The domestic chicken (Gallus gallus domesticus) is the globally most important source of commercially produced meat. While genetic approaches have played an important role in the development of chicken stocks, little is known about chicken epigenetics. We have systematically analyzed the chicken DNA methylation machinery and DNA methylation landscape. While overall DNA methylation distribution was similar to mammals, sperm DNA appeared hypomethylated, which correlates with the absence of the DNMT3L cofactor in the chicken genome. Additional analysis revealed the presence of low-methylated regions, which are conserved gene regulatory elements that show tissue-specific methylation patterns. We also used whole-genome bisulfite sequencing to generate 56 single-base resolution methylomes from various tissues and developmental time points to establish an LMR-based DNA methylation clock for broiler chicken age prediction. This clock was used to demonstrate epigenetic age acceleration in animals with experimentally induced inflammation. Our study provides detailed insights into the chicken methylome and suggests a novel application of the DNA methylation clock as a marker for livestock health.

Salmonella is a burden to the poultry, health, and food safety industries, resulting in illnesses, food contamination, and recalls. Salmonella enterica subspecies enterica Enteritidis (S. Enteritidis) is one of the most prevalent serotypes isolated from poultry. Salmonella enterica subspecies enterica Heidelberg (S. Heidelberg), which is becoming as prevalent as S. Enteritidis, is one of the five most isolated serotypes. Although S. Enteritidis and S. Heidelberg are almost genetically identical, they both are capable of inducing different immune and metabolic responses in host cells to successfully establish an infection. Therefore, using the kinome peptide array, we demonstrated that S. Enteritidis and S. Heidelberg infections induced differential phosphorylation of peptides on Rho proteins, caspases, toll-like receptors, and other proteins involved in metabolic- and immune-related signaling of HD11 chicken macrophages. Metabolic flux assays measuring extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) demonstrated that S. Enteritidis at 30 min postinfection (p.i.) increased glucose metabolism, while S. Heidelberg at 30 min p.i. decreased glucose metabolism. S. Enteritidis is more invasive than S. Heidelberg. These results show different immunometabolic responses of HD11 macrophages to S. Enteritidis and S. Heidelberg infections.

Significant changes in growth potential and feed conversion have been bred into the modern broiler chicken for well over 60 yr. These metabolic changes have had significant effects on the immune performance as well. To better understand these genetic differences in immunometabolism we studied the immune response of the modern broiler and the Athens Canadian Random Bred (ACRB) heritage broiler strain. We injected newly hatched modern broiler and ACRB chicks intraabdominally with CpG oligonucleotide, an immunostimulatory synthetic oligonucleotide. We conducted species-specific kinome array analysis and gene expression analysis on jejunum and cecal tonsil tissue. We also performed metabolic analysis of blood cells. In the modern birds, there is an initial inflammatory response to the injection at d 3 post-hatch with activation of PI3K-Akt, JAK-STAT, and NF-κB signaling, and IL-1β and IL-6 mRNA expression. By d 15 post-hatch this response changed to deactivation and downregulation of these immune responses in modern but not heritage broilers. Metabolic analysis showed an increase in glycolysis in peripheral blood mononuclear cells from modern birds given CpG, but no difference in ACRB. These results show that the ACRB birds may have a less inflammatory and more stable immune profile in response to immune stimulation than the modern broilers, possibly resulting in a more disease resistant phenotype overall.

Loss-of-function studies have shown that transcription factor T cell factor-1 (TCF1), encoded by the Tcf7 gene, is essential for T cell development in the thymus. We discovered that the Tcf7 expression level is regulated by E box DNA binding proteins, independent of Notch, and regulates αβ and γδ T cell development. Systematic interrogation of the five E protein binding elements (EPE1–5) in the Tcf7 enhancer region showed lineage-specific utilization. Specifically, loss-of-function analysis revealed that only EPE3 plays a critical role in supporting αβ T cell development, while EPE1, 3, and 5 regulate the γδ T cell maturation and functional cell fate decision. The importance of EPE3 in supporting both lineages may stem from its unique capacity to interact with the Tcf7 transcriptional start site. Together, these studies demonstrate that the precise dosage of TCF1 expression mediated by distinct EPEs generates a balanced output of T cells from the thymus.

This report by Lopes et al. provides the first clear insight into the impact of specific metabolic processes on the effector fates of gamma delta T cells. Specifically, the authors showed that IL-17 producing gamma delta T cells rely primarily on oxidative metabolism for energy production, whereas IFN gamma producers rely primarily on glycolysis. The authors illustrated that this distinction in metabolic activity is established early in gamma delta T cell development and that differences in mitochondrial membrane potential in CD24+ immature thymic precursors are predictive of maturation into the alterative IFN gamma IL-17 producing fates. This dichotomy between the IL-17 producers and the IFN gamma producers was also maintained in the periphery and the tumor microenvironment. Naïve alpha-beta T cells alter their preferred method of energy production upon TCR ligation and activation, switching from primarily oxidative phosphorylation in the thymus to glycolysis as they recognize antigen and differentiate into effector T cells {1}. The findings presented here show that, in contrast to the alpha-beta lineage cells, gamma delta T cells’ metabolic preferences, like their effector fates, are set very early in their development and are influenced by TCR signaling. These findings have important implications for disease pathogenesis. Specifically, IL-17 producing gamma delta T cells’ preferential uptake of lipids and the IFN-gamma producers’ dependence on glucose suggest that the lipid-rich, glucose-poor tumor microenvironment may be selectively permissive for IL-17 producing, pro-tumor gamma delta T cells. The findings also provide a potential mechanistic link between lipid-rich diets, obesity, and the risk of cancer and inflammatory diseases. The work presented in this manuscript will be instrumental for future studies into the use of gamma delta T cell-based immunotherapy.

In this study, the authors address the long-standing problem of how Aire induces tissue-restricted antigens (TRA) in medullary thymic epithelial cells (mTEC). mTECs express ectopic antigens which can potentially be recognized by developing T cells. Expression of TRA by mTEC is fundamental to the establishment of immunological tolerance. It has long been understood that Aire plays a key role in driving TRA expression in mTECs, and that in the absence of functional Aire, autoimmune disease develops in both mice and humans. However, the mechanism of Aire’s control over the expression of TRA has remained unclear. The authors hypothesized that Aire drives the formation of superenhancer-promoter loops, where superenhancers and promoters physically associate to allow for the initiation of transcription of a large gene set. First, the authors identified that Aire broadly impacts chromatin architecture, as Aire-induced genes preferentially moved from the repressive to activated chromatin compartment in the presence of Aire. More specifically, the presence of Aire also drove increased contacts between superenhancers and known Aire-induced genes, whereas there was no such impact on Aire-neutral genes. The authors then explored the relationship between Aire and protein complexes known to drive and shape chromatin organization; namely, mediator, cohesion, and CTCF. The authors determined that there was generally co-localization between Aire, MED1 (mediator component), and SMC1 (cohesin complex component). However, they never observed colocalization between Aire and CTCF. The authors then investigated whether Aire, MED1, and SMC1 colocalize at superenhancer sites previously identified in mTECS, and indeed they observed a significant increase in Aire, MED1, and SMC1 at superenhancers in mTECS from wildtype mice when compared to mTECS isolated from Aire knockout mice. They also saw that the presence of Aire led to decreased numbers of topologically-associating domains (TADs), and less insulated TADs, more interchromosomal contacts, and decreased CTCF binding at architectural domain boundaries. To clarify the mechanism of Aire occupancy at superenhancers, and subsequent increase in cohesin occupancy and decrease in CTCF binding, the authors identified through immunoprecipitation assays an association between Aire and NIPBL, the cohesin assembly loader. The authors found that NIPBL preferentially localized at superenhancers in Aire+ mTECs, and there was significant overlap between NIPBL and Aire at superenhancer locations. Finally, the authors clarified the necessity of cohesin for Aire’s downstream impacts on gene expression. In mice with decreased Stag2 expression, the gene that encodes SA-2, which is a subunit of the cohesin complex, there was a significant reduction in the expression of Aire-dependent genes, and this impact was not seen in Aire-neutral genes. Based on these findings, the authors propose a model in which Aire causes the eviction of CTCF from architectural domain binding sites, and the accumulation of cohesin and mediator protein complexes at superenhancers. This alters the overall chromatin structure and drives the expression of a broad range of disparate genes in mTECS. These structural changes in the chromatin due to loss of CTCF binding promote the formation of regulatory loops in which superenhancers can interact with promoters. Superenhancers are known to preferentially associate with cell-identity genes which encode TRAs, in addition to cell cycle genes. Therefore, transcriptional activation at superenhancers due to Aire’s influence leads to increased expression of TRA encoding genes in mTECs. This process of superenhancer-promoter interaction is also known to be highly dynamic, which explains the wide variation in TRA gene expression driven by Aire, even at the level of individual cells. The authors propose that Aire drives the formation of these regulatory loops in tandem with NIPBL, the cohesin loader, as both Aire and NIPBL are known to preferentially localize to double-stranded DNA breaks within superenhancers and associate with similar protein-binding partners. The relationship between Aire and NIPBL will need to be further examined, as the exact nature of their association remains unclear. The mechanism through which Aire evicts CTCF from its binding sites will need further clarification as well, although the authors provide a possible partial explanation that Aire prevents cohesin from associating with CTCF during DNA extrusion. The findings presented in this manuscript clarify part of the mechanism of how Aire could possibly regulate the expression of a wide array of TRA genes in mTECs, and how a disruption of Aire’s functionality leads to autoimmune disease. Additionally, this work offers more general insight into how regulatory and architectural chromatin loops differ in the protein complexes that drive their formation, and the specific impacts they may have on transcription or repression in a variety of settings.