Regulation of a remote Shh forebrain enhancer by the Six3 homeoprotein

Abstract

The secreted morphogen, Sonic hedgehog (Shh) is a significant determinant of brain size and craniofacial morphology1-4. In humans, SHH haploinsufficiency results in holoprosencephaly (HPE)5, a defect in anterior midline formation. 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 combination of genetic and biochemical experiments to uncover a critical pair of cis and trans acting determinants of Shh forebrain expression. A rare nucleotide variant located 460kb upstream of SHH was discovered 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 SBE2 sequence for DNA binding proteins and identified members of the Six3/Six6 homeodomain family as candidate regulators of Shh transcription. Six3 and Six6 showed reduced binding affinity for the mutant compared to wild type SBE2 sequence. Moreover, HPE causing mutations in Six3 failed to bind and activate SBE2, whereas, Shh forebrain expression was unaltered in Six6 −/− embryos. These data provide a direct link between Six3 and Shh regulation during normal forebrain development and in the pathogenesis of HPE. Previous efforts to address this issue focused on determining the genomic location of functional Shh regulatory elements13. These experiments identified six enhancers distributed over a 500 kb interval surrounding the Shh gene that directed reporter activity to most areas of Shh expression in the mouse CNS, including the ventral forebrain ( Fig. 1). In particular, the highly conserved Shh brain enhancer-2 (SBE2), located 460 kb upstream of the SHH coding sequence, was identified as unique in its ability to regulate Shh-like expression throughout the hypothalamus. KeywordsTo identify functionally relevant nucleotides in SBE2, we screened the 1.1 kb sequence mediating its activity for mutations in humans with HPE. We reasoned that HPE causing variants in SBE2 could aid in identifying critical cis and trans determinants of SHH expression in the forebrain. Similar resequencing approaches have been successful in identifying common and rare coding sequence variants in genes associated with common diseases, but have not been routinely applied to the study of remote noncoding regions in rare diseases such as HPE (1:16,000 livebirths)12,14.From 474 HPE patients, we identified one individual who was heterozygous for a C to T base change at nucleotide position 444 of the enhancer sequence. The C/T variant is situated within a block of 10 nucleotides that have been maintained in human, mouse, chicken and frog for over 350 million years ( Fig. 1). This C/T nucleotide variant was not observed in DNA samples from 450 unrelated control individuals. The affected female exhibited features of semilobar HPE including microcephaly, midfacial hypoplasia, cleft-lip and palate, diabetes insipidus, and moderate fus...

Six3 dosage mediates the pathogenesis of holoprosencephaly

“…Six3 directly regulates Shh expression in the RDVM Jeong et al, 2008). As the amount of functional Six3 decreases in Six3 +/-and Six3 +/ki embryos, it fails to activate Shh expression in the ventral forebrain and subsequently leads to HPElike phenotypes.…”

“…We previously showed that Six3 directly activates the expression of Shh in the rostral diencephalon ventral midline (RDVM) of mice Jeong et al, 2008). In turn, Shh maintains the expression of Six3 in the RDVM.…”

Proper differentiation of photoreceptors and amacrine cells depends on a regulatory loop between NeuroD and Six6

“…Both genes act as transcriptional repressors by interacting with members of the Groucho family of transcriptional co-repressors Marco-Ferreres et al, 2009) but there is also evidence for their role as transcriptional activators (Jeong et al, 2008;Liu et al, 2006;Reichman et al, 2010). In most vertebrate species, both genes are strongly expressed starting at the gastrula stages in the anterior neural plate, where they are required for early forebrain specification.…”

Zebrafishzic2apatterns the forebrain through modulation of Hedgehog-activated gene expression

“…Hh function results in transcriptional activation of several targets, many of which encode transcription factors (TFs). The homeobox TF gene six3 is transcriptionally activated by Hh signaling and directly activates shh transcription Jeong et al, 2008). six3 function is essential for correct development of all forebrain derivatives, as mice and human with compromised six3 or shh function develop with a profound forebrain defect termed holoprosencephaly (HPE) (Krauss, 2007).…”