Xenobiotic-induced activation of human aryl hydrocarbon receptor target genes in Drosophila is mediated by the epigenetic chromatin modifiers

Aryl hydrocarbon receptor (AHR) is the key transcription factor that controls animal development and various adaptive processes. The AHR\u27s target genes are involved in biodegradation of endogenous and exogenous toxins, regulation of immune response, organogenesis, and neurogenesis. Ligand binding is important for the activation of the AHR signaling pathway. Invertebrate AHR homologs are activated by endogenous ligands whereas vertebrate AHR can be activated by both endogenous and exogenous ligands (xenobiotics). Several studies using mammalian cultured cells have demonstrated that transcription of the AHR target genes can be activated by exogenous AHR ligands, but little is known about the effects of AHR in a living organism. Here, we examined the effects of human AHR and its ligands using transgenic Drosophila lines with an inducible human AhR gene. We found that exogenous AHR ligands can increase as well as decrease the transcription levels of the AHR target genes, including genes that control proliferation, motility, polarization, and programmed cell death. This suggests that AHR activation may affect the expression of gene networks that could be critical for cancer progression and metastasis. Importantly, we found that AHR target genes are also controlled by the enzymes that modify chromatin structure, in particular components of the epigenetic Polycomb Repressive complexes 1 and 2. Since exogenous AHR ligands (alternatively - xenobiotics) and small molecule inhibitors of epigenetic modifiers are often used as pharmaceutical anticancer drugs, our findings may have significant implications in designing new combinations of therapeutic treatments for oncological diseases. © Akishina et al

NAP Family CG5017 Chaperone Pleiotropically Regulates Human AHR Target Genes Expression in Drosophila Testis

To study the regulatory mechanism of the Aryl hydrocarbon receptor (AHR), target genes of transcription are necessary for understanding the normal developmental and pathological processes. Here, we examined the effects of human AHR ligands on male fecundity. To induce ectopic human AhR gene expression, we used Drosophila melanogaster transformed with human AhR under the control of a yeast UAS promoter element capable of activation in the two-component UAS-GAL4 system. We found that exogenous AHR ligands decrease the number of Drosophila gonadal Tj-positive cells. We also found both an increase and decrease of AHR target gene expression, including in genes that control homeostasis and testis development. This suggests that gonadal AHR activation may affect the expression of gene networks that control sperm production and could be critical for fertility not just in Drosophila but also in humans. Finally, we found that the activation of the expression for some AHR target genes depends on the expression of testis-specific chaperone CG5017 in gonadal cells. Since CG5017 belongs to the nucleosome assembly protein (NAP) family and may participate in epigenetic regulation, we propose that this nucleotropic chaperone is essential to provide the human AHR with access to only the defined set of its target genes during spermatogenesis

Wild type and mutant antenna and leg in flies in normal conditions and following irradiation.

<p>Simultaneous combination of mutations at both <i>ss</i> and <i>CG5017</i> loci increases sensitivity to even low doses of X-ray radiation (1 and 10 R), which is manifested as an increase in the mutant phenotype (photographs within the red frame).</p

Dynamics of acquisition and retention of conditioned courtship suppression in mutant males.

<p>Males from wild type <i>Canton S</i> (A-B), <i>milkah-1</i> (C–D), <i>ss^a40a</i> (E–F) and <i>ss^a40amilkah-1</i> (G–H) lines were tested. (A, C, E, G) – of tested males. (B, D, F, H) – of tested males. On the X-axis: time following training, in days; on the Y axis: – courtship index, – learning index, standard units. Open columns – of naive males, hatched columns – () of trained males. Each point represents 20 males. ** - or significantly lower than for wild type (two-sided randomization test, ) in similar condition; - in a test immediately following training or in a deferred test, significantly lower than the CI of naive males (two-sided randomization test, ); - in the delayed test significantly lower than in test immediately following training (two-sided randomization test,.</p

Wild type and mutant leg and antenna phenotypes.

<p>Micrographs show the normal morphology of the tarsal structures of the wild type <i>Canton S</i>, <i>ss^a40ahm</i> and <i>milkah-1</i> flies. Antennal structures of the <i>milkah-1</i> mutant flies do not show any difference from the wild type. The distal segment of the antenna – the arista - of the <i>ss^a40ahm</i> flies presents a certain thickening of the proximal end. The arista of the <i>ss^a40ahmmilkah-1</i> hybrid flies suffered homeotic transformation into an unsegmented tarsus. The tarsal structures of the <i>ss^a40ahmmilkah-1</i> hybrids show altered segment fusion.</p

Effect of X-rays and pharmacological agents on Cyp6g1, GST-theta (CG1681) and ss mRNA expression.

<p>The relative level of expression of mRNA was measured using real-time PCR. A, B, C – level of mRNA expressed in <i>Canton S</i>, <i>ss^a40a</i> and <i>ss^aSc</i> flies respectively. As a control, we used RNA from flies, which have not been exposed to radiation (cont.). F, 5-HT – level of mRNA expressed in flies, grown on medium with added furazidin (F) and 5-hydroxytryptamine (5-HT), respectively; R – radiation dose in Roentgens; 1-10R, level of expressed mRNA of the genes under study in flies irradiated with a dose of 1-10 R. F1-10R, 5-HT1-10R – level of expressed mRNA of the genes under study in flies grown on medium with added F or 5-HT and radiated with 1-10 R, respectively. The bars show the level of mRNA expression. The error bars represent the standard error of the mean of triplicate experiments. * - P<0.05, compared to control group, - P<0.05, compared to F or 5-HT group.</p