Epigenetic marks in the mature pollen of Quercus suber L. (Fagaceae)

We have analysed the distribution of epigenetic marks for histone modifications at lysine residues H3 and H4, and DNA methylation, in the nuclei of mature pollen cells of the Angiosperm tree Quercus suber; a monoecious wind pollinated species with a protandrous system, and a long post-pollination period. The ultrasonic treatment developed for the isolation of pollen nuclei proved to be a fast and reliable method, preventing the interference of cell wall autofluorescence in the in situ immunolabelling assays. In contrast with previous studies on herbaceous species with short progamic phases, our results are consistent with a high level of silent (5-mC and H3K9me2) epigenetic marks on chromatin of the generative nucleus, and the prevalence of active marks (H3K9me3 and H4Kac) in the vegetative nucleus. The findings are discussed in terms of the pollination/fertilization timing strategy adopted by this plant specie

New histone supply regulates replication fork speed and PCNA unloading

International audienc

Domain Model Explains Propagation Dynamics and Stability of Histone H3K27 and H3K36 Methylation Landscapes

Chromatin states must be maintained during cell proliferation to uphold cellular identity and genome integrity. Inheritance of histone modifications is central in this process. However, the histone modification landscape is challenged by incorporation of new unmodified histones during each cell cycle, and the principles governing heritability remain unclear. We take a quantitative computational modeling approach to describe propagation of histone H3K27 and H3K36 methylation states. We measure combinatorial H3K27 and H3K36 methylation patterns by quantitative mass spectrometry on subsequent generations of histones. Using model comparison, we reject active global demethylation and invoke the existence of domains defined by distinct methylation endpoints. We find that H3K27me3 on pre-existing histones stimulates the rate of de novo H3K27me3 establishment, supporting a read-write mechanism in timely chromatin restoration. Finally, we provide a detailed quantitative picture of the mutual antagonism between H3K27 and H3K36 methylation and propose that it stabilizes epigenetic states across cell division

Cmr1/WDR76 defines a nuclear genotoxic stress body linking genome integrity and protein quality control

DNA replication stress is a source of genomic instability. Here we identify ​changed mutation rate 1 (​Cmr1) as a factor involved in the response to DNA replication stress in Saccharomyces cerevisiae and show that ​Cmr1—together with ​Mrc1/​Claspin, ​Pph3, the chaperonin containing ​TCP1 (CCT) and 25 other proteins—define a novel intranuclear quality control compartment (INQ) that sequesters misfolded, ubiquitylated and sumoylated proteins in response to genotoxic stress. The diversity of proteins that localize to INQ indicates that other biological processes such as cell cycle progression, chromatin and mitotic spindle organization may also be regulated through INQ. Similar to ​Cmr1, its human orthologue ​WDR76 responds to proteasome inhibition and DNA damage by relocalizing to nuclear foci and physically associating with CCT, suggesting an evolutionarily conserved biological function. We propose that ​Cmr1/​WDR76 plays a role in the recovery from genotoxic stress through regulation of the turnover of sumoylated and phosphorylated proteins

Dynamic de novo heterochromatin assembly and disassembly at replication forks ensures fork stability

Chromatin is dynamically reorganized when DNA replication forks are challenged. However, the process of epigenetic reorganization and its implication for fork stability is poorly understood. Here we discover a checkpoint-regulated cascade of chromatin signalling that activates the histone methyltransferase EHMT2/G9a to catalyse heterochromatin assembly at stressed replication forks. Using biochemical and single molecule chromatin fibre approaches, we show that G9a together with SUV39h1 induces chromatin compaction by accumulating the repressive modifications, H3K9me1/me2/me3, in the vicinity of stressed replication forks. This closed conformation is also favoured by the G9a-dependent exclusion of the H3K9-demethylase JMJD1A/KDM3A, which facilitates heterochromatin disassembly upon fork restart. Untimely heterochromatin disassembly from stressed forks by KDM3A enables PRIMPOL access, triggering single-stranded DNA gap formation and sensitizing cells towards chemotherapeutic drugs. These findings may help in explaining chemotherapy resistance and poor prognosis observed in patients with cancer displaying elevated levels of G9a/H3K9me3.</p

DNA mediated chromatin pull-down for the study of chromatin replication

Chromatin replication involves duplicating DNA while maintaining epigenetic information. These processes are critical for genome stability and for preserving cell-type identity. Here we describe a simple experimental approach that allows chromatin to be captured and its content analysed after in vivo replication and labeling of DNA by cellular DNA polymerases. We show that this technique is highly specific and that proteins bound to the replicated DNA can be analyzed by both immunological techniques and large scale mass spectrometry. As proof of concept we have used this novel procedure to begin investigating the relationship between chromatin protein composition and the temporal programme of DNA replication in human cells. It is expected that this technique will become a widely used tool to address how chromatin proteins assemble onto newly replicated DNA after passage of a replication fork and how chromatin maturation is coupled to DNA synthesis

Effective, homogeneous and transient interference with cytosine methylation in plant genomic DNA by zebularine

Covalent modification by methylation of cytosine residues represents an important epigenetic hallmark. While sequence analysis after bisulphite conversion allows correlative analyses with single-base resolution, functional analysis by interference with DNA methylation is less precise, due to the complexity of methylation enzymes and their targets. A cytidine analogue, 5-azacytidine, is frequently used as an inhibitor of DNA methyltransferases, but its rapid degradation in aqueous solution is problematic for culture periods of longer than a few hours. Application of zebularine, a more stable cytidine analogue with a similar mode of action that is successfully used as a methylation inhibitor in Neurospora and mammalian tumour cell lines, can significantly reduce DNA methylation in plants in a dose-dependent and transient manner independent of sequence context. Demethylation is connected with transcriptional reactivation and partial decondensation of heterochromatin. Zebularine represents a promising new and versatile tool for investigating the role of DNA methylation in plants with regard to transcriptional control, maintenance and formation of (hetero-) chromatin

Developmental regulation of N-terminal H2B methylation in Drosophila melanogaster

Histone post-translational modifications play an important role in regulating chromatin structure and gene expression in vivo. Extensive studies investigated the post-translational modifications of the core histones H3 and H4 or the linker histone H1. Much less is known on the regulation of H2A and H2B modifications. Here, we show that a major modification of H2B in Drosophila melanogaster is the methylation of the N-terminal proline, which increases during fly development. Experiments performed in cultured cells revealed higher levels of H2B methylation when cells are dense, regardless of their cell cycle distribution. We identified dNTMT (CG1675) as the enzyme responsible for H2B methylation. We also found that the level of N-terminal methylation is regulated by dART8, an arginine methyltransferase that physically interacts with dNTMT and asymmetrically methylates H3R2. Our results demonstrate the existence of a complex containing two methyltransferases enzymes, which negatively influence each other’s activity