Highly conserved non-coding DNA regions (HCNR) occur frequently in vertebrate genomes, but their functional roles remain unclear. Here, we provide evidence that a large portion of HCNRs are enriched for binding sites for Sox, POU and Homeodomain transcription factors, and such HCNRs can act as cis-regulatory regions active in neural stem cells. Strikingly, these HCNRs are linked to several hundreds of genes expressed in the developing CNS and they may exert locus-wide regulatory effects on multiple genes flanking their genomic location. Moreover, these data imply a unifying transcriptional logic for a large set of CNS-expressed genes in which Sox and POU proteins act as generic promoters of transcription while Homeodomain proteins control the spatial expression of genes through active repression.