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.
Highlights
The functions in a genome are often conceptually divided into protein functions for coding DNA and regulatory functions for noncoding DNA
Conserved Coding Elements Act as Enhancers Conserved Coding Elements (CCEs) were identified using minimal criteria of .60% DNA sequence conservation between zebrafish and human, 100–1000 bp length, and occurrence within a set of developmental genes orthologous between zebrafish and human. These criteria were chosen to be similar to those used for identifying Conserved Noncoding Elements (CNEs) in a previous study of CNE enhancer activity [3] to allow for comparison of CCEs and CNEs
Activity and Tissue-Specificity In a previous study we reported that 76/101 of CNEs, chosen by criteria similar to those used for the CCEs, exhibited enhancer activity as measured using methods similar to those applied to CCEs [3]
Summary
The functions in a genome are often conceptually divided into protein functions for coding DNA and regulatory functions for noncoding DNA. This division is based on the intuition that constraints associated with encoding a protein would prevent the evolution of noncoding functions in a coding region. One important class of regulatory functional elements in noncoding DNA is enhancers These are DNA sequences classically found distal to gene promoters and associated with tissue- or temporally-specific transcriptional regulation of gene expression, especially for developmental genes [1,2,3,4,5,6,7,8].
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