Non-catalytic Roles of Tet2 Are Essential to Regulate Hematopoietic Stem and Progenitor Cell Homeostasis

The Ten-eleven translocation (TET) enzymes regulate gene expression by promoting DNA demethylation and partnering with chromatin modifiers. TET2, a member of this family, is frequently mutated in hematological disorders. The contributions of TET2 in hematopoiesis have been attributed to its DNA demethylase activity, and the significance of its nonenzymatic functions has remained undefined. To dissect the catalytic and non-catalytic requirements of Tet2, we engineered catalytically inactive Tet2 mutant mice and conducted comparative analyses of Tet2 mutant and Tet2 knockout animals. Tet2 knockout mice exhibited expansion of hematopoietic stem and progenitor cells (HSPCs) and developed myeloid and lymphoid disorders, while Tet2 mutant mice predominantly developed myeloid malignancies reminiscent of human myelodysplastic syndromes. HSPCs from Tet2 knockout mice exhibited distinct gene expression profiles, including downregulation of Gata2. Overexpression of Gata2 in Tet2 knockout bone marrow cells ameliorated disease phenotypes. Our results reveal the non-catalytic roles of TET2 in HSPC homeostasis

TET2 inhibits differentiation of embryonic stem cells but does not overcome methylation-induced gene silencing

TET2 is a methylcytosine dioxygenase that is frequently mutated in myeloid malignancies, notably myelodysplasia and acute myeloid leukemia. TET2 catalyses the conversion of 5′-methylcytosine to 5′-hydroxymethylcytosine within DNA and has been implicated in the process of genomic demethylation. However, the mechanism by which TET2 loss of function results in hematopoietic dysplasia and leukemogenesis is poorly understood. Here, we show that TET2 is expressed in undifferentiated embryonic stem cells and that its knockdown results in reduction of 5′-hydroxymethylcytosine in genomic DNA. We also present DNA methylation data from bone marrow samples obtained from patients with TET2-mutated myelodysplasia. Based on these findings, we sought to identify the role of TET2 in regulating pluripotency and differentiation. We show that overexpression of TET2 in a stably integrated transgene leads to increased alkaline phosphatase expression in differentiating ES cells and impaired differentiation in methylcellulose culture. We speculate that this effect is due to TET2-mediated expression of stem cell genes in ES cells via hydroxylation of 5′-methylcytosines at key promoter sequences within genomic DNA. This leads to relative hypomethylation of gene promoters as 5′-hydroxymethylcytosine is not a substrate for DNMT1-mediated maintenance methylation. We sought to test this hypothesis by cotransfecting the TET2 gene with methylated reporter genes. The results of these experiments are presented

Tet2 Rescues Age-Related Regenerative Decline and Enhances Cognitive Function in the Adult Mouse Brain.

Restoring adult stem cell function provides an exciting approach for rejuvenating the aging brain. However, molecular mechanisms mediating neurogenic rejuvenation remain elusive. Here we report that the enzyme ten eleven translocation methylcytosine dioxygenase 2 (Tet2), which catalyzes the production of 5-hydroxymethylcytosine (5hmC), rescues age-related decline in adult neurogenesis and enhances cognition in mice. We detected a decrease in Tet2 expression and 5hmC levels in the aged hippocampus associated with adult neurogenesis. Mimicking an aged condition in young adults by abrogating Tet2 expression within the hippocampal neurogenic niche, or adult neural stem cells, decreased neurogenesis and impaired learning and memory. In a heterochronic parabiosis rejuvenation model, hippocampal Tet2 expression was restored. Overexpressing Tet2 in the hippocampal neurogenic niche of mature adults increased 5hmC associated with neurogenic processes, offset the precipitous age-related decline in neurogenesis, and enhanced learning and memory. Our data identify Tet2 as a key molecular mediator of neurogenic rejuvenation

Analysis of the machinery and intermediates of the 5hmC-mediated DNA demethylation pathway in aging on samples from the MARKAGE Study

Gradual changes in the DNA methylation landscape occur throughout aging virtually in all human tissues. A widespread reduction of 5-methylcytosine (5mC), associated with highly reproducible site-specific hypermethylation, characterizes the genome in aging. Therefore, an equilibrium seems to exist between general and directional deregulating events concerning DNA methylation controllers, which may underpin the age-related epigenetic changes. In this context, 5mC-hydroxylases (TET enzymes) are new potential players. In fact, TETs catalyze the stepwise oxidation of 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC), driving the DNA demethylation process based on thymine DNA glycosylase (TDG)-mediated DNA repair pathway. The present paper reports the expression of DNA hydroxymethylation components, the levels of 5hmC and of its derivatives in peripheral blood mononuclear cells of age-stratified donors recruited in several European countries in the context of the EU Project ‘MARK-AGE’. The results provide evidence for an age-related decline of TET1, TET3 and TDG gene expression along with a decrease of 5hmC and an accumulation of 5caC. These associations were independent of confounding variables, including recruitment center, gender and leukocyte composition. The observed impairment of 5hmC-mediated DNA demethylation pathway in blood cells may lead to aberrant transcriptional programs in the elderly

cyTRON and cyTRON/JS: two Cytoscape-based applications for the inference of cancer evolution models

The increasing availability of sequencing data of cancer samples is fueling the development of algorithmic strategies to investigate tumor heterogeneity and infer reliable models of cancer evolution. We here build up on previous works on cancer progression inference from genomic alteration data, to deliver two distinct Cytoscape-based applications, which allow to produce, visualize and manipulate cancer evolution models, also by interacting with public genomic and proteomics databases. In particular, we here introduce cyTRON, a stand-alone Cytoscape app, and cyTRON/JS, a web application which employs the functionalities of Cytoscape/JS. cyTRON was developed in Java; the code is available at https://github.com/BIMIB-DISCo/cyTRON and on the Cytoscape App Store http://apps.cytoscape.org/apps/cytron. cyTRON/JS was developed in JavaScript and R; the source code of the tool is available at https://github.com/BIMIB-DISCo/cyTRON-js and the tool is accessible from https://bimib.disco.unimib.it/cytronjs/welcome

DNA hydroxymethylation levels are altered in blood cells from Down syndrome persons enrolled in the MARK-AGE project

Down syndrome (DS) is caused by the presence of part or an entire extra copy of chromosome 21, a phenomenon that can cause a wide spectrum of clinically defined phenotypes of the disease. Most of the clinical signs of DS are typical of the ageing process including dysregulation of immune system. Beyond the causative genetic defect, DS persons display epigenetic alterations, particularly aberrant DNA methylation patterns that can contribute to the heterogeneity of the disease. In the present work we investigated the levels of 5-hydroxymethylcytosine (5hmC) and of the TET dioxygenase enzymes, which are involved in DNA demethylation processes and are often deregulated in pathological conditions as well as in ageing. Analyses were carried out on peripheral blood mononuclear cells of DS volunteers enrolled in the context of the MARK-AGE study, a large-scale cross-sectional population study with subjects representing the general population in eight European countries. We observed a decrease of 5hmC, TET1 and other components of the DNA methylation/demethylation machinery in DS subjects, indicating that aberrant DNA methylation patterns in DS, which may have consequences on the transcriptional status of immune cells, may be due to a global disturbance of methylation control in DS

5-hydroxymethylcytosine is a key epigenetic regulator of keratinocyte stem cells during psoriasis pathogenesis

Epigenetic regulation is now known to play an important role in determining stem cell fate during normal tissue development and disease pathogenesis. In this study, we report loss of 5-hydroxymethylcytosine (5-hmC) mediated by ten-eleven translocation (TET) methylcytosine dioxygenases in keratinocyte stem cells (KSCs) and in their progenitor transit-amplifying (TA) cells of psoriatic lesions. We establish the DNA hydroxymethylation profile in both human psoriasis as well as in the imiquimod (IMQ)-induced mouse psoriasis model. Genome-wide mapping of 5-hmC in IMQ-treated mice epithelium revealed a loci-specific reduction of 5-hmC in genes associated with maintaining stem cell homeostasis including those involved in the RAR and Wnt/β-catenin signaling pathways. Restoration of TET expression in human KSC cultures via vitamin C treatment increased 5-hmC levels and induced more normal KSC differentiation. We found that by modulating 5-hmC levels in vitro, we could alter downstream expression of genes important in regulating stem cell homeostasis like nestin as well as IL-17R known to promote the psoriatic phenotype. Our findings demonstrate that loss of 5-hmC is a critical epigenomic phenomenon in KSCs and TA cells during psoriasis pathogenesis.2019-12-17T00:00:00

Short- and long-term effects of 56Fe irradiation on cognition and hippocampal DNA methylation and gene expression.

BackgroundAstronauts are exposed to 56Fe ions that may pose a significant health hazard during and following prolonged missions in deep space. We showed previously that object recognition requiring the hippocampus, a structure critical for cognitive function, is affected in 2-month-old mice irradiated with 56Fe ions. Here we examined object recognition in 6-month-old mice irradiated with 56Fe ions, a biological age more relevant to the typical ages of astronauts. Moreover, because the mechanisms mediating the detrimental effects of 56Fe ions on hippocampal function are unclear, we examined changes in hippocampal networks involved in synaptic plasticity and memory, gene expression, and epigenetic changes in cytosine methylation (5mC) and hydroxymethylation (5hmC) that could accompany changes in gene expression. We assessed the effects of whole body 56Fe ion irradiation at early (2 weeks) and late (20 weeks) time points on hippocampus-dependent memory and hippocampal network stability, and whether these effects are associated with epigenetic changes in hippocampal DNA methylation (both 5mC and 5hmC) and gene expression.ResultsAt the two-week time point, object recognition and network stability were impaired following irradiation at the 0.1 and 0.4 Gy dose, but not following irradiation at the 0.2 Gy dose. No impairments in object recognition or network stability were seen at the 20-week time point at any irradiation dose used. Consistent with this pattern, the significance of pathways for gene categories for 5hmC was lower, though not eliminated, at the 20-week time point compared to the 2-week time point. Similarly, significant changes were observed for 5mC gene pathways at the 2-week time point, but no significant gene categories were observed at the 20-week time point. Only the 5hmC changes tracked with gene expression changes.ConclusionsDose- and time-dependent epigenomic remodeling in the hippocampus following 56Fe ion exposure correlates with behavioral changes