Polycomb group protein complexes exchange rapidly in living Drosophila

Fluorescence recovery after photobleaching (FRAP) microscopy was used to determine the kinetic properties of Polycomb group (PcG) proteins in whole living Drosophila organisms (embryos) and tissues (wing imaginal discs and salivary glands). PcG genes are essential genes in higher eukaryotes responsible for the maintenance of the spatially distinct repression of developmentally important regulators such as the homeotic genes. Their absence, as well as overexpression, causes transformations in the axial organization of the body. Although protein complexes have been isolated in vitro, little is known about their stability or exact mechanism of repression in vivo. We determined the translational diffusion constants of PcG proteins, dissociation constants and residence times for complexes in vivo at different developmental stages. In polytene nuclei, the rate constants suggest heterogeneity of the complexes. Computer simulations with new models for spatially distributed protein complexes were performed in systems showing both diffusion and binding equilibria, and the results compared with our experimental data. We were able to determine forward and reverse rate constants for complex formation. Complexes exchanged within a period of 1-10 minutes, more than an order of magnitude faster than the cell cycle time, ruling out models of repression in which access of transcription activators to the chromatin is limited and demonstrating that long-term repression primarily reflects mass-action chemical equilibria

Genome-wide distribution of 5-formylcytosine in embryonic stem cells is associated with transcription and depends on thymine DNA glycosylase

This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.EAR is a Herchel Smith Fellow. MB and HB are supported by the Centre for Trophoblast Research, MB is a Next Generation Research Fellow. MJB is supported by a BBSRC studentship. The WR lab is supported by BBSRC, MRC, the Wellcome Trust, EU EpiGeneSys and BLUEPRINT. The SB lab is supported by core funding from Cancer Research UK

5-hydroxymethyl-cytosine enrichment of non-committed cells is not a universal feature of vertebrate development

5-hydroxymethyl-cytosine (5-hmc) is a cytosine modification that is relatively abundant in mammalian pre-implantation embryos and embryonic stem cells (Esc) derived from mammalian blastocysts. Recent observations imply that both 5-hmc and Tet1/2/3 proteins, catalyzing the conversion of 5-methyl-cytosine to 5-hmc, may play an important role in self renewal and differentiation of Escs. here we assessed the distribution of 5-hmc in zebrafish and chick embryos and found that, unlike in mammals, 5-hmc is immunochemically undetectable in these systems before the onset of organogenesis. In addition, Tet1/2/3 transcripts are either low or undetectable at corresponding stages of zebrafish development. however, 5-hmc is enriched in later zebrafish and chick embryos and exhibits tissue-specific distribution in adult zebrafish. Our findings show that 5-hmc enrichment of non-committed cells is not a universal feature of vertebrate development and give insights both into evolution of embryonic pluripotency and the potential role of 5-hmc in its regulation

Medulloblastoma and ependymoma cells display levels of 5-carboxylcytosine and elevated TET1 expression

Background Alteration of DNA methylation (5-methylcytosine, 5mC) patterns represents one of the causes of tumorigenesis and cancer progression. Tet proteins can oxidize 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine and 5-carboxylcytosine (5caC). Although the roles of these oxidised forms of 5mC (oxi-mCs) in cancer pathogenesis are still largely unknown, there are indications that they may be involved in the mechanisms of malignant transformation. Thus, reduction of 5hmC content represents an epigenetic hallmark of human tumours and, according to our recent report; 5caC is enriched in a proportion of breast cancers and gliomas. Nevertheless, the distribution of oxi-mCs in paediatric brain tumours has not been assessed. Findings Here we analyse the global levels and spatial distribution of 5hmC and 5caC in 4 brain tumour cell lines derived from paediatric sonic hedgehog (SHH) pathway activated medulloblastomas (Daoy and UW228-3) and ependymomas (BXD-1425EPN and DKFZEP1NS). We show that, unlike HeLa cells, the paediatric tumour cell lines possess both 5hmC and 5caC at immunochemically detectable levels, and demonstrate that both modifications display high degrees of spatial overlap in the nuclei of medulloblastomas and ependymomas. Moreover, although 5hmC levels are comparable in the 4 brain tumour cell lines, 5caC staining intensities differ dramatically between them with highest levels of this mark in a subpopulation of DKFZ-EP1NS cells. Remarkably, the 5caC enrichment does not correlate with 5hmC levels and is not associated with alterations in Thymine DNA Glycosylase (TDG) expression in SHH medulloblastoma and ependymoma cell lines, but corresponds to elevated levels of TET1 transcript in UW228-3 and DKFZ-EP1NS cells. Conclusions We demonstrate that both 5caC enrichment and elevated TET1 expression are observed in SHH medulloblastomas and ependymomas. Our results suggest that increased Tet-dependent 5mC oxidation may represent one of the epigenetic signatures of cancers with neural stem cell origin and, thus, may contribute to development of novel approaches for diagnosis and therapy of the brain tumours

Hippocampal TET1 and TET2 expression and DNA hydroxymethylation are affected by physical exercise in aged mice

The function of 5-hydroxymethylcytosine (5hmC) is poorly understood. 5hmC is an epigenetic modification of DNA, resulting from the oxidation of 5-methylcytosine (5mC) by the Fe2+, and 2-oxoglutarate-dependent, 10–11 translocation methylcytosine dioxygenases (TET1, TET2, and TET3). Recent evidence suggests that, in addition to being an intermediate in active demethylation, 5hmC may also have an epigenetic role. 5hmC is enriched in the adult brain, where it has been implicated in regulating neurogenesis. The rate of adult neurogenesis decreases with age, however physical exercise has been shown to counteract this deficit. Here, we investigated the impact of voluntary exercise on the age-related changes of TET1, TET2, expression and 5hmC content in the hippocampus and hypothalamus. For this purpose, we used voluntary exercise in young adult (3 months) and aged (18 months) mice as a rodent model of healthy brain aging. We measured the levels of hippocampal and hypothalamic TET1, TET2 mRNA, and 5hmC and memory [Object Location (OL) test] in mice that either exercised for 1 month or remained sedentary. While aging was associated with decreased TET1 and TET2 expression, voluntary exercise counteracted the decline in expression. Moreover, aged mice that exercised had higher hippocampal 5hmC content in the promoter region of miR-137, an miRNA involved in adult neurogenesis. Exercise improved memory in aged mice, and there was a positive correlation between 5hmC miR-137 levels and performance in the OL test. In the hypothalamus neither exercise nor aging affected TET1 or TET2 expression. These results suggest that exercise partially restores the age-related decrease in hippocampal TET1 and TET2 expression, which may be linked to the improvement in memory. Future studies should further determine the specific genes where changes in 5hmC levels may mediate the exercise-induced improvements in memory and neurogenesis in aged animals

The hypomethylating agent Decitabine causes a paradoxical increase in 5-hydroxymethylcytosine in human leukemia cells

The USFDA approved "epigenetic drug", Decitabine, exerts its effect by hypomethylating DNA, demonstrating the pivotal role aberrant genome-wide DNA methylation patterns play in cancer ontology. Using sensitive technologies in a cellular model of Acute Myeloid Leukemia, we demonstrate that while Decitabine reduces the global levels of 5-methylcytosine (5mC), it results in paradoxical increase of 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) levels. Hitherto, the only biological mechanism known to generate 5hmC, 5fC and 5caC, involving oxidation of 5mC by members of Ten-Eleven-Translocation (TET) dioxygenase family, was not observed to undergo any alteration during DAC treatment. Using a multi-compartmental model of DNA methylation, we show that partial selectivity of TET enzymes for hemi-methylated CpG dinucleotides could lead to such alterations in 5hmC content. Furthermore, we investigated the binding of TET1-catalytic domain (CD)-GFP to DNA by Fluorescent Correlation Spectroscopy in live cells and detected the gradual increase of the DNA bound fraction of TET1-CD-GFP after treatment with Decitabine. Our study provides novel insights on the therapeutic activity of DAC in the backdrop of the newly discovered derivatives of 5mC and suggests that 5hmC has the potential to serve as a biomarker for monitoring the clinical success of patients receiving DAC

TET1 is a tumor suppressor of hematopoietic malignancy

The methylcytosine dioxygenase TET1 (‘ten-eleven translocation 1’) is an important regulator of 5-hydroxymethylcytosine (5hmC) in embryonic stem cells. The diminished expression of TET proteins and loss of 5hmC in many tumors suggests a critical role for the maintenance of this epigenetic modification. Here we found that deletion of Tet1 promoted the development of B cell lymphoma in mice. TET1 was required for maintenance of the normal abundance and distribution of 5hmC, which prevented hypermethylation of DNA, and for regulation of the B cell lineage and of genes encoding molecules involved in chromosome maintenance and DNA repair. Whole-exome sequencing of TET1-deficient tumors revealed mutations frequently found in non-Hodgkin B cell lymphoma (B-NHL), in which TET1 was hypermethylated and transcriptionally silenced. Our findings provide in vivo evidence of a function for TET1 as a tumor suppressor of hematopoietic malignancy.National Institutes of Health (U.S.) (5RO1HD045022)National Institutes of Health (U.S.) (5R37CA084198

Histone modifications form a cell-type-specific chromosomal bar code that persists through the cell cycle.

Chromatin configuration influences gene expression in eukaryotes at multiple levels, from individual nucleosomes to chromatin domains several Mb long. Post-translational modifications (PTM) of core histones seem to be involved in chromatin structural transitions, but how remains unclear. To explore this, we used ChIP-seq and two cell types, HeLa and lymphoblastoid (LCL), to define how changes in chromatin packaging through the cell cycle influence the distributions of three transcription-associated histone modifications, H3K9ac, H3K4me3 and H3K27me3. We show that chromosome regions (bands) of 10-50 Mb, detectable by immunofluorescence microscopy of metaphase (M) chromosomes, are also present in G1 and G2. They comprise 1-5 Mb sub-bands that differ between HeLa and LCL but remain consistent through the cell cycle. The same sub-bands are defined by H3K9ac and H3K4me3, while H3K27me3 spreads more widely. We found little change between cell cycle phases, whether compared by 5 Kb rolling windows or when analysis was restricted to functional elements such as transcription start sites and topologically associating domains. Only a small number of genes showed cell-cycle related changes: at genes encoding proteins involved in mitosis, H3K9 became highly acetylated in G2M, possibly because of ongoing transcription. In conclusion, modified histone isoforms H3K9ac, H3K4me3 and H3K27me3 exhibit a characteristic genomic distribution at resolutions of 1 Mb and below that differs between HeLa and lymphoblastoid cells but remains remarkably consistent through the cell cycle. We suggest that this cell-type-specific chromosomal bar-code is part of a homeostatic mechanism by which cells retain their characteristic gene expression patterns, and hence their identity, through multiple mitoses

Developmental functions of the dynamic DNA methylome and hydroxymethylome in the mouse and zebrafish: similarities and differences

5-methylcytosine (5mC) is the best understood DNA modification and is generally believed to be associated with repression of gene expression. Over the last decade, sequentially oxidized forms of 5mC (oxi-mCs) have been discovered within the genomes of vertebrates. Their discovery was accompanied by that of the ten-eleven translocation (TET) methylcytosine dioxygenases, the enzymes that catalyse the formation of the oxi-mCs. Although a number of studies performed on different vertebrate models and embryonic stem cells demonstrated that both TET enzymes and oxi-mCs are likely to be important for several developmental processes it is currently unclear whether their developmental roles are conserved among vertebrates. Here, we summarise recent developments in this field suggesting that biological roles of TETs/oxi-mCs may significantly differ between mice and zebrafish. Thus, although the role of TET proteins in late organogenesis has been documented for both these systems; unlike in mice the enzymatic oxidation of 5mC does not seem to be involved in zygotic reprogramming or gastrulation in zebrafish. Our analysis may provide an insight into the general principles of epigenetic regulation of animal development and cellular differentiation