Systematic dissection and optimization of inducible enhancers in human cells using a massively parallel reporter assay

Abstract

Learning to read and write the transcriptional regulatory code is of central importance to progress in genetic analysis and engineering. Here, we describe a massively parallel reporter assay (MPRA) that enables systematic dissection of transcriptional regulatory elements by integrating microarray-based DNA synthesis and high-throughput tag sequencing. We apply MPRA to compare more than 27,000 distinct variants of two inducible enhancers in human cells: a synthetic cAMP-regulated enhancer and the virus-inducible interferon beta enhancer. We first show that the resulting data define accurate maps of functional transcription factor binding sites in both enhancers at single-nucleotide resolution. We then use the data to train quantitative sequence-activity models (QSAMs) of the two enhancers. We show that QSAMs from two cellular states can be combined to identify novel enhancer variants that optimize potentially conflicting objectives, such as maximizing induced activity while minimizing basal activity.

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

“…1C). We cloned these 3,000 sequences ("candidate enhancers") into plasmids containing a minimal promoter and an ORF containing a unique barcode that identifies its specific upstream enhancer (48). We transfected this plasmid pool into mouse 3T3-L1 adipocytes 7 d postdifferentiation; grew the cells for 16 h; and measured PPARγ binding by performing ChIP-seq and calculating the relative enrichment of reads corresponding to each sequence.…”

“…Oligonucleotide libraries containing the 145-or 150-bp candidate enhancers were synthesized (Agilent Technologies) and unique barcodes were added by PCR. The oligonucleotide libraries were cloned into a plasmid backbone with a minP-luc2 insert, as previously described (48,95).…”

“…MPRA involves testing huge collections of short regulatory sequences (≤150 bp) in parallel by coupling each to a transcription unit containing a matched DNA barcode (48) (Fig. 1 A and B).…”

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).…”