text: Traditionally, enhancers were identified by enhancer trap techniques using a reporter gene or by comparative sequence analysis and computational genomics. In genetically tractable models such as the fruit fly Drosophila melanogaster, for example, a reporter construct such as the lacZ gene can be randomly integrated into the genome using a P element transposon. If the reporter gene integrates near an enhancer, its expression will reflect the expression pattern driven by that enhancer. Thus, staining the flies for LacZ expression or activity and cloning the sequence surrounding the integration site allows the identification of the enhancer sequence.
  The development of genomic and epigenomic technologies, however, has dramatically changed the outlook for cis-regulatory modules (CRM) discovery. Next-generation sequencing (NGS) methods now enable high-throughput functional CRM discovery assays, and the vastly increasing amounts of available data, including large-scale libraries of transcription factor-binding site (TFBS) motifs, collections of annotated, validated CRMs, and extensive epigenetic data across many cell types, are making accurate computational CRM discovery an attainable goal. An example of NGS-based approach called DNase-seq have enabled identification of nucleosome-depleted, or open chromatin regions, which can contain CRM. More recently techniques such as ATAC-seq have been developed which require less starting material. Nucelosome depleted regions can be identified in vivo through expression of Dam methylase, allowing for greater control of cell-type specific enhancer identification.
  Computational methods include comparative genomics, clustering of known or predicted TF-binding sites, and supervised machine-learning approaches trained on known CRMs. 
  All of these methods have proven effective for CRM discovery, but each has its own considerations and limitations, and each is subject to a greater or lesser number of false-positive identifications.
  In the comparative genomics approach, sequence conservation of non-coding regions can be indicative of enhancers. Sequences from multiple species are aligned, and conserved regions are identified computationally. Identified sequences can then be attached to a reporter gene such as green fluorescent protein or lacZ to determine the in vivo pattern of gene expression produced by the enhancer when injected into an embryo. mRNA expression of the reporter can be visualized by in situ hybridization, which provides a more direct measure of enhancer activity, since it is not subjected to the complexities of translation and protein folding. Although much evidence has pointed to sequence conservation for critical developmental enhancers, other work has shown that the function of enhancers can be conserved with little or no primary sequence conservation. For example, the RET enhancers in humans have very little sequence conservation to those in zebrafish, yet both species' sequences produce nearly identical patterns of reporter gene expression in zebrafish. Similarly, in highly diverged insects (separated by around 350 million years), similar gene expression patterns of several key genes was found to be regulated through similarly constituted CRMs although these CRMs do not show any appreciable sequence conservation detectable by standard sequence alignment methods such as BLAST.