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Objective Hypomethylation of CD40-ligand (CD40L) in T-cells is associated with increased disease activity in systemic lupus erythematosus (SLE). We therefore investigated possible associations of dietary methyl donors and products with CD40L methylation status in SLE. Methods Food frequency questionnaires were employed to calculate methyl donor micronutrients in 61 female SLE patients (age 45.7 ± 12.0 years, disease duration 16.2 ± 8.4 years) and compared to methylation levels of previously identified key DNA methylation sites (CpG17 and CpG22) within CD40L promotor of T-cells using quantitative DNA methylation analysis on the EpiTYPER mass spectrometry platform. Disease activity was assessed by SLE Disease Activity Index (SLEDAI). Linear regression modelling was used. P values were adjusted according to Benjamini & Hochberg. Results Amongst the micronutrients assessed (g per day), methionine and cysteine were associated with methylation of CpG17 (β = 5.0 (95%CI: 0.6-9.4), p = 0.04; and β = 2.4 (0.6-4.1), p = 0.02, respectively). Methionine, choline, and cysteine were additionally associated with the mean methylation of the entire CD40L (β = 9.5 (1.0-18.0), p = 0.04; β = 1.6 (0.4-3.0), p = 0.04; and β = 4.3 (0.9-7.7), p = 0.02, respectively). Associations of the SLEDAI with hypomethylation were confirmed for CpG17 (β=-32.6 (-60.6 to -4.6), p = 0.04) and CpG22 (β=-38.3 (-61.2 to -15.4), p = 0.004), but not the mean methylation of CD40L. Dietary products with the highest impact on methylation included meat, ice cream, white bread, and cooked potatoes. Conclusions Dietary methyl donors may influence DNA methylation levels and thereby disease activity in SLE.

Objective To comprehensively assess associations of site-specific CD4+-T-cell hypomethylation of the CD40-Ligand gene ( CD40L) with disease activity of women with systemic lupus erythematosus (SLE). Methods CpG-sites within the DNA of the promotor and two enhancer regions (n = 22) of CD40L were identified and numbered consecutively. The rate of methylated DNA in isolated CD4+-T-cells of women with SLE were quantified for each methylation site by MALDI-TOF. Disease activity was assessed by SLE Disease Activity Index (SLEDAI). Associations of site-specific methylation rates with the SLEDAI scores were assessed by linear regression modelling. P values were adjusted according to Bonferroni-Holm as indicated. Results 60 female SLE patients participated in the study (age 45.7 ± 11.1 years, disease duration 17.0 ± 8.3 years). Significant associations to the SLEDAI were noted for CpG22 hypomethylation of the promotor (β = −40.1, p = 0.017, adjusted p = 0.027), trends were noted for CpG17 hypomethylation of the promotor (β = −30.5, p = 0.032, adjusted p = 0.6), and for CpG11 hypermethylation of the second enhancer (β = 15.0, p = 0.046, adjusted p = 0.8). Conclusion Site-specific hypomethylation of the CD40L promotor in CD4+-T-cells show associations with disease activity in female SLE patients.

Establishing gene regulatory networks during differentiation or reprogramming requires master or pioneer transcription factors (TFs) such as PU.1, a prototype master TF of hematopoietic lineage differentiation. To systematically determine molecular features that control its activity, here we analyze DNA-binding in vitro and genome-wide in vivo across different cell types with native or ectopic PU.1 expression. Although PU.1, in contrast to classical pioneer factors, is unable to access nucleosomal target sites in vitro, ectopic induction of PU.1 leads to the extensive remodeling of chromatin and redistribution of partner TFs. De novo chromatin access, stable binding, and redistribution of partner TFs both require PU.1’s N-terminal acidic activation domain and its ability to recruit SWI/SNF remodeling complexes, suggesting that the latter may collect and distribute co-associated TFs in conjunction with the non-classical pioneer TF PU.1., PU.1 is a master TF of hematopoietic lineage differentiation. Here the authors analyse properties of PU.1 DNA-binding in vitro and genome-wide in vivo across different cell types with native or ectopic PU.1 expression, and uncover the mechanisms governing the pioneering and redistribution capabilities of the non-classical pioneer PU.1.

A distinct group of colorectal carcinomas (CRCs) referred to as the “CpG island methylator phenotype” (CIMP) shows an extremely high incidence of de novo DNA methylation and may share common pathological, clinical or molecular features. However, there is limited consensus about which CpG islands (CGIs) define a CIMP, particularly in microsatellite stable (MSS) carcinomas. To study this phenotype in a systematic manner, we analyzed genome-wide CGI DNA methylation profiles of 19 MSS CRC using methyl-CpG immunoprecipitation (MCIp) and hybridization on 244K CGI oligonucleotide microarrays, determined KRAS and BRAF mutation status and compared disease-related DNA methylation changes to chromosomal instability as detected by microarray-based comparative genomic hybridization. Results were validated using mass spectrometry analysis of bisulfite-converted DNA at a subset of 76 individual CGIs in 120 CRC and 43 matched normal tissue samples. Both genome-wide profiling and CpG methylation fine mapping segregated a group of CRC showing pronounced and frequent de novo DNA methylation of a distinct group of CGIs that only partially overlapped with previously established classifiers. The CIMP group defined in our study revealed significant association with colon localization, either KRAS or BRAF mutation, and mostly minor chromosomal losses but no association with known histopathological features. Our data provide a basis for defining novel marker panels that may enable a more reliable classification of CIMP in all CRCs, independently of the MS status.

The differentiation of human blood monocytes (MO), the post-mitotic precursors of macrophages (MAC) and dendritic cells (moDC), is accompanied by the active turnover of DNA methylation, but the extent, consequences and mechanisms of DNA methylation changes remain unclear. Here, we profile and compare epigenetic landscapes during IL-4/GM-CSF-driven MO differentiation across the genome and detect several thousand regions that are actively demethylated during culture, both with or without accompanying changes in chromatin accessibility or transcription factor (TF) binding. We further identify TF that are globally associated with DNA demethylation processes. While interferon regulatory factor 4 (IRF4) is found to control hallmark dendritic cell functions with less impact on DNA methylation, early growth response 2 (EGR2) proves essential for MO differentiation as well as DNA methylation turnover at its binding sites. We also show that ERG2 interacts with the 5mC hydroxylase TET2, and its consensus binding sequences show a characteristic DNA methylation footprint at demethylated sites with or without detectable protein binding. Our findings reveal an essential role for EGR2 as epigenetic pioneer in human MO and suggest that active DNA demethylation can be initiated by the TET2-recruiting TF both at stable and transient binding sites., DNA methylation turnover is an essential epigenetic process during development. Here, the authors look at the changes in DNA methylation during the differentiation of post-mitotic human monocytes (MO), and find that EGR2 interacts with TET2 and is required for DNA demethylation at its binding sites; revealing EGR2 as an epigenetic pioneer factor in human MO.

Murine regulatory T (Treg) cells in tissues promote tissue homeostasis and regeneration. We sought to identify features that characterize human Treg cells with these functions in healthy tissues. Single-cell chromatin accessibility profiles of murine and human tissue Treg cells defined a conserved, microbiota-independent tissue-repair Treg signature with a prevailing footprint of the transcription factor BATF. This signature, combined with gene expression profiling and TCR fate mapping, identified a population of tissue-like Treg cells in human peripheral blood that expressed BATF, chemokine receptor CCR8 and HLA-DR. Human BATF+CCR8+ Treg cells from normal skin and adipose tissue shared features with nonlymphoid T follicular helper-like (Tfh-like) cells, and induction of a Tfh-like differentiation program in naive human Treg cells partially recapitulated tissue Treg regenerative characteristics, including wound healing potential. Human BATF+CCR8+ Treg cells from healthy tissue share features with tumor-resident Treg cells, highlighting the importance of understanding the context-specific functions of these cells.

The bone marrow niche has a pivotal role in progression, survival, and drug resistance of multiple myeloma cells. Therefore, it is important to develop means for targeting the multiple myeloma bone marrow microenvironment. Myeloma-associated macrophages (MAM) in the bone marrow niche are M2 like. They provide nurturing signals to multiple myeloma cells and promote immune escape. Reprogramming M2-like macrophages toward a tumoricidal M1 phenotype represents an intriguing therapeutic strategy. This is especially interesting in view of the successful use of mAbs against multiple myeloma cells, as these therapies hold the potential to trigger macrophage-mediated phagocytosis and cytotoxicity. In this study, we observed that MAMs derived from patients treated with the immunomodulatory drug (IMiD) lenalidomide skewed phenotypically and functionally toward an M1 phenotype. Lenalidomide is known to exert its beneficial effects by modulating the CRBN-CRL4 E3 ligase to ubiquitinate and degrade the transcription factor IKAROS family zinc finger 1 (IKZF1). In M2-like MAMs, we observed enhanced IKZF1 levels that vanished through treatment with lenalidomide, yielding MAMs with a bioenergetic profile, T-cell stimulatory properties, and loss of tumor-promoting capabilities that resemble M1 cells. We also provide evidence that IMiDs interfere epigenetically, via degradation of IKZF1, with IFN regulatory factors 4 and 5, which in turn alters the balance of M1/M2 polarization. We validated our observations in vivo using the CrbnI391V mouse model that recapitulates the IMiD-triggered IKZF1 degradation. These data show a role for IKZF1 in macrophage polarization and can provide explanations for the clinical benefits observed when combining IMiDs with therapeutic antibodies. See related Spotlight on p. 254

Epigenetic control of gene expression occurs within discrete spatial chromosomal units called topologically associating domains (TADs), but the exact spatial requirements of most genes are unknown; this is of particular interest for genes involved in cancer. We therefore applied high-resolution chromosomal conformation capture sequencing to map the three-dimensional (3D) organization of the human locus encoding the key myeloid transcription factor PU.1 in healthy monocytes and acute myeloid leukemia (AML) cells. We identified a dynamic ∼75-kb unit (SubTAD) as the genomic region in which spatial interactions between PU.1 gene regulatory elements occur during myeloid differentiation and are interrupted in AML. Within this SubTAD, proper initiation of the spatial chromosomal interactions requires PU.1 autoregulation and recruitment of the chromatin-adaptor protein LDB1 (LIM domain–binding protein 1). However, once these spatial interactions have occurred, LDB1 stabilizes them independently of PU.1 autoregulation. Thus, our data support that PU.1 autoregulates its expression in a “hit-and-run” manner by initiating stable chromosomal loops that result in a transcriptionally active chromatin architecture.

Due to the critical impact of active AP-1 transcription factors in melanoma, it is important to define their target genes and to identify and ultimately inhibit oncogenic signals. Here we mapped the genome-wide occupancy of the AP-1 family member c-Jun in different melanoma cells and correlated AP-1 binding with transcriptome data to detect genes in melanoma regulated by c-Jun. Our analysis shows that c-Jun supports the malignant phenotype by deregulating genes in cancer-relevant signaling pathways, such as mitogen-activated protein kinase (MAPK) and phosphatidylinositol-3-kinase (PI3K) pathways. Moreover, we demonstrate that the importance of c-Jun depends on melanoma stage and mutation status of the tumor suppressor PTEN. Our study reveals that activation of c-Jun overrules the tumor suppressive effect of PTEN in early melanoma development. These findings help to understand the relevance of c-Jun within cancer pathways in different melanoma cell types, especially in relation to MAPK and PI3K pathways, which are commonly deregulated in melanomas. Consequently, targeting c-Jun in PTEN+ melanoma cells may represent a promising therapeutic strategy to inhibit survival of melanoma cells to prevent the development of a metastatic phenotype.

Neutrophilic differentiation is dependent on CCAAT enhancer-binding protein a (C/EBP alpha), a transcription factor expressed in multiple organs including the bone marrow. Using functional genomic technologies in combination with clustered regularly-interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 genome editing and in vivo mouse modeling, we show that CEBPA is located in a 170-kb topological-associated domain that contains 14 potential enhancers. Of these, 1 enhancer located 142 kb from CEBPA is active and engages with the CEBPA promoter in myeloid cells only. Germ line deletion of the homologous enhancer in mice in vivo reduces Cebpa levels exclusively in hematopoietic stem cells (HSCs) and myeloid-primed progenitor cells leading to severe defects in the granulocytic lineage, without affecting any other Cebpa-expressing organ studied. The enhancer-deleted progenitor cells lose their myeloid transcription program and are blocked in differentiation. Deletion of the enhancer also causes loss of HSC maintenance. We conclude that a single +42-kb enhancer is essential for CEBPA expression in myeloid cells only.

Malignant transformation is frequently associated with disease-specific epigenetic alterations, but the underlying mechanisms and pathophysiological consequences remain poorly understood. Here, we used global comparative DNA methylation profiling at CG-rich regions of 27 acute myeloid leukemia (AML) samples to select a subset of aberrantly methylated CG-rich regions (~400 regions, ~15,000 CpGs) for quantitative DNA methylation profiling in a large cohort of AML patients (n = 196) using MALDI-TOF analysis of bisulfite-treated DNA. Meta-analysis separated a subgroup of CG-rich regions showing highly correlated DNA methylation changes that were marked by histone H3 lysine 27 trimethylation in normal hematopoietic progenitor cells. While the group of non-polycomb group (PcG) target regions displayed methylation patterns that correlated well with molecular and cytogenetic markers, PcG target regions displayed a much weaker association with genetic features. However, the degree of methylation gain across the latter panel showed significant correlation with active DNMT3A levels and with overall survival. Our study suggests that both epigenetic as well as genetic aberrations underlay AML-related changes in DNA methylation at CG-rich regions and that the former may provide a marker to improve classification and prognostication of adult AML patients.

T-cell receptor (TCR) redirected T cells are promising tools for adoptive cancer immunotherapy. Since not only CD8 but also CD4 T cells are key players for efficient antitumor responses, the targeted redirection of both subsets with the same antigen-specific TCR comes more and more into focus. Although rapidly evolving technologies enable the reliable genetic re-programming of T cells, the limited availability of TCRs that induce T-cell activation in both T-cell subsets without CD4/CD8 co-receptor contribution hampers the broad application of this approach. We developed a novel stimulation approach, which drives the activation and proliferation of CD4 T-cell populations capable of inducing effector functions in a CD4-independent manner. Naive-enriched CD4 T cells were stimulated against dendritic cells (DC) expressing allogeneic HLA-DP antigens upon RNA transfection and CD4/HLA interactions were blocked by the addition of CD4 binding antibody. Evolving CD4 T-cell populations were specifically activated independent of the CD4 co-signal and induced strong TCR-mediated IFN-γ secretion as well as cytolysis upon recognition of leukemia cells expressing HLA-DP antigen. Our novel stimulation approach may facilitate the generation of CD4 T cells as source for co-receptor independent TCRs for future immunotherapies.