Interrogation of the functionality of ERα binding sites with STARR-seq

Breast cancer (BCa) and Endometrial cancer (EnCa) are the most common cancer types worldwide in the female population. The common point of these cancer types is Estrogen Receptor α (ERα) activity which is a transcriptional factor controlling a variety of genes important for drug response, cell proliferation and survival. Active ERα translocates from cytoplasm to the nucleus where it can bind thousands of regions, ERα binding sites (ERBSs). These regions are known as regulatory elements controlling the genes related to the initiation and progression of BCa and EnCa. Considering the critical role of ERα in both cancer types, a better understanding of how ERα-bound enhancers drive the transcription of target genes is promising to clarify unique pathways and to develop effective treatment strategies. To characterize the ERBSs in BCa and EnCa, we aimed to assess the enhancer activity of clinical ERBSs in a quantitative manner with a novel massively parallel reporter assay, STARR-seq. This will allow us to generate the first functional "map" of transcriptional activity of ERα in two systems, from these maps ERα regulated enhancers can be classified as inducible ones, constitutively active ones, and inactive ones. As a first step of this collaborative project, clinically relevant ERBSs were identified by ERα ChIP-seq from ERα positive BCa and EnCa patients to create a custom STARR-Seq library. As a model, MCF7 cells (breast cancer) and Ishikawa cells (endometrial cancer) were used to assess the transcriptional activity of candidate ERBSs to assess pathologically important enhancer regions collected from patients. Within the scope of this thesis, we optimized the experimental conditions to deliver a custom STARR-Seq library to cell lines models, MCF7 and Ishikawa, and after optimization; we prepared samples required for next-generation sequencing and bioinformatic analysis. The bioinformatics analysis will be performed as the final step. Overall, the final goal is to identify and categorize the activity of clinically detected ERBSs and generate a functional mapping of ERα enhancer activity.