PRMT5 Promotes T follicular helper Cell Differentiation and Germinal Center Responses during Influenza Virus Infection.

Protein arginine methyltransferases (PRMTs) modify diverse protein targets and regulate numerous cellular processes; yet, their contributions to individual effector T cell responses during infections are incompletely understood. In this study, we identify PRMT5 as a critical regulator of CD4+ T follicular helper cell (Tfh) responses during influenza virus infection in mice. Conditional PRMT5 deletion in murine T cells results in an almost complete ablation of both Tfh and T follicular regulatory populations and, consequently, reduced B cell activation and influenza-specific Ab production. Supporting a potential mechanism, we observe elevated surface expression of IL-2Rα on non-T regulatory effector PRMT5-deficient T cells. Notably, IL-2 signaling is known to negatively impact Tfh differentiation. Collectively, our findings identify PRMT5 as a prominent regulator of Tfh programming, with potential causal links to IL-2 signaling.

CEMIG: prediction of the cis-regulatory motif using the de Bruijn graph from ATAC-seq.

Sequence motif discovery algorithms enhance the identification of novel deoxyribonucleic acid sequences with pivotal biological significance, especially transcription factor (TF)-binding motifs. The advent of assay for transposase-accessible chromatin using sequencing (ATAC-seq) has broadened the toolkit for motif characterization. Nonetheless, prevailing computational approaches have focused on delineating TF-binding footprints, with motif discovery receiving less attention. Herein, we present Cis rEgulatory Motif Influence using de Bruijn Graph (CEMIG), an algorithm leveraging de Bruijn and Hamming distance graph paradigms to predict and map motif sites. Assessment on 129 ATAC-seq datasets from the Cistrome Data Browser demonstrates CEMIG's exceptional performance, surpassing three established methodologies on four evaluative metrics. CEMIG accurately identifies both cell-type-specific and common TF motifs within GM12878 and K562 cell lines, demonstrating its comparative genomic capabilities in the identification of evolutionary conservation and cell-type specificity. In-depth transcriptional and functional genomic studies have validated the functional relevance of CEMIG-identified motifs across various cell types. CEMIG is available at https://github.com/OSU-BMBL/CEMIG, developed in C++ to ensure cross-platform compatibility with Linux, macOS and Windows operating systems.

Epigenetic remodeling by vitamin C potentiates plasma cell differentiation.

Ascorbate (vitamin C) is an essential micronutrient in humans. The severe chronic deficiency of ascorbate, termed scurvy, has long been associated with increased susceptibility to infections. How ascorbate affects the immune system at the cellular and molecular levels remained unclear. From a micronutrient analysis, we identified ascorbate as a potent enhancer for antibody response by facilitating the IL-21/STAT3-dependent plasma cell differentiation in mouse and human B cells. The effect of ascorbate is unique as other antioxidants failed to promote plasma cell differentiation. Ascorbate is especially critical during early B cell activation by poising the cells to plasma cell lineage without affecting the proximal IL-21/STAT3 signaling and the overall transcriptome. As a cofactor for epigenetic enzymes, ascorbate facilitates TET2/3-mediated DNA modification and demethylation of multiple elements at the Prdm1 locus. DNA demethylation augments STAT3 association at the Prdm1 promoter and a downstream enhancer, thus ensuring efficient gene expression and plasma cell differentiation. The results suggest that an adequate level of ascorbate is required for antibody response and highlight how micronutrients may regulate the activity of epigenetic enzymes to regulate gene expression. Our findings imply that epigenetic enzymes can function as sensors to gauge the availability of metabolites and influence cell fate decisions.

PERK is a critical metabolic hub for immunosuppressive function in macrophages.

Chronic inflammation triggers compensatory immunosuppression to stop inflammation and minimize tissue damage. Studies have demonstrated that endoplasmic reticulum (ER) stress augments the suppressive phenotypes of immune cells; however, the molecular mechanisms underpinning this process and how it links to the metabolic reprogramming of immunosuppressive macrophages remain elusive. In the present study, we report that the helper T cell 2 cytokine interleukin-4 and the tumor microenvironment increase the activity of a protein kinase RNA-like ER kinase (PERK)-signaling cascade in macrophages and promote immunosuppressive M2 activation and proliferation. Loss of PERK signaling impeded mitochondrial respiration and lipid oxidation critical for M2 macrophages. PERK activation mediated the upregulation of phosphoserine aminotransferase 1 (PSAT1) and serine biosynthesis via the downstream transcription factor ATF-4. Increased serine biosynthesis resulted in enhanced mitochondrial function and α-ketoglutarate production required for JMJD3-dependent epigenetic modification. Inhibition of PERK suppressed macrophage immunosuppressive activity and could enhance the efficacy of immune checkpoint programmed cell death protein 1 inhibition in melanoma. Our findings delineate a previously undescribed connection between PERK signaling and PSAT1-mediated serine metabolism critical for promoting immunosuppressive function in M2 macrophages.

Gene Regulatory Circuits in Innate and Adaptive Immune Cells.

Cell identity and function largely rely on the programming of transcriptomes during development and differentiation. Signature gene expression programs are orchestrated by regulatory circuits consisting of cis-acting promoters and enhancers, which respond to a plethora of cues via the action of transcription factors. In turn, transcription factors direct epigenetic modifications to revise chromatin landscapes, and drive contacts between distal promoter-enhancer combinations. In immune cells, regulatory circuits for effector genes are especially complex and flexible, utilizing distinct sets of transcription factors and enhancers, depending on the cues each cell type receives during an infection, after sensing cellular damage, or upon encountering a tumor. Here, we review major players in the coordination of gene regulatory programs within innate and adaptive immune cells, as well as integrative omics approaches that can be leveraged to decipher their underlying circuitry.

Micro but mighty-Micronutrients in the epigenetic regulation of adaptive immune responses.

Micronutrients are essential small molecules required by organisms in minute quantity for survival. For instance, vitamins and minerals, the two major categories of micronutrients, are central for biological processes such as metabolism, cell replication, differentiation, and immune response. Studies estimated that around two billion humans worldwide suffer from micronutrient deficiencies, also known as "hidden hunger," linked to weakened immune responses. While micronutrients affect the immune system at multiple levels, recent studies showed that micronutrients potentially impact the differentiation and function of immune cells as cofactors for epigenetic enzymes, including the 2-oxoglutarate-dependent dioxygenase (2OGDD) family involved in histone and DNA demethylation. Here, we will first provide an overview of the role of DNA methylation in T cells and B cells, followed by the micronutrients ascorbate (vitamin C) and iron, two critical cofactors for 2OGDD. We will discuss the emerging evidence of these micronutrients could regulate adaptive immune response by influencing epigenetic remodeling.

Roles of TET and TDG in DNA demethylation in proliferating and non-proliferating immune cells.

BACKGROUND: TET enzymes mediate DNA demethylation by oxidizing 5-methylcytosine (5mC) in DNA to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC). Since these oxidized methylcytosines (oxi-mCs) are not recognized by the maintenance methyltransferase DNMT1, DNA demethylation can occur through "passive," replication-dependent dilution when cells divide. A distinct, replication-independent ("active") mechanism of DNA demethylation involves excision of 5fC and 5caC by the DNA repair enzyme thymine DNA glycosylase (TDG), followed by base excision repair. RESULTS: Here by analyzing inducible gene-disrupted mice, we show that DNA demethylation during primary T cell differentiation occurs mainly through passive replication-dependent dilution of all three oxi-mCs, with only a negligible contribution from TDG. In addition, by pyridine borane sequencing (PB-seq), a simple recently developed method that directly maps 5fC/5caC at single-base resolution, we detect the accumulation of 5fC/5caC in TDG-deleted T cells. We also quantify the occurrence of concordant demethylation within and near enhancer regions in the Il4 locus. In an independent system that does not involve cell division, macrophages treated with liposaccharide accumulate 5hmC at enhancers and show altered gene expression without DNA demethylation; loss of TET enzymes disrupts gene expression, but loss of TDG has no effect. We also observe that mice with long-term (1 year) deletion of Tdg are healthy and show normal survival and hematopoiesis. CONCLUSIONS: We have quantified the relative contributions of TET and TDG to cell differentiation and DNA demethylation at representative loci in proliferating T cells. We find that TET enzymes regulate T cell differentiation and DNA demethylation primarily through passive dilution of oxi-mCs. In contrast, while we observe a low level of active, replication-independent DNA demethylation mediated by TDG, this process does not appear to be essential for immune cell activation or differentiation.

5-Azacytidine Transiently Restores Dysregulated Erythroid Differentiation Gene Expression in TET2-Deficient Erythroleukemia Cells.

DNA methyltransferase inhibitors (DNMTI) like 5-Azacytidine (5-Aza) are the only disease-modifying drugs approved for the treatment of higher-risk myelodysplastic syndromes (MDS), however less than 50% of patients respond, and there are no predictors of response with clinical utility. Somatic mutations in the DNA methylation regulating gene tet-methylcytosine dioxygenase 2 (TET2) are associated with response to DNMTIs, however the mechanisms responsible for this association remain unknown. Using bisulfite padlock probes, mRNA sequencing, and hydroxymethylcytosine pull-down sequencing at several time points throughout 5-Aza treatment, we show that TET2 loss particularly influences DNA methylation (5mC) and hydroxymethylation (5hmC) patterns at erythroid gene enhancers and is associated with downregulation of erythroid gene expression in the human erythroleukemia cell line TF-1. 5-Aza disproportionately induces expression of these down-regulated genes in TET2KO cells and this effect is related to dynamic 5mC changes at erythroid gene enhancers after 5-Aza exposure. We identified differences in remethylation kinetics after 5-Aza exposure for several types of genomic regulatory elements, with distal enhancers exhibiting longer-lasting 5mC changes than other regions. This work highlights the role of 5mC and 5hmC dynamics at distal enhancers in regulating the expression of differentiation-associated gene signatures, and sheds light on how 5-Aza may be more effective in patients harboring TET2 mutations. IMPLICATIONS: TET2 loss in erythroleukemia cells induces hypermethylation and impaired expression of erythroid differentiation genes which can be specifically counteracted by 5-Azacytidine, providing a potential mechanism for the increased efficacy of 5-Aza in TET2-mutant patients with MDS. VISUAL OVERVIEW: http://mcr.aacrjournals.org/content/molcanres/19/3/451/F1.large.jpg.

Circles of Life: linking metabolic and epigenetic cycles to immunity.

Metabolites are the essential substrates for epigenetic modification enzymes to write or erase the epigenetic blueprint in cells. Hence, the availability of nutrients and activity of metabolic pathways strongly influence the enzymatic function. Recent studies have shed light on the choreography between metabolome and epigenome in the control of immune cell differentiation and function, with a major focus on histone modifications. Yet, despite its importance in gene regulation, DNA methylation and its relationship with metabolism is relatively unclear. In this review, we will describe how the metabolic flux can influence epigenetic networks in innate and adaptive immune cells, with a focus on the DNA methylation cycle and the metabolites S-adenosylmethionine and α-ketoglutarate. Future directions will be discussed for this rapidly emerging field.