## 3 Results  
### PARP7 Expression is Induced by ER\(\upalpha\)  
To determine if PARP7 expression is regulated by E2, we treated MCF-7 cells with 10 nM E2 and prepared extracts at various time points from 15 min to 24 h and compared the mRNA levels of PARP7 to that of the E2-responsive gene, GREB1 (Figure 1A). This time course analysis revealed that PARP7 mRNA was induced by E2 treatment, but exhibited distinct temporal regulations compared with that of GREB1 (Figure 1A). The maximum PARP7 mRNA levels were observed between 1.5 and 2.5 h, whereas GREB1 mRNA levels reached a maximum at 24 h. We then determined if the E2-mediated regulation of PARP7 could be prevented by pharmacological inhibition of ER\(\upalpha\) and whether this regulation was independent of AHR, a well-known and potent regulator of PARP7 mRNA levels. We treated MCF-7 cells and MCF-7 AHR\({}^{\text{KO}}\) cells with E2 in the presence or absence the ER\(\upalpha\) antagonist, 4-hydroxytamoxifen (4-OHT), for 2 h. The relative levels of PARP7 mRNA were determined by RT-qPCR. E2-treatment alone resulted in a significant increase in PARP7 mRNA levels in both cell lines (Figure 1B). Treatment with 4-OHT alone did not increase PARP7 expression, but prevented the ability of E2 to induce PARP7 mRNA levels. Treatment with E2, but not the AHR agonist, 2,3,7,8-tetrachlorophenoxy-\(\upbeta\)-dioxin (TCDD), failed to induce PARP7 mRNA levels in ER\(\upalpha\) negative MDA-MB-231 cells (Figure 1C). ChIP assays confirmed ER\(\upalpha\) recruitment to the _PARP7_ promoter in an E2-dependent manner (Figure 1D). Taken together these data show that ER\(\upalpha\) regulated PARP7 expression in response to E2 independently of AHR. Figure 1: PARP7 is a target gene and repressor of ER\(\alpha\)**(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 ER\(\alpha\) target gene and its expression is regulated by ER\(\alpha\), independent of AHR. MCF-7 wildtype and AHR\({}^{\text{KO}}\) 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 ER\(\alpha\) levels in MCF-7 compared with MDA-MB-231 cells. The asterisk * denotes significant differences (\(p<0.05\)) from DMSO. **(D)** ER\(\alpha\) 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 ER\(\alpha\)-regulated reporter gene activity. **(E)** HuH-7 or **(F)** MCF-7 cells were transfected with ERE-TK-Luc reporter, pSG5-ER\(\alpha\), \(\beta\)-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 \({}^{\star}\) 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**) Overexpression GFP-PARP7, and GFP-PARP7H532A are recruited to _GREB1_ in response to E2. GFP-PARP7 but not GFP-PARP7H532A decreased (**H**) ER\(\alpha\) binding to _GREB1_ and (**I**) reduced GREB1 mRNA levels in transfected HuH-7 cells. Overexpression (**J**) GFP-PARP7 and GFP-PARP7H532A are recruited _TFF1_ in response to E2.