Capturing and stabilizing folded proteins in lattices formed with branched oligonucleotide hybrids

The encapsulation of folded proteins in stabilizing matrices is one of the challenges of soft‐matter materials science. Capturing such fragile bio‐macromolecules from aqueous solution, and embedding them in a lattice that stabilizes them against denaturation and decomposition is difficult. Here, we report that tetrahedral oligonucleotide hybrids as branching elements, and connecting DNA duplexes with sticky ends can assemble into materials. The material‐forming property was used to capture DNA‐binding proteins selectively from aqueous protein mixtures. The three‐dimensional networks also encapsulate guest molecules in a size‐selective manner, accommodating proteins up to a molecular weight of approximately 159 kDa for the connecting duplex lengths tested. Exploratory experiments with green fluorescent protein showed that, when embedded in the DNA‐based matrix, the protein is more stable toward denaturation than in the free form, and retains its luminescent properties for at least 90 days in dry form. The noncrystalline biohybrid matrices presented herein may be used for capturing other proteins or for producing functional materials

Synthetic epigenetics-towards intelligent control of epigenetic states and cell identity

Epigenetics is currently one of the hottest topics in basic and biomedical research. However, to date, most of the studies have been descriptive in nature, designed to investigate static distribution of various epigenetic modifications in cells. Even though tremendous amount of information has been collected, we are still far from the complete understanding of epigenetic processes, their dynamics or even their direct effects on local chromatin and we still do not comprehend whether these epigenetic states are the cause or the consequence of the transcriptional profile of the cell. In this review, we try to define the concept of synthetic epigenetics and outline the available genome targeting technologies, which are used for locus-specific editing of epigenetic signals. We report early success stories and the lessons we have learned from them, and provide a guide for their application. Finally, we discuss existing limitations of the available technologies and indicate possible areas for further development

Bisulfite sequencing Data Presentation and Compilation (BDPC) web server—a useful tool for DNA methylation analysis

During bisulfite genomic sequencing projects large amount of data are generated. The Bisulfite sequencing Data Presentation and Compilation (BDPC) web interface (http://biochem.jacobs-university.de/BDPC/) automatically analyzes bisulfite datasets prepared using the BiQ Analyzer. BDPC provides the following output: (i) MS-Excel compatible files compiling for each PCR product (a) the average methylation level, the number of clones analyzed and the percentage of CG sites analyzed (which is an indicator of data quality), (b) the methylation level observed at each CG site and (c) the methylation level of each clone. (ii) A methylation overview table compiling the methylation of all amplicons in all tissues. (iii) Publication grade figures in PNG format showing the methylation pattern for each PCR product embedded in an HMTL file summarizing the methylation data, the DNA sequence and some basic statistics. (iv) A summary file compiling the methylation pattern of different tissues, which is linked to the individual HTML result files, and can be directly used for presentation of the data in the internet. (v) A condensed file, containing all primary data in simplified format for further downstream data analysis and (vi) a custom track file for display of the results in the UCSC genome browser

Different forms of African cassava mosaic virus capsid protein within plants and virions

One geminiviral gene encodes the capsid protein (CP), which can appear as several bands after electrophoresis depending on virus and plant. African cassava mosaic virus-Nigeria CP in Nicotiana benthamiana, however, yielded one band (~ 30 kDa) in total protein extracts and purified virions, although its expression in yeast yielded two bands (~ 30, 32 kDa). Mass spectrometry of the complete protein and its tryptic fragments from virions is consistent with a cleaved start M1, acetylated S2, and partial phosphorylation at T12, S25 and S62. Mutants for additional potentially modified sites (N223A; C235A) were fully infectious and formed geminiparticles. Separation in triton acetic acid urea gels confirmed charge changes of the CP between plants and yeast indicating differential phosphorylation. If the CP gene alone was expressed in plants, multiple bands were observed like in yeast. A high turnover rate indicates that post-translational modifications promote CP decay probably via the ubiquitin-triggered proteasomal pathway

Sperm induce a secondary increase in ATP levels in mouse eggs that is independent of Ca2+ oscillations

Egg activation at fertilization in mouse eggs is caused by a series of cytosolic Ca2+ oscillations that are associated with an increase in ATP concentrations driven by increased mitochondrial activity. We have investigated the role of Ca2+ oscillations in these changes in ATP at fertilization by measuring the dynamics of ATP and Ca2+ in mouse eggs. An initial ATP increase started with the first Ca2+ transient at fertilization and then a secondary increase in ATP occurred about 1 hour later and this preceded a small and temporary increase in the frequency of Ca2+ oscillations. Other stimuli that caused Ca2+ oscillations such as PLCz1 or thimerosal, caused smaller or slower changes in ATP that failed to show the distinct secondary rise. Sperm induced Ca2+ oscillations in the egg also triggered changes in fluorescence of NADH which followed the pattern of Ca2+ spikes in a similar pattern to oscillations triggered by PLCz1 or thimerosal. When eggs were loaded with low concentrations of the Ca2+ chelator BAPTA, sperm triggered one small Ca2+ increase, but there were still extra phases of ATP increase that were similar to control fertilized eggs. Singular Ca2+ increases caused by thapsigargin were much less effective in elevating ATP levels. Together these data suggest that the secondary ATP increase at fertilization in mouse eggs is not caused by increases in cytosolic Ca2+. The fertilizing sperm may stimulate ATP production in eggs via both Ca2+ and by another mechanism that is independent of PLCz1 or Ca2+ oscillations

The lung microbiota in children with cystic fibrosis captured by induced sputum sampling

Background Spatial topography of the cystic fibrosis (CF) lung microbiota is poorly understood in childhood. How best to sample the respiratory tract in children for microbiota analysis, and the utility of microbiota profiling in clinical management of early infection remains unclear. By comparison with bronchoalveolar lavage (BAL), we assessed the ability of induced sputum (IS) sampling to characterise the lower airway microbiota. Methods Sample sets from IS and two or three matched BAL compartments were obtained for microbiota analysis as part of the CF-Sputum Induction Trial (UKCRN_14615, ISRCTNR_12473810). Microbiota profiles and pathogen detection were compared between matched samples. Results Twenty-eight patients, aged 1.1–17.7 years, provided 30 sample sets. Within-patient BAL comparisons revealed spatial heterogeneity in 8/30 (27%) sample sets indicating that the lower airway microbiota from BAL is frequently compartmentalised in children with CF. IS samples closely resembled one or more matched BAL compartments in 15/30 (50%) sets, and were related in composition in a further 9/30 (30%). IS detected 86.2% of the Top 5 genera found across matched BAL samples. The sensitivity of IS to detect specific CF-pathogens identified in matched BAL samples at relative abundance ≥5% varied between 43 and 100%, with negative predictive values between 73 and 100%. Conclusions Spatial heterogeneity of the lower airway microbiota was observed in BAL samples and presents difficulties for consistent lung sampling. IS captured a microbiota signature representative of the lower airway in 80% of cases, and is a straightforward, non-invasive intervention that can be performed frequently to aid pathogen diagnosis and understand microbiota evolution in children with CF

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

Efficient targeted DNA methylation with chimeric dCas9-Dnmt3a-Dnmt3L methyltransferase.

DNA methylation plays a critical role in the regulation and maintenance of cell-type specific transcriptional programs. Targeted epigenome editing is an emerging technology to specifically regulate cellular gene expression in order to modulate cell phenotypes or dissect the epigenetic mechanisms involved in their control. In this work, we employed a DNA methyltransferase Dnmt3a-Dnmt3L construct fused to the nuclease-inactivated dCas9 programmable targeting domain to introduce DNA methylation into the human genome specifically at the EpCAM, CXCR4 and TFRC gene promoters. We show that targeting of these loci with single gRNAs leads to efficient and widespread methylation of the promoters. Multiplexing of several guide RNAs does not increase the efficiency of methylation. Peaks of targeted methylation were observed around 25 bp upstream and 40 bp downstream of the PAM site, while 20-30 bp of the binding site itself are protected against methylation. Potent methylation is dependent on the multimerization of Dnmt3a/Dnmt3L complexes on the DNA. Furthermore, the introduced methylation causes transcriptional repression of the targeted genes. These new programmable epigenetic editors allow unprecedented control of the DNA methylation status in cells and will lead to further advances in the understanding of epigenetic signaling

Drosophila TET acts with PRC1 to activate gene expression independently of its catalytic activity

Enzymes of the ten-eleven translocation (TET) family play a key role in the regulation of gene expression by oxidizing 5-methylcytosine (5mC), a prominent epigenetic mark in many species. Yet, TET proteins also have less characterized noncanonical modes of action, notably in Drosophila, whose genome is devoid of 5mC. Here, we show that Drosophila TET activates the expression of genes required for larval central nervous system (CNS) development mainly in a catalytic-independent manner. Genome-wide profiling shows that TET is recruited to enhancer and promoter regions bound by Polycomb group complex (PcG) proteins. We found that TET interacts and colocalizes on chromatin preferentially with Polycomb repressor complex 1 (PRC1) rather than PRC2. Furthermore, PRC1 but not PRC2 is required for the activation of TET target genes. Last, our results suggest that TET and PRC1 binding to activated genes is interdependent. These data highlight the importance of TET noncatalytic function and the role of PRC1 for gene activation in the Drosophila larval CNS

Retinol and ascorbate drive erasure of epigenetic memory and enhance reprogramming to naïve pluripotency by complementary mechanisms

Epigenetic memory, in particular DNA methylation, is established during development in differentiating cells and must be erased to create naïve (induced) pluripotent stem cells. The ten-eleven translocation (TET) enzymes can catalyze the oxidation of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) and further oxidized derivatives, thereby actively removing this memory. Nevertheless, the mechanism by which the TET enzymes are regulated, and the extent to which they can be manipulated, are poorly understood. Here we report that retinoic acid (RA) or retinol (vitamin A) and ascorbate (vitamin C) act as modulators of TET levels and activity. RA or retinol enhances 5hmC production in naïve embryonic stem cells by activation of TET2 and TET3 transcription, whereas ascorbate potentiates TET activity and 5hmC production through enhanced Fe2+ recycling, and not as a cofactor as reported previously. We find that both ascorbate and RA or retinol promote the derivation of induced pluripotent stem cells synergistically and enhance the erasure of epigenetic memory. This mechanistic insight has significance for the development of cell treatments for regenenerative medicine, and enhances our understanding of how intrinsic and extrinsic signals shape the epigenome