PCR was performed using the following primers (IDT, standard desalting): forward (F) CTTGATCTGTGCCCTGCAT, reverse 1 (R1) ACCTCTGCTCTGATGGCTGT, and reverse 2 (R2) CCGAGGACACTCAAGAGAGC
July 27, 2021
PCR was performed using the following primers (IDT, standard desalting): forward (F) CTTGATCTGTGCCCTGCAT, reverse 1 (R1) ACCTCTGCTCTGATGGCTGT, and reverse 2 (R2) CCGAGGACACTCAAGAGAGC. by repressing effector T cell transcriptional programs. Constitutive and induced deficiency of Uhrf1 within Foxp3+ cells resulted in global yet nonuniform loss of DNA methylation, derepression of inflammatory transcriptional programs, destabilization of the Treg lineage, and spontaneous inflammation. These findings support a paradigm in which maintenance DNA methylation is required in distinct regions of the Treg genome for both lineage establishment and stability of identity and suppressive function. gene exhibit the scurfy phenotype, succumbing to multiorgan lymphoproliferative inflammation approximately 4 weeks after birth (4, 5). Humans with mutations develop comparable endocrine and enteral inflammation as part of immunodysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome (6). Treg dysfunction contributes to the pathogenesis of numerous autoimmune conditions, including systemic lupus erythematosus (7C11) and systemic sclerosis (12). In contrast, modern malignancy immunotherapy blocks Treg-suppressive function to disinhibit effector T cellCmediated killing of malignant cells (13C15). Thus, mechanisms involved with both development and stability of the Treg lineage represent targets for therapies aimed at amelioration of autoimmune and malignant diseases (3, 16). Tregs develop from CD4Csingle-positive autoreactive thymocytes that receive signals via CD28/Lck, the IL-2/CD25/Stat5 axis, and T cell receptor engagement by MHC self-peptide complexes (17). These events induce Foxp3 expression OTSSP167 and independently establish a Treg-specific cytosine-phospho-guanine (CpG) hypomethylation pattern at certain genomic loci, including the locus and other loci whose gene products are important for Treg lineage identity and suppressive function (18C20). Consistent with the Treg requirement for CpG hypomethylation, pharmacologic inhibition of DNA methyltransferase activity is sufficient to induce Foxp3 expression in mature standard CD4+ T cells and to potentiate Treg-suppressive function in multiple models of inflammation (21C24). In contrast, conditional constitutive genetic deletion of the DNA methyltransferase Dnmt1 but not Dnmt3a in developing Tregs diminishes their figures and suppressive function (25). Treg-specific Dnmt1 deficiency decreases global methyl-CpG content while maintaining the Treg-specific CpG hypomethylation pattern at results in embryologic lethality (34), phenocopying of homozygous loss of (39). Conditional loss of Uhrf1 in T cells using a sequences at the gene locus (promoter (mice with female mice generated F1 pups in statistically Mendelian ratios, although male offspring were underrepresented at the time of genotyping (approximately 3 weeks of age) (Supplemental Physique 1B). mice appeared normal at birth, but then exhibited spontaneous mortality, with a median survival of 28.5 days (Figure 1A). Beginning at approximately 3 weeks of age, mice were smaller than littermate control mice (= 9) and (= 14) mice Mouse monoclonal to CD95(FITC) compared using the log-rank (Mantel-Cox) test. (B) Gross photographs of 3- to 4-week-old littermate and mice along with photomicrographs of skin. Scale bars: 100 m. (C) Photomicrographic survey of organ pathology. Level bars: 100 m. (D) CD3+ T cell subsets in selected organs. For lung, = 5 (littermate) and OTSSP167 = 10 (= 6 (littermate) and = 4 (= 6 (littermate) and = 4 (= 3 (littermate and = 5 per group. Summary plots show all data points with mean and SD. *< 0.05; **< 0.01; ?< 0.001; ?< 0.0001; NS, not significant by the 2-stage linear step-up process of Benjamini, Krieger, and Yekutieli with = 5%; exact values are in Supplemental Data. Observe Supplemental Table 3 for fluorochrome abbreviations. Treg-specific Uhrf1-deficient mice showed other indicators of lymphocyte-driven immune system activation, including splenomegaly and splenic structural disarray characterized by architectural disruption OTSSP167 and lymphoid hyperplasia (Supplemental Physique 1, DCH). CD3+CD4+ T cells in the spleen displayed an activated profile, exhibiting an increased frequency and total number of CD44hiCD62Llo effector.