UC San Diego

Proteins perform their roles in the cell through interactions -- with DNA, with other proteins, or with other molecular substrates. Protein-protein interactions are especially important during the regulation of transcription, as many transcription factors come together to bind to each other and to DNA to form a transcriptional complex. Mutations can disrupt these interactions, leading to abnormal gene regulation, expression, and possibly disease. Here, we analyze the 3D structure of the core binding factor (CBF) complex to create a library of mutations for its subunits CBFB and RUNX1. We use ScalablE fUnctional Screening by Sequencing (SEUSS) to study the effects of the RUNX1 library in myelogenous leukemia K562 cells using single cell RNA sequencing. We then further investigate the effects of selected RUNX1 mutations on chromatin accessibility and gene expression using bulk ATAC and RNA sequencing. We find that our library design was able to select mutations that perturbed interfaces of the complex, resulting in loss of function-like and hypomorphic phenotypes with effects on distinct cellular pathways. Our bulk ATAC and RNA sequencing analysis reveals enrichment of RUNX1 binding in accessible DNA regions associated with regulation of differentially expressed genes. Our work shows the potential of targeting the binding interfaces of a protein to gain insight into how disruption of its function may cause downstream effects in the cell.