Modeling Enhancer Hijacking in Cancer via Genome Engineering and Single-Cell Sequencing

Structural variants (SVs) are a major contributing factor to human cancer. Non-coding structural variants were found to cause rearrangements of chromosomes, resulting in the rewiring of Enhancer-Promoter communication. Despite our knowledge of the function of enhancer-promoter re-wiring in gene regulation, recent studies in human cancer suggested that structural variants that altered gene expression nearby are rare. What distinguishes structural variants that alter nearby gene expression from those that do not remains unclear. Therefore, this gap in understanding gene regulation in the rearranged genomes prevents us from modeling gene regulation near structural variants. To address this, recent work from Dr. Xu engineered translocations near MYC and showed that the activation of MYC after rearrangements requires both adjacent enhancer activity and strong contact between enhancer and promoter. This finding suggests that the Activity-By-Contact (ABC) model can predict MYC activation after translocation. However, the ABC model is still not precise in predicting the accurate expression level. Further, it is unclear how well these models generalize to other genes in the genome, and whether additional chromatin regulatory machinery can improve the predictions. This proposal aims to investigate the role of trans-acting factors and cis-element sequences in oncogene activation in rearranged human genomes. The central hypothesis is that quantitative oncogene expression modeling should include the variable binding of trans-acting factors, as well as the promoter sequences. Dr. Xu will employ CRISPR/Cas9 genome engineering to introduce different chromosomal translocations near the oncogene, whose expression will be modeled from the single-cell multi-omic sequencing data (Aim 1). Subsequently, Dr. Xu will systematically identify TFs/co-activators specific to certain oncogenes or structural variants by performing CRISPR KO screens, followed by molecular mechanism study using a dTAG-based degron and genomic profiling (Aim 2). Finally, Dr. Xu will characterize promoter sequence features that render genes susceptible to enhancer-hijacking, by developing a novel method that interferes with sequences and simultaneously measures the genome conformation in single-cell (Aim 3). Overall, the results from this proposal will reveal new regulatory roles of TFs and other regulators in gene expression in the context of rearrangement. Dr. Xu’s career goal is to lead an independent research group devoted to applying novel computational and experimental approaches to investigate gene regulation in disease and evolution. In the year prior to K22 activation, he will continue to receive computational and experimental training from his postdoctoral mentor and consultants at the Salk Institute for Biological Studies and UT Southwestern Medical Center. The rigorous mentored support and results obtained will facilitate Dr. Xu’s transition to independence as an investigator in the K22 phase and lay the foundation for his future career. Project Number: 1K22CA293300-01A1 | Fiscal Year: 2025 | NIH Institute/Center: National Cancer Institute (NCI) | Principal Investigator: Zhichao Xu | Institution: UNIVERSITY OF TEXAS HLTH SCI CTR HOUSTON, HOUSTON, TX | Award Amount: $194,914 | Activity Code: K22 | Study Section: Special Emphasis Panel[ZCA1 RTRB-R (J1)] View on NIH RePORTER: https://reporter.nih.gov/project-details/11137314