Single Cell Accessibility Profiling of Murine DN3 Thymocytes

Abstract

Abstract Heterogeneity is critical for immune system health, else our defenses are easily evaded by pathogens. Likewise, epigenetic diversity likely governs lineage decisions during development. Some enhancers appear to open prior to the transcription of proximal genes, which we hypothesized would create gradients of epigenetic activity in developmentally intermediate, but otherwise uniform, cells. To evaluate regulatory diversity in a fixed, well defined population, we performed single cell accessibility measurements on Rag-deficient double negative 3 (DN3) thymocytes. Computationally defined models predicted epigenetic stratification within this homogenous population. Sequence-based imputations further separated the DN3 cells via enrichment for lineage-defining transcription factor motifs, suggesting the epigenetic spectra reflects early versus late DN3 stages. As antigen receptor (AgR) loci recombine within DN3 cells, we also examined if single cell accessibility of AgR promoters and recombination signal sequences could predict pre-selection T Cell Receptor Beta (TCRb) repertoire. Compared to algorithms that use bulk measurements of chromatin composition or DNA sequence, single cell accessibility had slightly improved accuracy in predicting Vb segment choice. Collectively, our results illustrate the application, and perhaps complexities, of interpreting single cell accessibility data, and identify possible regulatory dynamics within an otherwise homogenous DN3 population.