## 3 Results
### PARP7 represses ER\(\alpha\) Activity  
We have previously reported that PARP7 acts as a negative regulator of AHR activity via mono-ADP-ribosylation [31]. To determine whether PARP7 represses ER\(\alpha\) activity, we transfected HuH-7 cells with various amounts of PARP7, or the catalytically inactive mutant (PARP7H532A), together with ER\(\alpha\) and an ER\(\alpha\)-regulated luciferase reporter plasmid. Increasing amounts of PARP7 resulted in a dose- and ligand-dependent repression of ER\(\alpha\)-regulated reporter gene activity (Figure 1E). Transfection of PARP7H532A did not repress reporter gene activity, indicating that PARP7 must be catalytically active in order to repress ER\(\alpha\) signaling. Transfection of PARP7H532A also resulted in increased reporter gene activity compared with no PARP7. Similar findings were observed in transiently transfected MCF-7 cells (Figure 1F). In order to further understand how PARP7 affects ER\(\alpha\) transactivation, ChIP assays were carried out in HuH-7 cells transfected with GFP-PARP7, or GFP-PARP7H532A, and ER\(\alpha\). E2 induced recruitment of GFP-PARP7 and GFP-PARP7H532A to the regulatory region of _GREB1_. The recruitment levels of GFP-PARP7H532A were significantly greater than those of GFP-PARP7 (Figure 1G). Decreased ER\(\alpha\) binding and reduced GREB1 mRNA levels were observed in the presence of GFP-PARP7 but not GFP-PARP7H532A (Figure 1H,I). Similar findings were observed for TFF1 (Figure 1J-L). These data provide evidence that catalytically active PARP7 negatively regulated ER\(\alpha\) transactivation. The PARP7 Inhibitor, RBN-2397, Increases E2-Dependent GREB1 mRNA Levels and Stabilizes PARP7 and ER\(\alpha\) Proteins  
Since we had observed the ability of PARP7 to inhibit ER\(\alpha\) activity (Figure 1E,F), we investigated the effect of the small molecule PARP7 inhibitor, RBN-2397, on the PARP7-dependent regulation of ER\(\alpha\). ADP-ribosylation assays done on cell extracts isolated from COS-1 cells transfected with GFP-PARP7 and treated for 24 h with RBN-2397 confirmed RBN-2397's ability to inhibit PARP7 catalytic activity (Figure 2A). In agreement with our previous data showing that the introduction of the point mutation H532A destroys PARP7 catalytic activity but also stabilizes PARP7 protein levels [17], treatment with RBN-2397 stabilized transfected GFP-PARP7 protein levels (Figure 2A,B). However, RBN-2397 did not affect the protein levels of GFP-PARP7H532A (Figure 2B). We next determined the effect of RBN-2397 on the levels of endogenous PARP7 levels in _Parp7+/-_, _Parp7-/-_ and _Parp7H532A_ MEFs. Since we have been unable to identify a reliable commercially available anti-PARP7 antibody that detects endogenous protein, we generated a mouse monoclonal antibody against murine Parp7. Treatment of MEFs confirmed that RBN-2397 stabilizes endogenous Parp7 but does not affect the protein levels of Parp7H532A (Figure 2C). In support of these data, treatment with RBN-2397 also stabilized endogenous PARP7 in E0771 murine triple negative breast cancer cells. However, due to a lack of ER\(\alpha\) expression, co-treatment with E2 had no effect (Supplementary Figure S1A). Treatment of MCF-7 cells with E2 resulted in a significant increase in GREB1 mRNA levels. RBN-2397 treatment alone also significantly increased GREB1 mRNA levels compared with DMSO, but to a significantly lower level than those induced by E2 (Figure 2D). Co-treatment of E2+RBN-2397 resultedin a slight, but significantly higher increase in GREB1 mRNA levels compared with E2 alone (Figure 2D). To determine whether E2 treatment also results in increased endogenous PARP7 protein levels, MCF-7 cells were treated with 10 nM of E2 for 4 and 24 h. Transfection with different N-terminal truncations of GFP-PARP7 (human) revealed that our anti-PARP7 antibody recognizes a region within the N-terminus in PARP7 that includes 1-53 a.a. (Supplementary Figure S1B,C).