A functional screen for sonic hedgehog regulatory elements across a 1 Mb interval identifies long-range ventral forebrain enhancers

Abstract

The secreted protein sonic hedgehog (Shh) plays an integral role in forming the ventral midline of the vertebrate central nervous system (CNS). In the absence of Shh function, ventral midline development is perturbed resulting in holoprosencephaly (HPE), a structural malformation of the brain, as well as in neuronal patterning and path finding defects along the length of the anteroposterior neuraxis. Central to the understanding of ventral neural tube development is how Shh transcription is regulated in the CNS. To address this issue, we devised an enhancer trap assay to systematically screen 1 Mb of DNA surrounding the Shh locus for the ability to target reporter gene expression to sites of Shh transcription in transgenic mouse embryos. This analysis uncovered six enhancers distributed over 400 kb, the combined activity of which covered all sites of Shh expression in the mouse embryonic CNS from the ventral forebrain to the posterior extent of the spinal cord. To evaluate the relative contribution of these enhancers to the overall pattern of Shh expression, individual elements were deleted in the context of a transgenic Bac reporter assay. Redundant mechanisms were found to control Shh-like reporter activity in the ventral spinal cord, hindbrain and regions of the telencephalon, whereas unique elements regulated Shh-like expression in the ventral midbrain, the majority of the ventral diencephalon and parts of the telencephalon. Three ventral forebrain enhancers locate on the distal side of translocation breakpoints that occurred upstream of Shh in human cases of HPE, suggesting that displacement of these regulatory elements from the Shh promoter is a likely cause of HPE in these individuals.

An evolutionarily conserved three-dimensional structure in the vertebrate Irx clusters facilitates enhancer sharing and coregulation

“…To achieve this, their rate of transcription is controlled by multiple cis -regulatory elements, some of them functioning over very long distances 28,29 . Our analysis has identifi ed a large number of cis -regulatory elements, located in HCNRs and distributed throughout the 2-Mbp IrxA cluster, and that altogether appear to recapitulate In most cell types, CTCF is bound to the Irx promoters (coloured boxes).…”

Multiple layers of complexity in cis‐regulatory regions of developmental genes

“…In recent years, numerous cis-regulatory regions of animal genes have been shown to contain multiple enhancers with overlapping or very similar expression patterns (Jeong et al, 2006;Werner et al, 2007;Hong et al, 2008;McGaughey et al, 2008;Corbo et al, 2010;Frankel et al, 2010;Kalay and Wittkopp, 2010;L'Honore et al, 2010;Naranjo et al, 2010;Perry et al, 2010Perry et al, , 2011Franchini et al, 2011;Galindo et al, 2011;Ghiasvand et al, 2011;Lee et al, 2011;McBride et al, 2011;Watts et al, 2011;Pauls et al, 2012). Yet, the first observations of overlapping expression from different enhancers date back from the 1990s (Hoch et al, 1990;Kassis, 1990;Tolias and Kafatos, 1990).…”

Identification of EphA7 BAC clone containing a long-range dorsal midline-specific enhancer

“…For the enhancer trap assay, a transposable vector carrying a β-globin promoter, LacZ gene containing SV40 poly(A) site, and a kanamycin resistance gene was introduced into each BAC clone according to the method of Spitz et al (8,9). Briefly, 40 ng of transposable vector was mixed with 200 ng of BAC DNA, GPS buffer, and TnsABC (NEB), followed by incubation at 37 o C for 10 minutes.…”