Abstract
Enhancers are key transcriptional drivers of gene expression. The identification of enhancers in the genome is central for understanding gene-expression programs. Although transposon-mediated enhancer trapping (ET) is a powerful approach to the identification of enhancers in zebrafish, its efficiency varies considerably. To improve the ET efficiency, we constructed Tol2-mediated ET vectors with a reporter gene (mCherry) expression box driven by four minimal promoters (Gata, Myc, Krt4 and Oct4), respectively. The ET efficiency and expression background were compared among the four promoters by zebrafish embryo injection at the one-cell stage. The results showed that the Gata minimal promoter yielded the lowest basic expression and the second-highest trapping efficiency (44.6% at 12 hpf (hour post-fertilization) and 23.1% at 72 hpf, n = 305 and n = 307). The Krt4 promoter had the highest trapping efficiency (64% at 12 hpf and 67.1% at 72 hpf, n = 302 and n = 301) and the strongest basic expression. To detect enhancer activity, chicken 5′HS4 double insulators were cloned into the two ET vectors with the Gata or Krt4 minimal promoter, flanking the mCherry expression box. The resulting detection vectors were injected into zebrafish embryos. mCherry expression driven by the Gata promoter (about 5%, n = 301) was decreased significantly compared with that observed for embryos injected with the ET vectors (23% at 72 hpf, n = 308). These results suggest that the insulators block the genome-position effects and that this vector is fit for enhancer-activity evaluation. To assess the compatibility between the enhancers and the minimal promoters, four enhancers (CNS1, Z48, Hand2 and Hs769) were cloned upstream of the Gata or Beta-globin minimal promoter in the enhancer-activity-detection vectors. The resulting recombinant vectors were assayed by zebrafish embryo injection. We found that Z48 and CNS1 responded to the Gata minimal promoter, and that Hand2 only responded to the Beta-globin minimal promoter. In contrast, Hs769 did not respond to either the Gata or Beta-globin minimal promoters. These results suggest the existence of compatibility between enhancers and minimal promoters. This study represents a systematic approach to the discovery of optional ET and enhancer-detection vectors. We are eager to provide a superior tool for understanding functional genomics.
Highlights
Enhancers are among the most important cis-regulatory elements that play a major role in cell-type-specific gene expression (Guenther et al, 2007; Heintzman et al, 2009), which widely reflects developmental patterning (Sagai et al, 2005) or human genetic disease (Grosveld et al, 1987)
The basic expression level was the highest in the Krt4 group compared with the remaining three groups, which suggests that the Krt4 minimal promoter itself drives mCherry, resulting in a high background expression that disturbs enhancer trapping (ET) (Trinh & Fraser, 2013)
The rate of mCherry-positive embryos at 12 or 72 hpf was second highest in the Gata group, while the basic expression noise was negligible in this group, which suggests that the Gata minimal promoter can be used in ET
Summary
Enhancers are among the most important cis-regulatory elements that play a major role in cell-type-specific gene expression (Guenther et al, 2007; Heintzman et al, 2009), which widely reflects developmental patterning (Sagai et al, 2005) or human genetic disease (Grosveld et al, 1987). Transgenic mice with lentiviral vectors carrying single-copy enhancer-detector probes encoding either the marker gene lacZ or Cre recombinase were generated and used as an enhancer-detection strategy in mammals (Kelsch, Stolfi & Lois, 2012). Among these methods, transposon-mediated ET represents the most effective insertion in vertebrate systems and has been developed as a gene-delivery tool for gene therapy and insertional mutagenesis (Kebriaei et al, 2017).
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