Enhancer-promoter specificity mediated by DPE or TATA core promoter motifs

Abstract

To investigate the basis for enhancer-promoter specificity, we compared the ability of enhancers to activate transcription in vivo from core promoters containing either downstream promoter element (DPE) or TATA box motifs. To eliminate position effects, we generated and analyzed pairs of sister Drosophila lines that contain a DPE- or TATA-dependent reporter gene at precisely the same genomic position relative to each enhancer. These studies revealed transcriptional enhancers that are specific for promoters that contain either DPE or TATA box elements. Thus, the core promoter not only mediates the initiation of transcription, but also functions as a regulatory element.

Figure 1

A model for enhancer-core promoter specificity. This diagram depicts transcriptional enhancers that function specifically with DPE- or TATA-dependent core promoters.

Figure 2

The use of the waffle vector to generate allelic transgenes that contain DPE- or TATA-dependent reporter genes. (A) Selective excision of the TATA–GFP or DPE–GFP reporter genes from the P-TATA/DPE transposon. The P-TATA/DPE transposon (top) contains both TATA–GFP and DPE–GFP reporter genes. FLP recombinase recognizes the FRT sites and excises the mini-white and TATA–GFP genes. Cre recombinase recognizes the loxP sites and excises the DPE–GFP and mini-white genes. The two resulting sister lines thus contain either DPE–GFP or TATA–GFP at precisely the same genomic position. (B) Sequences of analogous DPE- or TATA-containing core promoters. The segment from the Inr through the DPE is from the Drosophila Antennapedia P2 core promoter, whereas the upstream TATA box is from the adenovirus major late promoter. The two promoters are identical except for 7 bp at the TATA region (−31 to −25) and 7 bp at the DPE region (+28 to +34). The lower case letters denote mutations in the TATA or DPE motifs. (C) In vitro transcription analysis of core promoters containing a DPE, a TATA box, or neither a DPE nor a TATA box.

Figure 3

Identification of DPE- and TATA-specific enhancers. (A,B) The jb23 and jb5 fly lines have DPE-specific trapped enhancers. (C) The jb9 line has a TATA-specific trapped enhancer. (D) The jb2 line has a trapped enhancer that has no apparent specificity for DPE or TATA motifs.

Figure 4

The DPE-specific enhancer in the jb23 fly line initiates transcription from the correct +1 start site in vivo. Poly A+ mRNA was purified from embryos of the DPE and TATA sister lines and then subjected to primer extension analysis. There is no detectable specific transcription from the TATA–GFP reporter gene. As a control for the integrity of the poly A+ RNA, transcripts derived from the Drosophila actin 5C gene were analyzed in parallel, as shown at the bottom.