Direct and concentration-dependent regulation of the proneural geneNeurogenin2by Pax6

Abstract

Expression of the proneural gene Neurogenin2 is controlled by several enhancer elements, with the E1 element active in restricted progenitor domains in the embryonic spinal cord and telencephalon that express the homeodomain protein Pax6. We show that Pax6 function is both required and sufficient to activate this enhancer, and we identify one evolutionary conserved sequence in the E1 element with high similarity to a consensus Pax6 binding site. This conserved sequence binds Pax6 protein with low affinity both in vitro and in vivo, and its disruption results in a severe decrease in E1 activity in the spinal cord and in its abolition in the cerebral cortex. The regulation of Neurogenin2 by Pax6 is thus direct. Pax6 is expressed in concentration gradients in both spinal cord and telencephalon. We demonstrate that the E1 element is only activated by high concentrations of Pax6 protein, and that this requirement explains the restriction of E1 enhancer activity to domains of high Pax6 expression levels in the medioventral spinal cord and lateral cortex. By modifying the E1 enhancer sequence, we also show that the spatial pattern of enhancer activity is determined by the affinity of its binding site for Pax6. Together, these data demonstrate that direct transcriptional regulation accounts for the coordination between mechanisms of patterning and neurogenesis. They also provide evidence that Pax6 expression gradients are involved in establishing borders of gene expression domains in different regions of the nervous system.

An Allelic Series at the Paired Box Gene 6 (Pax6) Locus Reveals the Functional Specificity of Pax Genes

“…In addition, we confirmed the previous finding that GST-Pax6-PD weakly bound the Ngn2 E1 enhancer element probe E1.1 but not the mutated E1 enhancer probe mtE1.1 (Fig. 8A) (44). Although GST-Pax6-PD could also recognize the 5aCON control probe, GST-Pax6(5a)-PD was unable to bind the P6CON site effectively ( Fig.…”

“…Previous studies have shown that the canonical Pax6 isoform through its PD binds to a low affinity site on the Ngn2 E1 enhancer, which promotes Ngn2 expression only in the telencephalic domains of high Pax6 expression (44). To assess whether there could exist a direct physical interaction between the Ngn2 E1 enhancer and Pax6(5a) or Pax2 PD, we performed DNA EMSAs using oligonucleotide probes containing sequences corresponding to the Pax6 binding sites.…”

Conserved regulation of Math5 and Math1 revealed by Math5-GFP transgenes

“…Interestingly, all of these genes, except NeuroD, genetically require Pax6 (Marquardt et al, 2001;Blader et al, 2004). The activation of Ngn1 and Ngn2 is directly regulated by Pax6 binding to particular CNS enhancers (Marquardt et al, 2001;Scardigli et al, 2003;Blader et al, 2004).…”

Pax6 3′ deletion results in aniridia, autism and mental retardation

“…It is well known that PAX6 influences development of the nervous system and brain through regulation of proneural genes such as neurogenin 2 (Ngn2) and achaete-scute complex homolog-like 1 (Mash-1) (van Heyningen and Williamson 2002;Scardigli et al (2003). In human populations, recent studies have identified individuals with PAX6 mutations who present only with mental retardation and aniridia (Malandrini et al 2001;Ticho et al (2006;Graziano et al (2007).…”