Gene Regulatory Programs that Specify Age-Related Differences during Thymocyte Development

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. We identify transcription factors whose activities likely underlie alternative gene expression programs and phenotypes in adults and neonatal thymocytes. In particular, we identify and focus on a conserved transcriptional regulator, Zbtb20, which is preferentially expressed in adult HSCs and during thymocyte development. We employ a CRISPR-based perturbation approach coupled with single-cell RNA sequencing as a readout to validate the role of Zbtb20 in thymocyte development. To compare T cell development in control and Zbtb20 knockdown ATOs, we generated ATOs from EGFP-Cas9 HSCs (Lineage−, Sca1(hi), c-Kit(hi) HSCs) that either received no gRNA, gRNAs against EGFP (sgGFP) or against Zbtb20 (sgZbtb20-1 or 2), and harvested thymocytes at different timepoints.