Background

Genes make proteins through two steps: transcription and translation. However, specific expression of genes in particular cells is largely regulated by transcription, which is, in general, controlled by millions of promoters and enhancers. Both promoters and enhancers contain short DNA sequences that serve as cis-regulatory elements (cis-Res), which regulate gene transcription by recruiting regulatory proteins (Mikhaylichenko et al., 2018; Schoenfelder and Fraser, 2019). In addition, gene transcription can also be regulated on the epigenetic level, by opening the chromatin structure to alter DNA accessibility, thereby making the underlying cis-REs accessible to regulatory proteins (Crawford et al., 2006; Angeloni and Bogdanovic, 2019; Zhang et al., 2021). Therefore, identification of cis-REs is the key for understanding of biology.

To date, a number of high-throughput (HTP) approaches have been developed to identify cis-REs. Some identify cis-REs directly, by determining the functional impact of each DNA fragment using HTP reporter assays, such as massive parallel reporter assay (MPRA) (Ulirsch et al., 2016; Tewhey et al., 2016), multiplexed editing regulatory assay (MERA) (Rajagopal et al., 2016), clustered regularly interspaced short palindromic repeats interference, flow cytometry and RNA fluorescence in situ hybridization (CRISPRi-FlowFISH) (Fulco et al., 2019), and cis-regulatory element scan by tiling-deletion and sequencing (CREST-seq) (Diao et al., 2017). Some use antibodies specifically against transcriptional factors or epigenetic markers, to identify cis-REs based on protein-DNA interactions, such as chromatin immunoprecipitation DNA-sequencing (ChIP-seq) (Park et al., 2009), and chromatin immunocleavage sequencing (ChIC-seq) (Schmid et al., 2004). Others use the open chromatin regions that are indicative of active regulatory sites, to identify cis-Res, such as DNase-seq (Song and Crawford, 2010), and the assay for transposase-accessible chromatin sequencing (ATAC-seq) (Buenrostro et al., 2015). While all these approaches have their advantages and specific applications in identifying cis-REs, the technical complexity of these methods limits their routine application. In addition, some of these approaches do not have the high resolution to define each single cis-RE, whereas others do not have enough sensitivity and fidelity to reveal cis-REs. Recently, we developed a simple and time-effective technique that not only complements the pre-existing approaches, but can also be used to prioritize candidate cis-REs (Wu et al., 2022). We refer to this technique as regulatory element-sequencing (Reel-seq). Reel-seq is an in vitro HTP screen technique, designed to identify cis-REs based on protein-DNA interaction that is detected by an electrophoresis mobility shift assay (EMSA). For screening, a Reel-seq construct will be engineered by placing a 35-bp DNA fragment between two primers for PCR amplification, as well as for next-generation sequencing (see Graphic abstract A). A Reel-seq library (e.g., Library 1) will be generated by hundreds of thousands of these constructs synthesized by massive parallel oligonucleotide synthesis, so that an entire DNA region will be reconstructed. To cover the break points between all 35-bp fragments, another Reel-seq library (e.g., Library 2) will be generated in the same fashion, except that each 35-bp fragment in this library will bridge the break point of the two contiguous 35-bp fragments in Library 1. For screening, each Reel-seq library will be first mixed with and without nuclear extract (NE, source of regulatory proteins) isolated from relevant types of cells or tissues, and then analyzed on a TBE native gel. After electrophoresis, unshifted bands from both the buffer-treated control and the NE-treated samples will be isolated, and amplified by PCR. Amplified DNA will then be used for another round of gel shifting. In total, ten rounds of EMSA will be performed so that, if a DNA fragment in the library is a cis-RE, it will be shifted in each round of the gel shift assay. After ten screening rounds, a cis-RE can be identified by its decreased percentage in the unshifted DNA library (see Graphic abstract B). Since Reel-seq is an EMSA-based assay, it is highly specific and sensitive in identifying cis-REs. Moreover, since Reel-seq identifies cis-REs at a high resolution, within a defined 35-bp fragment, each identified cis-RE can be used as a “bait” to pull down the regulatory proteins, by using flanking restriction enhanced DNA pulldown-mass spectrometry (FREP-MS) (Li et al., 2018; Wu et al., 2022), another technique recently developed in our lab. Here, we present the detailed protocol of Reel-seq, for the convenient application of this method to identify cis-REs.