Cutting Edge: CRISPR-Based Transcriptional Regulators Reveal Transcription-Dependent Establishment of Epigenetic Memory of Foxp3 in Regulatory T Cells

Transcription factor Foxp3 specifies and maintains regulatory T cell (Treg) identity. During Treg differentiation, a CpG-rich Foxp3 intronic enhancer, conserved noncoding sequence 2 (CNS2), is activated via DNA demethylation to establish epigenetic memory of Foxp3 expression to protect Treg identity. However, it is unclear how this epigenetic memory of Foxp3 expression is established, as CNS2 is thought to be demethylated independently of Foxp3 expression. In this article, we uncover an unexpected causal relationship between Foxp3 -transcriptional activation and CNS2 demethylation in mice. CRISPR/dCas9-mediated Foxp3 -transcriptional activation elicits CNS2 demethylation. Sustaining Foxp3 -transcriptional activation in induced Tregs also promotes CNS2 demethylation, enhancing Treg lineage stability and suppressive function. Importantly, CRISPR-mediated silencing of Foxp3 ranscription, but not protein expression, abolishes CNS2 demethylation. The novel finding tha Foxp3 -transcriptional activation promotes CNS2 demethylation may facilitate the development of Treg-based therapies and represent a general mechanism for the establishment of epigenetic memory of immune gene expression.