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Abstract

Improving understanding of transcriptional regulation is important in T cells, given the growing impact of genetically engineered T cells in gene therapy. Although many transcription factors (TFs) central to T-cell identity have been extensively studied, far less is known about the broader set of genomic features that may contribute to T-cell-specific gene expression. Defining these features will advance understanding of T-cell biology and support the development of cell-type-specific synthetic promoters for therapeutic applications. We performed a comprehensive and unbiased search for genomic features enriched in regulatory elements associated with T-cell-specific genes compared to those linked to broadly expressed T-cell genes. We identified 2,036 previously uncharacterised motifs and 629 known transcription factor binding sites (TFBS) that are enriched both individually and in specific combinations, in the regulatory elements for genes exhibiting T-cell-specific gene expression. These novel motifs, which are absent from current models of T-cell gene regulation, emerged as strong candidates for modulating gene transcription in T cells. To determine whether these novel motifs can directly modulate gene transcription in T cells, we constructed a synthetic self-transcribing active regulatory region sequencing (STARR-seq) library of ~60,000 synthetic regulatory elements. This library captured all possible three-way combinations of a subset of 18 candidate motifs. Overall, our results revealed novel motifs that are capable of modulating gene transcription in T cells, with some exhibiting stronger regulatory effects than TFBS for TFs with established roles in T cells. Further, we demonstrate that the regulatory activity of these motifs is influenced by variables known to dictate a regulatory grammar for TFBS, such as orientation, copy number, and their order. Overall, these results highlight our incomplete understanding of the relationship between sequence composition and T-cell gene regulation and indicate that previously annotated TFBS represent only a subset of motifs capable of modulating transcription in T cells.