High-throughput and quantitative assessment of enhancer activity in mammals by CapStarr-seq

Abstract

Cell-type specific regulation of gene expression requires the activation of promoters by distal genomic elements defined as enhancers. The identification and the characterization of enhancers are challenging in mammals due to their genome complexity. Here we develop CapStarr-Seq, a novel high-throughput strategy to quantitatively assess enhancer activity in mammals. This approach couples capture of regions of interest to previously developed Starrseq technique. Extensive assessment of CapStarr-seq demonstrates accurate quantification of enhancer activity. Furthermore, we find that enhancer strength is associated with binding complexity of tissue-specific transcription factors and super-enhancers, while additive enhancer activity isolates key genes involved in cell identity and function. The CapStarr-Seq thus provides a fast and cost-effective approach to assess the activity of potential enhancers for a given cell type and will be helpful in decrypting transcription regulation mechanisms.

Enhancer RNA profiling predicts transcription factor activity

“…1E; Supplemental Table S2). In the same vein, TF binding sites that overlap a region with strong enhancer activity-as measured by a CapStarr-seq enhancer assay (Vanhille et al 2015) -are five times more likely to associate with eRNAs than regions Table S1) were grouped according to eRNA association. A box-and-whiskers displays the median/variability in proportion of histone mark association between the groups across all TFs (Supplemental Table S1).…”

“…We next examined regions evaluated by a functional assay, namely, CapStarr-seq (Vanhille et al 2015), for their co-occurance with eRNA origins. In CapStarr-seq, they utilized mouse 3T3 cells, selected TF-bound regions (by ChIP), and determined whether the bound regions functioned as an enhancer using a GFP expression assay.…”

Multiplex enhancer-reporter assays uncover unsophisticated TP53 enhancer logic

“…However, considering that the human genome is 25 times larger, it presents a considerable challenge toward genome-wide assays. 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.…”

“…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).…”

Complex cis-regulatory landscape of the insulin receptor gene underlies the broad expression of a central signaling regulator

“…The transcriptional significance of other regions, including extensive introns, remains largely unexplored. Genomic surveys of the mammalian gene reveal functionally uncharacterized chromatin marks and structures consistent with enhancers, and putative intronic enhancers for the gene were identified in mouse T cells (Pasquali et al, 2014;Kundaje et al, 2015;Vanhille et al, 2015).…”