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The potential for early thymic developmental events to program epigenetic states that influence adult T cell physiology remains an important question in health. Herein using the Cd4 locus as a paradigm for early developmental programming, we demonstrate that DNA demethylation during thymic development is critical for the licensing of a novel stimulus-responsive element that serves to maintain CD4 gene expression in effector T cells. We document the importance of maintaining high CD4 expression during parasitic infection and show that by driving transcription, this stimulus-responsive element allows for the maintenance of H3K4me3 levels during T cell replication, which is critical for repelling de novo DNA methylation at the Cd4 promoter. A failure to undergo epigenetic programming during development leads to gene silencing during effector T cell replication, thus providing evidence that early development can program stimulus-responsive elements to propagate a stable epigenetic state in effector T cells, with important biological consequences.

The repressive effect of DNA methylation at promoters is well-known. However, its role within conserved sequences in intragenic and intergenic regions is less clear. Using Cd4 as a model gene, here we show that DNA methylation regulates the function of stimulus-responsive regulatory elements in effector T cells. Two cis-elements orchestrate intra-and intergenic DNA demethylation of the Cd4 gene during thymic development, which in turn licenses a stimulus-responsive element, E4a, for its later function in effector cells. Deficiency in DNA demethylation leads to impaired E4a function, reduced H3K4me3 promoter levels and an inability to repel de novo DNA methylation during replication, ultimately leading to gene silencing. This physiological reduction in CD4 expression leads to a defect in Th1 polarization during cutaneous Leishmaniasis. Similar patterns of regulation were observed in a broad number of genes, highlighting an essential role for DNA demethylation during thymic development in modulating the function of stimulus-responsive elements.

While the pathophysiology of schizophrenia has been extensively investigated using homogenized postmortem brain samples, few studies have examined changes in brain samples with techniques that may attribute perturbations to specific cell types. To fill this gap, we performed microarray assays on mRNA isolated from anterior cingulate cortex (ACC) superficial and deep pyramidal neurons from 12 schizophrenia and 12 control subjects using laser-capture microdissection. Among all the annotated genes, we identified 134 significantly increased and 130 decreased genes in superficial pyramidal neurons, while 93 significantly increased and 101 decreased genes were found in deep pyramidal neurons, in schizophrenia compared to control subjects. In these differentially expressed genes, we detected lamina-specific changes of 55 and 31 genes in superficial and deep neurons in schizophrenia, respectively. Gene set enrichment analysis (GSEA) was applied to the entire pre-ranked differential expression gene lists to gain a complete pathway analysis throughout all annotated genes. Our analysis revealed overrepresented groups of gene sets in schizophrenia, particularly in immunity and synapse-related pathways, suggesting the disruption of these pathways plays an important role in schizophrenia. We also detected other pathways previously demonstrated in schizophrenia pathophysiology, including cytokine and chemotaxis, postsynaptic signaling, and glutamatergic synapses. In addition, we observed several novel pathways, including ubiquitin-independent protein catabolic process. Considering the effects of antipsychotic treatment on gene expression, we applied a novel bioinformatics approach to compare our differential expression gene profiles with 51 antipsychotic treatment datasets, demonstrating that our results were not influenced by antipsychotic treatment. Taken together, we found pyramidal neuron-specific changes in neuronal immunity, synaptic dysfunction, and olfactory dysregulation in schizophrenia, providing new insights for the cell-subtype specific pathophysiology of chronic schizophrenia.

The inheritance of gene expression patterns is dependent on epigenetic regulation, but the establishment and maintenance of epigenetic landscapes during T cell differentiation are incompletely understood. Here we show that two stage-specific Cd4 cis-elements, the previously characterized enhancer E4p and a novel enhancer E4m, coordinately promote Cd4 transcription in mature thymic MHC-II-specific T cells, in part through the canonical Wnt pathway. Specifically, E4p licenses E4m to orchestrate DNA demethylation by TET1 and TET3, which in turn poises the Cd4 locus for transcription in peripheral T cells. Cd4 locus demethylation is important for subsequent Cd4 transcription in activated peripheral T cells wherein these cis-elements become dispensable. By contrast, in developing thymocytes the loss of TET1/3 does not affect Cd4 transcription, highlighting an uncoupled event between transcription and epigenetic modifications. Together our findings reveal an important function for thymic cis-elements in governing gene expression in the periphery via a heritable epigenetic mechanism., The expression of CD4, a critical co-receptor providing T cell help in adaptive immunity, is finely tuned during development. Here the authors show that two enhancer elements, E4p and the newly-defined E4m, coordinate the expression and heritable demethylation of Cd4 in thymocytes but are dispensable for its sustained expression in peripheral T cells.

SummaryTargeting glycolysis has been considered therapeutically intractable owing to its essential housekeeping role. However, the context-dependent requirement for individual glycolytic steps has not been fully explored. We show that CRISPR-mediated targeting of glycolysis in T cells in mice results in global loss of Th17 cells, whereas deficiency of the glycolytic enzyme glucose phosphate isomerase (Gpi1) selectively eliminates inflammatory encephalitogenic and colitogenic Th17 cells, without substantially affecting homeostatic microbiota-specific Th17 cells. In homeostatic Th17 cells, partial blockade of glycolysis upon Gpi1 inactivation was compensated by pentose phosphate pathway flux and increased mitochondrial respiration. In contrast, inflammatory Th17 cells experience a hypoxic microenvironment known to limit mitochondrial respiration, which is incompatible with loss of Gpi1. Our study suggests that inhibiting glycolysis by targeting Gpi1 could be an effective therapeutic strategy with minimum toxicity for Th17-mediated autoimmune diseases, and, more generally, that metabolic redundancies can be exploited for selective targeting of disease processes.

The transcriptional repression of alternative lineage genes is critical for cell fate commitment. Mechanisms by which locus-specific gene silencing is initiated and heritably maintained during cell division are not clearly understood. To study the maintenance of silent gene states, we investigated how the Cd4 gene is stably repressed in CD8+ T cells. Through CRISPR and shRNA screening, we identified the histone chaperone CAF-1 as a critical component for Cd4 repression. We found that the large subunit of CAF-1, Chaf1a, requires the N-terminal KER domain to associate with the histone deacetylases HDAC1/2 and the histone demethylase LSD1, enzymes that also participate in Cd4 silencing. When CAF-1 was lacking, Cd4 derepression was markedly enhanced in the absence of the de novo DNA methyltransferase Dnmt3a but not the maintenance DNA methyltransferase Dnmt1. In contrast to Dnmt1, Dnmt3a deficiency did not significantly alter levels of DNA methylation at the Cd4 locus. Instead, Dnmt3a deficiency sensitized CD8+ T cells to Cd4 derepression mediated by compromised functions of histone-modifying factors, including the enzymes associated with CAF-1. Thus, we propose that the heritable silencing of the Cd4 gene in CD8+ T cells exploits cooperative functions among the DNA methyltransferases, CAF-1, and histone-modifying enzymes.

Background Mental and physical health treatment should be delivered together for children and young people with epilepsy. Training healthcare professionals (HCPs) in epilepsy services to deliver mental health interventions is an important way to facilitate integrated care. Objective To determine the feasibility of remotely delivered assessment and psychological treatment for mental health difficulties delivered by HCPs in pediatric epilepsy clinics with limited formal training in psychological interventions. We hypothesized that it would be (i) feasible to train HCPs to deliver the psychological intervention and (ii) that participants receiving the psychological therapy would report reductions in symptoms of mental health difficulties including anxiety, depression, and behavior difficulties and improve quality of life. Methods Thirty-four children and young people with epilepsy who had impairing symptoms of a common mental health difficulty (anxiety, depression, disruptive behavior, and/or trauma) were allocated to receive 6 months of a modular cognitive behavioral intervention delivered by a HCP with limited formal psychological therapy experience. Thirteen HCPs were trained in delivery of the intervention. Healthcare professional competence was assessed in a two-stage process. Parent-reported measures of mental health symptoms and quality of life were completed at baseline and following the intervention. Paired t-tests were used to analyze changes in symptoms over time. Results All thirteen HCPs who participated in the training were considered competent in therapeutic delivery by the end of the training period. Twenty-three patients completed pre- and post-intervention measures and were included in the analysis. There were statistically significant improvements in: symptoms of mental health problems (p = 0.01; Cohen’s d = 0.62), total impact of mental health problems (p = 0.03; Cohen’s d = 0.52), anxiety and depression symptoms (p = 0.02; Cohen’s d = 0.57) and quality of life (p = 0.01; Cohen’s d = 0.57). Conclusion A modular cognitive behavioral treatment delivered over the telephone by HCPs with limited experience of psychological therapy was feasible and effective in treating mental health problems in children and young people with epilepsy. Health-related Quality of Life also improved over the duration of treatment. A randomized controlled trial (RCT) is needed to demonstrate efficacy of the intervention.

Summary Targeting glycolysis has been considered therapeutically intractable owing to its essential housekeeping role. However, the context-dependent requirement for individual glycolytic steps has not been fully explored. We show that CRISPR-mediated targeting of glycolysis in T cells in mice results in global loss of Th17 cells, whereas deficiency of the glycolytic enzyme glucose phosphate isomerase (Gpi1) selectively eliminates inflammatory encephalitogenic and colitogenic Th17 cells, without substantially affecting homeostatic microbiota-specific Th17 cells. In homeostatic Th17 cells, partial blockade of glycolysis upon Gpi1 inactivation was compensated by pentose phosphate pathway flux and increased mitochondrial respiration. In contrast, inflammatory Th17 cells experience a hypoxic microenvironment known to limit mitochondrial respiration, which is incompatible with loss of Gpi1. Our study suggests that inhibiting glycolysis by targeting Gpi1 could be an effective therapeutic strategy with minimum toxicity for Th17-mediated autoimmune diseases, and, more generally, that metabolic redundancies can be exploited for selective targeting of disease processes.

The transcriptional repression of alternative lineage genes is critical for cell fate commitment. Mechanisms by which locus-specific gene silencing is initiated and heritably maintained during cell division are not clearly understood. To study the maintenance of silent gene states, we investigated how the Cd4 gene is stably repressed in CD8(+) T cells. Through CRISPR and shRNA screening, we identified the histone chaperone CAF-1 as a critical component for Cd4 repression. We found that the large subunit of CAF-1, Chaf1a, requires the N-terminal KER domain to associate with the histone deacetylases HDAC1/2 and the histone demethylase LSD1, enzymes that also participate in Cd4 silencing. When CAF-1 was lacking, Cd4 derepression was markedly enhanced in the absence of the de novo DNA methyltransferase Dnmt3a but not the maintenance DNA methyltransferase Dnmt1. In contrast to Dnmt1, Dnmt3a deficiency did not significantly alter levels of DNA methylation at the Cd4 locus. Instead, Dnmt3a deficiency sensitized CD8(+) T cells to Cd4 derepression mediated by compromised functions of histone-modifying factors, including the enzymes associated with CAF-1. Thus, we propose that the heritable silencing of the Cd4 gene in CD8(+) T cells exploits cooperative functions among the DNA methyltransferases, CAF-1, and histone-modifying enzymes.

Both microbial and host genetic factors contribute to the pathogenesis of autoimmune disease1-4. Accumulating evidence suggests that microbial species that potentiate chronic inflammation, as in inflammatory bowel disease (IBD), often also colonize healthy individuals. These microbes, including the Helicobacter species, have the propensity to induce autoreactive T cells and are collectively referred to as pathobionts4-8. However, an understanding of how such T cells are constrained in healthy individuals is lacking. Here we report that host tolerance to a potentially pathogenic bacterium, Helicobacter hepaticus (H. hepaticus), is mediated by induction of RORγt+Foxp3+ regulatory T cells (iTreg) that selectively restrain pro-inflammatory TH17 cells and whose function is dependent on the transcription factor c-Maf. Whereas H. hepaticus colonization of wild-type mice promoted differentiation of RORγt-expressing microbe-specific iTreg in the large intestine, in disease-susceptible IL-10-deficient animals there was instead expansion of colitogenic TH17 cells. Inactivation of c-Maf in the Treg compartment likewise impaired differentiation of bacteria-specific iTreg, resulting in accumulation of H. hepaticus-specific inflammatory TH17 cells and spontaneous colitis. In contrast, RORγt inactivation in Treg only had a minor effect on bacterial-specific Treg-TH17 balance, and did not result in inflammation. Our results suggest that pathobiont-dependent IBD is a consequence of microbiota-reactive T cells that have escaped this c-Maf-dependent mechanism of iTreg-TH17 homeostasis.

Both microbial and host genetic factors contribute to the pathogenesis of autoimmune diseases. There is accumulating evidence that microbial species that potentiate chronic inflammation, as in inflammatory bowel disease, often also colonize healthy individuals. These microorganisms, including the Helicobacter species, can induce pathogenic T cells and are collectively referred to as pathobionts. However, how such T cells are constrained in healthy individuals is not yet understood. Here we report that host tolerance to a potentially pathogenic bacterium, Helicobacter hepaticus, is mediated by the induction of RORγt+FOXP3+ regulatory T (iTreg) cells that selectively restrain pro-inflammatory T helper 17 (TH17) cells and whose function is dependent on the transcription factor c-MAF. Whereas colonization of wild-type mice by H. hepaticus promoted differentiation of RORγt-expressing microorganism-specific iTreg cells in the large intestine, in disease-susceptible IL-10-deficient mice, there was instead expansion of colitogenic TH17 cells. Inactivation of c-MAF in the Treg cell compartment impaired differentiation and function, including IL-10 production, of bacteria-specific iTreg cells, and resulted in the accumulation of H. hepaticus-specific inflammatory TH17 cells and spontaneous colitis. By contrast, RORγt inactivation in Treg cells had only a minor effect on the bacteria-specific Treg and TH17 cell balance, and did not result in inflammation. Our results suggest that pathobiont-dependent inflammatory bowel disease is driven by microbiota-reactive T cells that have escaped this c-MAF-dependent mechanism of iTreg-TH17 homeostasis.

In this Letter, the ‘Competing interests’ statement should have stated: ‘D.R.L. consults for and has equity in Vedanta Biosciences.’ The original Letter has not been corrected.

The assembly of the preinitiation complex (PIC) occurs upstream of the +1 nucleosome which, in yeast, obstructs the transcription start site and is frequently assembled with the histone variant H2A.Z. To understand the contribution of the transcription machinery in the disassembly of the +1 H2A.Z nucleosome, conditional mutants were used to block PIC assembly. A quantitative ChIP-seq approach, which allows detection of global occupancy change, was employed to measure H2A.Z occupancy. Blocking PIC assembly resulted in promoter-specific H2A.Z accumulation, indicating that the PIC is required to evict H2A.Z. By contrast, H2A.Z eviction was unaffected upon depletion of INO80, a remodeler previously reported to displace nucleosomal H2A.Z. Robust PIC-dependent H2A.Z eviction was observed at active and infrequently transcribed genes, indicating that constitutive H2A.Z turnover is a general phenomenon. Finally, sites with strong H2A.Z turnover precisely mark transcript starts, providing a new metric for identifying cryptic and alternative sites of initiation. DOI: http://dx.doi.org/10.7554/eLife.14243.001, eLife digest To fit the genetic information of an animal, yeast or other eukaryote into cells, DNA is tightly wound around proteins called histones to form repeating units known as nucleosomes. However, this tight winding prevents proteins from accessing the DNA, and so prevents gene transcription – the first stage of producing the molecules encoded by a gene. For transcription to take place, nucleosomes at DNA sequences called promoters must be reorganized and disassembled, thereby allowing proteins to bind to and engage these sequences and to turn nearby genes on. H2A is a histone protein that is found in the majority of nucleosomes in yeast cells. A different form of this histone – called H2A.Z – is found in nucleosomes near the promoter of almost every gene. It is thought that nucleosomes that contain H2A.Z are recognized and disassembled as the gene turns on, but it is unclear how this happens. To investigate how H2A.Z nucleosomes are disassembled, Tramantano et al. depleted yeast cells of various proteins thought to play a role in the disassembly process. This indicated that the proteins that transcribe genes play crucial roles in the process of disassembling the H2A.Z nucleosomes, because H2A.Z accumulated at promoters in cells that are depleted of these proteins. Further investigation revealed that disassembled H2A.Z nucleosomes are reassembled with H2A histones, before being converted back to the H2A.Z form by an enzyme called SWR1. This turnover of H2A.Z was seen at active genes and those that are infrequently transcribed, suggesting that it is a general phenomenon. Tramantano et al. also found that the turnover rate of H2A.Z can be used to accurately predict the sites in the DNA where transcription starts. This observation could therefore help to identify previously unknown transcription start sites. Future work could address further questions about how H2A.Z nucleosomes are disassembled. For example, what is the mechanical force that drives this process? And at what step of the transcription process does it occur? DOI: http://dx.doi.org/10.7554/eLife.14243.002