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

A screen for hydroxymethylcytosine and formylcytosine binding proteins suggests functions in transcription and chromatin regulation

MI is supported by the People Programme (Marie Curie Actions) of the European Union’s Seventh Framework Programme FP7/2007-2013/under REA grant agreement no. 290123 and was supported by Unipharma-Graduates 7 Da Vinci Programme. MJB is supported by a BBRSC studentship

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

A Temporal Threshold for Formaldehyde Crosslinking and Fixation

Formaldehyde crosslinking is in widespread use as a biological fixative for microscopy and molecular biology. An assumption behind its use is that most biologically meaningful interactions are preserved by crosslinking, but the minimum length of time required for an interaction to become fixed has not been determined.Using a unique series of mutations in the DNA binding protein MeCP2, we show that in vivo interactions lasting less than 5 seconds are invisible in the microscope after formaldehyde fixation, though they are obvious in live cells. The stark contrast between live cell and fixed cell images illustrates hitherto unsuspected limitations to the fixation process. We show that chromatin immunoprecipitation, a technique in widespread use that depends on formaldehyde crosslinking, also fails to capture these transient interactions.Our findings for the first time establish a minimum temporal limitation to crosslink chemistry that has implications for many fields of research

Low HER2 expression in normal breast epithelium enables dedifferentiation and malignant transformation via chromatin opening.

Overexpression of the HER2 protein in breast cancer patients is a predictor of poor prognosis and resistance to therapies. We used an inducible breast cancer transformation system that allows investigation of early molecular changes. HER2 overexpression to similar levels as those observed in a subtype of HER2-positive breast cancer patients induced transformation of MCF10A cells and resulted in gross morphological changes, increased anchorage-independent growth of cells, and altered the transcriptional programme of genes associated with oncogenic transformation. Global phosphoproteomic analysis during HER2 induction predominantly detected an increase in protein phosphorylation. Intriguingly, this correlated with chromatin opening, as measured by ATAC-seq on acini isolated from 3D cell culture. HER2 overexpression resulted in opening of many distal regulatory regions and promoted reprogramming-associated heterogeneity. We found that a subset of cells acquired a dedifferentiated breast stem-like phenotype, making them likely candidates for malignant transformation. Our data show that this population of cells, which counterintuitively enriches for relatively low HER2 protein abundance and increased chromatin accessibility, possesses transformational drive, resulting in increased anchorage-independent growth in vitro compared to cells not displaying a stem-like phenotype

The Ageing Brain: Effects on DNA Repair and DNA Methylation in Mice

The Centre for Ageing & Vitality is funded by the MRC and BBSRC (Grant Reference MR/L016354/1). This work was further supported by the Centre for Integrated Systems Biology of Ageing and Nutrition funded by the BBSRC and EPSRC (G0700718). Part of the work was supported by BBSRC Grant BB/K010867/1

The Influence of Hydroxylation on Maintaining CpG Methylation Patterns: A Hidden Markov Model Approach

German Research Council (DFG) as part of the Collaborative Research Center "Physical modeling of non-equilibrium processes in biological systems" (SFB 1027) and the Cluster of Excellence on Multimodal Computing and Interaction at Saarland Universit

Integrating 5-Hydroxymethylcytosine into the Epigenomic Landscape of Human Embryonic Stem Cells

Covalent modification of DNA distinguishes cellular identities and is crucial for regulating the pluripotency and differentiation of embryonic stem (ES) cells. The recent demonstration that 5-methylcytosine (5-mC) may be further modified to 5-hydroxymethylcytosine (5-hmC) in ES cells has revealed a novel regulatory paradigm to modulate the epigenetic landscape of pluripotency. To understand the role of 5-hmC in the epigenomic landscape of pluripotent cells, here we profile the genome-wide 5-hmC distribution and correlate it with the genomic profiles of 11 diverse histone modifications and six transcription factors in human ES cells. By integrating genomic 5-hmC signals with maps of histone enrichment, we link particular pluripotency-associated chromatin contexts with 5-hmC. Intriguingly, through additional correlations with defined chromatin signatures at promoter and enhancer subtypes, we show distinct enrichment of 5-hmC at enhancers marked with H3K4me1 and H3K27ac. These results suggest potential role(s) for 5-hmC in the regulation of specific promoters and enhancers. In addition, our results provide a detailed epigenomic map of 5-hmC from which to pursue future functional studies on the diverse regulatory roles associated with 5-hmC