Transcriptional enhancers in protein-coding exons of vertebrate developmental genes

Abstract

Many conserved noncoding sequences function as transcriptional enhancers that regulate gene expression. Here, we report that protein-coding DNA also frequently contains enhancers functioning at the transcriptional level. We tested the enhancer activity of 31 protein-coding exons, which we chose based on strong sequence conservation between zebrafish and human, and occurrence in developmental genes, using a Tol2 transposable GFP reporter assay in zebrafish. For each exon we measured GFP expression in hundreds of embryos in 10 anatomies via a novel system that implements the voice-recognition capabilities of a cellular phone. We find that 24/31 (77%) exons drive GFP expression compared to a minimal promoter control, and 14/24 are anatomy-specific (expression in four anatomies or less). GFP expression driven by these coding enhancers frequently overlaps the anatomies where the host gene is expressed (60%), suggesting self-regulation. Highly conserved coding sequences and highly conserved noncoding sequences do not significantly differ in enhancer activity (coding: 24/31 vs. noncoding: 105/147) or tissue-specificity (coding: 14/24 vs. noncoding: 50/105). Furthermore, coding and noncoding enhancers display similar levels of the enhancer-related histone modification H3K4me1 (coding: 9/24 vs noncoding: 34/81). Meanwhile, coding enhancers are over three times as likely to contain an H3K4me1 mark as other exons of the host gene. Our work suggests that developmental transcriptional enhancers do not discriminate between coding and noncoding DNA and reveals widespread dual functions in protein-coding DNA.

Figure 1. Overview of EGFP expression scoring process.

(A) Zebrafish with eGFP expression are scored using a (B) limited anatomy corresponding to (C) numerical values. (D) These are interpreted using the iPhone app Dragon Dictation. (E) A PERL script transforms text into numerical strings representing embryo expression in each anatomy. These data are analyzed to determine anatomical regions with significant expression for each CCE via a proportions test and a Wilcoxon rank-sum test (see Methods and Supplementary Data File S2).

Figure 2. Specific and Non-Specific CCE Activity.

(A) Examples of Specific CCE Activity. CCEs from the genes gria3b, rab11fip4a, prim1, and abca1a each drove robust expression in a finely localized anatomical region. Overall, 14 CCEs produced this type of specific expression (defined as expression in 4 or fewer anatomical regions). (B) This behavior contrasts with CCEs that drove robust but non-specific expression, such as CCE-ephb3a. 6 of the active CCEs drove nonspecific expression.

Figure 3. Comparison of CCE-lmo1 Stable and Transient Transgenic Expression.

(A) Stable transgenic F1 embryos from two independently generated lines displaying strong forebrain and hindbrain expression. Supplementary Figure S3 shows this behavior in a larger group of stable transgenic embryos. (B) Similarly, three transient transgenic embryos injected with CCE-lmo1 display analogous forebrain and hindbrain expression.

Figure 4. CCE and CNE Activity and Tissue Specificity.

(A) Comparison of the fraction of enhancers active in conserved coding elements (CCEs) and conserved non-coding elements (CNEs). CCEs and CNEs exhibit similar enhancer activity levels, with no significant difference in activity. (B) Comparison of the fraction of enhancers exhibiting tissue specificity in CCEs and CNEs. While CNEs are marginally less tissue-specific, the difference is not statistically significant.

Figure 5. Representative images of CCE expression and host gene expression (mRNA in situ hybridization data from ZFIN) for 4 CCEs, showing overlap between CCE activity and host gene expression.

Figure 6. CCE-fez1 drives expression in multiple anatomies, with significant concurrent activity in forebrain, mid/hindbrain and eye. 4 representative embryos are shown.

Figure 7. CCE-sfrs3b, an alternatively spliced exon, is shown here to drive enhancer expression in the eye and brain, despite poison cassette activity of the exon.