A Search for Enhancers: Analysis of Sequence Conservation Near patched (ptc) Gene

Abstract

Enhancers are sequences of DNA within the genome that control gene expression. They regulate transcription by binding transcription factor proteins via binding site sequences embedded within them. Enhancer structure and function is shaped by three important binding sites characteristics: the transcription factors they recruit, spacing of the sites within the enhancer, and binding site affinity (Swanson et. al, 2010; Parker and Barolo, under review). Binding site affinity describes the intensity of attraction between a transcription factor and a sequence of nucleotides in the DNA. Expression of patched (ptc) gene is regulated by an enhancer that contains binding sites for the transcription factor protein Cubitus interruptus (Ci). Ci is the transcription factor responsible for communicating between the Hedgehog (Hh) cell-signaling pathway, a pathway that controls crucial developmental steps, and its target genes. Key cell-fate decisions that occur during development rely on the ability of ptc to respond to the Hh signal correctly. Therefore, characterizing the enhancer used to communicate the Hh signal to ptc is important. Alexandre et. al, (2003) described the ptc enhancer as a cluster of high-affinity Ci binding sites contained within the 2.5 kb region upstream of ptc. Previous data showed, however, that this enhancer is incomplete (Forbes et al., 1993). The complete ptc enhancer is located somewhere in the region upstream of the previously described ptc enhancer. To search for possible ptc enhancers in the DNA in the vicinity of ptc - the 5’ region upstream of ptc as well as the first intron of ptc – we identified potential Ci binding sites in the Drosophila Melanogaster sequence that were highly conserved in the remaining 11 Drosophila species. We found a few regions containing clusters of highly conserved Ci binding sites of varying affinity. These results will help to locate DNA segments that are more likely to contain regulatory elements that respond to the Hh signal that others.