Z-DNA underlies the target choice of Aire by facilitating promoter poising [scRNA-Seq]
Aire is an unconventional transcription factor (TF) that enhances the expression of thousands of genes in medullary thymic epithelial cells (mTECs), which promotes clonal deletion or phenotypic diversion of self-reactive T cells. The biological logic of Aire's target specificity remains largely unknown as, unlike many TFs, it does not bind to a particular DNA-sequence motif. We implemented two orthogonal approaches to investigate Aire's cis-regulatory mechanisms: construction of a convolutional neural network and leveraging of natural genetic variation via analysis of F1-hybrid mice. Both approaches nominated Z-DNA and Nfe2•Maf as putative positive influences on Aire's target choices. Genome-wide mapping studies revealed that Z-DNA-forming and Nfe2l2-binding motifs were positively associated with the inherent ability of a gene's promoter to generate DNA double-strand breaks (DSBs), and promoters showing strong DSB generation were more likely to enter a poised state with accessible chromatin and already-assembled transcriptional machinery. Consequently, Aire preferentially targeted genes with poised promoters. We propose a model whereby Z-DNA anchors the Aire-mediated transcriptional program by enhancing DSB generation and promoter poising. Beyond resolving a long-standing mechanistic conundrum, these findings suggest previously unexplored routes of manipulating T-cell tolerance. Overall design: We studied how spermidine injection influences the transcriptome of mTECs. To do so, 4-week-old female B6 WT mice were ip-injected with 15mg/kg spermidine (Sigma-Aldrich) in PBS, or just PBS as control for seven consecutive days. mTEChi and post-Aire mTEClo were then sorted. Samples were hashed using the TotalSeq-A anti-mouse (anti-CD45/MHC class I) hashtags (BioLegend). Cells were then submitted to the Broad Institute Genomics Platform for encapsulation and library preparation following 10X Genomics protocols. Two replicates were prepared for each condition.