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Tregs require Foxp3 expression and induction of a specific DNA hypomethylation signature during development, after which Tregs persist as a self-renewing population that regulates immune system activation. Whether maintenance DNA methylation is required for Treg lineage development and stability and how methylation patterns are maintained during lineage self-renewal remain unclear. Here, we demonstrate that the epigenetic regulator ubiquitin-like with plant homeodomain and RING finger domains 1 (Uhrf1) is essential for maintenance of methyl-DNA marks that stabilize Treg cellular identity 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.

The COMPASS protein family catalyzes histone H3 Lys 4 (H3K4) methylation and its members are essential for regulating gene expression. MLL2/COMPASS methylates H3K4 on many developmental genes and bivalent clusters. To understand MLL2-dependent transcriptional regulation, we performed a CRISPR-based screen with an MLL2-dependent gene as a reporter in mouse embryonic stem cells. We found that MLL2 functions in gene expression by protecting developmental genes from repression via repelling PRC2 and DNA methylation machineries. Accordingly, repression in the absence of MLL2 is relieved by inhibition of PRC2 and DNA methyltransferases. Furthermore, DNA demethylation on such loci leads to reactivation of MLL2-dependent genes not only by removing DNA methylation but also by opening up previously CpG methylated regions for PRC2 recruitment, diluting PRC2 at Polycomb-repressed genes. These findings reveal how the context and function of these three epigenetic modifiers of chromatin can orchestrate transcriptional decisions and demonstrate that prevention of active repression by the context of the enzyme and not H3K4 trimethylation underlies transcriptional regulation on MLL2/COMPASS targets.

People with ataxia-telangiectasia (A-T) display phenotypic variability with regard to progression of immunodeficiency, sino-pulmonary disease, and neurologic decline. To determine the association between differential gene expression, epigenetic state, and phenotypic variation among people with A-T, we performed transcriptional and genome-wide DNA methylation profiling in patients with mild and classic A-T progression as well as healthy controls. RNA and genomic DNA were isolated from peripheral blood mononuclear cells for transcriptional and DNA methylation profiling with RNA-sequencing and modified reduced representation bisulfite sequencing, respectively. We identified 555 genes that were differentially expressed among the control, mild A-T, and classic A-T groups. Genome-wide DNA methylation profiling revealed differential promoter methylation in cis with 146 of these differentially expressed genes. Functional enrichment analysis identified significant enrichment in immune, growth, and apoptotic pathways among the methylation-regulated genes. Regardless of clinical phenotype, all A-T participants exhibited downregulation of critical genes involved in B cell function (PAX5, CD79A, CD22, and FCRL1) and upregulation of several genes associated with senescence and malignancy, including SERPINE1. These findings indicate that gene expression differences may be associated with phenotypic variability and suggest that DNA methylation regulates expression of critical immune response genes in people with A-T.

Pediatric acute lung injury, usually because of pneumonia, has a mortality rate of more than 20% and an incidence that rivals that of all childhood cancers combined. CD4 + T-cells coordinate the immune response to pneumonia but fail to function robustly among the very young, who have poor outcomes from lung infection. We hypothesized that DNA methylation represses a mature CD4 + T-cell transcriptional program in neonates with pneumonia. Here, we found that neonatal mice (3-4 days old) aspirated with Escherichia coli bacteria had a higher mortality rate than juvenile mice (11-14 days old). Transcriptional profiling with an unsupervised RNA-Seq approach revealed that neonates displayed an attenuated lung CD4 + T-cell transcriptional response to pneumonia compared with juveniles. Unlike neonates, juveniles up-regulated a robust set of canonical T-cell immune response genes. DNA methylation profiling with modified reduced representation bisulfite sequencing revealed 44,119 differentially methylated CpGs, which preferentially clustered around transcriptional start sites and CpG islands. A methylation difference-filtering algorithm detected genes with a high likelihood of differential promoter methylation regulating their expression; these 731 loci encoded important immune response and tissue-protective T-cell pathway components. Disruption of DNA methylation with the hypomethylating agent decitabine induced plasticity in the lung CD4 + T-cell marker phenotype. Altogether, multidimensional profiling suggested that DNA methylation within the promoters of a core set of CD4 + T-cell pathway genes contributes to the hyporesponsive neonatal immune response to pneumonia. These findings also suggest that DNA methylation could serve as a mechanistic target for disease-modifying therapies in pediatric lung infection and injury., (© 2018 McGrath-Morrow et al.)