Massively parallel in vivo enhancer assay reveals that highly local features determine thecis-regulatory function of ChIP-seq peaks

Abstract

Transcription factors (TFs) recognize short sequence motifs that are present in millions of copies in large eukaryotic genomes. TFsmust distinguish their target binding sites from a vast genomic excess of spurious motif occurrences; however, it is unclear whether functional sites are distinguished from nonfunctional motifs by local primary sequence features or by the larger genomic context in which motifs reside. We used a massively parallel enhancer assay in living mouse retinas to compare 1,300 sequences bound in the genome by the photoreceptor transcription factor Cone-rod homeobox (Crx), to 3,000 control sequences. We found that very short sequences bound in the genome by Crx activated transcription at high levels, whereas unbound genomic regions with equal numbers of Crx motifs did not activate above background levels, even when liberated from their larger genomic context. High local GC content strongly distinguishes bound motifs from unbound motifs across the entire genome. Our results show that the cis-regulatory potential of TF-bound DNA is determined largely by highly local sequence features and not by genomic context. (1), yet the sequence features that distinguish functional cis-regulatory sites from the millions of spurious motif occurrences in large eukaryotic genomes are poorly understood (2-6). Several models have been proposed to explain how TFs distinguish between functional cis-regulatory elements (CREs) and nonfunctional motif occurrences (3, 6, 7). In one model, large-scale chromatin context directs TF binding to target sites while limiting TF access to spurious motif occurrences (7-9). This model is supported by recent analyses of genomic DNaseI hypersensitivity, which show that only 1% of the genome typically resides in open chromatin in any given cell type (1, 10), suggesting that most spurious motif occurrences are inaccessible. A second model states that target sites are recognized through cooperative TF binding to highly specific combinations of sequence motifs, which are unlikely to occur by chance in nonregulatory regions of the genome (6, 11). This model is supported by evidence that the binding specificity of many TFs is affected by cooperative interactions with cofactors (12). A third model states that most TF binding is promiscuous, low occupancy, and nonfunctional, whereas functional CREs are characterized by high TF occupancy, achieved through either a permissive chromatin context or high affinity for TFs (3,6). This model is motivated by recent genomewide binding studies demonstrating that binding locations of functionally diverse TFs overlap substantially (13, 14), a result that suggests binding is unlikely to be primarily determined by rare, specific combinations of cooperative interactions. Regardless of the mechanisms by which TFs select functional CREs, the distinction between functional and nonfunctional motif occurrences must ultimately depend on information encoded either locally or within the larger sequence context surrounding functional CREs.To distinguish between t...

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.…”

“…A solution to this is to preselect the input, as has been done in the preset study, as well as recently in mice, using a STARR-seq-like approach, called CapStarr-seq (Vanhille et al 2015). The possibilities that these methodologies provide with regard to enhancer validation, both in vitro and in vivo (White et al 2013;Shen et al 2015), underscore the value and the need for such approaches in the field of regulatory genomics today.…”

“…Such enhancers are generally classified under the enhanceosome or billboard models, depending on whether or not the order and spacing of motifs is important (Arnosti and Kulkarni 2005). Other local features in the DNA sequence of an enhancer have also been shown to contribute to the discrimination of bound versus unbound sites, such as GC content (White et al 2013), preferential sequences for nucleosome positioning (Lidor Nili et al 2010), DNA shape features (Chiu et al 2015), and dinucleotide repeat motifs (Yáñez-Cuna et al 2014). However, to gain further insight into the cis-regulatory enhancer code and identify those sites that are truly bound and functional, validation assays are needed.…”

“…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).…”

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).…”