Cis-regulatory architecture of a brain signaling center predates the origin of chordates

Abstract

Genomic approaches have predicted hundreds of thousands of tissue specific cis-regulatory sequences, but the determinants critical to their function and evolutionary history are mostly unknown1–4. Here, we systematically decode a set of brain enhancers active in the zona limitans intrathalamica (zli), a signaling center essential for vertebrate forebrain development via the secreted morphogen, Sonic hedgehog (Shh)5,6. We apply a de novo motif analysis tool to identify six position-independent sequence motifs together with their cognate transcription factors that are essential for zli enhancer activity and Shh expression in the mouse embryo. Using knowledge of this regulatory lexicon, we discover novel Shh zli enhancers in mice, and a functionally equivalent element in hemichordates, indicating an ancient origin of the Shh zli regulatory network that predates the chordate phylum. These findings support a strategy for delineating functionally conserved enhancers in the absence of overt sequence homologies, and over extensive evolutionary distances.

SBE6: a novel long-range enhancer involved in driving sonic hedgehog expression in neural progenitor cells

“…Others have been identified through genetics in mouse and man when mutations in Shh enhancers cause phenotypes that result from aberrant control of specific aspects of Shh expression in development. Most recently, information on transcription factor motifs in known Shh brain enhancers has been used to search for other similar patterns of motifs in the Shh regulatory domain and has identified a new enhancer that drives Shh expression in a discrete region of the brain [7,13]. …”

“…Consistent with this, ENCODE and Roadmap data also indicate that this region of the mammalian genome has active enhancer chromatin marks in neural tissue, as well as in the liver (figure 6 a ; electronic supplementary material, figure S3) and gastric tissue (figure 6 b ). Important sequences required for enhancer function work as assemblies of transcription factor motifs [13]. SBE6.1 and SLGE motifs may be intermingled but still specific to a precise tissue and stage of development, or may be overlapping to various extents.…”

“…An enhancer trap assay—using BAC transgenes to screen the Shh regulatory region—identified SBE2, SBE3 and SBE4 that drive Shh expression in the diencephalon (SBE2) and in the telencephalon (SBE4) [7]. Most recently, a combined informatics and experimental study identified SBE5 that drives expression in the zli [13]. …”

Barhl2 Determines the Early Patterning of the Diencephalon by Regulating Shh

“…Since Barhl2 expression begins at E10.5 in the developing diencephalon and expression of Shh begins at E9 in the ZLI [29], it is likely that loss of Barhl2 restricts the dorsal expansion of ZLI. Recently published studies have identified a Shh ZLI enhancer of evolutionary origin and have demonstrated that BARHL2 directly binds to this enhancer in E10.5 mouse embryo brains [30]. Moreover, studies in Xenopus have shown that Barhl2 restricts the dorsal expansion of the ZLI by affecting the competence of neuroepithelial cells to respond to the secreted form of SHH from the alar plate [31].…”

Deep homology in the age of next-generation sequencing

“…TAD conservation could thus reveal deep homology of entire regulatory landscapes, just as deep conservation of enhancer activities might inform us about evolutionary relationships of different morphological structures that they help to pattern [76]. Clearly, however, the most instructive insights at the gene regulatory level would come from the integration of different NGS technologies to first describe the regulatory architecture at loci of interest, and then test them in reciprocal, cross-species enhancer reporter experiments [83][84][85].…”