A systematic approach to identify functional motifs within vertebrate developmental enhancers

Abstract

Uncovering the cis-regulatory logic of developmental enhancers is critical to understanding the role of non-coding DNA in development. However, it is cumbersome to identify functional motifs within enhancers, and thus few vertebrate enhancers have their core functional motifs revealed. Here we report a combined experimental and computational approach for discovering regulatory motifs in developmental enhancers. Making use of the zebrafish gene expression database, we computationally identified conserved non-coding elements (CNEs) likely to have a desired tissue-specificity based on the expression of nearby genes. Through a high throughput and robust enhancer assay, we tested the activity of ~100 such CNEs and efficiently uncovered developmental enhancers with desired spatial and temporal expression patterns in the zebrafish brain. Application of de novo motif prediction algorithms on a group of forebrain enhancers identified five top-ranked motifs, all of which were experimentally validated as critical for forebrain enhancer activity. These results demonstrate a systematic approach to discover important regulatory motifs in vertebrate developmental enhancers. Moreover, this dataset provides a useful resource for further dissection of vertebrate brain development and function.

Stable Gene Silencing in Zebrafish with Spatiotemporally Targetable RNA Interference

“…Although the mosaic expression is an inevitable feature associated with transient transgenesis, this can be alleviated by coexpression of the Tol2 transposase (Li et al 2010) and selection of high expressers with the visible reporter TdTomato ( Figure S4). DNA plasmids carrying the Pef-1a-Gal4 (elongation factor 1a promoter-driven Gal4) and UAS-miR-shRNA together with the transposase were co-injected into one-cell-stage zebrafish embryos.…”

Conserved non-coding elements and cis regulation: actions speak louder than words

“…Since this discovery, numerous studies have shown that conserved sequences can act as CRMs and drive gene expression in embryos (e.g. Nobrega et al, 2003;Johnson et al, 2004;Teng et al, 2004;Woolfe et al, 2005;Pennacchio et al, 2006;Li et al, 2010), suggesting that these sequences may be conserved because of a consistent gene regulatory function. These observations are the basis for phylogenetic footprinting (see Glossary, Box 1) as a means to identify functional regulatory sequences.…”

“…Although the terminology used in the literature is intended to highlight the extent to which a genomic sequence is conserved, the range of criteria to which these terms are applied may be misleading. The most typically used labels are conserved non-coding elements [CNEs, 60-70% sequence identity over 100 bp (see Dermitzakis and Clark, 2002;Li et al, 2010)]; highly conserved non-coding elements [HCNEs, 70-98% sequence identity over 30-100 bp (see Sandelin et al, 2004;Woolfe et al, 2005;Engström et al, 2007;Kikuta et al, 2007;Engström et al, 2008)]; and ultra-conserved regions/elements [UCEs, 95-100% sequence identity over 200 bp (see Bejerano et al, 2004;Pennacchio et al, 2006)]. Although UCEs appear to exist in the order of hundreds in vertebrate genomes, and HCNEs are of the order of tens of thousands, a common feature -regardless of identification criteria -is that they cluster around key developmental genes.…”

“…Although numerous studies have used this approach to show that, indeed, many CNEs drive specific patterns of expression (e.g. Nobrega et al, 2003;Johnson et al, 2004;Teng et al, 2004;Woolfe et al, 2005;Pennacchio et al, 2006;Li et al, 2010), other studies also demonstrate that where conserved sequence drives expression, the expression pattern is not always conserved across multiple species, and lineage-specific changes in cis or trans regulation have occurred (e.g. Gordon and Ruvinsky, 2012).…”

Identification of Biologically Relevant Enhancers in Human Erythroid Cells

“…Enhancers demonstrate tissue, cell, and developmental stage specificity. Phylogenetic conservation has been used as an indicator for functional conservation of enhancers throughout evolution (16,(27)(28)(29)(30)(31)(32); however, varying degrees of sequence conservation between cell types in different species have been observed. In some human tissues, such as forebrain, enhancers are subject to stringent evolutionary constraint, whereas in others, such as heart, they are under weak evolutionary constraint (12,16,(33)(34)(35)(36)(37)(38).…”