Paul J. Hurd

The methyl-CpG-binding protein MeCP2 links DNA methylation to histone methylation.

DNA methylation plays an important role in mammalian development and correlates with chromatin-associated gene silencing. The recruitment of MeCP2 to methylated CpG dinucleotides represents a major mechanism by which DNA methylation can repress transcription. MeCP2 silences gene expression partly by recruiting histone deacetylase (HDAC) activity, resulting in chromatin remodeling. Here, we show that MeCP2 associates with histone methyltransferase activity in vivo and that this activity is directed against Lys9 of histone H3. Two characterized repression domains of MeCP2 are involved in tethering the histone methyltransferase to MeCP2. We asked if MeCP2 can deliver Lys9 H3 methylation to the H19 gene, whose activity it represses. We show that the presence of MeCP2 on nucleosomes within the repressor region of the H19 gene (the differentially methylated domain) coincides with an increase in H3 Lys9methylation. Our data provide evidence that MeCP2 reinforces a repressive chromatin state by acting as a bridge between two global epigenetic modifications, DNA methylation and histone methylation.

Zebularine: a novel DNA methylation inhibitor that forms a covalent complex with DNA methyltransferases.

Mechanism-based inhibitors of enzymes, which mimic reactive intermediates in the reaction pathway, have been deployed extensively in the analysis of metabolic pathways and as candidate drugs. The inhibition of cytosine-[C5]-specific DNA methyltransferases (C5 MTases) by oligodeoxynucleotides containing 5-azadeoxycytidine (AzadC) and 5-fluorodeoxycytidine (FdC) provides a well-documented example of mechanism-based inhibition of enzymes central to nucleic acid metabolism. Here, we describe the interaction between the C5 MTase from Haemophilus haemolyticus (M.HhaI) and an oligodeoxynucleotide duplex containing 2-H pyrimidinone, an analogue often referred to as zebularine and known to give rise to high-affinity complexes with MTases. X-ray crystallography has demonstrated the formation of a covalent bond between M.HhaI and the 2-H pyrimidinone-containing oligodeoxynucleotide. This observation enables a comparison between the mechanisms of action of 2-H pyrimidinone with other mechanism-based inhibitors such as FdC. This novel complex provides a molecular explanation for the mechanism of action of the anti-cancer drug zebularine.

Advantages of next-generation sequencing versus the microarray in epigenetic research

Several recent studies from the field of epigenetics have combined chromatin-immunoprecipitation (ChIP) with next-generation high-throughput sequencing technologies to describe the locations of histone post-translational modifications (PTM) and DNA methylation genome-wide. While these reports begin to quench the chromatin biologists thirst for visualizing where in the genome epigenetic marks are placed, they also illustrate several advantages of sequencing based genomics compared to microarray analysis. Accordingly, next-generation sequencing (NGS) technologies are now challenging microarrays as the tool of choice for genome analysis. The increased affordability of comprehensive sequence-based genomic analysis will enable new questions to be addressed in many areas of biology. It is inevitable that massively-parallel sequencing platforms will supercede the microarray for many applications, however, there are niches for microarrays to fill and interestingly we may very well witness a symbiotic relationship between microarrays and high-throughput sequencing in the future.

Phosphorylation of Histone H3 Thr-45 Is Linked to Apoptosis

Numerous post-translational modifications have been identified in histones. Most of these occur within the histone tails, but a few have been identified within the histone core sequences. Histone core post-translational modifications have the potential to directly modulate nucleosome structure and consequently DNA accessibility. Here, we identify threonine 45 of histone H3 (H3T45) as a site of phosphorylation in vivo. We find that phosphorylation of H3T45 (H3T45ph) increases dramatically in apoptotic cells, around the time of DNA nicking. To further explore this connection, we analyzed human neutrophil cells because they are short-lived cells that undergo apoptosis in vivo. Freshly isolated neutrophils contain very little H3T45ph, whereas cells cultured for 20 h possess significant amounts; the kinetics of H3T45ph induction closely parallel those of caspase-3 activation. Cytokine inhibition of neutrophil apoptosis leads to reduced levels of H3T45ph. We identify protein kinase C-δ as the kinase responsible for H3T45ph in vitro and in vivo. Given the nucleosomal position of H3T45, we postulate that H3T45ph induces structural change within the nucleosome to facilitate DNA nicking and/or fragmentation.

Extensive histone post-translational modification in honey bees

Histone post-translational modifications (PTMs) play a key role in regulating a variety of cellular processes including the establishment, maintenance and reversal of transcriptional programmes in eukaryotes. However, little is known about such modifications in the economically and ecologically important insect pollinator, the honey bee (Apis mellifera). Using mass spectrometry approaches, we show that histone H3.1, H3.3 and H4 of the honey bee are extensively modified by lysine acetylation and lysine methylation. We analysed histones isolated from queen ovaries and 96 hr-old larvae, in toto we quantified 23 specific modification states on 23 distinct peptides. In addition, we have identified and characterised patterns of histone PTMs that reside on the same peptide, generating detailed combinatorial information. Overall, we observed similar profiles of histone PTMs in both samples, with combinatorial patterns of lysine methylations on H3K27 and H3K36 more frequently identified in histones extracted from queen ovaries than from larvae. To our knowledge, this comprehensive dataset represents the first identification and quantitation of histone PTMs in this eusocial insect and emerging epigenetic model.

Systems Biology for Ecology: From Molecules to Ecosystems

Summary Ecology stands on the edge of a true paradigm shift, fuelled by a recent technological revolution in our ability to measure both taxonomic and functional biodiversity via the application of metagenomics and transcriptomics. The advent of ‘next generation sequencing’ (NGS) in molecular biology is rapidly opening the black box of microbial ecology, providing us with some of the first glimpses of a previously hidden world. This is now enabling microbial ecology to become firmly embedded as a core subdiscipline within ecology, and to test general theories about biodiversity, biogeography and ecosystem functioning using a combination of molecular and more traditional techniques. In addition, NGS offers a means of not only measuring the abundance and diversity of the main drivers of many of the planets key biogeochemical processes, but also of linking the microscopic and macroscopic worlds that have, until now, been largely studied in isolation. We provide a detailed review of the rise of NGS, as well as highlighting areas that offer special promise for addressing general ecological questions across a range of levels of organisation, from individuals to ecosystems: essentially, how a ‘systems biology for ecology’ might be developed. We consider the current limitations and future prospects for NGS, and also how it offers potential economic benefits, for instance via bioprospecting the environment for commercially valuable genes and their products within the metagenome of natural ecosystems.

Heritable Gene Repression through the Action of a Directed DNA Methyltransferase at a Chromosomal Locus

The ability to exogenously impose targeted epigenetic changes in the genome represents an attractive route for the simulation of genomic de novo epigenetic events characteristic of some diseases and for the study of their downstream effects and also provides a potential therapeutic approach for the heritable repression of selected genes. Here we demonstrate for the first time the ability of zinc finger peptides to deliver DNA cytosine methylation in vivo to a genomic integrated target promoter when expressed as fusions with a mutant prokaryotic DNA cytosine methyltransferase enzyme, thus mimicking cellular genomic de novo methylation events and allowing a direct analysis of the mechanics of de novo DNA methylation-mediated gene silencing at a genomic locus. We show that targeted methylation leads to gene silencing via the initiation of a repressive chromatin signature at the targeted genomic locus. This repression is maintained after the loss of targeted methyltransferase enzyme from the cell, confirming epigenetic maintenance purely through the action of cellular enzymes. The inherited DNA methylation pattern is restricted only to targeted sites, suggesting that the establishment of repressive chromatin structure does not drive further de novo DNA methylation in this system. As well as demonstrating the potential of these enzymes as tools for the exogenous, heritable control of cellular gene expression, this work also provides the most definitive confirmation to date for a transcriptionally repressive role for de novo DNA methylation in the cell and lends some weight to the hypothesis that the aberrant methylation associated with certain diseases may well be a cause rather than a consequence of transcriptional gene repression.

Chapter 3 - Systems Biology for Ecology: From Molecules to Ecosystems

Summary Ecology stands on the edge of a true paradigm shift, fuelled by a recent technological revolution in our ability to measure both taxonomic and functional biodiversity via the application of metagenomics and transcriptomics. The advent of ‘next generation sequencing’ (NGS) in molecular biology is rapidly opening the black box of microbial ecology, providing us with some of the first glimpses of a previously hidden world. This is now enabling microbial ecology to become firmly embedded as a core subdiscipline within ecology, and to test general theories about biodiversity, biogeography and ecosystem functioning using a combination of molecular and more traditional techniques. In addition, NGS offers a means of not only measuring the abundance and diversity of the main drivers of many of the planets key biogeochemical processes, but also of linking the microscopic and macroscopic worlds that have, until now, been largely studied in isolation. We provide a detailed review of the rise of NGS, as well as highlighting areas that offer special promise for addressing general ecological questions across a range of levels of organisation, from individuals to ecosystems: essentially, how a ‘systems biology for ecology’ might be developed. We consider the current limitations and future prospects for NGS, and also how it offers potential economic benefits, for instance via bioprospecting the environment for commercially valuable genes and their products within the metagenome of natural ecosystems.

Inactive or moderately active human promoters are enriched for inter-individual epialleles

BackgroundInter-individual epigenetic variation, due to genetic, environmental or random influences, is observed in many eukaryotic species. In mammals, however, the molecular nature of epiallelic variation has been poorly defined, partly due to the restricted focus on DNA methylation. Here we report the first genome-scale investigation of mammalian epialleles that integrates genomic, methylomic, transcriptomic and histone state information.ResultsFirst, in a small sample set, we demonstrate that non-genetically determined inter-individual differentially methylated regions (iiDMRs) can be temporally stable over at least 2 years. Then, we show that iiDMRs are associated with changes in chromatin state as measured by inter-individual differences in histone variant H2A.Z levels. However, the correlation of promoter iiDMRs with gene expression is negligible and not improved by integrating H2A.Z information. We find that most promoter epialleles, whether genetically or non-genetically determined, are associated with low levels of transcriptional activity, depleted for housekeeping genes, and either depleted for H3K4me3/enriched for H3K27me3 or lacking both these marks in human embryonic stem cells. The preferential enrichment of iiDMRs at regions of relative transcriptional inactivity validates in a larger independent cohort, and is reminiscent of observations previously made for promoters that undergo hypermethylation in various cancers, in vitro cell culture and ageing.ConclusionsOur work identifies potential key features of epiallelic variation in humans, including temporal stability of non-genetically determined epialleles, and concomitant perturbations of chromatin state. Furthermore, our work suggests a novel mechanistic link among inter-individual epialleles observed in the context of normal variation, cancer and ageing.

Arginine methylation and citrullination of splicing factor proline- and glutamine-rich (SFPQ/PSF) regulates its association with mRNA.

Splicing factor proline- and glutamine-rich (SFPQ) also commonly known as polypyrimidine tract-binding protein-associated-splicing factor (PSF) and its binding partner non-POU domain-containing octamer-binding protein (NONO/p54nrb), are highly abundant, multifunctional nuclear proteins. However, the exact role of this complex is yet to be determined. Following purification of the endogeneous SFPQ/NONO complex, mass spectrometry analysis identified a wide range of interacting proteins, including those involved in RNA processing, RNA splicing, and transcriptional regulation, consistent with a multifunctional role for SFPQ/NONO. In addition, we have identified several sites of arginine methylation in SFPQ/PSF using mass spectrometry and found that several arginines in the N-terminal domain of SFPQ/PSF are asymmetrically dimethylated. Furthermore, we find that the protein arginine N-methyltransferase, PRMT1, catalyzes this methylation in vitro and that this is antagonized by citrullination of SFPQ. Arginine methylation and citrullination of SFPQ/PSF does not affect complex formation with NONO. However, arginine methylation was shown to increase the association with mRNA in mRNP complexes in mammalian cells. Finally we show that the biochemical properties of the endogenous complex from cell lysates are significantly influenced by the ionic strength during purification. At low ionic strength, the SFPQ/NONO complex forms large heterogeneous protein assemblies or aggregates, preventing the purification of the SFPQ/NONO complex. The ability of the SFPQ/NONO complex to form varying protein assemblies, in conjunction with the effect of post-translational modifications of SFPQ modulating mRNA binding, suggests key roles affecting mRNP dynamics within the cell.