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

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.

Multiplex enhancer-reporter assays uncover unsophisticated TP53 enhancer logic

“…We, and others before us, have shown that massively parallel enhancer-reporter assays can relatively quickly lead to such training sets, and usually lead to exciting new insight into the cis-regulatory logic of enhancers (Kwasnieski et al 2012;Melnikov et al 2012;Arnold et al 2013;Kheradpour et al 2013;White et al 2013). In our study, we tested long enhancer sequences, of several hundreds of base pairs.…”

“…Whereas classically enhancer-reporter assays consist of cloning each enhancer one by one, first in vitro, later in vivo (Banerji et al 1981;O'Kane and Gehring 1987;Chiocchetti et al 1997;Dailey 2015), now hundreds to thousands of enhancers can be tested in parallel (Patwardhan et al 2009(Patwardhan et al , 2012Kwasnieski et al 2012;Melnikov et al 2012;Arnold et al 2013;Kheradpour et al 2013;Smith et al 2013;White et al 2013;Vanhille et al 2015). These methods can be broadly categorized in two groups, namely, massively parallel reporter assays (MPRA) utilizing barcodes as a measure of activity of synthesized enhancer fragments (Patwardhan et al 2009(Patwardhan et al , 2012Kwasnieski et al 2012;Melnikov et al 2012;Kheradpour et al 2013;Smith et al 2013;White et al 2013) and self-transcribing active regulatory region sequencing (STARR-seq) (Arnold et al 2013;Vanhille et al 2015).…”

Conserved non-AUG uORFs revealed by a novel regression analysis of ribosome profiling data

“…Future work is needed to determine the functions of more of these uORFs. Testing large numbers of uORFs will require new high-throughput assays, perhaps similar to those used recently to test transcription factor binding sites (Sharon et al 2012;Kheradpour et al 2013). In addition, translation regulation is known to be particularly sensitive to environmental conditions, cell-cycle phases, and developmental transitions.…”

Systematic dissection of genomic features determining transcription factor binding and enhancer function

“…In the past decade, experimental advances have enabled characterization of the binding motifs for hundreds of TFs in vitro (5)(6)(7)(8)(9), mapping of the genome-wide binding sites of TFs in vivo (10)(11)(12)(13)(14), and functional characterization of the enhancer activity of thousands of genomic sequences (15)(16)(17)(18)(19). Comparisons between these experiments, however, have revealed that only a small fraction of the potential TF-binding sites (TFBSs) in eukaryotic genomes are actually occupied by TFs in any given cell type, and that these sites vary substantially across cell types and conditions (3,(19)(20)(21).…”