Analysis of Long Range Gene Regulation in Drosophila; Insights from the dPax2 sparking Enhancer.

Abstract

Enhancers, a subclass of cis-regulatory sequences control gene expression and patterning during development. As enhancers are often located at considerable genomic distances from their target genes, distal enhancer promoter interactions are a crucial part of transcriptional regulation, yet surprisingly little is known about how these interactions are facilitated. To better understand this essential enhancer function, we are characterizing the ability of, sparkling, the dPax2 cone cell-specific enhancer, ability to activate gene expression from both a promoter proximal, and distal position. Using this approach, we have identified a sequence within spa required when the enhancer is placed at a distance from the promoter, but is dispensable in a promoter proximal position. As this DNA sequence appears to convey long-range enhancer activity, we refer to it as the “remote control” element or RCE. Subsequent analysis has analyzed the distance at which sparkling requires the RCE to function and demonstrated that the RCE can function as a separable unit from the enhancer that can be moved to new locations and still facilitate sparkling action. Furthermore experimental observations support a role for the RCE in facilitating the ability of the enhancer to communicate with specific promoters. This work as also shown that additional regulator sequences, both outside of, and from within the enhancer can also support long-range enhancer activity. This study reveled that the regulatory sequences within in spa can be rearranged globally, but some local restrictions must be obeyed. We have also attempted to identify proteins that interact with the spa enhancer and have found that the Six family protein, Sine Oculis, can interact in vitro with at least two regions of the enhancer capable of conveying long distance gene regulation - the RCE and region 4. This work has contributed to the study of gene regulation by identifying the first sequence with an enhancer to regulate long-range gene transcription without requiring specific promoter elements as well. The knowledge gained from this work can ultimately aid in identifying novel enhancers, designing tissue/temporal specific synthetic enhancers, and when broadly applied a can give a clearer view of basic transcriptional mechanisms, signal transduction, and development.