biostudies-arrayexpressChristopher BennerMus musculushttps://www.ebi.ac.uk/biostudies/studies/E-GEOD-57272Regulatory T (Treg) cells play an indispensable role in immune homeostasis. The development and function of Tregs are dependent on transcriptional factor Foxp3, but how constant expression of Foxp3 is maintained in Tregs is not clear. Here we show that ablation of the conserved non-coding DNA sequence 2 (CNS2) at the Foxp3 locus in mice led to spontaneous lymphoproliferative disease and exacerbation of experimental autoimmune encephalomyelitis (EAE). CNS2 is required for activated Treg cells to maintain elevated Foxp3 expression, which is critical for their suppressor function and lineage stability. Mechanistically, upon TCR stimulation, NFAT binds to both CNS2 and Foxp3 promoter and mediates the interaction between CNS2 and Foxp3 promoter. Our findings demonstrated an essential role for CNS2 in maintaining the stability and function of activated Treg cells and identified NFAT as a key mediator of its function. Gene expression was profiled in T regulatory cells (Treg) in WT and CNS2 knockout mice. CNS2 knockout mice lack a conserved non-coding DNA sequence 2 (CNS2) at the Foxp3 locus. Treg cells were further sorted into Foxp3-high and Foxp3-low populations based on the expression level of Foxp3. mRNA was profiled using RNA-Seq (unstranded, polyA+, SE100) in replicate for each conditionbiostudies-arrayexpressGrowth Protocol - Total CD4+ T cells were purified from mouse lymph nodes and spleens with the Dynabeads FlowComp Mouse CD4 kit (Invitrogen). Foxp3GFP+ CD4+ T cells and naive CD4+ T cells (CD62L+ CD44- CD4+) were further purified from total CD4+ T cells by sorting on a BD FACSAria cell sorter. Intracellular staining for cytokines and Foxp3 used the Foxp3 staining kit (eBiosciences). Cells were stimulated for 4 h with PMA (phorbol 12-myristate 13-acetate; 50 ng/ml; Sigma) and ionomycin (1 µg/ml; Sigma) and treated with for an additional 1 h with Golgi-Plug (BD Biosciences) before intracellular staining for cytokines.Library Construction - RNA was extracted with the TRIzol Reagent (Life Technologies) from Foxp3hi CD4+ and Foxp3lo CD4+ Treg cells FACS-purified from male CNS2- mice and age-matched male Foxp3GFP mice. Multiplexed libraries were constructed using the Illumina TruSeq RNA Sample Prep Kit. Libraries were sequenced on the Illumina HiSeq 2500 according to the manufacturer's instructions.OrganizationMINSEQE ScoreAssays and DataMAGE-TAB FilesData Transformation - Reads from all libraries were mapped to the mouse genome (mm9) using STAR (v2.2.0c). To calculate gene expression, read counts in the exons of RefSeq transcripts where calculated using HOMER Genome_build: mm9 Supplementary_files_format_and_content: rpkm.txt.gz - tab-delimited text file with RPKM values from all experiments and gene annotation information for RefSeq transcriptsUnknownTranscriptomicsGenomicsProteomicsThe homeostasis of multicellular organisms requires terminally differentiated cells to preserve their lineage specificity. However, it is unclear whether mechanisms exist to actively protect cell identity in response to environmental cues that confer functional plasticity. Regulatory T (Treg) cells, specified by the transcription factor Foxp3, are indispensable for immune system homeostasis. Here, we report that conserved noncoding sequence 2 (CNS2), a CpG-rich Foxp3 intronic cis-element specifically demethylated in mature Tregs, helps maintain immune homeostasis and limit autoimmune disease development by protecting Treg identity in response to signals that shape mature Treg functions and drive their initial differentiation. In activated Tregs, CNS2 helps protect Foxp3 expression from destabilizing cytokine conditions by sensing TCR/NFAT activation, which facilitates the interaction between CNS2 and Foxp3 promoter. Thus, epigenetically marked cis-elements can protect cell identity by sensing key environmental cues central to both cell identity formation and functional plasticity without interfering with initial cell differentiation.RNA-seq of coding RNAMus musculusFunction of a Foxp3 cis-element in protecting regulatory T cell identity.Li X, Liang Y, LeBlanc M, Benner C, Zheng YYe ZhengChristophter BennerChristopher BennerMathias LeBlancXudong LiYuqiong LiangfalseConserved non-coding sequence 2 (CNS2) of the Foxp3 genomic locus is essential for the function and stability of regulatory T cells during their activationRegulatory T (Treg) cells play an indispensable role in immune homeostasis. The development and function of Tregs are dependent on transcriptional factor Foxp3, but how constant expression of Foxp3 is maintained in Tregs is not clear. Here we show that ablation of the conserved non-coding DNA sequence 2 (CNS2) at the Foxp3 locus in mice led to spontaneous lymphoproliferative disease and exacerbation of experimental autoimmune encephalomyelitis (EAE). CNS2 is required for activated Treg cells to maintain elevated Foxp3 expression, which is critical for their suppressor function and lineage stability. Mechanistically, upon TCR stimulation, NFAT binds to both CNS2 and Foxp3 promoter and mediates the interaction between CNS2 and Foxp3 promoter. Our findings demonstrated an essential role for CNS2 in maintaining the stability and function of activated Treg cells and identified NFAT as a key mediator of its function. Gene expression was profiled in T regulatory cells (Treg) in WT and CNS2 knockout mice. CNS2 knockout mice lack a conserved non-coding DNA sequence 2 (CNS2) at the Foxp3 locus. Treg cells were further sorted into Foxp3-high and Foxp3-low populations based on the expression level of Foxp3. mRNA was profiled using RNA-Seq (unstranded, polyA+, SE100) in replicate for each condition2014-09-04T00:00:00Z2023-09-15T13:22:30.058Z2022-03-15T05:01:32.525ZE-GEOD-5727225126782SRP041655EFO_000373810.1016/j.cell.2014.07.030