Li Y et al. (2021), Identification of estrogen receptor target gene...

XB-ART-57762

Aquat Toxicol 2021 Jan 21;232:105760. doi: 10.1016/j.aquatox.2021.105760.

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Identification of estrogen receptor target genes involved in gonadal feminization caused by estrogen in Xenopus laevis.

Li Y , Li J , Shen Y , Xiong Y , Li X , Qin Z .

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Estrogens and estrogenic endocrine disrupting chemicals can cause gonadal feminization in some vertebrates mainly through estrogen receptor (ER), but the underlying molecular mechanisms are unclear. The present study aimed to identify ER target genes involved in estrogen-caused gonadal feminization in Xenopus laevis. Based on our recent transcriptomic data that 10 nM 17β-estradiol (E2) altered gene transcription in feminizing gonads of male X. laevis at NF stages 48, 50, and 52, we searched estrogen response element (ERE) using the Dragon ERE Finder software in the promoter region of all the E2-regulated genes. As a result, 163 genes containing ERE sequence were identified as predicted ER target genes at NF stage 50 (on the 14th day postfertilization), a crucial stage for gonadal feminization. Then, some of these predicted ER target genes were further investigated, mainly including the genes that were suggested to be involved in E2-caused gonadal feminization and genes being dramatically up or down-regulated by E2. Fifteen genes were demonstrated to be responsive to E2, in turn ER antagonist blocked the E2-regulated transcription. Finally, we identified 10 genes that can bind to ERα by a chromatin immunoprecipitation-qPCR. Taken together, we identified the 10 genes that contain predicted ERE sequences, are responsive to estrogen and ER antagonist, and have ability to bind to ER as ER target genes, including pglyrp2, apoa1, fgb, tdo2, ca6, nags, cpb2, tmprss6, nudc, zwilch. Our results could help to improve the understanding of the molecular mechanisms for gonadal feminization caused by estrogenic endocrine disrupting chemicals in X. laevis, and even in other species.

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Species referenced: Xenopus laevis
Genes referenced: a2m akr1c3 amy2a apoa1 asic4 aurka c5 ca6 cdca5 cdca8 cdkn2c cndp1 cpb2 crim1 ctsz ddr1 dm-w dnajc12 dok1 ercc6l esr1 esr2 f13b f5 fbxl12 fgb haus8 hspb1 itih3 klhl41 krt18.1 lrrcc1 mmp19 mtss1 muc2 myom2 myoz1 nags nr4a1 nudc pglyrp2 prim1 prss1 pvalb rbpms2 rgmb rpl8 serpini2 sftpd sik1 slco1c1 tdo2 tf tm4sf1 tmprss13 tmprss6 tpx2 txnl4a ybx3 zwilch

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	Fig. 1. Predicted ER target genes containing estrogen response element (ERE) sequences among all 17β-estradiol (E2)-regulated genes in male Xenopus laevis at NF stages 48, 50, and 52. A Numbers of predicted ER target genes among all E2-regulated genes at NF stages 48, 50, and 52. B The number of E2-upregulated or E2-downregulated predicted ER target genes in estrogen-treated male (ETM) compared with control male (CM) at NF stages 48, 50, and 52. C Protein–protein interaction (PPI) network that depicts the connections among proteins encoded by all predicted ER target genes and their interacted genes at NF stage 50. The green dots indicate E2-downregulated potential ER target genes and the magenta dots indicate E2-upregulated potential ER target genes. The green background depicts PPI network associated with cell proliferation, the light magenta background depicts PPI network associated with extracellular matrix (ECM) and the complement and coagulation cascade, and the light pink background depicts PPI network associated with cell motility.
	Fig. 2. Responses of predicted estrogen receptor (ER) target genes to both estrogen and ER antagonist in male Xenopus laevis at NF stage 50. A. RT-qPCR analysis confirmed the transcriptional regulation of predicted ER target genes by 10 nM E2 in male gonad-mesonephros complexes (GMCs) at NF stage 50. B. E2 and ER antagonist (ICI 182780) co-exposure identified 15 ER-mediated genes in male GMCs at NF stage 50. Gene expression was reported as the fold change relative to controls. Error bars represent standard deviations. The ligatures in B indicate significant differences between E2 groups and co-exposure groups (two-tailed independent-sample t test). n = 3 replicated aquaria, each containing two pooled samples. * indicates significant differences in E2 compared with control (two-tailed independent-sample t test, p < 0.05).
	Fig. 3. The expression and the binding of 15 predicted estrogen receptor (ER) target genes to ER in froglet testes. A. The expression (CT value) of 15 predicted ER target genes (pglyrp2, a2m, apoa1, fgb, tdo2, ca6, itih3, tf-b, nags, c5, cpb2, tmprss6, nudc, zwilch, csda), coupled with esr1 (erα), esr2 (erβ), vtg and rpl8, in the testis of froglets. B. Quantitative enrichment of 15 predicted ER target genes after chromatin immunoprecipitation (ChIP) with ERα antibody and immunoglobulin G (IgG) in froglet testes. A threshold of 5-fold enrichment was set as the positive result.