Regulation of a remote Shh forebrain enhancer by the Six3 homeoprotein

Publisher Website
Google Scholar
Abstract
Doug Epstein and colleagues present biochemical and transgenic studies implicating Six3 in the regulation of Shh forebrain expression through direct binding to a remote enhancer. The work was driven by the discovery of a rare variant in this enhancer in an individual with holoprosencephaly In humans, SHH haploinsufficiency results in holoprosencephaly (HPE), a defect in anterior midline formation1,2. Despite the importance of maintaining SHH transcript levels above a critical threshold, we know little about the upstream regulators of SHH expression in the forebrain. Here we describe a rare nucleotide variant located 460 kb upstream of SHH in an individual with HPE that resulted in the loss of Shh brain enhancer-2 (SBE2) activity in the hypothalamus of transgenic mouse embryos. Using a DNA affinity-capture assay, we screened the SBE2 sequence for DNA-binding proteins and identified members of the Six3 and Six6 homeodomain family as candidate regulators of Shh transcription. Six3 showed reduced binding affinity for the mutant compared to the wild-type SBE2 sequence. Moreover, Six3 with HPE-causing alterations failed to bind and activate SBE2. These data suggest a direct link between Six3 and Shh regulation during normal forebrain development and in the pathogenesis of HPE.