library_construction_protocol: For T-bet and  Foxp3 CUT&Tag assay, 2×10^5 WT and V408M Treg cells sorted as described  above from WT and V408M mice were prepared for each reaction. Treg cells were isolated and subjected to CUT&Tag profiling using the Vazyme CUT&Tag Kit (TD904-01) according to the manufacturer's instructions. Briefly, purified Treg cells were incubated with primary antibodies, followed by binding of secondary antibody and pA-Tn5 transposase complex to enable site-specific tagmentation. After activation of transposase, genomic DNA fragments containing sequencing adapters were released and purified. For ATAC-seq, freshly isolated Treg cells were processed for nuclei preparation, followed by transposition using Tn5 transposase to simultaneously fragment accessible chromatin and insert sequencing adapters. Transposed DNA fragments were then purified and PCR-amplified to construct sequencing libraries. CUT&Tag DNA samples were quantified using a Qubit 3.0 Fluorometer (Invitrogen) and qualified by Agilent Bioanalyzer 2100(Agilent Technologies). Next generation sequencing library preparations were constructed following the manufacturer's protocol. For each sample, CUT&Tag product was used for library preparation. The CUT&Tag product was treated with End Prep Enzyme Mix for end repairing, 5' Phosphorylation and dAtailing in one reaction, followed by ligation to adaptors with a “T” base overhang. Adaptor-ligated DNA was then recovered using Beads. Each sample was then amplified by PCR using P5 and P7 primers, with both primers carrying sequences which can anneal with flowcell to perform bridge PCR and P7 primer carrying index allowing for multiplexing. The PCR products were cleaned up using beads, validated using an Agilent 2100 Bioanalyzer, and quantified by Qubit 3.0 Fluorometer. Then libraries with different indices were multiplexed and loaded on an Illumina Novaseq instrument according to manufacturer's instruction.