## 3 Results
### PARP7 Knockout MCF-7 Cells Display Increased ER\(\alpha\) Activity and E2-Induced Proliferation  
We next determined the E2-dependent ER\(\alpha\) recruitment to GREB1 and TFF1 in MCF-7 wildtype and PARP7\({}^{\text{KO}}\) cells. Cells were treated with DMSO or E2 for one hour prior to doing ChIP qPCR assays. We observed increased E2-induced recruitment of ER\(\alpha\) to the promoter region of _GREB1_ (Figure 4A) and _TFF1_ (Figure 4B) in MCF-7 PARP7\({}^{\text{KO}}\) compared with wildtype cells. Cells were then treated with DMSO or E2 for 24 h and the relative amounts of GREB1 and TFF1 mRNA levels were determined. Significantly higher E2-dependent increases GREB1 (Figure 4C) and TFF1 (Figure 4D) mRNA levels were observed in MCF-7 PARP7\({}^{\text{KO}}\) compared with wildtype cells. Interestingly, mRNA levels of TFF1 in the DMSO-treated MCF-7 PARP7\({}^{\text{KO}}\) cells were significantly higher than those of MCF-7 wildtype cells. This could indicate that PARP7 also negatively regulates ER\(\alpha\) in the absence of ligand. Figure 4: MCF-7 PARP7\({}^{\text{KO}}\) cells display increased ER\(\alpha\) activity. Recruitment of ER\(\alpha\) to the regulatory regions of (**A**) _GREB1_ and (**B**) _TFF1_ was increased in MCF-7 PARP7\({}^{\text{KO}}\) cells compared with MCF-7 wildtype cells. Cells were treated with 0.1% DMSO or 10 nM E2 for one hour. Chromatin immunoprecipitation was carried out with no antibody (control) or anti-ER\(\alpha\). Recruitment differences significantly greater than the control (\(p<0.05\)) are denoted with the letter “a”, differences due to PARP7 (\(p<0.05\)) are denoted with the hash mark #. Significant differences greater than DMSO (\(p<0.05\)) are denoted with the asterisk *. (**C**) GREB1 and (**D**) TFF1 mRNA levels were significantly higher in MCF-7 PARP7\({}^{\text{KO}}\) cells treated with 10 nM E2 for 24 h compared with MCF-7 wildtype cells. The asterisk * denotes statistical significance (\(p<0.05\)) when compared to theDMSO-treated wildtype, and hash mark # denotes significant (\(p<0.05\)) differences due to PARP7. (**E**) MCF-7 PARP7 KO cells exhibited increased proliferation in response to E2 compared with wildtype cells. Cells were treated with 0.1% DMSO or 10 nM E2 every day for 4 days. Cell proliferation was normalized to baseline (Day 1) and to the DMSO-treated samples. The hash mark # denotes statistical significance (\(p<0.05\)) between the cell lines. (**F**) MCF7 cells expressing a Tet-ON regulated HA-tagged PARP7 were cultured in the presence or absence of doxycycline (DOX) to induce (**F**) PARP7 mRNA and protein levels (inset F). Cells were then incubated with or without DOX and treated with DMSO or 10 nM of E2 for 20 h. The mRNA expression levels of (**G**) GREB1 and (**H**) TFF1 were reduced in the presence of DOX. The data are representative of three independent experiments. The asterisk \({}^{*}\) denotes significant difference (\(p<0.05\)) greater than DMSO, and the hash mark # denotes differences due to DOX. Data are shown as means \(\pm\) SEM for three independent experiments. Since E2 supports proliferation of ER positive breast cancer cells [33], we hypothesized that MCF-7 PARP7 KO cells would exhibit increased proliferation in response to E2. To test this, the proliferation of MCF-7 wildtype and PARP7 KO cells was measured after being exposed to E2 for 4 days (Figure 4E). Consistent with our hypothesis, our data showed significantly increased proliferation in the MCF-7 PARP7 KO cells compared to wildtype cells, indicating that endogenous PARP7 negatively affects breast cancer cell proliferation, possibly due to repression of ER\(\alpha\). To determine the effect of PARP7 expression on endogenous ER\(\alpha\) signalling, we used a Tet-ON PARP7 overexpression cell line, in which the expression of HA-tagged PARP7 is regulated by doxycycline (DOX). Treatment with 1.5 \(\upmu\)g/mL DOX resulted in an approximate 12-fold increase in PARP7 mRNA levels (Figure 4F) and increase in PARP7-HA protein levels (inset Figure 4F).