## 4 Discussion  
Here we provide evidence that PARP7 is part of a negative feedback loop regulating ER\(\alpha\) activity. Inhibition of PARP7 resulted in increased ER\(\alpha\) protein levels and signaling, while overexpression of PARP7 repressed ER\(\alpha\) activity and decreased its recruitment to its target gene promoters. In support of these data, PARP7 knockout cells displayed increased ER\(\alpha\) activity, as shown by increased mRNA expression levels of and recruitment of ER\(\alpha\) to target genes, and increased cell proliferation in response to E2. ER\(\alpha\) was mono-ADP-ribosylated by PARP7 in response to E2, and PARP7's ability to repress ER\(\alpha\) was dependent on its catalytic activity. Taken together, these data illustrate the importance of PARP7 and mono-ADP-ribosylation in the regulation of ER\(\alpha\) activity, and possibly cell proliferation in ER positive breast cancers. Inhibiting PARP7 catalytic activity stabilized its protein levels but also those of ER\(\alpha\). PARP7 has also been reported to regulate AHR protein levels [17; 31], and more recently PARP7 has been shown to recruit both HIF-1\(\alpha\) and an E3 ubiquitin ligase HUWE1 to nuclear bodies to promote the ubiquitination and degradation of HIF-1\(\alpha\)[19]. We provide evidence implicating mono-ADP-ribosylation of ER\(\alpha\) as a mechanism to regulate its protein stability. However, the events leading to degradation of ER\(\alpha\) and whether PARP7 recruits ER\(\alpha\) together with an E3 ubiquitin ligase to nuclear bodies remain elusive. AHR functions as an E3 ligase to regulate ER\(\alpha\) and other oncogenic transcription factor levels [36]. Given the importance of PARP7 in AHR signaling it is tantalizing to speculate that PARP7 functions in concert with AHR to regulate the protein levels of these and other oncogenic transcription factors. Due to the low levels of detected mono-ADP-ribosylated peptides, we were unable to identify target residues in ER\(\alpha\), but three mono-ADP-ribosylated peptides in ER\(\alpha\) were mapped to the receptor's ligand independent transactivation domain, AF-1. In vitro ADP-ribosylation assays failed to confirm the mono-ADP-ribosylation in AF-1 (AB domains) without the presence of the D domain. The truncated CDEF variant (AF-1 deficient) was not mono-ADP-ribosylated by PARP7, suggesting that the D domain is not mono-ADP-ribosylated. How the D domain influences the ability of PARP7 to modify AF-1 region of ER\(\alpha\) is unknown. We cannot, however, exclude the possibility that there are additional mono-ADP-ribosylated residues in ER\(\alpha\) that we were unable to identify using purified ER\(\alpha\) and PARP7 proteins. Moreover, it is possible that mono-ADP-ribosylated peptides identified in heterologous expressed and purified ER\(\alpha\) may not reflect peptides or amino acid residues that are mono-ADP-ribosylated in vivo. Recent studies using enrichment of ADP-ribosylated proteins by incubation with the macrodomain protein, AF1521, have revealed that ADP-ribosylation occurs on a number of distinct amino acids, including acidic residues (Glu/Asp), arginine (Arg), serine (Ser), tyrosine (Tyr), histidine (His), and cysteine (Cys) [37; 38]. Many of these residues are present in the identified peptides and could represent potential ADP-ribose acceptor sites in ER\(\alpha\). The application of ADP enrichment strategies, which have been utilized to characterize the ADP-ribosylome, could be used to map mono-ADP-ribosylation sites in ER\(\alpha\). It is important to note, however, that the in vitro ADP-ribosylation studies done using purified proteins may not accurately reflect mono-ADP-ribosylation sites in PARP7 that occur in vivo. Based on the results presented in here, we hypothesize that PARP7 functions as a tumor suppressor in E2 responsive breast cancer cells by repressing the oncogenic actions of ER\(\alpha\). In support of this, a recent study reported that PARP7 knockdown promoted tumor growth in an MCF-7 xenograft model [19].