Examining the interaction partners of essential chromatin regulators in triple- negative breast cancer

Triple Negative Breast Cancer (TNBC) is a highly invasive breast cancer subtype, which occurs with the absence of HER2, Estrogen and Progesterone receptors. The lack of specific hormone receptors causes the conventional targeted therapies to fail. The absence of such therapeutic options and the complexity of the genetic pathways led us to study the epigenetics of TNBC. From our previous studies, a novel epigenetic regulator SS18L2 was identified to be affecting TNBC cell viability and cell cycle. SS18L2 (Synovial Sarcoma Translocation Gene on Chromosome 18- Like Protein 2) is a homolog of SS18 and SS18L1, chromatin regulators found in BAF (SWI/SNF) complex. There is limited knowledge on SS18L2 and the roles of SS18L2 in cancer are elusive. To decipher the roles of SS18L2 in TNBC cell survival, we aimed to reveal the interaction partners of SS18L2 in a TNBC cell line model. To examine the interactome of SS18L2, BioID (Proximity Dependent Biotin Identification) method was utilized. The BioID fusion proteins were engineered for SS18L2 and its homologs, SS18 and SS18L1; which were all further examined with cell-based assays. First, western blot experiments revealed efficient and specific production of engineered BioID fusion proteins. The SS18L2, SS18L1, and SS18 fusion proteins were mainly localized to nucleus, as gauged by immunofluorescence staining. Biotinylation efficiencies of the cell lines overexpressing the fusion proteins and the pull-down efficiency were all tested; and N-BioID fusion constructs were chosen for further experiments. To identify the unique interaction partners of SS18L2, different than its homologs, SS18 and SS18L1, mass spectrometry was performed for all three homologs. The results showed 169 interacting partners of SS18L2, with LogFC higher than 2 and 15 hit proteins with LogFC higher than 5 and p value smaller than 0.001. These interactions were mainly from the BAF (SWI/SNF) complex, such as SMARCC1, SMARCD1, SMARCA2/4, ARID1B, GLTSCR1, DPF2, ARID1A, SMARCB1, SMARCC2 and BCL7. The interactomes of SS18 and SS18L1 were similar to that of SS18L2, although a higher number of proteins were identified (374 and 267 respectively). There were 17 unique interacting proteins for SS18L2, however the significance values for these interactions were low, prompting further studies. Overall, the study shows that SS18L2 forms complexes within BAF complex in TNBC cells. The mechanisms behind the selective cell survival regulatory role of SS18L2 remains unanswered, prompting more studies with different approaches. This thesis provides a strong groundwork for deciphering the role of SS18L2 in cancer.