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Objectives: Better disease management can be achieved with earlier detection through robust, sensitive, and easily accessible biomarkers. The aim of the current study was to identify novel epigenetic biomarkers determining the risk of type 2 diabetes (T2D). Methods: Livers of 10-week-old female New Zealand Obese (NZO) mice, slightly differing in their degree of hyperglycemia and liver fat content and thereby in their diabetes susceptibility were used for expression and methylation profiling. We screened for differences in hepatic expression and DNA methylation in diabetes-prone and -resistant mice, and verified a candidate (HAMP) in human livers and blood cells. Hamp expression was manipulated in primary hepatocytes and insulin-stimulated pAKT was detected. Luciferase reporter assays were conducted in a murine liver cell line to test the impact of DNA methylation on promoter activity. Results: In livers of NZO mice, the overlap of methylome and transcriptome analyses revealed a potential transcriptional dysregulation of 12 hepatokines. The strongest effect with a 52% decreased expression in livers of diabetes-prone mice was detected for the Hamp gene, mediated by elevated DNA methylation of two CpG sites located in the promoter. Hamp encodes the iron-regulatory hormone hepcidin, which had a lower abundance in the livers of mice prone to developing diabetes. Suppression of Hamp reduces the levels of pAKT in insulin-treated hepatocytes. In liver biopsies of obese insulin-resistant women, HAMP expression was significantly downregulated along with increased DNA methylation of a homologous CpG site. In blood cells of incident T2D cases from the prospective EPIC-Potsdam cohort, higher DNA methylation of two CpG sites was related to increased risk of incident diabetes. Conclusions: We identified epigenetic changes in the HAMP gene which may be used as an early marker preceding T2D.

Objective: Liver mitochondria adapt to high-calorie intake. We investigated how exercise alters the early compensatory response of mitochondria, thus preventing fatty liver disease as a long-term consequence of overnutrition. Methods: We compared the effects of a steatogenic high-energy diet (HED) for six weeks on mitochondrial metabolism of sedentary and treadmill-trained C57BL/6N mice. We applied multi-OMICs analyses to study the alterations in the proteome, transcriptome, and lipids in isolated mitochondria of liver and skeletal muscle as well as in whole tissue and examined the functional consequences by high-resolution respirometry. Results: HED increased the respiratory capacity of isolated liver mitochondria, both in sedentary and in trained mice. However, proteomics analysis of the mitochondria and transcriptomics indicated that training modified the adaptation of the hepatic metabolism to HED on the level of respiratory complex I, glucose oxidation, pyruvate and acetyl-CoA metabolism, and lipogenesis. Training also counteracted the HED-induced glucose intolerance, the increase in fasting insulin, and in liver fat by lowering diacylglycerol species and c-Jun N-terminal kinase (JNK) phosphorylation in the livers of trained HED-fed mice, two mechanisms that can reverse hepatic insulin resistance. In skeletal muscle, the combination of HED and training improved the oxidative capacity to a greater extent than training alone by increasing respiration of isolated mitochondria and total mitochondrial protein content. Conclusion: We provide a comprehensive insight into the early adaptations of mitochondria in the liver and skeletal muscle to HED and endurance training. Our results suggest that exercise disconnects the HED-induced increase in mitochondrial substrate oxidation from pyruvate and acetyl-CoA-driven lipid synthesis. This could contribute to the prevention of deleterious long-term effects of high fat and sugar intake on hepatic mitochondrial function and insulin sensitivity.

Objective: Technological advances have brought a steady increase in the availability of various types of omics data, from genomics to metabolomics. Integrating these multi-omics data is a chance and challenge for systems biology; yet, tools to fully tap their potential remain scarce. Methods: We present here a fully unsupervised and versatile correlation-based method – termed Correlation guided Network Integration (CoNI) – to integrate multi-omics data into a hypergraph structure that allows for the identification of effective modulators of metabolism. Our approach yields single transcripts of potential relevance that map to specific, densely connected, metabolic subgraphs or pathways. Results: By applying our method on transcriptomics and metabolomics data from murine livers under standard Chow or high-fat diet, we identified eleven genes with potential regulatory effects on hepatic metabolism. Five candidates, including the hepatokine INHBE, were validated in human liver biopsies to correlate with diabetes-related traits such as overweight, hepatic fat content, and insulin resistance (HOMA-IR). Conclusion: Our method's successful application to an independent omics dataset confirmed that the novel CoNI framework is a transferable, entirely data-driven, flexible, and versatile tool for multiple omics data integration and interpretation.

Objective: Hundreds of missense mutations in the coding region of PDX1 exist; however, if these mutations predispose to diabetes mellitus is unknown. Methods: In this study, we screened a large cohort of subjects with increased risk for diabetes and identified two subjects with impaired glucose tolerance carrying common, heterozygous, missense mutations in the PDX1 coding region leading to single amino acid exchanges (P33T, C18R) in its transactivation domain. We generated iPSCs from patients with heterozygous PDX1P33T/+, PDX1C18R/+ mutations and engineered isogenic cell lines carrying homozygous PDX1P33T/P33T, PDX1C18R/C18R mutations and a heterozygous PDX1 loss-of-function mutation (PDX1+/−). Results: Using an in vitro β-cell differentiation protocol, we demonstrated that both, heterozygous PDX1P33T/+, PDX1C18R/+ and homozygous PDX1P33T/P33T, PDX1C18R/C18R mutations impair β-cell differentiation and function. Furthermore, PDX1+/− and PDX1P33T/P33T mutations reduced differentiation efficiency of pancreatic progenitors (PPs), due to downregulation of PDX1-bound genes, including transcription factors MNX1 and PDX1 as well as insulin resistance gene CES1. Additionally, both PDX1P33T/+ and PDX1P33T/P33T mutations in PPs reduced the expression of PDX1-bound genes including the long-noncoding RNA, MEG3 and the imprinted gene NNAT, both involved in insulin synthesis and secretion. Conclusions: Our results reveal mechanistic details of how common coding mutations in PDX1 impair human pancreatic endocrine lineage formation and β-cell function and contribute to the predisposition for diabetes. Keywords: PDX1, Transactivation domain, β-Cell differentiation, Insulin secretion, PDX1-Bound genes

Background: Chronic Obstructive Pulmonary Disease (COPD) is the third leading cause of death worldwide with no curative therapy. A non-canonical Notch ligand, DNER, has been recently identified in GWAS to associate with COPD severity, but its function and contribution to COPD is unknown. Methods: DNER localisation was assessed in lung tissue from healthy and COPD patients, and cigarette smoke (CS) exposed mice. Microarray analysis was performed on WT and DNER deficient M1 and M2 bone marrow-derived macrophages (BMDM), and gene set enrichment undertaken. WT and DNER deficient mice were exposed to CS or filtered air for 3 day and 2 months to assess IFNγ-expressing macrophages and emphysema development. Notch and NFKB active subunits were quantified in WT and DNER deficient LPS-treated and untreated BMDM. Findings: Immunofluorescence staining revealed DNER localised to macrophages in lung tissue from COPD patients and mice. Human and murine macrophages showed enhanced DNER expression in response to inflammation. Interestingly, pro-inflammatory DNER deficient BMDMs exhibited impaired NICD1/NFKB dependent IFNγ signalling and reduced nuclear NICD1/NFKB translocation. Furthermore, decreased IFNγ production and Notch1 activation in recruited macrophages from CS exposed DNER deficient mice were observed, protecting against emphysema and lung dysfunction. Interpretation: DNER is a novel protein induced in COPD patients and 6 months CS-exposed mice that regulates IFNγ secretion via non-canonical Notch in pro-inflammatory recruited macrophages. These results provide a new pathway involved in COPD immunity that could contribute to the discovery of innovative therapeutic targets. Funding: This work was supported from the Helmholtz Alliance ‘Aging and Metabolic Programming, AMPro’. Keywords: COPD, Macrophages, DNER, Notch signalling, IFNγ, Cigarette smoke, Lung, NFkB

Summary: Exhausted immune responses to chronic diseases represent a major challenge to global health. We study CD4+ T cells in a mouse model with regulatable antigen presentation. When the cells are driven through the effector phase and are then exposed to different levels of persistent antigen, they lose their T helper 1 (Th1) functions, upregulate exhaustion markers, resemble naturally anergic cells, and modulate their MAPK, mTORC1, and Ca2+/calcineurin signaling pathways with increasing dose and time. They also become unable to help B cells and, at the highest dose, undergo apoptosis. Transcriptomic analyses show the dynamic adjustment of gene expression and the accumulation of T cell receptor (TCR) signals over a period of weeks. Upon antigen removal, the cells recover their functionality while losing exhaustion and anergy markers. Our data suggest an adjustable response of CD4+ T cells to different levels of persisting antigen and contribute to a better understanding of chronic disease.

Objective: Homozygous loss-of-function mutations in the gene coding for the homeobox transcription factor (TF) PDX1 leads to pancreatic agenesis, whereas heterozygous mutations can cause Maturity-Onset Diabetes of the Young 4 (MODY4). Although the function of Pdx1 is well studied in pre-clinical models during insulin-producing β-cell development and homeostasis, it remains elusive how this TF controls human pancreas development by regulating a downstream transcriptional program. Also, comparative studies of PDX1 binding patterns in pancreatic progenitors and adult β-cells have not been conducted so far. Furthermore, many studies reported the association between single nucleotide polymorphisms (SNPs) and T2DM, and it has been shown that islet enhancers are enriched in T2DM-associated SNPs. Whether regions, harboring T2DM-associated SNPs are PDX1 bound and active at the pancreatic progenitor stage has not been reported so far. Methods: In this study, we have generated a novel induced pluripotent stem cell (iPSC) line that efficiently differentiates into human pancreatic progenitors (PPs). Furthermore, PDX1 and H3K27ac chromatin immunoprecipitation sequencing (ChIP-seq) was used to identify PDX1 transcriptional targets and active enhancer and promoter regions. To address potential differences in the function of PDX1 during development and adulthood, we compared PDX1 binding profiles from PPs and adult islets. Moreover, combining ChIP-seq and GWAS meta-analysis data we identified T2DM-associated SNPs in PDX1 binding sites and active chromatin regions. Results: ChIP-seq for PDX1 revealed a total of 8088 PDX1-bound regions that map to 5664 genes in iPSC-derived PPs. The PDX1 target regions include important pancreatic TFs, such as PDX1 itself, RFX6, HNF1B, and MEIS1, which were activated during the differentiation process as revealed by the active chromatin mark H3K27ac and mRNA expression profiling, suggesting that auto-regulatory feedback regulation maintains PDX1 expression and initiates a pancreatic TF program. Remarkably, we identified several PDX1 target genes that have not been reported in the literature in human so far, including RFX3, required for ciliogenesis and endocrine differentiation in mouse, and the ligand of the Notch receptor DLL1, which is important for endocrine induction and tip-trunk patterning. The comparison of PDX1 profiles from PPs and adult human islets identified sets of stage-specific target genes, associated with early pancreas development and adult β-cell function, respectively. Furthermore, we found an enrichment of T2DM-associated SNPs in active chromatin regions from iPSC-derived PPs. Two of these SNPs fall into PDX1 occupied sites that are located in the intronic regions of TCF7L2 and HNF1B. Both of these genes are key transcriptional regulators of endocrine induction and mutations in cis-regulatory regions predispose to diabetes. Conclusions: Our data provide stage-specific target genes of PDX1 during in vitro differentiation of stem cells into pancreatic progenitors that could be useful to identify pathways and molecular targets that predispose for diabetes. In addition, we show that T2DM-associated SNPs are enriched in active chromatin regions at the pancreatic progenitor stage, suggesting that the susceptibility to T2DM might originate from imperfect execution of a β-cell developmental program. Keywords: iPSC, T2DM, ChIP-seq, PDX1, SNPs, PP, GWAS

Summary: Zebrafish have a high capacity to replace lost neurons after brain injury. New neurons involved in repair are generated by a specific set of glial cells, known as ependymoglial cells. We analyze changes in the transcriptome of ependymoglial cells and their progeny after injury to infer the molecular pathways governing restorative neurogenesis. We identify the aryl hydrocarbon receptor (AhR) as a regulator of ependymoglia differentiation toward post-mitotic neurons. In vivo imaging shows that high AhR signaling promotes the direct conversion of a specific subset of ependymoglia into post-mitotic neurons, while low AhR signaling promotes ependymoglial proliferation. Interestingly, we observe the inactivation of AhR signaling shortly after injury followed by a return to the basal levels 7 days post injury. Interference with timely AhR regulation after injury leads to aberrant restorative neurogenesis. Taken together, we identify AhR signaling as a crucial regulator of restorative neurogenesis timing in the zebrafish brain. : Zebrafish have a high capacity to replace lost neurons after brain injury. Di Giaimo et al. identify the aryl hydrocarbon receptor (AhR) as a crucial regulator of restorative neurogenesis timing in the zebrafish brain. Interference with timely AhR regulation after injury leads to aberrant restorative neurogenesis. Keywords: neurogenesis, regeneration, aryl hydrocarbon receptor, direct conversion, zebrafish, live imaging

Objective: Serum concentrations of the hepatokine fibroblast growth factor (FGF) 21 are elevated in obesity, type-2 diabetes, and the metabolic syndrome. We asked whether FGF21 levels differ between subjects with metabolically healthy vs. unhealthy obesity (MHO vs. MUHO), opening the possibility that FGF21 is a cross-talker between liver and adipose tissue in MUHO. Furthermore, we studied the effects of chronic FGF21 treatment on adipocyte differentiation, lipid storage, and adipokine secretion. Methods: In 20 morbidly obese donors of abdominal subcutaneous fat biopsies discordant for their whole-body insulin sensitivity (hereby classified as MHO or MUHO subjects), serum FGF21 was quantified. The impact of chronic FGF21 treatment on differentiation, lipid accumulation, and adipokine release was assessed in isolated preadipocytes differentiated in vitro. Results: Serum FGF21 concentrations were more than two-fold higher in MUHO as compared to MHO subjects (457 ± 378 vs. 211 ± 123 pg/mL; p

Master regulators of the epithelial-mesenchymal transition such as Twist1 and Snail1 have been implicated in invasiveness and the generation of cancer stem cells, but their persistent activity inhibits stem-cell-like properties and the outgrowth of disseminated cancer cells into macroscopic metastases. Here, we show that Twist1 activation primes a subset of mammary epithelial cells for stem-cell-like properties, which only emerge and stably persist following Twist1 deactivation. Consequently, when cells undergo a mesenchymal-epithelial transition (MET), they do not return to their original epithelial cell state, evidenced by acquisition of invasive growth behavior and a distinct gene expression profile. These data provide an explanation for how transient Twist1 activation may promote all steps of the metastatic cascade; i.e., invasion, dissemination, and metastatic outgrowth at distant sites.

The paracaspase Malt1 is a central regulator of antigen receptor signaling that is frequently mutated in human lymphoma. As a scaffold, it assembles protein complexes for NF-κB activation, and its proteolytic domain cleaves negative NF-κB regulators for signal enforcement. Still, the physiological functions of Malt1-protease are unknown. We demonstrate that targeted Malt1-paracaspase inactivation induces a lethal inflammatory syndrome with lymphocyte-dependent neurodegeneration in vivo. Paracaspase activity is essential for regulatory T cell (Treg) and innate-like B cell development, but it is largely dispensable for overcoming Malt1-dependent thresholds for lymphocyte activation. In addition to NF-κB inhibitors, Malt1 cleaves an entire set of mRNA stability regulators, including Roquin-1, Roquin-2, and Regnase-1, and paracaspase inactivation results in excessive interferon gamma (IFNγ) production by effector lymphocytes that drive pathology. Together, our results reveal distinct threshold and modulatory functions of Malt1 that differentially control lymphocyte differentiation and activation pathways and demonstrate that selective paracaspase blockage skews systemic immunity toward destructive autoinflammation.