ChIP samples were prepared using a SimpleChIP Plus Enzymatic Chromatin IP Kit (9005S, Cell Signaling) as previously described. Treg cells (2-8 x 105 per ChIP-seq or 1-4 x 105 per ChIP-PCR) were activated with anti-CD3 antibody and anti-CD28 antibody in the presence of IL-2 for 60 h, crosslinked in culture medium containing 1% formaldehyde at room temperature for 10 min, and glycine solution was added to stop the reaction. After lysing cells, nuclei were isolated and treated with micrococcal nuclease (0.00313 mL mL-1) for 20 min at 37oC, and the reaction was stopped by adding 0.05 M EGTA. Samples were then sonicated to disrupt nuclear membranes and centrifuged to collect supernatants containing chromatin. Chromatin solutions were incubated with 1 mg of antibodies overnight at 4oC with rotation, and complexes of antibodies and chromatin were collected with Protein G magnetic beads (Veritas; DB10003). Beads were washed five times with low-salt wash solution and three times with high-salt wash solution buffer (incubated for 5 min for each washing) at 4oC. Chromatin was eluted, de-crosslinked following the manufacturer's instructions, purified by phenol/chloroform extraction, and used for ChIP-sequencing . To prepare ChIP-seq libraries, immunoprecipitated DNA was blunt-ended and ligated with adaptors using a KAPA Hyper Prep Kit (KAPA Biosystems; KK8500). DNA was then cleaned up with an Agencort AMPure XP (Beckman Coulter; A63880) at a 1.8x DNA ratio, amplified by PCR, and purified using the AMPure XP at a 1.2x DNA ratio. Library DNA was size-selected using a 2% agarose gel cassette of Blue Pippin (Sage Science) for a target size range 150-300 bp, quantified with droplet digital PCR (Bio-Rad), and then sequenced on an Illumina HiSeq 4000 to obtain 10 million uniquely aligned reads.