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

Abstract

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.

Keywords: Aryl hydrocarbon receptor; CG5017; Drosophila; nucleosome assembly protein; spermatogenesis; xenobiotic.

Figure A1

Merged confocal immunofluorescence images of apical tips of testis stained with SytoxGreen to highlight DNA (green) and anti-Tj to visualize Tj-positive cells (red). Testes are from Tj-Gal4/+ males: (left) raised on standard medium; (right) fed for 3 days with indirubin. A number of Tj-positive cells in testis of flies developed on standard medium or fed with xenobiotic was the same (100.1 ± 5.2; n = 22 and 104.8 ± 7.7; n = 19 correspondently). Scale bars, 30 µm.

Figure 1

Daily effect of exogenous ligands on the proportion of undeveloped eggs from wild-type female after crossing with UAS-AhR/Tj-GAL4 male without exposure to ligands (control, orange), exposed to indirubin (blue), beta-Naphthoflavone (azure), indinol (green). Data correspond to the mean ± SD of three independent experiments. Asterisks mean the significant difference compared to the control group. Statistical analysis was performed using Student’s t-test (* p ≤ 0.05).

Figure 2

Xenobiotic-mediated activation of human AHR in cells of Drosophila testis causes a decrease in number of Tj-positive cells. (A–D) Confocal immunofluorescence sections of the apical tip of testes stained with SytoxGreen to highlight DNA (green) and anti-Tj to visualize Tj-positive cells (red). The third column represents merged images. (A) Testes are from flies raised on standard medium or fed for 3 days with (B) indirubin, (C) beta-Naphthoflavone, (D) indinol. All tested males were of the same UAS-AhR/Tj-Gal4 genotype. Note that apical tips of males fed with xenobiotics are smaller than apical tip of male fed with standard medium. Scale bars, 30 µm. (E) Quantification of Tj-positive cells in testes of UAS-AhR/Tj-GAL4 flies raised on standard medium (No ligand) and on medium with indirubin, beta-Naphthoflavone and indinol for 3 days counted at the 4^th day after feeding. Error bars represent 90% confidence interval of the mean. Asterisks indicate statistically significant difference using Student’s t-test (* p ≤ 0.01; ** p ≤ 0.001).

Figure 3

Different effects of human AHR exogenous ligands on AHR target gene mRNA levels in testes of UAS-AhR/Tj-GAL4 flies. Relative mRNA levels were analyzed by real-time PCR in testes dissected from UAS-AhR/Tj-GAL4 flies fed with indirubin, beta-Naphthoflavone or indinol for 2 days. UAS-AhR/Tj-GAL4 flies developed on standard medium were used as a control. Transcript levels are represented as means ± SD (error bars). * p < 0.05, compared to the control. Statistical analysis was performed using Student’s t-test.

Figure 4

Decreased expression of CG5017 nucleotropic chaperone leads to ligand dependent activation in transcription of some AHR target genes. Relative mRNA levels were analyzed by real-time PCR in testes dissected from UAS-AhR/Tj-GAL4; ss^aSc flies fed with indirubin, beta-Naphthoflavone or indinol for 2 days. UAS-AhR/Tj-GAL4; ss^aSc flies developed on standard medium were used as a control. Transcript levels are represented as means ± SD (error bars). * p < 0.05, compared to the control. Statistical analysis was performed using Student’s t-test.