The identification of ultraconserved noncoding sequences in vertebrates has been 
associated with developmental regulators and DNA-binding proteins. One of the 
first of these was identified in the intergenic region between the Dlx-5 and 
Dlx-6 genes, members of the Dlx/dll homeodomain-containing protein family. In 
previous experiments, we showed that Sonic hedgehog treatment of forebrain 
neural explants results in the activation of Dlx-2 and the novel noncoding RNA 
(ncRNA), Evf-1. In this report, we show that the Dlx-5/6 ultraconserved region 
is transcribed to generate an alternatively spliced form of Evf-1, the ncRNA 
Evf-2. Evf-2 specifically cooperates with Dlx-2 to increase the transcriptional 
activity of the Dlx-5/6 enhancer in a target and homeodomain-specific manner. A 
stable complex containing the Evf-2 ncRNA and the Dlx-2 protein forms in vivo, 
suggesting that the Evf-2 ncRNA activates transcriptional activity by directly 
influencing Dlx-2 activity. These experiments identify a novel mechanism whereby 
transcription is controlled by the cooperative actions of an ncRNA and a 
homeodomain protein. The possibility that a subset of vertebrate ultraconserved 
regions may function at both the DNA and RNA level to control key developmental 
regulators may explain why ultraconserved sequences exhibit 90% or more 
conservation even after 450 million years of vertebrate evolution.