TABLE 2.

Genomic approaches for enhancer mapping.

Method category	Method strategy	Description	References
ChIP-seq (detect DNA-binding factor occupancy and histone modification profiles)	Co-factors (EP300, Mediator)	Assays enhancers mediated by specific co-factors; TFs need not be specified in advance.	Blow et al., 2010; He et al., 2011; May et al., 2012
	Co-occupancy of multiple TFs	Reveals specific trans factors but requires specific antibodies for each factor and often each species. Typically requires large numbers of nuclei.	He et al., 2011, 2014; Luna-Zurita et al., 2016; Akerberg et al., 2019
	Active histone marks (H3K27ac, H3K4me1)	Robust antibodies that work across metazoans; reveals enhancer states;requires less input than for TFs.	Wamstad et al., 2012; Nord et al., 2013; He et al., 2014
Enzyme tethering ChIP alternative (use factor-mediated in-situ genome fragmentation to profile epigenome)	CUT&RUN (pA-MNase fusion protein)	Unfixed in-situ procedure, requires lower cell numbers (∼100 for histone modification) and less sequencing reads	Skene and Henikoff, 2017; Meers et al., 2019
	CUT&Tag (pA-Tn5)	Similar to CUT&RUN with a simpler barcoding step; streamlined workflow in a single tube; works on low cell numbers or even single cells	Kaya-Okur et al., 2019; Henikoff et al., 2020
	CUTAC (pA-Tn5, low salt)	Similar to CUT&Tag with a small modification that detects accessible chromatin in parallel with adjacent histone modifications	Henikoff et al., 2020
Accessible chromatin profiling (detect nucleosome-depleted regions that are enriched for enhancers)	DNase-seq	High quality TF footprintscan be generated.	Thurman et al., 2012; Vierstra et al., 2014, 2020
	ATAC-seq	Simple and robust method that requires low cell numbers, widely applied; can be used on frozen sections; produces a comprehensive list of where CREs may be located.	Buenrostro et al., 2013; Corces et al., 2017
Nascent RNA sequencing run-on assays (depict the real-time activity of RNA polymerases and detect eRNAs)	GRO-seq	Detect actively transcribed eRNAs which is a hallmark of active enhancers	Core et al., 2008
	PRO-seq	Refined version of GRO-seq that uses biotinylated nucleotide to reach nucleotide-resolution, low background, and large dynamic ranges	Kwak et al., 2013; Core et al., 2014
	ChRO-seq	Similar to PRO-seq but use chromatin as starting materials; can be applied to solid tissues and samples with degraded RNAs	Chu et al., 2018
Chromosome conformation capture (use proximity ligation and detect enhancer-promoter interaction)	Hi-C	Maps genome-wide chromatin contacts (‘all-to-all’); requires substantial sequencing to reveal local enhancer-promoter interactions	Lieberman-Aiden et al., 2009
	Promoter capture Hi-C	Maps promoter-centric chromatin interactions; requires less reads for detecting promoter-enhancer interactions	Mifsud et al., 2015; Schoenfelder et al., 2015
	ChIA-PET	Detect chromatin interactions mediated by a specific DNA-binding factor; can enrich rare factor-specific chromatin interactions	Fullwood et al., 2009; Grubert et al., 2020
	HiChIP& PLAC-seq (Use in-situ Hi-C followed by ChIP)	Detects factor-centric chromatin interaction similar to ChIA-PET but require 10-fold to 100-fold fewer cells, also more robust and less time-consuming	Fang et al., 2016; Mumbach et al., 2016
	4C	Identifies all genomic regions that interacts a reference locus (‘one-to-all’); can be used for studying specific enhancers	Simonis et al., 2006