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. 2021 Mar 11;10(3):623. doi: 10.3390/cells10030623

Figure 1.

Figure 1

PARP7 is a target gene and repressor of ERα (A) PARP7 expression is induced by E2. MCF-7 cells were treated with E2, and RNA was isolated at various time points ranging from 15 min to 24 h. The relative mRNA levels of PARP7 (left axis) and GREB1 (right axis) were determined with RT-qPCR. (B) PARP7 is an ERα target gene and its expression is regulated by ERα, independent of AHR. MCF-7 wildtype and AHRKO cells were treated with 0.1% DMSO, 10 nM E2 or/and 100 nM 4-OHT for 2 h. The co-treated samples were treated with 4-OHT 2 h prior to E2 treatment. The relative mRNA levels were determined with RT-qPCR. The asterisk * denotes significant differences (p < 0.05) from DMSO. (C) PARP7 mRNA levels in MDA-MB-231 cells treated with 0.1% DMSO, 10 nM E2 or 10 nM TCDD for 2 h. Insert western blot of ERα levels in MCF-7 compared with MDA-MB-231 cells. The asterisk * denotes significant differences (p < 0.05) from DMSO. (D) ERα is recruited to the PARP7 promoter in an E2-dependent manner. Wildtype MCF-7 cells were treated with 0.1% DMSO or 10 nM E2 for one hour. The letter “a” denotes recruitment differences significantly greater than the control (p < 0.05), and significant differences (p < 0.05) from DMSO is denoted with the asterisk *. PARP7, but not the catalytically inactive H532A mutant, repressed ERα-regulated reporter gene activity. (E) HuH-7 or (F) MCF-7 cells were transfected with ERE-TK-Luc reporter, pSG5-ERα, β-galactosidase, PARP7, or the catalytically inactive mutant. Six hours after transfection, cells were treated with 0.1% DMSO or 10 nM E2 for 18 h. Changes in reporter gene activity are shown as normalized relative light units (RLU). The asterisk * denotes significant differences (p < 0.05) from DMSO. Hash mark # denotes significant differences (p < 0.05) when compared to the response to E2 treatment in the absence of PARP7. (G) Overexpressed GFP-PARP7, and GFP-PARP7H532A are recruited to GREB1 in response to E2. GFP-PARP7 but not GFP-PARP7H532A decreased (H) ERα binding to GREB1 and (I) reduced GREB1 mRNA levels in transfected HuH-7 cells. Overexpressed (J) GFP-PARP7 and GFP-PARP7H532A are recruited TFF1 in response to E2. GFP-PARP7 but not GFP-PARP7H532A decreased (K) ERα binding to TFF1 and reduced (L) TFF1 mRNA levels in transfected HuH-7 cells. For G, H, J and K, recruitment differences significantly greater than the control (p < 0.05) are denoted with the letter “a”. Significant differences greater than antibody matched DMSO (p < 0.05) are denoted with the asterisk *. Significant differences greater than treatment matched GFP (p < 0.05) are denoted with the hash mark #. For I and L, the asterisk * denotes significant differences (p < 0.05) from transfection matched DMSO. Significant differences greater than transfection matched E2 (p < 0.05) are denoted with the hash mark #. Data are shown as means ± S.E.M. from three independent experiments.