The histone lysine methyltransferase MLL1 regulates the activation and functional specialization of regulatory T cells [ChIP-seq]

The activation and specialization of regulatory T cells (Tregs) are crucial for maintaining immune self-tolerance, but the epigenetic regulation of these processes remains largely unexplored. Here, we show that T-cell-specific deletion of the lysine methyltransferase MLL1 results in a spontaneous lymphocyte proliferation phenotype in aged mice without disturbing the development of conventional T cells and Tregs. Treg-specific MLL1 ablation leads to a systemic autoimmune disease associated with Treg dysfunction. RNA sequencing demonstrated that the induction of multiple genes involved in Treg activation, functional specialization and tissue immigration is defective in MLL1-deficient Tregs. This dysregulation is associated with defects in H3K4 trimethylation at the transcription start sites (TSSs) of these genes. Finally, using a T-bet fate-mapping mouse system, we determined that MLL1 is required to establish stable Th1-type Tregs. Thus, MLL1 plays an essential role in optimal Treg function by providing a coordinated epigenetic context for activation and specialization. Lymphocytes were isolated from the spleen and lymph nodes of 6- to 8-week-old Foxp3-Cre.RosaYFP and Foxp3-Cre.RosaYFPMll1fl/fl male mice or Foxp3RFP-Cre.RosaYFPMll1fl/fl male mice and enriched for CD4+ T cells using magnetic beads. YFP+Tregs or RFP+YFP+ and RFP+YFP- Tregs (1 x 105) were sorted to a typical purity of > 95 for CUT&TAG sequencing. Histone H3K4me3 CUT&TAG sequencing (anti-H3K4me3 CUT&TAG-seq) was performed as described in regents kits of Hyperactive pG-MNase CUT&RUN Assay Kit for Illumina (Vazyme) as described previously.Briefly, 50,000 cells for each CUT&Tag library were incubated with 100 μL balanced concanavalin A-coated magnetic beads at RT for 10 min. The bead-bound cells were resuspended with 50 μL of antibody buffer. Then added 1 ug anti-H3K4me3 antibody (Abcam, ab8580) to the bead-bound cells. After 2 h incubation at RT, the primary antibody was discarded carefully and 0.5 μg goat anti-rabbit IgG (Vazyme, Ab206-10-AA) diluted with 50μL Dig-wash buffer was added to the cells at RT for 1 h. Afterward, samples were washed gently with 800 μL Dig-wash buffer, and 2 μL pG–Tn5 together with 100 μL Dig-300 buffer was added to the samples. After 1 h incubation at RT, samples were washed gently with 800 μL Dig-300 buffer. 10 μL tagmentation buffer together with 40 μL Dig-300 buffer was then added to each sample and the samples were incubated at 37°c for 1 h. The interactions were quenched by adding 10 μL 0.5 M EDTA, 3 μL 10% SDS, and 2.5 μL 20 mg/mL Proteinase K and incubating the samples at 50°c for 1 h. Phenol–chloroform and ethanol precipitation, as well as PCR and size selection, were performed as described in Hi-C library construction. All CUT&Tag libraries were sequenced on Illumina Nova Seq 6000 platform at PE150 mode subsequently.