Lineage-determining Transcription Factors Research Articles

Optimization of the Irf8 +32-kb enhancer disrupts dendritic cell lineage segregation.

Autoactivation of lineage-determining transcription factors mediates bistable expression, generating distinct cell phenotypes essential for complex body plans. Classical type 1 dendritic cell (cDC1) and type 2 dendritic cell (cDC2) subsets provide nonredundant functions for defense against distinct immune challenges. Interferon regulatory factor 8 (IRF8), the cDC1 lineage-determining transcription factor, undergoes autoactivation in cDC1 progenitors to establish cDC1 identity, yet its expression is downregulated during cDC2 differentiation by an unknown mechanism. This study reveals that the Irf8 +32-kb enhancer, responsible for IRF8 autoactivation, is naturally suboptimized with low-affinity IRF8 binding sites. Introducing multiple high-affinity IRF8 sites into the Irf8 +32-kb enhancer causes a gain-of-function effect, leading to erroneous IRF8 autoactivation in specified cDC2 progenitors, redirecting them toward cDC1 and a novel hybrid DC subset with mixed-lineage phenotypes. Further, this also causes a loss-of-function effect, reducing Irf8 expression in cDC1s. These developmental alterations critically impair both cDC1-dependent and cDC2-dependent arms of immunity. Collectively, our findings underscore the significance of enhancer suboptimization in the developmental segregation of cDCs required for normal immune function.

Interplay of Transcriptional Factors In Beta Cells Development and Maturation

Diabetes is a group of metabolic disorders resulting from defects in insulin secretion, insulin action, or both. It is characterized by high blood sugar levels over a prolonged period of time with disturbances of carbohydrate, protein, and fat metabolism. It is a global disorder affecting about half a billion or 536.6 million people worldwide, which is said to rise to 783.2 million by 2045. To maintain normoglycemia, pancreatic β-cells release insulin in proportion to the amounts of nutrients in the blood. Through a process of postnatal development, β-cells learn to connect insulin release to food cues. The insulin secretory response in mature β-cells adjusts to alterations in nutritional status. The interaction between transcriptional programs specific to each cell type and external stimuli is necessary for both β-cell maturation and functional adaptation. In this review, we examine the growing data that suggests lineage-determining and signal-dependent transcription factors (LDTFs and SDTFs, respectively) work together to regulate β-cell activity during development and homeostasis. In-depth knowledge of β-cell SDTFs and their corresponding signals would clarify the processes involved in β-cell maturation and functional adaptation, directly affecting diabetes treatments and the production of mature β-cells from stem cells.

Genomic transcription factor binding site selection is edited by the chromatin remodeling factor CHD4.

Biologically precise enhancer licensing by lineage-determining transcription factors enables activation of transcripts appropriate to biological demand and prevents deleterious gene activation. This essential process is challenged by the millions of matches to most transcription factor binding motifs present in many eukaryotic genomes, leading to questions about how transcription factors achieve the exquisite specificity required. The importance of chromatin remodeling factors to enhancer activation is highlighted by their frequent mutation in developmental disorders and in cancer. Here, we determine the roles of CHD4 inenhancer licensing and maintenance in breast cancer cells and during cellular reprogramming. In unchallenged basal breast cancer cells, CHD4 modulates chromatin accessibility. Its depletion leads to redistribution of transcription factors to previously unoccupied sites. During cellular reprogramming induced by the pioneer factor GATA3, CHD4 activity is necessary to prevent inappropriate chromatin opening. Mechanistically, CHD4 promotes nucleosome positioning over GATA3 binding motifs to compete with transcription factor-DNA interaction. We propose that CHD4 acts as a chromatin proof-reading enzyme that prevents unnecessary gene expression by editing chromatin binding activities of transcription factors.

Single-cell multiomics decodes regulatory programs for mouse secondary palate development

Perturbations in gene regulation during palatogenesis can lead to cleft palate, which is among the most common congenital birth defects. Here, we perform single-cell multiome sequencing and profile chromatin accessibility and gene expression simultaneously within the same cells (n = 36,154) isolated from mouse secondary palate across embryonic days (E) 12.5, E13.5, E14.0, and E14.5. We construct five trajectories representing continuous differentiation of cranial neural crest-derived multipotent cells into distinct lineages. By linking open chromatin signals to gene expression changes, we characterize the underlying lineage-determining transcription factors. In silico perturbation analysis identifies transcription factors SHOX2 and MEOX2 as important regulators of the development of the anterior and posterior palate, respectively. In conclusion, our study charts epigenetic and transcriptional dynamics in palatogenesis, serving as a valuable resource for further cleft palate research.

Lhx3/4 initiates a cardiopharyngeal-specific transcriptional program in response to widespread FGF signaling.

Individual signaling pathways, such as fibroblast growth factors (FGFs), can regulate a plethora of inductive events. According to current paradigms, signal-dependent transcription factors (TFs), such as FGF/MapK-activated Ets family factors, partner with lineage-determining factors to achieve regulatory specificity. However, many aspects of this model have not been rigorously investigated. One key question relates to whether lineage-determining factors dictate lineage-specific responses to inductive signals or facilitate these responses in collaboration with other inputs. We utilize the chordate model Ciona robusta to investigate mechanisms generating lineage-specific induction. Previous studies in C. robusta have shown that cardiopharyngeal progenitor cells are specified through the combined activity of FGF-activated Ets1/2.b and an inferred ATTA-binding transcriptional cofactor. Here, we show that the homeobox TF Lhx3/4 serves as the lineage-determining TF that dictates cardiopharyngeal-specific transcription in response to pleiotropic FGF signaling. Targeted knockdown of Lhx3/4 leads to loss of cardiopharyngeal gene expression. Strikingly, ectopic expression of Lhx3/4 in a neuroectodermal lineage subject to FGF-dependent specification leads to ectopic cardiopharyngeal gene expression in this lineage. Furthermore, ectopic Lhx3/4 expression disrupts neural plate morphogenesis, generating aberrant cell behaviors associated with execution of incompatible morphogenetic programs. Based on these findings, we propose that combinatorial regulation by signal-dependent and lineage-determinant factors represents a generalizable, previously uncategorized regulatory subcircuit we term "cofactor-dependent induction." Integration of this subcircuit into theoretical models will facilitate accurate predictions regarding the impact of gene regulatory network rewiring on evolutionary diversification and disease ontogeny.

Repression of latent NF-κB enhancers by PDX1 regulates β cell functional heterogeneity

Interactions between lineage-determining and activity-dependent transcription factors determine single-cell identity and function within multicellular tissues through incompletely known mechanisms. By assembling a single-cell atlas of chromatin state within human islets, we identified β cell subtypes governed by either high or low activity of the lineage-determining factor pancreatic duodenal homeobox-1 (PDX1). β cells with reduced PDX1 activity displayed increased chromatin accessibility at latent nuclear factor κB (NF-κB) enhancers. Pdx1 hypomorphic mice exhibited de-repression of NF-κB and impaired glucose tolerance at night. Three-dimensional analyses in tandem with chromatin immunoprecipitation (ChIP) sequencing revealed that PDX1 silences NF-κB at circadian and inflammatory enhancers through long-range chromatin contacts involving SIN3A. Conversely, Bmal1 ablation in β cells disrupted genome-wide PDX1 and NF-κB DNA binding. Finally, antagonizing the interleukin (IL)-1β receptor, an NF-κB target, improved insulin secretion in Pdx1 hypomorphic islets. Our studies reveal functional subtypes of single β cells defined by a gradient in PDX1 activity and identify NF-κB as a target for insulinotropic therapy.

Abundant binary promoter switches in lineage-determining transcription factors indicate a digital component of cell fate determination

Previous studies of the murine Ly49 and human KIR gene clusters implicated competing sense and antisense promoters in the control of variegated gene expression. In the current study, an examination of transcription factor genes defines an abundance of convergent and divergent sense/antisense promoter pairs, suggesting that competing promoters may control cell fate determination. Differentiation of CD34+ hematopoietic progenitors invitro shows that cells with GATA1 antisense transcription have enhanced GATA2 transcription and a mast cell phenotype, whereas cells with GATA2 antisense transcription have increased GATA1 transcripts and an erythroblast phenotype. Detailed analyses of the AHR and RORC genes demonstrate the ability of competing promoters to act as binary switches and the association of antisense transcription with an immature/progenitor cell phenotype. These data indicate that alternative cell fates generated by promoter competition in lineage-determining transcription factors contribute to the programming of cell differentiation.

Discrimination of cell-intrinsic and environment-dependent effects of natural genetic variation on Kupffer cell epigenomes and transcriptomes

Noncoding genetic variation drives phenotypic diversity, but underlying mechanisms and affected cell types are incompletely understood. Here, investigation of effects of natural genetic variation on the epigenomes and transcriptomes of Kupffer cells derived from inbred mouse strains identified strain-specific environmental factors influencing Kupffer cell phenotypes, including leptin signaling in Kupffer cells from a steatohepatitis-resistant strain. Cell-autonomous and non-cell-autonomous effects of genetic variation were resolved by analysis of F1 hybrid mice and cells engrafted into an immunodeficient host. During homeostasis, non-cell-autonomous trans effects of genetic variation dominated control of Kupffer cells, while strain-specific responses to acute lipopolysaccharide injection were dominated by actions of cis-acting effects modifying response elements for lineage-determining and signal-dependent transcription factors. These findings demonstrate that epigenetic landscapes report on trans effects of genetic variation and serve as a resource for deeper analyses into genetic control of transcription in Kupffer cells and macrophages in vitro.

In vivo screening characterizes chromatin factor functions during normal and malignant hematopoiesis

Cellular differentiation requires extensive alterations in chromatin structure and function, which is elicited by the coordinated action of chromatin and transcription factors. By contrast with transcription factors, the roles of chromatin factors in differentiation have not been systematically characterized. Here, we combine bulk ex vivo and single-cell in vivo CRISPR screens to characterize the role of chromatin factor families in hematopoiesis. We uncover marked lineage specificities for 142 chromatin factors, revealing functional diversity among related chromatin factors (i.e. barrier-to-autointegration factor subcomplexes) as well as shared roles for unrelated repressive complexes that restrain excessive myeloid differentiation. Using epigenetic profiling, we identify functional interactions between lineage-determining transcription factors and several chromatin factors that explain their lineage dependencies. Studying chromatin factor functions in leukemia, we show that leukemia cells engage homeostatic chromatin factor functions to block differentiation, generating specific chromatin factor–transcription factor interactions that might be therapeutically targeted. Together, our work elucidates the lineage-determining properties of chromatin factors across normal and malignant hematopoiesis.

Integrative Single-Cell Transcriptomics and Epigenomics Mapping of the Fetal Retina Developmental Dynamics.

The underlying mechanisms that determine gene expression and chromatin accessibility in retinogenesis are poorly understood. Herein, single-cell RNA sequencing and single-cell assay for transposase-accessible chromatin sequencing are performed on human embryonic eye samples obtained 9-26 weeks after conception to explore the heterogeneity of retinal progenitor cells (RPCs) and neurogenic RPCs. The differentiation trajectory from RPCs to 7 major types of retinal cells are verified. Subsequently, diverse lineage-determining transcription factors are identified and their gene regulatory networks are refined at the transcriptomic and epigenomic levels. Treatment of retinospheres, with the inhibitor of RE1 silencing transcription factor, X5050, induces more neurogenesis with the regular arrangement, and a decrease in Müller glial cells. The signatures of major retinal cells and their correlation with pathogenic genes associated with multiple ocular diseases, including uveitis and age-related macular degeneration are also described. A framework for the integrated exploration of single-cell developmental dynamics of the human primary retina is provided.

TRIB2 safeguards naive T cell homeostasis during aging

Naive CD4+ Tcells are more resistant to age-related loss than naive CD8+ Tcells, suggesting mechanisms that preferentially protect naive CD4+ Tcells during aging. Here, we show that TRIB2 is more abundant in naive CD4+ than CD8+ Tcells and counteracts quiescence exit by suppressing AKT activation. TRIB2 deficiency increases AKT activity and accelerates proliferation and differentiation in response to interleukin-7 (IL-7) in humans and during lymphopenia in mice. TRIB2 transcription is controlled by the lineage-determining transcription factors ThPOK and RUNX3. Ablation of Zbtb7b (encoding ThPOK) and Cbfb (obligatory RUNT cofactor) attenuates the difference in lymphopenia-induced proliferation between naive CD4+ and CD8+ cells. In older adults, ThPOK and TRIB2 expression wanes in naive CD4+ Tcells, causing loss of naivety. These findings assign TRIB2 a key role in regulating Tcell homeostasis and provide a model to explain the lesser resilience of CD8+ Tcells to undergo changes with age.

Differential regulation of lineage-determining transcription factor expression in innate lymphoid cell and adaptive T helper cell subsets.

CD4 T helper (Th) cell subsets, including Th1, Th2 and Th17 cells, and their innate counterparts innate lymphoid cell (ILC) subsets consisting of ILC1s, ILC2s and ILC3s, display similar effector cytokine-producing capabilities during pro-inflammatory immune responses. These lymphoid cell subsets utilize the same set of lineage-determining transcription factors (LDTFs) for their differentiation, development and functions. The distinct ontogeny and developmental niches between Th cells and ILCs indicate that they may adopt different external signals for the induction of LDTF during lineage commitment. Increasing evidence demonstrates that many conserved cis-regulatory elements at the gene loci of LDTFs are often preferentially utilized for the induction of LDTF expression during Th cell differentiation and ILC development at different stages. In this review, we discuss the functions of lineage-related cis-regulatory elements in inducing T-bet, GATA3 or RORγt expression based on the genetic evidence provided in recent publications. We also review and compare the upstream signals involved in LDTF induction in Th cells and ILCs both in vitro and in vivo. Finally, we discuss the possible mechanisms and physiological importance of regulating LDTF dynamic expression during ILC development and activation.

Transcriptional and Epigenetic Regulation of Context-Dependent Plasticity in T-Helper Lineages.

Th cell lineage determination and functional specialization are tightly linked to the activation of lineage-determining transcription factors (TFs) that bind cis-regulatory elements. These lineage-determining TFs act in concert with multiple layers of transcriptional regulators to alter the epigenetic landscape, including DNA methylation, histone modification and three-dimensional chromosome architecture, in order to facilitate the specific Th gene expression programs that allow for phenotypic diversification. Accumulating evidence indicates that Th cell differentiation is not as rigid as classically held; rather, extensive phenotypic plasticity is an inherent feature of T cell lineages. Recent studies have begun to uncover the epigenetic programs that mechanistically govern T cell subset specification and immunological memory. Advances in next generation sequencing technologies have allowed global transcriptomic and epigenomic interrogation of CD4+ Th cells that extends previous findings focusing on individual loci. In this review, we provide an overview of recent genome-wide insights into the transcriptional and epigenetic regulation of CD4+ T cell-mediated adaptive immunity and discuss the implications for disease as well as immunotherapies.

Integrated genomics approaches identify transcriptional mediators and epigenetic responses to Afghan desert particulate matter in small airway epithelial cells.

Military Deployment to Southwest Asia and Afghanistan and exposure to toxic airborne particulates have been associated with an increased risk of developing respiratory disease, collectively termed deployment-related respiratory diseases (DRRDs). Our knowledge about how particulates mediate respiratory disease is limited, precluding the appropriate recognition or management. Central to this limitation is the lack of understanding of how exposures translate into dysregulated cell identity with dysregulated transcriptional programs. The small airway epithelium is involved in both the pathobiology of DRRD and fine particulate matter deposition. To characterize small airway epithelial cell epigenetic and transcriptional responses to Afghan desert particulate matter (APM) and investigate the functional interactions of transcription factors that mediate these responses, we applied two genomics assays, the assay for transposase accessible chromatin with sequencing (ATAC-seq) and Precision Run-on sequencing (PRO-seq). We identified activity changes in a series of transcriptional pathways as candidate regulators of susceptibility to subsequent insults, including signal-dependent pathways, such as loss of cytochrome P450 or P53/P63, and lineage-determining transcription factors, such as GRHL2 loss or TEAD3 activation. We further demonstrated that TEAD3 activation was unique to APM exposure despite similar inflammatory responses when compared with wood smoke particle exposure and that P53/P63 program loss was uniquely positioned at the intersection of signal-dependent and lineage-determining transcriptional programs. Our results establish the utility of an integrated genomics approach in characterizing responses to exposures and identifying genomic targets for the advanced investigation of the pathogenesis of DRRD.

Abstract 5732: p53 deficiency causes Myc dysregulation through a novel TGFβ-facilitated promoter switching mechanism to instigate osteosarcoma development

Abstract p53 deficiency and Myc dysregulation, driven by genetic and epigenetic alterations, respectively, are frequently associated with cancer. Previously, we reported that the combination of p53 inactivation and Myc overexpression, mediated by Runx transcription factors (TFs), leads to osteosarcoma (OS), the most common primary bone cancer. However, the mechanisms leading to Myc upregulation by Runx in a p53-deficient microenvironment remain unclear. TGFβ is a main driver of the epithelial-to-mesenchymal transition and its signal-dependent TFs, Smads, are known to associate with lineage-determining TFs including Runx. As TGFβ paracrine signalling is reportedly increased during human OS development, we immunodetected increased co-expression of TGFβ and phospho-Smad2 in the bone marrow of osteoprogenitor-specific p53 knockout mice, Osterix (Osx)/Sp7-Cre; p53fl/fl mice, which are widely used as an animal model of human OS. Heterozygous deletion of the type II TGFβ receptor in those mice weakened their susceptibility to OS, phenocopying the overall life-extending effect of Myc depletion in the same model. p53 disruption caused TGFβ to aberrantly induce Myc in osteoprogenitors, rendering the cells tumorigenic upon transplantation into immunocompromised mice. Within the 3-Mb Myc regulatory region, we identified a TGFβ-responsive element dubbed m340, which contains binding sites for multiple TFs including Runx. The m340 enhancer caused Myc upregulation upon p53 disruption by switching its associated promoter from the lncRNA Pvt1 to Myc. In support of this promoter switching mechanism, a specific disruption of the evolutionarily conserved p53 site within the Pvt1 promoter recapitulated TGFβ-induced Myc overexpression. At m340, TGFβ promoted its occupancy by a transcriptional complex consisting of Runx, its cooperative factors Smads and AP1, and CBP, further activating the enhancer by increasing H3K27ac deposition. Runx inhibition not only repressed the aberrant upregulation of Myc by reducing Smad occupancy at m340, but also suppressed OS development in vivo. These results suggest that p53 deficiency causes Myc dysregulation through a novel promoter switching programme that is facilitated by TGFβ signalling and reveals a novel epigenetic mechanism underlying the interaction between cancer-predisposing genetic alterations (p53 deficiency) and environmental cues (TGFβ) in tumorigenesis. Considering the mediating role Runx plays in m340 activation, our study may provide a clinical rationale for targeting Runx in p53-deficient malignancies. Citation Format: Yuki Date, Tomoya Ueno, Kosei Ito. p53 deficiency causes Myc dysregulation through a novel TGFβ-facilitated promoter switching mechanism to instigate osteosarcoma development [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5732.

Detecting critical transition signals from single-cell transcriptomes to infer lineage-determining transcription factors.

Analyzing single-cell transcriptomes promises to decipher the plasticity, heterogeneity, and rapid switches in developmental cellular state transitions. Such analyses require the identification of gene markers for semi-stable transition states. However, there are nontrivial challenges such as unexplainable stochasticity, variable population sizes, and alternative trajectory constructions. By advancing current tipping-point theory-based models with feature selection, network decomposition, accurate estimation of correlations, and optimization, we developed BioTIP to overcome these challenges. BioTIP identifies a small group of genes, called critical transition signal (CTS), to characterize regulated stochasticity during semi-stable transitions. Although methods rooted in different theories converged at the same transition events in two benchmark datasets, BioTIP is unique in inferring lineage-determining transcription factors governing critical transition. Applying BioTIP to mouse gastrulation data, we identify multiple CTSs from one dataset and validated their significance in another independent dataset. We detect the established regulator Etv2 whose expression change drives the haemato-endothelial bifurcation, and its targets together in CTS across three datasets. After comparing to three current methods using six datasets, we show that BioTIP is accurate, user-friendly, independent of pseudo-temporal trajectory, and captures significantly interconnected and reproducible CTSs. We expect BioTIP to provide great insight into dynamic regulations of lineage-determining factors.

Interferon regulatory factor 1 (IRF1) controls the metabolic programmes of low-grade pancreatic cancer cells

ObjectivePancreatic ductal adenocarcinomas (PDACs) include heterogeneous mixtures of low-grade cells forming pseudoglandular structures and compact nests of high-grade cells organised in non-glandular patterns. We previously reported that low-grade PDAC cells...

Enhancer regulation by H3K4me1 methyltransferases MLL3/MLL4

Enhancers control cell type‐specific gene expression and are marked by H3K4me1. Active enhancers are further marked by H3K27ac. We identified MLL3/MLL4 as major H3K4me1 methyltransferases and CBP/p300 as the H3K27 acetyltransferases in mammalian cells (EMBO J 2011; eLife 2013). During differentiation of adipocytes, myocytes and ES cells, MLL3/MLL4 co‐localize with lineage‐determining transcription factors (LDTFs), CBP/p300, Brd4 and MED1 on active enhancers. MLL3/MLL4 are required for enhancer activation, enhancer‐promoter interaction, cell type‐specific gene expression, and cell differentiation (eLife 2013; PNAS 2016; Nat Comm 2017). MLL3/MLL4 control cell differentiation by orchestrating CBP/p300‐mediated enhancer activation (NAR 2017). Ectopic expression of H3.3K4M, an inhibitor of H3K4 methylation, or deletion of the enzymatic SET domain, destabilizes MLL3/MLL4 proteins, prevents enhancer activation, and impairs cell differentiation and tissue development (NAR 2019).Using proteomic approaches, we found that endogenous MLL4 complex associates with SWI/SNF chromatin remodeling complex components in cells. Using adipogenesis as a model system, we establish an interdependent relationship between SWI/SNF complex BAF and MLL4 in promoting cell type‐specific enhancer activation by LDTFs, which rectifies seemingly conflicting results from previous studies (Nat Comm 2021).We showed that MLL4 protein, rather than MLL4‐mediated H3K4me1, controls p300 recruitment to enhancers during ES cell differentiation, suggesting that MLL4 regulates enhancer activation independent of H3K4me1 (PNAS 2016). We have generated enzyme‐dead MLL3/MLL4 double knockin ES cells and mice by CRISPR. Initial analyses indicate that MLL3/MLL4 regulate ES cell differentiation and mouse embryonic development through both enzymatic activity‐dependent and ‐independent mechanisms.

BCG vaccination impacts the epigenetic landscape of progenitor cells in human bone marrow

Abstract The BCG vaccine, administered to more than 4 billion people worldwide, is designed to protect against Mycobacterium tuberculosis infection. Interestingly, clinical studies suggest that BCG also provides a degree of protection against heterologous infections, implicating other facets of the immune system apart from adaptive immunity. It has been hypothesized that BCG vaccination can leave immunological “scars” within hematopoietic stem and progenitor cells (HSPCs) that further impact downstream innate immune cell function. This is supported by studies in mice demonstrating that exposure to BCG leads to expansion and differential gene expression within hematopoietic stem cells and multipotent progenitors (HSCs/MPPs). However, very little is known about the impact of BCG vaccination on human bone marrow. Here we performed droplet-based scRNA- and scATAC-sequencing on human bone marrow aspirates from 20 healthy individuals, both before and 90 days after intradermal BCG vaccination or placebo. Over 1000 sites of differential chromatin accessibility across multiple CD34 subpopulations were present 90 days following BCG vaccination. Peaks of differential chromatin accessibility were enriched for binding of lineage determining and stress transcription factors, some of which were upregulated on the gene expression level. Expression levels of a subset of BCG-induced genes were found to significantly correlate with increased Il1b secretion of donor paired PBMCs in response to a C. albicans challenge. These data indicate that BCG vaccination re-wires transcription factor activity in human bone marrow in a way that may be linked to responses of PBMCs to secondary immune challenge with non-mycobacterial pathogens.

Whole-genome profiling of DNA methylation and hydroxymethylation identifies distinct regulatory programs among innate lymphocytes.

Innate lymphocytes encompass a diverse array of phenotypic identities with specialized functions. DNA methylation and hydroxymethylation are essential for epigenetic fidelity and fate commitment. The landscapes of these modifications are unknown in innate lymphocytes. Here, we characterized the whole-genome distribution of methyl-CpG and 5-hydroxymethylcytosine in mouse ILC3, ILC2, and NK cells. We identified differentially methylated and hydroxymethylated DNA regions between ILC-NK subsets and correlated them with transcriptional signatures. We associated lineage-determining transcription factors with demethylation and demonstrated unique patterns of DNA methylation/hydroxymethylation in relationship to open chromatin regions, histone modifications, and transcription factor binding sites. We further discovered a novel association between hydroxymethylation and NK cell super-enhancers. Using mice lacking DNA hydroxymethylase TET2, we showed its requirement for optimal production of hallmark cytokines by ILC3 and IL-17A by inflammatory ILC2. These findings provide a powerful resource for studying innate lymphocyte epigenetic regulation and decode the regulatory logic governing their identity.