Gene Regulatory Programs that Specify Age-Related Differences during Thymocyte Development (ATAC-Seq)

T cell development is fundamental to immune system establishment, yet how this development changes with age remains poorly understood. Here, we construct a transcriptional and epigenetic atlas of T cell developmental programs in neonatal and adult mice, revealing the ontogeny of divergent gene regulatory programs. We identify a gene module that diverges with age from the earliest stages of genesis and includes programs that govern effector response and cell cycle regulation. Interestingly, the epigenetic atlas reveals that neonates possess more accessible chromatin during early thymocyte development, likely establishing poised gene expression programs that manifest later in thymocyte development. Finally, we leverage this atlas, employing a CRISPR-based perturbation approach coupled with single-cell RNA sequencing as a readout to uncover a conserved transcriptional regulator, Zbtb20, that contributes to age-dependent differences in T cell development. Taken together, our study defines transcriptional and epigenetic programs that regulate age-specific differences in T-cell development. Overall design: We set out to determine how chromatin accessibilty profiles change between adults and neonates during thymocyte development. To this end, we isolated cells from thymi and spleens of neonatal (5-7 days post-birth) and adult (12 weeks) mice and subjected these cells to ATAC-seq. Antibodies to CD4, CD8, CD44 and CD25 were utilized for flow cytometry to isolate thymocytes at different stages of development. We obtained cells from four stages of thymocyte development: pooled Double Negative (DN1-3 cells; referred to as DN here), double-positive and single-positive CD8 cells (DP and SP8, respectively) and mature splenic naïve CD8+ T cells. We generated four biological replicates of ATAC-seq profiles from both adult and neonatal mice for all four developmental stages (DN, DP, SP8 and CD8).