DNA methylation and histone modifications together shape the cell-type-specific epigenomic landscape. To enhance genome-wide annotation, we developed EpiSegMixMeth (ESMM), the first integrative segmentation model combining chromatin marks and DNA methylation. ESMM improves upon hidden Markov models by incorporating flexible read count distributions and state duration modeling. Applied to 154 high-quality human epigenomes from the IHEC EpiATLAS, ESMM enhances the annotation of broad heterochromatic regions-over 60% of the genome-that are often missed by chromatin-only models. It accurately defines narrow regulatory element boundaries and captures local chromatin state transitions during cell differentiation. Notably, we show that DNA methylation can replace missing repressive histone marks in segmentation, ensuring robust results across various cell types. In developing memory B cells, ESMM reveals chromatin shifts that align with 3D genome architecture changes, providing a valuable resource for studying cell-type-specific epigenomic regulation.

Genes whose expression is affected, in a consistent manner, by GWAS-identified risk variants and the disease process, constitute preferred drug targets. We herein combine cis-eQTL analysis in 27 sorted blood cell populations and 43 intestinal cell types identified by single cell RNA-Seq in the ileum, colon and rectum, with information on gene expression in patients, to search for putative drug targets for inflammatory bowel disease. We detect >95 K cis-eQTL that affect >13 K e-genes and cluster in >24 K regulatory modules. We uncover matching regulatory modules for 140 risk loci, implicating >300 e-genes not previously connected with inflammatory bowel disease, and find 152 matching e-genes whose expression is perturbed in the blood or gut of patients. We identify entrectinib, a small molecule inhibiting the NRLP3 inflammasome by binding NEK7, as a possible repurposing candidate. Inflammatory bowel disease risk variants can identify genes and pathways involved in disease mechanisms. Here, the authors integrate blood and intestinal eQTL maps with patient gene expression data to prioritise candidate drug targets and identify entrectinib as a potential repurposing candidate.

While insulin-like growth factor 2 mRNA-binding protein 2 (IGF2BP2) has been extensively studied in tumor cells, its role in immune cells within the tumor microenvironment, particularly in macrophages, remains largely unknown. Here, we reveal a critical function of IGF2BP2 in macrophages, demonstrating that myeloid-specific deletion of IGF2BP2 profoundly alters macrophage metabolism and polarization, and markedly impairs tumor progression. Bulk RNA sequencing of IGF2BP2 knockout (KO) macrophages revealed significant alterations in gene expression profiles, particularly impacting pathways associated with glycolysis, mitochondrial function, cell motility, and cell migration. Functional assays confirmed increased glycolytic activity and a concomitant reduction in maximal respiration and reserve respiratory capacity, indicating a metabolic shift towards glycolysis. Furthermore, IGF2BP2 deficiency impaired tumor-associated macrophage (TAM)-like polarization in vitro, as evidenced by decreased expression of TAM markers, such as Mrc1, Mmp2, and Il10. Lipidomic profiling revealed distinct lipid signatures in IGF2BP2 KO TAM-like macrophages, including alterations in triglycerides and cardiolipins, crucial for mitochondrial integrity. In vivo, deletion of IGF2BP2 specifically in the myeloid lineage was sufficient to reduce tumor growth in a subcutaneous Lewis lung carcinoma model, accompanied by decreased TAM infiltration and a shift towards a pro-inflammatory macrophage phenotype. Additionally, IGF2BP2-deficient macrophages showed impaired migratory capacity both in vitro and in vivo. These findings underscore the critical role of IGF2BP2 in controlling macrophage metabolism, polarization, and tumor-supporting functions within the tumor microenvironment, and identify myeloid IGF2BP2 as a potential therapeutic target in cancer.

Abstract Background Persistent airway epithelial abnormalities contribute to chronic obstructive pulmonary disease (COPD), but it remains unclear whether smoking- and COPD-associated epithelial remodeling is retained in airway basal cells and transmitted during differentiation. We determined whether current smoking and COPD are associated with methylation-linked regulatory programs in airway basal cells that shape epithelial differentiation in patient-derived bronchial organoids. Methods We integrated DNA methylation profiling and bulk transcriptomics in patient-derived airway basal cells and matched three-dimensional bronchial organoids. Methylation-defined gene sets were mapped to organoid epithelial cell states using single-cell RNA-seq and contextualized with publicly available airway epithelial datasets. Results In this exploratory cohort, current smoking was associated with a predominant shift toward promoter hypomethylation in airway basal cells and matched organoids. Hypomethylated promoters were enriched for genes preferentially expressed in secretory epithelial cells, including BPIFB1 , BPIFA2 , MSMB and GALNT6 . These genes showed little smoking-associated expression difference in basal-cell culture but were upregulated after organoid differentiation, indicating a differentiation-dependent epithelial memory of smoking. In COPD-derived basal cells, promoter methylation changes involved reduced xenobiotic metabolism and enhanced immune- and infection-related programs. Consistently, COPD-derived organoids showed reduced expression of detoxification-associated pathways and increased lysosomal, endocytic and host-defense programs. Conclusions Current smoking and COPD are associated with persistent methylation-linked regulatory alterations in airway basal cells that become functionally apparent during epithelial differentiation. These findings support a model in which airway basal-cell memory contributes to secretory, inflammatory and host-defense remodeling in chronic airway disease.

The sequence of the human genome provides a foundation for understanding cellular processes in health and disease1. The organisation of this primary genetic information into cell-specific structure and function is critical to understanding the cell type-specific interpretation and execution of the genome. Epigenetic processes are essential for packaging and higher-level functional organisation of the genome, and changes therein are increasingly recognised as contributors to human disease. Building on primary data generated by multinational consortia, the International Human Epigenome Consortium2 (IHEC) has uniformly processed a collection of more than 2000 comprehensive human reference epigenomes, collectively referred to as EpiATLAS. This effort involved the development of standardised molecular and bioinformatics protocols, metadata models, and analytical tools to manage, integrate, display, and share vast amounts of epigenomic data. This includes the creation of a publicly available Epigenome Reference Registry, which provides a system for accessing protected human subject datasets and facilitates open searching of de-identified samples and experimental data. The integrated EpiATLAS ecosystem and its comprehensive human reference epigenome maps provide an unprecedented resource for the biosciences, expanding the annotated epigenomic landscape while uncovering previously unappreciated relationships among regulatory layers and revealing how epigenetic inputs underpin fundamental cellular functions and disease associations.

ABSTRACT Ten‐eleven translocation (TET) enzymes are critical epigenetic regulators, which oxidize the methylated cytosine nucleobase 5‐methyl‐dC (mdC) in the genome to 5‐hydroxymethyl‐dC (hmdC) in an α‐ketoglutarate‐dependent manner. Because the presence of mdC in the promoter region of a given gene silences its expression, this oxidation goes in hand with the reactivation of such silenced genes. In different highly aggressive cancers such as acute myeloid leukemia (AML) and glioblastoma, loss of TET enzyme function, and therefore reduced hmdC levels pave the way for tumor development. Impairment of TET activity can occur through metabolic inhibition, through loss‐of‐function mutations in TET genes themselves, and finally through suppression of TET‐expression via epigenetic silencing. Reactivation of TET enzyme expression represents a major aim of epigenetic cancer therapy. Here we show that the carbocyclic antimetabolite 5‐aza‐2′deoxycytidine (cAzadC), which is supposed to suppress the methylation of DNA during replication, leads to a substantial increase of TET2 expression and strongly increasing hmdC levels. We show that the treatment with cAzadC goes in hand with the broad reactivation of the cellular antitumor responses. With patient‐derived xenograft AML‐mouse models, we show that this translates into a strongly improved anticancer effect in vivo.

SetDB1 is best known for catalyzing H3K9me3, but it also influences H3K27me3 deposition, CTCF-binding, and DNA methylation (DNAme). Given the interplay between DNAme and the other epigenetic features, we profiled DNAme following Setdb1 knockout (KO) in ground-state and serum-grown mouse embryonic stem cells (ESCs) to illuminate DNAme-dependent and -independent functions of SetDB1. Time-course whole-genome bisulfite sequencing of serum-grown ESCs shows that nearly half of SetDB1 binding sites are enriched with DNAme and H3K9me3, primarily at retrotransposons. Upon Setdb1 KO, both H3K9me3 and DNAme are reduced, with DNAme rapidly removed at many sites by TET enzymes. Some retrotransposons, primarily IAPs, are TET-resistant and lose DNAme slowly via passive dilution. Notably, SetDB1-mediated regulation of H3K27me3, CTCF-binding, and SMAD3 are uncoupled from the DNAme-H3K9me3 axis, and from each other. AlphaFold modeling and co-immunoprecipitation mass spectrometry suggest this uncoupling involves competitive binding to distinct SetDB1 protein domains, highlighting the complex coordination underlying SetDB1 functions.

DNA methylation is a widely studied epigenetic mark and a powerful biomarker of cell type, age, environmental exposures, and disease. Whole-genome sequencing following selective conversion of unmethylated cytosines into thymines via bisulfite treatment or enzymatic methods remains the reference method for DNA methylation profiling genome-wide. While numerous software tools facilitate processing of DNA methylation sequencing reads, a comprehensive benchmarking study has been lacking. In this study, we systematically compared complete computational workflows for processing DNA methylation sequencing data using a dedicated benchmarking dataset generated with five whole-genome profiling protocols. As an evaluation reference, we employed accurate locus-specific measurements from our previous benchmark of targeted DNA methylation assays. Based on this experimental gold-standard assessment and multiple performance metrics, we identified workflows that consistently demonstrated superior performance and revealed major workflow development trends. To ensure the long-term utility of our benchmark, we implemented an interactive workflow execution and data presentation platform, adaptable to user-defined criteria and readily expandable to future software.

Adaptive immunity relies on antibodies and memory B and T cells, with memory T cells providing "reactive memory". These cells either circulate in the blood or remain as tissue-resident memory T cells, yet the epigenetic mechanisms underlying their recall function and maintenance are not well understood. Here, we present a comprehensive analysis of 56 reduced representation bisulfite sequencing (RRBS) datasets from 22 memory CD4 and CD8 T-cell populations isolated from human bone marrow, intestine, spleen, lung, skin, and peripheral blood, including surface CD69-positive and CD69-negative cells. Our study reveals unique DNA hypomethylation patterns in tissue-resident memory T cells, particularly in regions associated with genes involved in tissue homing, residency, and transcription factors regulating recall effector memory. The methylomes and differential methylation signatures identified here serve as a valuable resource for understanding the epigenetic program of memory T lymphocytes, their roles in immunological recall, and their maintenance within specific tissues.

Induced pluripotent stem cells (iPSCs) are rapidly emerging as a transformative resource in regenerative medicine. In a previous study, our laboratory achieved a significant milestone by successfully reprograming jaw periosteal cells (JPCs) into iPSCs, which were then differentiated into iPSC-derived mesenchymal stem cells (iMSCs). Using an optimized protocol, we generated iMSCs with a remarkable osteogenic potential while exhibiting lower expression levels of the senescence markers p16 and p21 compared to the original JPCs. This study aimed to explore the epigenetic landscape by comparing the DNA methylation and transcription profiles of iMSCs with their JPC precursors, seeking to uncover key differences. Additionally, this analysis provided an opportunity for us to investigate the potential rejuvenation effects associated with cellular reprogramming. To assess the safety of the generated cells, we evaluated their ability to form teratomas through subcutaneous injection into immunodeficient mice. Our findings revealed that, while the methylation profile of iMSCs closely mirrored that of JPCs, distinct iMSC-specific methylation patterns were evident. Strikingly, the application of DNA methylation (DNAm) clocks for biological age estimation showed a dramatic reduction in DNAm age to approximately zero in iPSCs—a rejuvenation effect that persisted in the derived iMSCs. This profound reset in biological age, together with our transcriptome data, indicate that iMSCs could possess an enhanced regenerative potential compared to adult MSCs. Future in vivo studies should validate this hypothesis.

Dynamic changes of the DNA methylation (DNAmeth) landscape drive differentiation of mature T lymphocytes. However, the role of DNAmeth-mediated regulation in thymocyte development remains unclear. Thus, we generated genome-wide DNAmeth profiles of eight defined human thymocyte subsets, revealing two waves of DNAmeth remodeling: first before TCR rearrangement, then during final maturation. Transcriptomic changes occurred also during phases of DNAmeth stability, indicating decoupled dynamics of epigenetic and transcriptional regulation. In contrast to mature T cells, thymocytes were protected from proliferation-induced changes in the DNA methylome. Finally, the DNAmeth profiles were useful as biomarkers to assess the quality of an in vivo mouse model of human T cell development. This study revealed unexpected dynamics of DNAmeth-mediated control of human thymocyte development and displayed the capacity of DNAmeth profiles to serve as biomarkers for successful T cell generation in model systems and, as a possible future application, during manufacturing of therapeutic cell products.

Chromosomal instability (CIN) drives tumor heterogeneity, complicating cancer therapy. Although Polo-like kinase 1 (PLK1) overexpression induces CIN, direct inhibition of PLK1 has shown limited clinical benefits. We therefore performed a genome-wide synthetic dosage lethality (SDL) screen to identify effective alternative targets and validated over 100 candidates using in vivo and in vitro secondary CRISPR screens. We employed direct-capture Perturb-seq to assess the transcriptional consequences and viability of each SDL perturbation at a single-cell resolution. This revealed IGF2BP2 as a critical genetic dependency that, when targeted, downregulated PLK1 and significantly restricted tumor growth. Mechanistic analyses showed that IGF2BP2 loss disrupted cellular energy metabolism and mitochondrial ATP production by downregulating PLK1 levels as well as genes associated with oxidative phosphorylation. Consistent with this, pharmacological inhibition of IGF2BP2 severely impacts the viability of PLK1-overexpressing cancer cells addicted to higher metabolic rates. Our work offers a novel therapeutic strategy against PLK1-driven heterogeneous malignancies.

DNA-methylation is a key epigenetic mark in chromatin that attenuates chromatin accessibility during transcription, implying a crucial role in gene regulation. Its symmetrical distribution and function is thought to be linked to the periodicity of the DNA helix and the positioning of DNA wrapped around the nucleosome. Epigenomic data suggest that DNA methyltransferases (DNMTs) can methylate DNA when wrapped around a histone octamer. Yet, how this is precisely linked to positioning and periodicity is yet to be elucidated. It has been hypothesized that the observed methylation patterns may be related to the changing accessibility of nucleosome-bound DNA to DNMTs. Here, incorporating NOMe-Seq data, which simultaneously measures nucleosome positioning and DNA methylation at CpG sites across the genome, the interaction of DNMT1 with nucleosomal DNA could be mechanistically modeled and compared to hypothesized dependencies. Furthermore, X-ray structures of DNMT1 were superimposed onto those of nucleosome core complexes at base resolution to determine which histone-bound DNA positions would be sterically accessible or inaccessible to DNMTs. Statistical comparison with experimental NOMe-Seq data revealed that structurally computed DNA accessibility scores can indeed explain DNA methylation patterns in actively transcribed regions with positioned high nucleosome density.

The generation of functionally mature hepatocytes from human induced pluripotent stem cells (iPSC) has the potential to replace primary human hepatocytes (PHH) as the gold standard in vitro model for drug screening, studies of hepatotoxicity as well as liver disease, and is considered a gateway technology to future cell therapy applications. However, we recently reported that current protocols for deriving hepatocyte-like cells (HLC) from iPSC fail to restrict differentiation to the hepatic lineage. Instead, single cell transcriptomics and protein expression analysis uncovered that current methods induce hybrid differentiation, leading to the establishment of abundant hepatic and intestinal gene expression signatures within individual HLC, thereby compromising hepatocyte functionality and phenotype. Differentiation of iPSC to HLC was performed and followed by analysis of transcriptome changes during iPSC to HLC differentiation in comparison to PHH. Differentiation pattern clustering (DiPaC) of differentially expressed genes (DEG) and downstream bioinformatic analysis identified the gene regulatory networks (GRN) involved in establishing hybrid differentiation signatures in HLC, indicating a major influence of caudal-domain type 2 (CDX2) in HLC-IEC hybrid differentiation. CRISPR Cas9-based genetic engineering was used to generate CDX2−/− iPSC, followed by HLC differentiation, transcriptomics and intestinal and hepatic protein expression analysis. The observed phenotypic changes in CDX2−/− HLC were verified by functional analysis of bile canaliculus transport kinetics as well as intestinal enzyme activity and compared to PHH. Transcriptome analysis of WT iPSC to HLC differentiation in comparison to PHH confirmed the establishment of intestinal differentiation gene expression signatures in HLC and indicated a major role of CDX2 in erroneous lineage decision making of HLC. Absence of CDX2 during iPSC to HLC differentiation promoted hepatic specification through the induction of HHEX and PROX1, while drastically reducing intestinal differentiation signatures. CDX2−/− HLC displayed phenotypic maturation of bile canaliculi, including loss of intestine-associated membrane proteins (e.g. IBAT, SI, LCT), increase in hepatic transporters (e.g. BSEP) and size reduction, leading to closer functional and architectural resemblance to bile canaliculi formed by PHH.

Active DNA demethylation depends on Ten-Eleven-Translocation (TET) enzymes, which oxidize 5-methylcytosine (mC) to 5-hydroxymethylcytosine (hmC) and further derivatives. Mutations in TET3 , encoding the predominant neuronal isoform, lead to Beck-Fahrner syndrome, a neurodevelopmental disorder. Using human iPSC-derived neurons, we show that TET3 is dispensable for neuronal specification but critical for subsequent maturation. Differentiating TET3 -deficient neurons exhibit delayed transcriptional and proteomic transitions, altered synaptic signatures, and impaired network activity, indicating delayed functional maturation. Mechanistically, we identified an interaction between TET3 and the mC/hmC-binding protein MECP2, pathogenic variants of which cause Rett syndrome. MECP2 negatively regulates TET3 activity, as demonstrated in functional assays and by inverse hmC patterns in MECP2 - and TET3 -deficient neurons. Despite this, MECP2 - and TET3 -deficient neurons exhibit highly similar phenotypes later in differentiation. Our findings uncover a functional interplay between TET3 and MECP2 that coordinates DNA methylation and chromatin dynamics during neuronal maturation, suggesting a shared pathogenic mechanism in Beck-Fahrner and Rett syndromes. ### Competing Interest Statement The authors have declared no competing interest. Deutsche Forschungsgemeinschaft, https://ror.org/018mejw64, 325871075, 563452213, 531985111, 390873048 Volkswagen Foundation, https://ror.org/03bsmfz84, Freigeist-A110720 Deutsches Zentrum für Luft- und Raumfahrt e. V. (DLR), https://ror.org/04bwf3e34, SANSRETINA (50WB2516) Münchener Universitätsgesellschaft e.V.

ABSTRACT To date, no drugs are approved for BK polyomavirus (BKPyV) reactivation, a major cause of nephropathy after kidney transplantation. Recently, tumor necrosis factor‐α (TNF‐α) blockade has been proposed as a promising therapy, however, the effect of TNF‐α on the clinically most common archetype (ww) BKPyV remained unclear. Assays in primary renal proximal tubule epithelial cells (RPTEC) allowed efficient replication only of BKPyV strains with rearranged (rr) non‐coding control regions (NCCR), which may develop at later disease stages, but not of ww‐BKPyV. Here, we optimized culture conditions allowing robust replication of patient‐derived ww‐BKPyV, while efficiently preserving their ww‐NCCR. TNF‐α promoted rr‐BKPyV replication, while the T H 1 cytokine IFN‐γ suppressed it, also in the presence of TNF‐α. Surprisingly, TNF‐α alone was sufficient to suppress all ww‐BKPyV strains tested. Comprehensive analysis using siRNAs, and chimeric or mutated BKPyV‐strains revealed that the response to TNF‐α depends on the NCCR type, and that the NF‐κB p65 pathway but not the conserved NF‐κB binding site is essential for the TNF‐α‐induced enhancement of rr‐BKPyV replication. Our data suggest that in immunosuppressed patients with archetype‐dominated infections, TNF‐α blockade could interfere with natural TNF‐α‐mediated anti‐BKPyviral control, and this could be detrimental when IFN‐γ‐driven T H 1 responses are impaired. Ongoing inflammation, however, could lead to the selection of rearrangements responding to NCCR‐activating pathways downstream of NF‐κB p65 signaling, that may overcome the initial TNF‐α‐mediated suppression. Our findings also highlight the importance of using clinically relevant BKPyV isolates for drug testing and discovery, for which this new assay paves the way.

Mucus plugging kommt bei Patienten mit schwerem Asthma vor und bezeichnet eine bronchiale Teil- oder Komplettverlegung mit Mucus. Einzelne Studien weisen auf einen positiven Effekt einer Biologika-Therapie auf das Mucus plugging bei Asthma hin.

Hintergrund Thymuskarzinome sind seltene maligne Tumoren, die im klinischen Alltag eine komplexe Herausforderung darstellen. Begleiterkrankungen können Therapie und Überleben erheblich beeinflussen. Diese Studie untersucht das Auftreten von Krebsvorerkrankung und Myasthenia gravis bei Thymuskarzinom sowie die Auswirkungen auf den Krankheitsverlauf und die Therapie.