Analysis of the interaction between the E74A transcription factor and the regulatory region of the D. melanogaster CGs 7599 and 7336 using trans-activating assays in yeast

Abstract

Our laboratory investigates mechanisms controlling gene expression in metazoans. As a model we employ the DNA puff gene BhC4-1, which is expressed at the end of the larval development of the sciarid Bradysia hygida. Functional assays in the model organism Drosophila melanogaster led to the identification of a salivary gland 129 bp (-186/-58) enhancer in the BhC4-1 promoter. Although the data obtained from gene interaction experiments and bioinformatics analysis indicate that the E74A transcription factor regulates BhC4-1 expression in the salivary gland, it is not possible at the moment to determine whether protein-DNA interactions occur between the transcription factor E74A and the BhC4-1 (-186/-58) salivary gland enhancer. A master's project currently underway in our laboratory aims to determine whether in the context of the yeast one-hybrid system yeast the D. melanogaster E74A transcription factor directly interacts with the BhC4-1 (-186/-58) salivary gland enhancer. This project aims to carry out experiments in the yeast one-hybrid system designed to verify the interaction between the E74A transcription factor and a D. melanogaster 420 bp genomic fragment located between the L71-5 (CG7599) and L71-6 (CG7336) genes, which contains five binding sites for the transcription factor E74A. The binding of E74A to these binding sites has been demonstrated by DNase I protection experiments and functional assays have revealed that these binding sites are required for the proper induction of a L71-6: lacZ transgene by E74A. The results obtained from the development of this project will reveal whether the E74A transcription factor interacts with a DNA fragment, for which the interaction with E74A has previously been demonstrated, in the context of the yeast one-hybrid system, which will be an important positive control for ongoing experiments in the laboratory. (AU)