BMI1 Maintains the Regulatory T cell Epigenomic Landscape to Prevent Inflammatory Bowel Disease (ChIP-Seq)

FOXP3+ Treg cells are expanded within the inflamed intestine of human Crohn’s disease, yet FOXP3-mediated gene repression within these cells is lost. The Polycomb repressive complexes play a role in FOXP3 target gene regulation, but deeper mechanistic insight is incomplete. We have now specifically identified the Polycomb Repressive Complex 1 (PRC1) family member, BMI1 in the regulation of a pro-inflammatory enhancer network in both human and murine Tregs. Using human Tregs and lamina propria T cells, we inferred PRC1 to regulate Crohn’s associated gene networks through assays of chromatin accessibility. Conditional deletion of BMI1 in murine FOXP3+ cells led to systemic inflammation. BMI1-deficient Tregs beared a TH1/TH17-like phenotype as assessed by assays of genome wide transcription, chromatin accessibility and proteomic techniques. Finally, BMI1 mutant FOXP3+ cells did not suppress colitis in the adoptive transfer model of human inflammatory bowel disease. We propose that BMI1 plays an important role in enforcing Treg identity in vitro and in vivo. Loss of Treg identity via genetic or transient BMI1 depletion perturbs the epigenome and converts Tregs into Th1/Th17-like proinflammatory cells, a transition relevant to human Crohn’s disease associated CD4+ T cells. Cultured Jurkat cells were harvested from suspension and pelleted down. Jurkat T cells were transiently transfected with indicated plasmids by electroporation at 315 mV for 10 ms and harvested 36 to 48 hours later. 2 x 10^6 Jurkat cells were transfected with 120 nM of ON-TARGETplus Human BMI1 siRNA (648) (Dharmacon cat# L-005230-01-0005) or 100 nM of ON-TARGETplus Human EZH2 siRNA (2146) (Dharmacon, cat# L-004218-00-0005). After treatment, he cells were incubated with 2 μg of histone modification-specific antibodies overnight at 4 °C. The following antibodies were used: anti-H3K27ac (Abcam, Cat# ab4729), anti-H3K27me3 (Cell Signaling Technology, Cat# 9733S). ChIP DNA was purified using the Mini-Elute PCR purification kit (Qiagen) after treatment with DNase-free RNase A (10 mg/mL, Thermo Scientific Cat #EN0531) and Proteinase K (20 mg/mL, Ambion, Cat #AM2546). Purified DNA was subjected to targeted real-time PCR to confirm enrichment. ChIP DNA library preparation was performed using 10 ng per reaction, and input DNA using the Ovation Ultralow DR Multiplex System (NuGen). The ChIP-seq libraries were subjected to sequencing by the Mayo Clinic Medical Genomics Core. These were sequenced to 50 base pairs from both ends using the Illumina HiSeq 2000. After adding 30 μL of protein G-agarose magnetic beads, the reactions were incubated for another 3 h. Beads were washed extensively with ChIP buffer, high-salt buffer, LiCl2 buffer, and TE buffer. Bound chromatin was eluted and reverse-crosslinked at 65 °C overnight. DNA was purified using the Mini-Elute PCR purification kit (Qiagen) after treatment with RNase A and proteinase K. Enrichment was confirmed by targeted real-time PCR in positive and negative genomic loci. For next-generation sequencing, ChIP-seq libraries were prepared from 10 ng of ChIP, and input DNA with the Ovation Ultralow DR Multiplex system (NuGEN). The ChIP-seq libraries were sequenced to 51 base pairs from both ends using the Illumina HiSeq 2000 in the Mayo Clinic Medical Genomics Core.