Potential Direct Regulators of theDrosophila yellowGene Identified by Yeast One-Hybrid and RNAi Screens

Abstract

The regulation of gene expression controls development, and changes in this regulation often contribute to phenotypic evolution. Drosophila pigmentation is a model system for studying evolutionary changes in gene regulation, with differences in expression of pigmentation genes such as yellow that correlate with divergent pigment patterns among species shown to be caused by changes in cis- and trans-regulation. Currently, much more is known about the cis-regulatory component of divergent yellow expression than the trans-regulatory component, in part because very few trans-acting regulators of yellow expression have been identified. This study aims to improve our understanding of the trans-acting control of yellow expression by combining yeast-one-hybrid and RNAi screens for transcription factors binding to yellow cis-regulatory sequences and affecting abdominal pigmentation in adults, respectively. Of the 670 transcription factors included in the yeast-one-hybrid screen, 45 showed evidence of binding to one or more sequence fragments tested from the 5′ intergenic and intronic yellow sequences from D. melanogaster, D. pseudoobscura, and D. willistoni, suggesting that they might be direct regulators of yellow expression. Of the 670 transcription factors included in the yeast-one-hybrid screen, plus another TF previously shown to be genetically upstream of yellow, 125 were also tested using RNAi, and 32 showed altered abdominal pigmentation. Nine transcription factors were identified in both screens, including four nuclear receptors related to ecdysone signaling (Hr78, Hr38, Hr46, and Eip78C). This finding suggests that yellow expression might be directly controlled by nuclear receptors influenced by ecdysone during early pupal development when adult pigmentation is forming.

Using Drosophila pigmentation traits to study the mechanisms of cis-regulatory evolution

“…For example, the D. melanogaster genome has around 750 transcription factor genes [43] whose expression patterns are generally poorly characterized. Thus, connecting a single transcription factor to a functional binding event in a CRE remains laborious to establish [44]. In the face of these challenges though, the diversity of Drosophila pigmentation traits and their underlying CREs present an excellent model system to better understand transcriptional regulatory evolution, and are likely to lead the way into the mechanistic understanding regulatory logic evolution.…”

Redundant and cryptic enhancer activities of the Drosophilayellowgene

“…Finally, we compared results from this study to data from a prior study using a yeast-onehybrid assay to test 670 transcription factors for evidence of binding to these same yellow 5' intergenic and intronic fragments (Kalay et al 2016). Only one transcription factor, Hr78, showed evidence of binding to fragments from all three species in this prior work (mel_A2, mel_A3, mel_A4, mel_A5, pse_B1, and will_C1).…”

“…Each of these ~1 kb fragments was cloned upstream of a nuclear Green Fluorescent Protein (nGFP) to form a reporter gene, and each reporter gene was inserted into the attP40 landing site on chromosome arm 2L. This schematic was modified fromKalay et al (2016). Pictures of Drosophila species generously provided by Dr. Nicolas Gompel (Ludwig-Maximilians-University of Munich).…”

Molecular Mechanisms of Larval Color Pattern Switch in the Swallowtail Butterfly

“…In D. melanogaster, Yellow is known to be a prepattern factor as well as a pigmentation factor in adult body patterning (Massey and Wittkopp 2016). Recently, comprehensive yeast one-hybrid and RNAi screens were carried out by Kalay et al (2016). They screened and identified four ecdysone-induced nuclear reporters (Hr78, Hr38, Hr46, and Eip78C) that showed a statistically significant interaction with at least one Yellow enhancer.…”

“…They screened and identified four ecdysone-induced nuclear reporters (Hr78, Hr38, Hr46, and Eip78C) that showed a statistically significant interaction with at least one Yellow enhancer. In an RNAi experiment, all four caused altered pigmentation when knocked down (Kalay et al 2016). In Bombyx mori, Yamaguchi et al (2013) used a type of L (multi lunar) mutant with twin-spot markings on the sequential segments and proved that the gene responsible for this phenotype (BmWnt1) can be induced by high concentrations of 20E in vitro (Yamaguchi et al 2013).…”