California Los Angeles, Los Angeles, CA 90095, USA.

Abstract

Genome-wide chromatin annotations have permitted the mapping of putative regulatory elements across multiple human cell types. However, their experimental dissection by directed regulatory motif disruption has remained unfeasible at the genome scale. Here, we use a massively parallel reporter assay (MPRA) to measure the transcriptional levels induced by 145-bp DNA segments centered on evolutionarily conserved regulatory motif instances within enhancer chromatin states. We select five predicted activators (HNF1, HNF4, FOXA, GATA, NFE2L2) and two predicted repressors (GFI1, ZFP161) and measure reporter expression in erythroleukemia (K562) and liver carcinoma (HepG2) cell lines. We test 2104 wild-type sequences and 3314 engineered enhancer variants containing targeted motif disruptions, each using 10 barcode tags and two replicates. The resulting data strongly confirm the enhancer activity and cell-type specificity of enhancer chromatin states, the ability of 145-bp segments to recapitulate both, the necessary role of regulatory motifs in enhancer function, and the complementary roles of activator and repressor motifs. We find statistically robust evidence that (1) disrupting the predicted activator motifs abolishes enhancer function, while silent or motif-improving changes maintain enhancer activity; (2) evolutionary conservation, nucleosome exclusion, binding of other factors, and strength of the motif match are predictive of enhancer activity; (3) scrambling repressor motifs leads to aberrant reporter expression in cell lines where the enhancers are usually inactive. Our results suggest a general strategy for deciphering _cis_-regulatory elements by systematic large-scale manipulation and provide quantitative enhancer activity measurements across thousands of constructs that can be mined to develop predictive models of gene expression.

Footnotes

• ↵6 Corresponding author
  E-mail manoli{at}mit.edu

• [Supplemental material is available for this article.]

• Article published online before print. Article, supplemental material, and publication date are at http://www.genome.org/cgi/doi/10.1101/gr.144899.112.
  Freely available online through the Genome Research Open Access option.

• Received June 26, 2012.

• Accepted March 14, 2013.

• © 2013, Published by Cold Spring Harbor Laboratory Press

This article is distributed exclusively by Cold Spring Harbor Laboratory Press for the first six months after the full-issue publication date (see http://genome.cshlp.org/site/misc/terms.xhtml). After six months, it is available under a Creative Commons License (Attribution-NonCommercial 3.0 Unported License), as described at http://creativecommons.org/licenses/by-nc/3.0/.