Control of Foxp3 induction and maintenance by sequential histone acetylation and DNA demethylation [ChIP,CutRun]

Regulatory T (Treg) cells play crucial roles in suppressing deleterious immune response. Here, we investigate how mechanistically Treg cells are induced and stabilized via transcriptional regulation of Treg lineage–specifying factor Foxp3. Acetylation of histone tails in the Foxp3 locus is induced during Treg cell development and required in cis for the initiation of Foxp3 transcription. Upon induction, histone acetylation signal largely acts in trans to sustain Foxp3 transcription via multiple factors. Subsequently, Tet-mediated DNA demethylation of Foxp3 cis-regulatory elements particularly enhancer CNS2 increases chromatin accessibility and protein binding, drastically stabilizing Foxp3 transcription, although histone acetylation still regulates global gene expression. These processes transform stochastic Treg cell induction into a stable cell fate, with the former sensitive and latter resistant to genetic and environmental perturbations. Thus, our study reveals distinct roles of histone acetylation in Foxp3 induction and maintenance, reflecting sequential mechanical switches governing Treg cell lineage specification. Overall design: Examination of chromatin accessibility, Stat5 binding, histone modifications, DNA methylation, RNA Polymerase II occupation, and nascent RNA expression in naive CD4 T cells, effector CD4 T cells, regulatory T cells, and in vitro induced regulatory T cells with or without ascorbic acid treatment, or in combination with JQ1 treatment.