Paternal diet defines offspring chromatin state and intergenerational obesity.

The global rise in obesity has revitalized a search for genetic and epigenetic factors underlying the disease. We present a Drosophila model of paternal-diet-induced intergenerational metabolic reprogramming (IGMR) and identify genes required for its encoding in offspring. Intriguingly, we find that as little as 2 days of dietary intervention in fathers elicits obesity in offspring. Paternal sugar acts as a physiological suppressor of variegation, desilencing chromatin-state-defined domains in both mature sperm and in offspring embryos. We identify requirements for H3K9/K27me3-dependent reprogramming of metabolic genes in two distinct germline and zygotic windows. Critically, we find evidence that a similar system may regulate obesity susceptibility and phenotype variation in mice and humans. The findings provide insight into the mechanisms underlying intergenerational metabolic reprogramming and carry profound implications for our understanding of phenotypic variation and evolution.

Mammalian Su(var) genes in chromatin control.

Genetic screens in Drosophila have been instrumental in distinguishing approximately 390 loci involved in position effect variegation and heterochromatin stabilization. Most of the identified genes [so-called Su(var) and E(var) genes] are also conserved in mammals, where more than 50 of their gene products are known to localize to constitutive heterochromatin. From these proteins, approximately 12 core heterochromatin components can be inferred. In addition, there are approximately 30 additional Su(var) and 10 E(var) factors that can, under distinct developmental options, interchange with constitutive heterochromatin and participate in the partitioning of the genome into repressed and active chromatin domains. A significant fraction of the Su(var) and E(var) factors are enzymes that respond to environmental and metabolic signals, thereby allowing both the variation and propagation of epigenetic states to a dynamic chromatin template. Moreover, the misregulation of human SU(VAR) and E(VAR) function can advance cancer and many other human diseases including more complex disorders. As such, mammalian Su(var) and E(var) genes and their products provide a rich source of novel targets for diagnosis of and pharmaceutical intervention in many human diseases.

Heterochromatin formation in Drosophila requires genome-wide histone deacetylation in cleavage chromatin before mid-blastula transition in early embryogenesis.

Su(var) mutations define epigenetic factors controlling heterochromatin formation and gene silencing in Drosophila. Here, we identify SU(VAR)2-1 as a novel chromatin regulator that directs global histone deacetylation during the transition of cleavage chromatin into somatic blastoderm chromatin in early embryogenesis. SU(VAR)2-1 is heterochromatin-associated in blastoderm nuclei but not in later stages of development. In larval polytene chromosomes, SU(VAR)2-1 is a band-specific protein. SU(VAR)2-1 directs global histone deacetylation by recruiting the histone deacetylase RPD3. In Su(var)2-1 mutants H3K9, H3K27, H4K8 and H4K16 acetylation shows elevated levels genome-wide and heterochromatin displays aberrant histone hyper-acetylation. Whereas H3K9me2- and HP1a-binding appears unaltered, the heterochromatin-specific H3K9me2S10ph composite mark is impaired in heterochromatic chromocenters of larval salivary polytene chromosomes. SU(VAR)2-1 contains an NRF1/EWG domain and a C2HC zinc-finger motif. Our study identifies SU(VAR)2-1 as a dosage-dependent, heterochromatin-initiating SU(VAR) factor, where the SU(VAR)2-1-mediated control of genome-wide histone deacetylation after cleavage and before mid-blastula transition (pre-MBT) is required to enable heterochromatin formation.

Ash1 counteracts Polycomb repression independent of histone H3 lysine 36 methylation.

Polycomb repression is critical for metazoan development. Equally important but less studied is the Trithorax system, which safeguards Polycomb target genes from the repression in cells where they have to remain active. It was proposed that the Trithorax system acts via methylation of histone H3 at lysine 4 and lysine 36 (H3K36), thereby inhibiting histone methyltransferase activity of the Polycomb complexes. Here we test this hypothesis by asking whether the Trithorax group protein Ash1 requires H3K36 methylation to counteract Polycomb repression. We show that Ash1 is the only Drosophila H3K36-specific methyltransferase necessary to prevent excessive Polycomb repression of homeotic genes. Unexpectedly, our experiments reveal no correlation between the extent of H3K36 methylation and the resistance to Polycomb repression. Furthermore, we find that complete substitution of the zygotic histone H3 with a variant in which lysine 36 is replaced by arginine does not cause excessive repression of homeotic genes. Our results suggest that the model, where the Trithorax group proteins methylate histone H3 to inhibit the histone methyltransferase activity of the Polycomb complexes, needs revision.

Proteome Analysis of Human Perilymph Using an Intraoperative Sampling Method.

The knowledge about the etiology and pathophysiology of sensorineural hearing loss (SNHL) is still very limited. This study aims at the improvement of understanding different types of SNHL by proteome analysis of human perilymph. Sampling of perilymph was established during inner ear surgeries (cochlear implantation, vestibular schwannoma surgeries), and safety of the sampling method was determined by checking hearing threshold with pure-tone audiometry postoperatively. An in-depth shot-gun proteomics approach was performed to identify cochlear proteins and the individual proteome in perilymph of patients. This method enables the identification and quantification of protein composition of perilymph. The proteome of 41 collected perilymph samples with volumes of 1-12 µL was analyzed by data-dependent acquisition, resulting in overall 878 detected protein groups. At least 203 protein groups were solely identified in perilymph, not in reference samples (serum, cerebrospinal fluid), displaying a specific protein pattern for perilymph. Samples were grouped by patient's age and surgery type, leading to the identification of some proteins specific to particular subgroups. Proteins with different abundances between different sample groups were subjected to classification by gene ontology annotations. The identified proteins might serve as biomarkers to develop tools for noninvasive inner ear diagnostics and to elucidate molecular profiles of SNHL.

Mechanism and biological role of Dnmt2 in Nucleic Acid Methylation.

A group of homologous nucleic acid modification enzymes called Dnmt2, Trdmt1, Pmt1, DnmA, and Ehmet in different model organisms catalyze the transfer of a methyl group from the cofactor S-adenosyl-methionine (SAM) to the carbon-5 of cytosine residues. Originally considered as DNA MTases, these enzymes were shown to be tRNA methyltransferases about a decade ago. Between the presumed involvement in DNA modification-related epigenetics, and the recent foray into the RNA modification field, significant progress has characterized Dnmt2-related research. Here, we review this progress in its diverse facets including molecular evolution, structural biology, biochemistry, chemical biology, cell biology and epigenetics.

Sound of silence: the beauvericin cluster in Fusarium fujikuroi is controlled by cluster-specific and global regulators mediated by H3K27 modification.

In this study, we compared the secondary metabolite profile of Fusarium fujikuroi and the histone deacetylase mutant ΔHDA1. We identified a novel peak in ΔHDA1, which was identified as beauvericin (BEA). Going in line with a 1000-fold increased BEA production, the respective non-ribosomal peptide synthetase (NRPS)-encoding gene (BEA1), as well as two adjacent genes (BEA2-BEA3), were significantly up-regulated in ΔHDA1 compared to the wild type. A special role was revealed for the ABC transporter Bea3: deletion of the encoding gene resulted in significant up-regulation of BEA1 and BEA2 and drastically elevated product yields. Furthermore, mutation of a conserved sequence motif in the promoter of BEA1 released BEA repression and resulted in elevated product levels. Candidate transcription factors (TFs) that could bind to this motif are the cluster-specific TF Bea4 as well as a homolog of the global mammalian Kruppel-like TF Yin Yang 1 (Yy1), both acting as repressors of BEA biosynthesis. In addition to Hda1, BEA biosynthesis is repressed by the activity of the H3K27 methyltransferase Kmt6. Consistently, Western blot analyses revealed a genome-wide enrichment of H3K27 acetylation (H3K27ac) in the ΔHDA1 and KMT6 knock-down mutants. Subsequent chromatin immunoprecipitation (ChIP) experiments showed elevated H3K27ac modification levels at the BEA cluster.

The Dnmt2 RNA methyltransferase homolog of Geobacter sulfurreducens specifically methylates tRNA-Glu.

Dnmt2 enzymes are conserved in eukaryotes, where they methylate C38 of tRNA-Asp with high activity. Here, the activity of one of the very few prokaryotic Dnmt2 homologs from Geobacter species (GsDnmt2) was investigated. GsDnmt2 was observed to methylate tRNA-Asp from flies and mice. Unexpectedly, it had only a weak activity toward its matching Geobacter tRNA-Asp, but methylated Geobacter tRNA-Glu with good activity. In agreement with this result, we show that tRNA-Glu is methylated in Geobacter while the methylation is absent in tRNA-Asp. The activities of Dnmt2 enzymes from Homo sapiens, Drosophila melanogaster, Schizosaccharomyces pombe and Dictyostelium discoideum for methylation of the Geobacter tRNA-Asp and tRNA-Glu were determined showing that all these Dnmt2s preferentially methylate tRNA-Asp. Hence, the GsDnmt2 enzyme has a swapped transfer ribonucleic acid (tRNA) specificity. By comparing the different tRNAs, a characteristic sequence pattern was identified in the variable loop of all preferred tRNA substrates. An exchange of two nucleotides in the variable loop of murine tRNA-Asp converted it to the corresponding variable loop of tRNA-Glu and led to a strong reduction of GsDnmt2 activity. Interestingly, the same loss of activity was observed with human DNMT2, indicating that the variable loop functions as a specificity determinant in tRNA recognition of Dnmt2 enzymes.

Alterations of histone modifications at the senescence-associated gene HvS40 in barley during senescence.

The barley gene HvS40, encoding a putative regulator of leaf senescence, is strongly induced during leaf senescence. As shown by chromatin immunoprecipitation, euchromatic histone modification H3K9ac is added at promoter close to ATG and coding sequence of HvS40 after onset of senescence. In parallel, level of heterochromatic H3K9me2 decreases at this gene. Bisulfite sequencing revealed no DNA-methylation in this region, but a heavily methylated DNA-island, starting 664 bp upstream from translational start site in both, mature and senescent leaves. A decrease in DNA methylation in senescing leaves could be shown at one specific CpG motif at the end of this methylation island. In addition, global changes in chromatin structure during senescence were analyzed via immunocytology, revealing senescence-associated changes in spatial distribution of heterochromatic H3K9me2 patterns in the nuclei. Our results prove a senescence-specific mechanism, altering histone modification marks at distinct sequences of the senescence-associated gene HvS40 and altering distribution of heterochromatic areas in the nuclei.

New, non-quinone fluorogeldanamycin derivatives strongly inhibit Hsp90.

Streptomyces hygroscopicus is a natural producer of geldanamycin. Mutasynthetic supplementation of an AHBA-blocked mutant with all possible monofluoro 3-aminobenzoic acids provided new fluorogeldanamycins. These showed strong antiproliferative activity and inhibitory effects on human heat shock protein Hsp90. Binding to Hsp90 in the low nanomolar range was determined from molecular modelling, AFM analysis and by calorimetric studies.

Deciphering the cryptic genome: genome-wide analyses of the rice pathogen Fusarium fujikuroi reveal complex regulation of secondary metabolism and novel metabolites.

The fungus Fusarium fujikuroi causes "bakanae" disease of rice due to its ability to produce gibberellins (GAs), but it is also known for producing harmful mycotoxins. However, the genetic capacity for the whole arsenal of natural compounds and their role in the fungus' interaction with rice remained unknown. Here, we present a high-quality genome sequence of F. fujikuroi that was assembled into 12 scaffolds corresponding to the 12 chromosomes described for the fungus. We used the genome sequence along with ChIP-seq, transcriptome, proteome, and HPLC-FTMS-based metabolome analyses to identify the potential secondary metabolite biosynthetic gene clusters and to examine their regulation in response to nitrogen availability and plant signals. The results indicate that expression of most but not all gene clusters correlate with proteome and ChIP-seq data. Comparison of the F. fujikuroi genome to those of six other fusaria revealed that only a small number of gene clusters are conserved among these species, thus providing new insights into the divergence of secondary metabolism in the genus Fusarium. Noteworthy, GA biosynthetic genes are present in some related species, but GA biosynthesis is limited to F. fujikuroi, suggesting that this provides a selective advantage during infection of the preferred host plant rice. Among the genome sequences analyzed, one cluster that includes a polyketide synthase gene (PKS19) and another that includes a non-ribosomal peptide synthetase gene (NRPS31) are unique to F. fujikuroi. The metabolites derived from these clusters were identified by HPLC-FTMS-based analyses of engineered F. fujikuroi strains overexpressing cluster genes. In planta expression studies suggest a specific role for the PKS19-derived product during rice infection. Thus, our results indicate that combined comparative genomics and genome-wide experimental analyses identified novel genes and secondary metabolites that contribute to the evolutionary success of F. fujikuroi as a rice pathogen.

The SUVR4 histone lysine methyltransferase binds ubiquitin and converts H3K9me1 to H3K9me3 on transposon chromatin in Arabidopsis.

Chromatin structure and gene expression are regulated by posttranslational modifications (PTMs) on the N-terminal tails of histones. Mono-, di-, or trimethylation of lysine residues by histone lysine methyltransferases (HKMTases) can have activating or repressive functions depending on the position and context of the modified lysine. In Arabidopsis, trimethylation of lysine 9 on histone H3 (H3K9me3) is mainly associated with euchromatin and transcribed genes, although low levels of this mark are also detected at transposons and repeat sequences. Besides the evolutionarily conserved SET domain which is responsible for enzyme activity, most HKMTases also contain additional domains which enable them to respond to other PTMs or cellular signals. Here we show that the N-terminal WIYLD domain of the Arabidopsis SUVR4 HKMTase binds ubiquitin and that the SUVR4 product specificity shifts from di- to trimethylation in the presence of free ubiquitin, enabling conversion of H3K9me1 to H3K9me3 in vitro. Chromatin immunoprecipitation and immunocytological analysis showed that SUVR4 in vivo specifically converts H3K9me1 to H3K9me3 at transposons and pseudogenes and has a locus-specific repressive effect on the expression of such elements. Bisulfite sequencing indicates that this repression involves both DNA methylation-dependent and -independent mechanisms. Transcribed genes with high endogenous levels of H3K4me3, H3K9me3, and H2Bub1, but low H3K9me1, are generally unaffected by SUVR4 activity. Our results imply that SUVR4 is involved in the epigenetic defense mechanism by trimethylating H3K9 to suppress potentially harmful transposon activity.

Lysine-specific histone demethylase LSD1 and the dynamic control of chromatin.

The flavin adenine dinucleotide-dependent amine oxidase LSD1 is the first molecularly defined histone demethylase, which specifically demethylates H3K4me1/me2. The enzyme dynamically controls a large variety of biological processes and is associated with protein complexes controlling transcriptional repression and activation. Molecular analysis of the Drosophila LSD1 homolog revealed new insights into the epigenetic control of heterochromatin formation during early embryogenesis, the establishment of transcriptional gene silencing and the epigenetic mechanisms associated with the maintenance of stem cell identity in primordial germline cells. This review summarizes our recent knowledge about the control of enzymatic activity and molecular function of LSD1 enzyme complexes in different model organisms including Schizosaccharomyces pombe, Drosophila and mammals. Finally, new developments in applied cancer research based on molecular analysis of LSD1 in cancer cells are discussed.

Crystal structures of glutaminyl cyclases (QCs) from Drosophila melanogaster reveal active site conservation between insect and mammalian QCs.

Glutaminyl cyclases (QCs), which catalyze the formation of pyroglutamic acid (pGlu) at the N-terminus of a variety of peptides and proteins, have attracted particular attention for their potential role in Alzheimer's disease. In a transgenic Drosophila melanogaster (Dm) fruit fly model, oral application of the potent competitive QC inhibitor PBD150 was shown to reduce the burden of pGlu-modified Aß. In contrast to mammals such as humans and rodents, there are at least three DmQC species, one of which (isoDromeQC) is localized to mitochondria, whereas DromeQC and an isoDromeQC splice variant possess signal peptides for secretion. Here we present the recombinant expression, characterization, and crystal structure determination of mature DromeQC and isoDromeQC, revealing an overall fold similar to that of mammalian QCs. In the case of isoDromeQC, the putative extended substrate binding site might be affected by the proximity of the N-terminal residues. PBD150 inhibition of DromeQC is roughly 1 order of magnitude weaker than that of the human and murine QCs. The inhibitor binds to isoDromeQC in a fashion similar to that observed for human QCs, whereas it adopts alternative binding modes in a DromeQC variant lacking the conserved cysteines near the active center and shows a disordered dimethoxyphenyl moiety in wild-type DromeQC, providing an explanation for the lower affinity. Our biophysical and structural data suggest that isoDromeQC and human QC are similar with regard to functional aspects. The two Dm enzymes represent a suitable model for further in-depth analysis of the catalytic mechanism of animal QCs, and isoDromeQC might serve as a model system for the structure-based design of potential AD therapeutics.

The SUUR protein is involved in binding of SU(VAR)3-9 and methylation of H3K9 and H3K27 in chromosomes of Drosophila melanogaster.

In the present work, we found that the SUUR protein is required for the SU(VAR)3-9 enzyme to bind to the salivary gland polytene chromosomes. The SuUR mutation results in loss of SU(VAR)3-9 on the chromosomes, whereas artificial expression of the SuUR gene restores its binding. The SUUR protein is also involved in methylation of the residues H3K9 and H3K27. However, mono-, di-, and tri-methylated forms of H3K9 and H3K27 behave differently in various chromosomal domains in response to the SuUR mutation. Euchromatin and chromosome 4 are almost completely deprived of mono-, di-, and tri-methylation of H3K9. In the chromocenter, mono-methylation is reduced, di-methylation shows no noticeable changes, and tri-methylation is lost. Furthermore, mono- and di-methylation of H3K27 are not influenced by the SuUR mutation, whereas tri-methylation is lost in the chromocenter. Artificial expression of the SuUR gene on the SuUR (-) background restores the pattern of methylated residues characteristic for the wild type.

Epigenetic programming via histone methylation at WRKY53 controls leaf senescence in Arabidopsis thaliana.

Leaf senescence, the final step of leaf development, involves extensive reprogramming of gene expression. Here, we show that these processes include discrete changes of epigenetic indexing, as well as global alterations in chromatin organization. During leaf senescence, the interphase nuclei show a decondensation of chromocenter heterochromatin, and changes in the nuclear distribution of the H3K4me2, H3K4me3, and the H3K27me2 and H3K27me3 histone modification marks that index active and inactive chromatin, respectively. Locus-specific epigenetic indexing was studied at the WRKY53 key regulator of leaf senescence. During senescence, when the locus becomes activated, H3K4me2 and H3K4me3 are significantly increased at the 5' end and at coding regions. Impairment of these processes is observed in plants overexpressing the SUVH2 histone methyltransferase, which causes ectopic heterochromatization. In these plants the transcriptional initiation of WRKY53 and of the senescence-associated genes SIRK, SAG101, ANAC083, SAG12 and SAG24 is inhibited, resulting in a delay of leaf senescence. In SUVH2 overexpression plants, significant levels of H3K27me2 and H3K27me3 are detected at the 5'-end region of WRKY53, resulting in its transcriptional repression. Furthermore, SUVH2 overexpression inhibits senescence-associated global changes in chromatin organization. Our data suggest that complex epigenetic processes control the senescence-specific gene expression pattern.

Aurora1 phosphorylation activity on histone H3 and its cross-talk with other post-translational histone modifications in Arabidopsis.

The enzymological properties of AtAurora1, a kinase responsible for the cell cycle-dependent phosphorylation of histone H3 at S10, and its cross-talk with other post-translational histone modifications, were determined. In vitro phosphorylation of H3S10 by AtAurora1 is strongly increased by K9 acetylation, and decreased by K14 acetylation and T11 phosphorylation. However, S10 phosphorylation activity is unaltered by mono-, di- or trimethylation of K9. An interference of H3K9 dimethylation by SUVR4 occurs by a pre-existing phosphorylation at S10. Hence, cross-talk in plants exists between phosphorylation of H3S10 and methylation, acetylation or phosphorylation of neighbouring amino acid residues. AtAurora1 undergoes autophosphorylation in vivo regardless of the presence of substrate, and forms dimers in planta. Of the three ATP-competitive Aurora inhibitors tested, Hesperadin was most effective in reducing the in vivo kinase activity of AtAurora1. Hesperadin consistently inhibited histone H3S10 phosphorylation during mitosis in Arabidopsis cells, but did not affect other H3 post-translational modifications, suggesting a specific inhibition of AtAurora in vivo. Inactivation of AtAurora also caused lagging chromosomes in a number of anaphase cells, but, unlike the situation in mammalian cells, Hesperadin did not influence the microtubule dynamics in dividing cells.

Human DNMT2 methylates tRNA(Asp) molecules using a DNA methyltransferase-like catalytic mechanism.

Although their amino acid sequences and structure closely resemble DNA methyltransferases, Dnmt2 proteins were recently shown by Goll and colleagues to function as RNA methyltransferases transferring a methyl group to the C5 position of C38 in tRNA(Asp). We observe that human DNMT2 methylates tRNA isolated from Dnmt2 knock-out Drosophila melanogaster and Dictyostelium discoideum. RNA extracted from wild type D. melanogaster was methylated to a lower degree, but in the case of Dictyostelium, there was no difference in the methylation of RNA isolated from wild-type and Dnmt2 knock-out strains. Methylation of in vitro transcribed tRNA(Asp) confirms it to be a target of DNMT2. Using site directed mutagenesis, we show here that the enzyme has a DNA methyltransferase-like mechanism, because similar residues from motifs IV, VI, and VIII are involved in catalysis as identified in DNA methyltransferases. In addition, exchange of C292, which is located in a CFT motif conserved among Dnmt2 proteins, strongly reduced the catalytic activity of DNMT2. Dnmt2 represents the first example of an RNA methyltransferase using a DNA methyltransferase type of mechanism.

The DrosDel deletion collection: a Drosophila genomewide chromosomal deficiency resource.

We describe a second-generation deficiency kit for Drosophila melanogaster composed of molecularly mapped deletions on an isogenic background, covering approximately 77% of the Release 5.1 genome. Using a previously reported collection of FRT-bearing P-element insertions, we have generated 655 new deletions and verified a set of 209 deletion-bearing fly stocks. In addition to deletions, we demonstrate how the P elements may also be used to generate a set of custom inversions and duplications, particularly useful for balancing difficult regions of the genome carrying haplo-insufficient loci. We describe a simple computational resource that facilitates selection of appropriate elements for generating custom deletions. Finally, we provide a computational resource that facilitates selection of other mapped FRT-bearing elements that, when combined with the DrosDel collection, can theoretically generate over half a million precisely mapped deletions.

The Arabidopsis SUVR4 protein is a nucleolar histone methyltransferase with preference for monomethylated H3K9.

Proteins containing the evolutionarily conserved SET domain are involved in regulation of eukaryotic gene expression and chromatin structure through their histone lysine methyltransferase (HMTase) activity. The Drosophila SU(VAR)3-9 protein and related proteins of other organisms have been associated with gene repression and heterochromatinization. In Arabidopsis there are 10 SUVH and 5 SUVR genes encoding proteins similar to SU(VAR)3-9, and 4 SUVH proteins have been shown to control heterochromatic silencing by its HMTase activity and by directing DNA methylation. The SUVR proteins differ from the SUVH proteins in their domain structure, and we show that the closely related SUVR1, SUVR2 and SUVR4 proteins contain a novel domain at their N-terminus, and a SUVR specific region preceding the SET domain. Green fluorescent protein (GFP)-fusions of these SUVR proteins preferably localize to the nucleolus, suggesting involvement in regulation of rRNA expression, in contrast to other SET-domain proteins studied so far. A novel HMTase specificity was demonstrated for SUVR4, in that monomethylated histone H3K9 is its preferred substrate in vitro.