Identification Of Cis-regulatory Elements Located Upstream Of Bristle Patterning Gene Extramacrochaetae In Drosophila

Co-evolution can occur at different biological levels. Although there are some reports about cis- and trans-regulation of microscopic co-evolution of gene expression, yet, there are rare research evidences in co-evolution of multi-gene regulation, in which, the same transcription factor can combine with different cis-regulatory elements. Present study explored the co-evolution of multi-gene regulation using notum macrochaetae as morphologic marker. The macrochaetae development progress is mainly determined by the proneural genes achaete-scute (ac-sc). In Drosophila, ac-sc expression is regulated by transcriptional activation at sites where bristle precursors develop. In gene regulatory network of ac-sc, extramacrochaetae(emc) can antagonize proneural sc function, thus emc negatively regulate macrochaetae progression and express at sites where no bristles develop. Pannier (pnr) and iro complex genes (iro-C) have already been proved to activate sc by binding positional enhancers. However the transcription-based mechanisms controlling emc expression during this process are not well understood. Previous reports with D. melanogaster indicate that emc expression is altered in pnr and iro-C mutants. Considering the fact that expression pattern of emc is fairly complementary to that of sc, we can speculate that emc is also controlled by pnr and iro-C genes at transcriptional level. If so, this could mean that emc and sc responds to the same trans- regulatory pattern and regulation of emc and sc will co-evolve during macrochaetae development.To test this hypothesis that pnr and iro-C are involved in the transcription regulation of emc, here we mainly focused on the identification and functional study of cis-regulatory sequences upstream of the emc in Drosophila. This study comprised of a series of experiments to test whether emc is regulated by pnr and iro-C gene in Drosophila. The main results of present study are as follows:(1) Firstly, by bioinformatics methods, we have analyzed putative emc regulatory sequences containing GATA, TAAT and ACAnnTGT sites, which have been predicted as potential binding sites for Pnr and Iro-C;(2) Then conserved non-coding elements upstream of emc gene were isolated and cloned into luciferase reporter vector. We have assayed the possible cis-regulatory elements upstream of emc for regulatory activities using transfection and luciferase reporter assays in vitro. Luciferase reporter assay revealed the presence of an upstream enhancer located about 2.3kb away from emc gene in Drosophila;(3) This study has resulted in the construction of emcE6-EGFP transgenic recombination vector and generated enhancer emcE6 transgenic Drosophila by microinjection. In vivo reporter assays have tested the expression pattern and regulatory activity of potential cis-regulatory sequence emcE6;(4) Luciferase reporter assay of GATA mutants revealed that a potential Pnr-binding site in emcE6 has the regulatory activity. Pnr can be involved in the transcription regulation of emc;(5) Luciferase reporter assay of TAAT mutants suggested that four potential Iro-C binding sites don’t have the regulatory activity. This study revealed that the Iro-C transcript factor is not involved in transcription and expression of emc.In conclusion, these findings reveal the presence of an upstream enhancer emcE6 located about 2.3kb away from emc gene, which contributes to transcriptional regulation of emc in Drosophila; A potential Pnr-binding site in emcE6 has the regulatory activity; Moreover, Iro-C is not found to be involved in transcription and expression of emc in this study. This study delineates the mechanism for transcription regulation of emc at the molecular level and reveal the co-evolution between regulation of emc and sc during development of DC bristles. Furthermore, this research provides a new case and has a certain innovation in theory.