Developmental Bias in Transcription Factor Activity Organizes Ectopic Gene Expression in Thymic Epithelial Cells

Medullary thymic epithelial cells (mTECs) ectopically express thousands of genes that are normally restricted to other tissues to support the selection of a T cell repertoire that can distinguish self from non-self, thereby preventing autoimmune disease. mTECs have been observed to express tissue-specific genes (TSGs) in patterns that are transient, stochastic, probabilistic, and heterogeneous; however, the mechanisms that govern how patterns of TSG expression emerge throughout development in individual mTECs remain unknown. Here, we investigated how developmental progression shapes the regulatory logic of TSG expression in individual mTECs. To address this, we leveraged joint profiling of chromatin accessibility and gene expression in single mTECs and found that distinct stages of mTEC development establish discrete regulatory programs that restrict which TSG modules can be expressed. Underlying the potential to express each subset of genes was stage-specific transcription factor activity, highlighted by EOMES which emerged as a candidate regulator of the late developmental programs. Conditional genetic deletions of EOMES selectively disrupted the expression of its associated subset of tissue-specific genes and the accessibility of neighboring cis-regulatory regions without impairing overall mTEC development nor the potential to activate other tissue-specific gene modules. Analysis of transcription factor binding from additional candidate regulators further highlighted selective tissue-specific gene association. These data define a developmental trajectory in mTECs that couples each maturation stage with orthogonal drivers of ectopic transcription that act transiently on the chromatin landscape to limit the range of genes that can be expressed. These findings reveal how developmental progression constrains ectopic gene expression in mTECs to promote immune tolerance and provides a general framework to understand how cells deviate from their established cell identity to access alternative transcriptional programs.