Visualization of the Drosophila dKeap1-CncC interaction on chromatin illumines cooperative, xenobiotic-specific gene activation

Abstract

Interactions among transcription factors control their physiological functions by regulating their binding specificities and transcriptional activities. We implement a strategy to visualize directly the genomic loci that are bound by multi-protein complexes in single cells in Drosophila. This method is based on bimolecular fluorescence complementation (BiFC) analysis of protein interactions on polytene chromosomes. Drosophila Keap1 (dKeap1)-CncC complexes localized to the nucleus and bound chromatin loci that were not bound preferentially by dKeap1 or CncC when they were expressed separately. dKeap1 and CncC binding at these loci was enhanced by phenobarbital, but not by tert-butylhydroquinone (tBHQ) or paraquat. Endogenous dKeap1 and CncC activated transcription of the Jheh (Jheh1, Jheh2, Jheh3) and dKeap1 genes at these loci, whereas CncC alone activated other xenobiotic response genes. Ectopic dKeap1 expression increased CncC binding at the Jheh and dKeap1 gene loci and activated their transcription, whereas dKeap1 inhibited CncC binding at other xenobiotic response gene loci and suppressed their transcription. The combinatorial chromatin-binding specificities and transcriptional activities of dKeap1-CncC complexes mediated the selective activation of different sets of genes by different xenobiotic compounds, in part through feed-forward activation of dKeap1 transcription.

Mechanisms and functions of Nrf2 signaling in Drosophila

“…The function of Drosophila Keap1 as a selective inhibitor of CncC has been confirmed in reporter gene studies in transgenic flies and in cell culture where Keap1 over-expression suppresses CncC dependent gene activation (Chatterjee and Bohmann, 2012). Finally co-immunoprecipitation and bimolecular fluorescence complementation (BiFC) experiments confirmed a physical interaction between CncC and Drosophila Keap1 ((Deng and Kerppola, 2014), M. Tian and D. Bohmann, unpublished observations).…”

Increasing autophagy and blocking Nrf2 suppress laminopathy‐induced age‐dependent cardiac dysfunction and shortened lifespan

“…Nrf2 and its cytoplasmic binding partner Keap1 function in cellular detoxification and are conserved in Drosophila (Deng & Kerppola, 2013, 2014). In a Drosophila model of noncardiac muscle laminopathies, Cap‐and‐collar C (CncC) [the orthologue of mammalian nuclear erythroid 2‐related factor 2 (Nrf2)] redox transcriptional regulator (Deng & Kerppola, 2013, 2014) accumulated in myonuclei and activated cellular detoxification genes (Dialynas et al., 2015). To determine whether CncC accumulated in this cardiac model of laminopathies, hearts expressing wild‐type and mutant LamC were stained with antibody to CncC (Deng & Kerppola, 2013, 2014).…”

“…Nuclear CncC (Nrf2) is a characteristic of redox imbalance (Deng & Kerppola, 2013, 2014). To determine the redox status of the hearts, reduced (GSH) and oxidized (GSSH) glutathione were measured (Anderson, 1985; Dialynas et al., 2015).…”

Zinc oxide nanoparticles exhibit cytotoxicity and genotoxicity through oxidative stress responses in human lung fibroblasts and <em>Drosophila melanogaster</em>

“…33 For in vivo viability assay, wild-type adult flies were placed in vials containing food media supplemented with either 0, 0.1, 0.25, 0.5 and 1 mg/mL ZnO NPs. The parent flies were then removed after 5 days, and the viability of the laid eggs to adult stage was monitored.…”