Regulatory Role of Alu Elements on Enhancer Activity and Estrogen-Responsive Gene Expression in Breast Cancer Cells
Richi Thakral
Mentor: Dr. Sreejith Nair, Department of Oncology, Georgetown University Medical Center.
Date/Time: August 23rd, 2024 at 1:00pm.
Abstract: Alu elements are short interspersed nuclear elements (SINEs) which influence the genome through insertional mutations and recombination events. These repetitive elements span up to 300 base pairs and are implicated in various human diseases, such as breast cancer. Approximately 80% of breast cancers are estrogen receptor-positive; thus, the MCF-7 breast cancer cell line, characterized by the presence estrogen receptors (ERalpha), provides a model for investigating the mechanisms by which estrogen (E2) signals drive gene expression. It is demonstrated that enhancers, non-coding DNA regulatory elements, facilitate the transcription of target genes and play a pivotal role in E2-induced gene regulatory programs. Despite their potential significance, the specific role of Alu elements in modulating enhancer activity and E2-mediated gene expression is not well understood. My project aims to explore the spatial relationships between different enhancer types, Alu elements, and E2-activated gene regulation in MCF-7 cells to reveal novel mechanisms underlying hormone-responsive gene expression.
This study utilized lab-generated and publicly available datasets comprising E2 and vehicle-treated samples. Separate informatics pipelines were created to process ATAC-seq, ChIP-seq, and GRO-seq data, and reads were aligned to the human genome hg38 assembly. Peak calling was performed using HOMER to identify regions of interest, followed by overlapping of ERalpha, RNA polymerase II (RNApolII), and histone-modified ChIP-seq peaks and removal of promoter-proximal regions. This approach allowed for the identification of 1,185 estrogen-activated enhancers, as well as 3,165 ERalpha-bound non-active enhancers and 11,792 non-ERalpha-bound enhancers. A Python-based informatic algorithm was developed to further refine this enhancer list to calculate the log2 fold change between vehicle and estrogen-treated conditions from GRO-Seq tags, and subsequent classification of active enhancers based on E2 induction. Differential analysis of GRO-seq data identified a curated list of E2 upregulated genes. Additionally, a publicly available list of hg38 Alu elements was integrated into the analysis. Overlaps between Alu elements, ATAC-seq, and RNApolII peaks were used to identify E2-active Alu elements. Bioinformatic tools such as bedtools suite were used to identify the closest active Alu elements to each enhancer type. Additionally, the distance between the nearest E2 upregulated genes and active Alu elements was identified and compared to the nearest enhancer coordinates. Statistical significance between distances was calculated using a pairwise Mann-Whitney U test, and median distances between enhancer types, Alu elements, and E2 upregulated genes were assessed.
The analysis revealed that the distances between strong and weak active enhancers based on E2 induction was not statistically significant. Therefore, enhancer types were consolidated into three categories: ‘strong’, ‘primed (ERalpha-bound non-active enhancers)’ and ‘non ERalpha bound.’ The median distance between Alu elements and enhancers was lowest in the active group, followed by non-ERalpha bound enhancers. A significant difference was observed between the active and primed groups in terms of median distance to the nearest Alu elements (p = 1.2e-08). Similarly, the proximity of Alu-linked E2 upregulated genes to enhancers mirrored these results, with active and non-ER_ bound enhancers exhibiting significantly lower median distances compared to primed enhancers. These findings suggest that Alu elements may play a role in the regulation of enhancer activity, particularly in the context of estrogen-responsive gene expression in breast cancer cells. Future work may focus on extending this analysis using CRISPR interference (CRISPRi) to silence specific enhancer regions identified in this study. Investigating the resulting impact on tumor suppressor genes and oncogenes in breast cancer cells may offer valuable insights for developing novel therapeutic strategies.