To identify transcription factors (TFs) that may be implicated in regulating these cells, we searched for overrepresented DNA sequence motifs in the set of CD8+ effector cell-specific peaks (Methods). This revealed a strong overrepresentation of EOMES, TBX21 and TBX2 TF-binding motifs. However, the motifs for each of these TFs are nearly identical (Fig. 2e) and displayed the same patterns of accessibility among the cells (Fig. 2f), making it difficult to correctly identify the TF involved in binding these motifs in effector T cells. To identify putative regulatory TFs, we examined gene expression data in these cells. While EOMES and TBX21 were both expressed in T cells, TBX2 was not detected (Fig. 2g). This indicated that EOMES and TBX21 likely regulate these sites49, rather than TBX2 and highlights the ability of combined gene expression and DNA accessibility data to improve identification of TFs involved in regulating different cell states. We further examined enrichment of Tn5 integration events surrounding EOMES and TBX21 motifs sites by performing TF footprinting50, revealing a strong enrichment of integration events flanking the TF motif in CD8+ effector cells compared to CD8+ naive cells (Fig. 2h).
