FoxP3 associates with enhancer-promoter loops to regulate Treg-specific gene expression [HiChIP]

We applied proximity-ligation with chromatin immunoprecipitation (HiChIP) in Treg and closely related conventional CD4+ T cells (Tconv) to map the H3K27Ac, FoxP3 and Yy1 chromatin structures. Overall design: For analyses in FoxP3 proficient mice, splenic CD4 T cells from FoxP3-Thy1.1 hemizygous male mice were first enriched using CD4 untouched kit (Miltenyi), followed by secondary purification with anti-CD90.1 microbeads (Miltenyi) according to manufacturer's instructions. Tconv (FoxP3-Thy1.1-Tcrb+CD4+CD45+CD19-CD8-CD11b-CD11c-) and Treg cells (FoxP3-Thy1.1+Tcrb+CD4+, CD45+CD19-CD8-CD11b-CD11c-) were sorted on a FACS-Aria to 96% and 94% purity, respectively. For experiments in FoxP3-deficient Tregs (Fig. 3), CD4+ T cells from spleen and peripheral lymph nodes from 6 week-old heterozygous females were first enriched as above by negative magnetic selection (Stem Cell), and Treg-like cells sorted as CD19-TCRß+CD4+Thy1.1-GFP+ on a Moflo Astrios (Beckman Coulter).