Genomic distribution of H3K9me2 and DNA methylation in a maize genome

DNA methylation and dimethylation of lysine 9 of histone H3 (H3K9me2) are two chromatin modifications that can be associated with gene expression or recombination rate. The maize genome provides a complex landscape of interspersed genes and transposons. The genome-wide distribution of DNA methylation and H3K9me2 were investigated in seedling tissue for the maize inbred B73 and compared to patterns of these modifications observed in Arabidopsis thaliana. Most maize transposons are highly enriched for DNA methylation in CG and CHG contexts and for H3K9me2. In contrast to findings in Arabidopsis, maize CHH levels in transposons are generally low but some sub-families of transposons are enriched for CHH methylation and these families exhibit low levels of H3K9me2. The profile of modifications over genes reveals that DNA methylation and H3K9me2 is quite low near the beginning and end of genes. Although elevated CG and CHG methylation are found within gene bodies, CHH and H3K9me2 remain low. Maize has much higher levels of CHG methylation within gene bodies than observed in Arabidopsis and this is partially attributable to the presence of transposons within introns for some maize genes. These transposons are associated with high levels of CHG methylation and H3K9me2 but do not appear to prevent transcriptional elongation. Although the general trend is for a strong depletion of H3K9me2 and CHG near the transcription start site there are some putative genes that have high levels of these chromatin modifications. This study provides a clear view of the relationship between DNA methylation and H3K9me2 in the maize genome and how the distribution of these modifications is shaped by the interplay of genes and transposons.The research was supported by a grant from the National Science Foundation (IOS-1237931) to MWV and NMS. This work also used resources or cyberinfrastructure provided by iPlant Collaborative. The iPlant Collaborative is funded by a grant from the National Science Foundation (DBI-0735191; www. iplantcollaborative.org). Start-up funds from the University of Georgia and a research grant from the National Science Foundation (IOS-1339194) to RJS supported aspects of this study

DNA cytosine hydroxymethylation levels are distinct among non-overlapping classes of peripheral blood leukocytes

AbstractBackgroundPeripheral blood leukocytes are the most commonly used surrogates to study epigenome-induced risk and epigenomic response to disease-related stress. We considered the hypothesis that the various classes of peripheral leukocytes differentially regulate the synthesis of 5-methylcytosine (5mCG) and its removal via Ten-Eleven Translocation (TET) dioxygenase catalyzed hydroxymethylation to 5-hydroxymethylcytosine (5hmCG), reflecting their responsiveness to environment. Although it is known that reductions in TET1 and/or TET2 activity lead to the over-proliferation of various leukocyte precursors in bone marrow and in development of chronic myelomonocytic leukemia and myeloproliferative neoplasms, the role of 5mCG hydroxymethylation in peripheral blood is less well studied.ResultsWe developed simplified protocols to rapidly and reiteratively isolate non-overlapping leukocyte populations from a single small sample of fresh or frozen whole blood. Among peripheral leukocyte types we found extreme variation in the levels of transcripts encoding proteins involved in cytosine methylation (DNMT1, 3A, 3B), the turnover of 5mC by demethylation (TET1, 2, 3), and DNA repair (GADD45A, B, G) and in the global and gene-region-specific levels of DNA 5hmCG (CD4+ T cells≫CD14+ monocytes>CD16+ neutrophils>CD19+ B cells>CD56+ NK cells>Siglec8+ eosinophils>CD8+ T cells).ConclusionsOur data taken together suggest a potential hierarchy of responsiveness among classes of leukocytes with CD4+, CD8+ T cells and CD14+ monocytes being the most distinctly poised for a rapid methylome response to physiological stress and disease

Histone H1 Limits DNA Methylation in Neurospora crassa

Histone H1 variants, known as linker histones, are essential chromatin components in higher eukaryotes, yet compared to the core histones relatively little is known about their in vivo functions. The filamentous fungus Neurospora crassa encodes a single H1 protein that is not essential for viability. To investigate the role of N. crassa H1, we constructed a functional FLAG-tagged H1 fusion protein and performed genomic and molecular analyses. Cell fractionation experiments showed that H1-3XFLAG is a chromatin binding protein. Chromatin-immunoprecipitation combined with sequencing (ChIP-seq) revealed that H1-3XFLAG is globally enriched throughout the genome with a subtle preference for promoters of expressed genes. In mammals, the stoichiometry of H1 impacts nucleosome repeat length. To determine if H1 impacts nucleosome occupancy or nucleosome positioning in N. crassa, we performed micrococcal nuclease digestion in the wild-type and the ΔhH1 strain followed by sequencing (MNase-seq). Deletion of hH1 did not significantly impact nucleosome positioning or nucleosome occupancy. Analysis of DNA methylation by whole-genome bisulfite sequencing (MethylC-seq) revealed a modest but global increase in DNA methylation in the ΔhH1 mutant. Together, these data suggest that H1 acts as a nonspecific chromatin binding protein that can limit accessibility of the DNA methylation machinery in N. crassa

The Genome and Methylome of a Beetle with Complex Social Behavior,Nicrophorus vespilloides(Coleoptera: Silphidae)

Testing for conserved and novelmechanisms underlying phenotypic evolution requires a diversity of genomes available for comparisonspanning multiple independent lineages. For example, complex social behavior in insects has been investigated primarily witheusocial lineages, nearly all of which are Hymenoptera. If conserved genomic influences on sociality do exist, we need data from awider range of taxa that also vary in their levels of sociality. Here,we present the assembled and annotated genome of the subsocialbeetle Nicrophorus vespilloides, a species long used to investigate evolutionary questions of complex social behavior. We used thisgenome to address two questions. First, do aspects of life history, such as using a carcass to breed, predict overlap in gene modelsmore strongly than phylogeny? We found that the overlap in gene models was similar between N. vespilloides and all other insectgroups regardless of life history. Second, like other insects with highly developed social behavior but unlike other beetles, doesN. vespilloides have DNA methylation?We found strong evidence for an active DNA methylation system. The distribution of methylationwassimilar to other insects with exons having themostmethylatedCpGs. Methylation status appears highly conserved; 85%of themethylated genes in N. vespilloides are alsomethylated in the hymentopteran Nasonia vitripennis. The addition of this genomeadds a coleopteran resource to answer questions about the evolution and mechanistic basis of sociality and to address questionsabout the potential role of methylation in social behavior

The LSH/DDM1 Homolog MUS-30 Is Required for Genome Stability, but Not for DNA Methylation in <i>Neurospora crassa</i>

<div><p>LSH/DDM1 enzymes are required for DNA methylation in higher eukaryotes and have poorly defined roles in genome maintenance in yeast, plants, and animals. The filamentous fungus <i>Neurospora crassa</i> is a tractable system that encodes a single LSH/DDM1 homolog (NCU06306). We report that the Neurospora LSH/DDM1 enzyme is encoded by <i>mutagen sensitive-30</i> (<i>mus-30</i>), a locus identified in a genetic screen over 25 years ago. We show that MUS-30-deficient cells have normal DNA methylation, but are hypersensitive to DNA damaging agents. MUS-30 is a nuclear protein, consistent with its predicted role as a chromatin remodeling enzyme, and levels of MUS-30 are increased following DNA damage. MUS-30 co-purifies with Neurospora WDR76, a homolog of yeast Changed Mutation Rate-1 and mammalian WD40 repeat domain 76. Deletion of <i>wdr76</i> rescued DNA damage-hypersensitivity of Δ<i>mus-30</i> strains, demonstrating that the MUS-30-WDR76 interaction is functionally important. DNA damage-sensitivity of Δ<i>mus-30</i> is partially suppressed by deletion of <i>methyl adenine glycosylase-1</i>, a component of the base excision repair machinery (BER); however, the rate of BER is not affected in Δ<i>mus-30</i> strains. We found that MUS-30-deficient cells are not defective for DSB repair, and we observed a negative genetic interaction between Δ<i>mus-30</i> and Δ<i>mei-3</i>, the Neurospora RAD51 homolog required for homologous recombination. Together, our findings suggest that MUS-30, an LSH/DDM1 homolog, is required to prevent DNA damage arising from toxic base excision repair intermediates. Overall, our study provides important new information about the functions of the LSH/DDM1 family of enzymes.</p></div

The evolution of CHROMOMETHYLASES and gene body DNA methylation in plants

Abstract Background The evolution of gene body methylation (gbM), its origins, and its functional consequences are poorly understood. By pairing the largest collection of transcriptomes (>1000) and methylomes (77) across Viridiplantae, we provide novel insights into the evolution of gbM and its relationship to CHROMOMETHYLASE (CMT) proteins. Results CMTs are evolutionary conserved DNA methyltransferases in Viridiplantae. Duplication events gave rise to what are now referred to as CMT1, 2 and 3. Independent losses of CMT1, 2, and 3 in eudicots, CMT2 and ZMET in monocots and monocots/commelinids, variation in copy number, and non-neutral evolution suggests overlapping or fluid functional evolution of this gene family. DNA methylation within genes is widespread and is found in all major taxonomic groups of Viridiplantae investigated. Genes enriched with methylated CGs (mCG) were also identified in species sister to angiosperms. The proportion of genes and DNA methylation patterns associated with gbM are restricted to angiosperms with a functional CMT3 or ortholog. However, mCG-enriched genes in the gymnosperm Pinus taeda shared some similarities with gbM genes in Amborella trichopoda. Additionally, gymnosperms and ferns share a CMT homolog closely related to CMT2 and 3. Hence, the dependency of gbM on a CMT most likely extends to all angiosperms and possibly gymnosperms and ferns. Conclusions The resulting gene family phylogeny of CMT transcripts from the most diverse sampling of plants to date redefines our understanding of CMT evolution and its evolutionary consequences on DNA methylation. Future, functional tests of homologous and paralogous CMTs will uncover novel roles and consequences to the epigenome