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.

Photochemical coating of Kapton® with hydrophilic polymers for the improvement of neural implants.

The polyimide Kapton® was coated photochemically with hydrophilic polymers to prevent undesirable cell growth on the polyimide surface. The polymer coatings were generated using photochemically reactive polymers synthesized by a simple and modular strategy. Suitable polymers or previously synthesized copolymer precursors were functionalized with photoactive arylazide groups by a polymer analogous amide coupling reaction with 4-azidobenzoic acid. A photoactive chitosan derivative (chitosan-Az) and photochemically reactive copolymers containing DMAA, DEAA or MTA as primary monomers were synthesized using this method. The amount of arylazide groups in the polymers was adjusted to approximately 5%, 10% and 20%. As coating on Kapton® all polymers effect a significantly reduced water contact angle (WCA) and consequently a rise of the surface hydrophilicity compared to the untreated Kapton®. The presence of the polymer coatings was also proven by ATR-IR spectroscopy. Coatings with chitosan-Az and the DEAA copolymer cause a distinct inhibition of the growth of fibroblasts. In the case of the DMAA copolymer even a strong anti-adhesive behavior towards fibroblasts was verified. Biocompatibility of the polymer coatings was proven which enables their utilization in biomedical applications.

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.

Magnetic Beads Enhance Adhesion of NIH 3T3 Fibroblasts: A Proof-of-Principle In Vitro Study for Implant-Mediated Long-Term Drug Delivery to the Inner Ear.

INTRODUCTION: Long-term drug delivery to the inner ear may be achieved by functionalizing cochlear implant (CI) electrodes with cells providing neuroprotective factors. However, effective strategies in order to coat implant surfaces with cells need to be developed. Our vision is to make benefit of electromagnetic field attracting forces generated by CI electrodes to bind BDNF-secreting cells that are labelled with magnetic beads (MB) onto the electrode surfaces. Thus, the effect of MB-labelling on cell viability and BDNF production were investigated. MATERIALS AND METHODS: Murine NIH 3T3 fibroblasts-genetically modified to produce BDNF-were labelled with MB. RESULTS: Atomic force and bright field microscopy illustrated the internalization of MB by fibroblasts after 24 h of cultivation. Labelling cells with MB did not expose cytotoxic effects on fibroblasts and allowed adhesion on magnetic surfaces with sufficient BDNF release. DISCUSSION: Our data demonstrate a novel approach for mediating enhanced long-term adhesion of BDNF-secreting fibroblasts on model electrode surfaces for cell-based drug delivery applications in vitro and in vivo. This therapeutic strategy, once transferred to cells suitable for clinical application, may allow the biological modifications of CI surfaces with cells releasing neurotrophic or other factors of interest.

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.

Overexpression of Arabidopsis thaliana ERI, the homolog of C. elegans Enhancer of RNAinterference, leads to enhanced growth.

Organisms adopt a wide range of strategies to adapt to change. Gene silencing describes the ability of organisms to modulate the expression of susceptible genes at certain times at the transcriptional or the translational level. In all known eukaryotic organisms 21-nt long short interfering RNAs (siRNAs) are the effector molecules of post-transcriptional gene silencing (PTGS), while 24-nt long siRNAs are involved in PTGS in plants. Mutant studies in Caenorhabditis elegans lead to the identification of the enzyme ERI (Enhancer of RNAinterference) with enhanced PTGS. Although the genes involved in growth vigor and growth rate are still unknown, it becomes clearer that the population of small RNAs plays a role in the very early phase of plant development. To pinpoint the link between growth and siRNAs, the expression of Arabidopsis uni-gene Enhancer of RNAi (ERI) homolog from C. elegans was modulated. Increased degradation of small RNAs was achieved by ectopic AtERI overexpression in planta. Based on global small RNA analysis, AtERI overexpression affects mainly the population of 21 mers, excluding miRNAs. To identify target genes, AtERI gain-of-function mutants were analyzed, and differentially abundant small RNAs were identified. Plants with an elevated level of AtERI were bigger in all three light intensities analyzed, indicating an inhibitory function of particular small RNAs in plant growth, with differences in relative growth rates depending on developmental stage and light intensity. Understanding the role of these siRNAs could open new avenues for enhancing plant growth.

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.

Hydrogel coated and dexamethasone releasing cochlear implants: quantification of fibrosis in guinea pigs and evaluation of insertion forces in a human cochlea model.

The insertion of cochlear implants (CIs) often causes fibrous tissue growth around the electrode, which leads to attenuation of function of CIs. Inhibition of fibrosis in vivo using dexamethasone (Dex) released from the implant base material (polydimethylsiloxane [PDMS]) coated with a protein repelling hydrogel (star-shaped polyethylene glycol prepolymer, sPEG) was, therefore, the aim of the study. PDMS filaments with Dex or sPEG were implanted into guinea pigs. The hearing status after implantation did not differ significantly in the treated groups. Using confocal laser scanning microscopy in transparent whole mount preparations, Dex, Dex/sPEG, as well as sPEG showed a tendency toward reduced formation of connective tissue around the implant. To apply such coatings for glass fibers for optical stimulation of the inner ear, insertion forces were measured into a human scala tympani model using fibers with sPEG coating. The results show that the hydrogel did not reduce insertion forces compared to the uncoated samples. However, PDMS-embedded fibers provide comparable insertion forces and depth to those measured with conventional CI electrodes, demonstrating the suitability of laser fibers for a minimal traumatic cochlear implantation.

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.

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.

Nanosecond laser pulse stimulation of spiral ganglion neurons and model cells.

Optical stimulation of the inner ear has recently attracted attention, suggesting a higher frequency resolution compared to electrical cochlear implants due to its high spatial stimulation selectivity. Although the feasibility of the effect is shown in multiple in vivo experiments, the stimulation mechanism remains open to discussion. Here we investigate in single-cell measurements the reaction of spiral ganglion neurons and model cells to irradiation with a nanosecond-pulsed laser beam over a broad wavelength range from 420 nm up to 1950 nm using the patch clamp technique. Cell reactions were wavelength- and pulse-energy-dependent but too small to elicit action potentials in the investigated spiral ganglion neurons. As the applied radiant exposure was much higher than the reported threshold for in vivo experiments in the same laser regime, we conclude that in a stimulation paradigm with nanosecond-pulses, direct neuronal stimulation is not the main cause of optical cochlea stimulation.

Dexamethasone released from cochlear implant coatings combined with a protein repellent hydrogel layer inhibits fibroblast proliferation.

The insertion of cochlear implants into the inner ear often causes inflammation and fibrosis inside the scala tympani and thus growth of fibrous tissue on the implant surface. This deposition leads to the loss of function in both electrical and laser-based implants. The design of this study was to realize fibroblast growth inhibition by dexamethasone (Dex) released from the base material of the implant [polydimethylsiloxane (PDMS)]. To prevent cell and protein adhesion, the PDMS was coated with a hydrogel layer [star-shaped polyethylene glycol prepolymer (sPEG)]. Drug release rates were studied over 3 months, and surface characterization was performed. It was observed that the hydrogel slightly smoothened the surface roughened by the Dex crystals. The hydrogel coating reduced and prolonged the release of the drug over several months. Unmodified, sPEG-coated, Dex-loaded, and Dex/sPEG-equipped PDMS filaments were cocultivated in vitro with fluorescent fibroblasts, analyzed by fluorescent microscopy, and quantified by cell counting. Compared to the unmodified PDMS, cell growth on all modified filaments was averagely 95% ±standard deviation (SD) less, while cell growth on the bottom of the culture dishes containing Dex-loaded filaments was reduced by 70% ±SD. Both, Dex and sPEG prevented direct cell growth on the filament surfaces, while drug delivery was maintained for the duration of several months.

Dissociated neurons and glial cells derived from rat inferior colliculi after digestion with papain.

The formation of gliosis around implant electrodes for deep brain stimulation impairs electrode-tissue interaction. Unspecific growth of glial tissue around the electrodes can be hindered by altering physicochemical material properties. However, in vitro screening of neural tissue-material interaction requires an adequate cell culture system. No adequate model for cells dissociated from the inferior colliculus (IC) has been described and was thus the aim of this study. Therefore, IC were isolated from neonatal rats (P3_5) and a dissociated cell culture was established. In screening experiments using four dissociation methods (Neural Tissue Dissociation Kit [NTDK] T, NTDK P; NTDK PN, and a validated protocol for the dissociation of spiral ganglion neurons [SGN]), the optimal media, and seeding densities were identified. Thereafter, a dissociation protocol containing only the proteolytic enzymes of interest (trypsin or papain) was tested. For analysis, cells were fixed and immunolabeled using glial- and neuron-specific antibodies. Adhesion and survival of dissociated neurons and glial cells isolated from the IC were demonstrated in all experimental settings. Hence, preservation of type-specific cytoarchitecture with sufficient neuronal networks only occurred in cultures dissociated with NTDK P, NTDK PN, and fresh prepared papain solution. However, cultures obtained after dissociation with papain, seeded at a density of 2×10(4) cells/well and cultivated with Neuro Medium for 6 days reliably revealed the highest neuronal yield with excellent cytoarchitecture of neurons and glial cells. The herein described dissociated culture can be utilized as in vitro model to screen interactions between cells of the IC and surface modifications of the electrode.

Position-effect variegation, heterochromatin formation, and gene silencing in Drosophila.

Position-effect variegation (PEV) results when a gene normally in euchromatin is juxtaposed with heterochromatin by rearrangement or transposition. When heterochromatin packaging spreads across the heterochromatin/euchromatin border, it causes transcriptional silencing in a stochastic pattern. PEV is intensely studied in Drosophila using the white gene. Screens for dominant mutations that suppress or enhance white variegation have identified many conserved epigenetic factors, including the histone H3 lysine 9 methyltransferase SU(VAR)3-9. Heterochromatin protein HP1a binds H3K9me2/3 and interacts with SU(VAR)3-9, creating a core memory system. Genetic, molecular, and biochemical analysis of PEV in Drosophila has contributed many key findings concerning establishment and maintenance of heterochromatin with concomitant gene silencing.

Spiral ganglion neuron quantification in the guinea pig cochlea using Confocal Laser Scanning Microscopy compared to embedding methods.

Neuron counting in the cochlea is a crucial but time-consuming operation for which various methods have been developed. To improve simplicity and efficiency, we tested an imaging method of the cochlea, and based on Confocal Laser Scanning Microscopy (CLSM), we visualised Rosenthal's Canal and quantified the spiral ganglion neurons (SGN) within. Cochleae of 8 normal hearing guinea pigs and one implanted with a silicone filament were fixed in paraformaldehyde (PFA), decalcified, dehydrated and cleared in Spalteholz solution. Using the tissue's autofluorescence, CLSM was performed at 100 fold magnification generating z-series stacks of about 20 slices of the modiolus. In 5 midmodiolar slices per cochlea the perimeters of the Rosenthal's Canal were surveyed, representative neuron diameters were measured and the neurons first counted manually and then software-assisted. For comparison, 8 normal hearing guinea pig cochleae were embedded in paraffin and examined similarly. The CLSM method has the advantage that the cochleae remain intact as an organ and keep their geometrical structure. Z-stack creation is nearly fully-automatic and frequently repeatable with various objectives and step sizes and without visible bleaching. The tissue shows minimal or no shrinking artefacts and damage typical of embedding and sectioning. As a result, the cells in the cleared cochleae reach an average diameter of 21 µm and a density of about 18 cells/10,000 µm(2) with no significant difference between the manual and the automatical counts. Subsequently we compared the CLSM data with those generated using the established method of paraffin slides, where the SGN reached a mean density of 9.5 cells/10,000 µm(2) and a mean soma diameter of 13.6 µm. We were able to prove that the semi-automatic CLSM method is a simple and effective technique for auditory neuron count. It provides a high grade of tissue preservation and the automatic stack-generation as well as the counter software reduces the effort considerably. In addition this visualisation technique offers the potential to detect the position and orientation of cochlear implants (CI) within the cochlea and tissue growing in the scala tympani around the CI and at the position of the cochleostomy due to the fact that the implant does not have to be removed to perform histology as in case of the paraffin method.

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.

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.