Characterization of PvuRts1I endonuclease as a tool to investigate genomic 5–hydroxymethylcytosine

In mammalian genomes a sixth base, 5-hydroxymethylcytosine ( hm C), is generated by enzymatic oxidation of 5-methylcytosine ( m C). This discovery has raised fundamental questions about the functional relevance of hm C in mammalian genomes. Due to their very similar chemical structure, discrimination of the rare hm C against the far more abundant m C is technically challenging and to date no methods for direct sequencing of hm C have been reported. Here, we report on a purified recombinant endonuclease, PvuRts1I, which selectively cleaves hm C-containing sequences. We determined the consensus cleavage site of PvuRts1I as hm CN 11–12 /N 9–10 G and show first data on its potential to interrogate hm C patterns in mammalian genomes

Metal oxides based electrochemical pH sensors: Current progress and future perspectives

Electrochemical pH sensors are on high demand in numerous applications such as food processing, health monitoring, agriculture and nuclear sectors, and water quality monitoring etc., owing to their fast response (<10 s), wide pH sensing range (2–12), superior sensitivity (close to Nernstian response of 59.12 mV/pH), easy integration on wearable/flexible substrates, excellent biocompatibility and low cost of fabrication. This article presents an in-depth review of the wide range of MOx materials that have been utilized to develop pH sensors, based on various mechanisms (e.g. potentiometric, conductimetric, chemi-resistors, ion sensitive field effect transistor (ISFET) and extended-gate field effect transistor etc.). The tools and techniques such as potentiometric and electrochemical impedance spectroscopic that are commonly adopted to characterize these metal oxide-based pH sensors are also discussed in detail. Concerning materials and design of sensors for various practical application, the major challenges are toxicity of materials, interfernce of other ions or analytes, cost, and flexibility of materials. In this regard, this review also discusses the metal oxide-based composite sensing (active) material, designs of pH sensors and their applications in flexible/wearable biosensors for medical application are examined to present their suitability for these futuristic applications

Systematic analysis of the binding behaviour of UHRF1 towards different methyl- and carboxylcytosine modification patterns at CpG dyads

The multi-domain protein UHRF1 is essential for DNA methylation maintenance and binds DNA via a base-flipping mechanism with a preference for hemi-methylated CpG sites. We investigated its binding to hemi- and symmetrically modified DNA containing either 5-methylcytosine (mC), 5-hydroxymethylcytosine (hmC), 5-formylcytosine (fC), or 5-carboxylcytosine (caC). Our experimental results indicate that UHRF1 binds symmetrically carboxylated and hybrid methylated/carboxylated CpG dyads in addition to its previously reported substrates. Complementary molecular dynamics simulations provide a possible mechanistic explanation of how the protein could differentiate between modification patterns. First, we observe different local binding modes in the nucleotide binding pocket as well as the protein's NKR finger. Second, both DNA modification sites are coupled through key residues within the NKR finger, suggesting a communication pathway affecting protein-DNA binding for carboxylcytosine modifications. Our results suggest a possible additional function of the hemi-methylation reader UHRF1 through binding of carboxylated CpG sites. This opens the possibility of new biological roles of UHRF1 beyond DNA methylation maintenance and of oxidised methylcytosine derivates in epigenetic regulation

Characterization of PvuRts1I endonuclease as a tool to investigate genomic 5–hydroxymethylcytosine

In mammalian genomes a sixth base, 5-hydroxymethylcytosine (hmC), is generated by enzymatic oxidation of 5-methylcytosine (mC). This discovery has raised fundamental questions about the functional relevance of hmC in mammalian genomes. Due to their very similar chemical structure, discrimination of the rare hmC against the far more abundant mC is technically challenging and to date no methods for direct sequencing of hmC have been reported. Here, we report on a purified recombinant endonuclease, PvuRts1I, which selectively cleaves hmC-containing sequences. We determined the consensus cleavage site of PvuRts1I as hmCN11–12/N9–10G and show first data on its potential to interrogate hmC patterns in mammalian genomes

A Low-Cost pH Sensor Based on RuO2 Resistor Material

Fresh water deficiency caused by climate change calls for employing novel measures to ensure safety of drinking water supply. Wireless sensor networks can be used for monitoring hydrological conditions across wide area, allowing flow forecasting and early detection of pollutants. While there are no fundamental technological obstacles to implementation of large area sensor networks, their feasibility is constrained by unit cost of sensing nodes. This paper describes a low-cost pH sensor, intended for use in fresh water monitoring. The sensor was fabricated in a standard thick film process, and an off-the-shelf resistive paste was used as a sensing material. For the fabrication of sensor, RuO2 resistive paste was screen printed on the alumina substrate with silver conducting layer. Test solutions with pH ranging from 2 to 10 were prepared from HCl or KOH solutions. The potential difference between reference and sensing electrode (electromotive force emf of an electrochemical cell) should be proportional to the pH of a solution according to the Nernst equation. The fabricated sensor exhibits Nernstian response to pH. Influence of storage conditions on sensing performance was also investigated

Sensitive enzymatic quantification of 5-hydroxymethylcytosine in genomic DNA

The recent discovery of genomic 5-hydroxymethylcytosine (hmC) and mutations affecting the respective Tet hydroxylases in leukemia raises fundamental questions about this epigenetic modification. We present a sensitive method for fast quantification of genomic hmC based on specific transfer of radiolabeled glucose to hmC by a purified glucosyltransferase. We determined hmC levels in various adult tissues and differentiating embryonic stem cells and show a correlation with differential expression of tet genes

5-Hydroxymethylation marks a class of neuronal gene regulated by intragenic methylcytosine levels.

AbstractWe recently identified a class of neuronal gene inheriting high levels of intragenic methylation from the mother and maintaining this through later development. We show here that these genes are implicated in basic neuronal functions such as post-synaptic signalling, rather than neuronal development and inherit high levels of 5mC, but not 5hmC, from the mother. 5mC is distributed across the gene body and appears to facilitate transcription, as transcription is reduced in DNA methyltransferase I (Dnmt1) knockout embryonic stem cells as well as in fibroblasts treated with a methyltransferase inhibitor. However in adult brain, transcription is more closely associated with a gain in 5hmC, which occurs without a measurable loss of 5mC. These findings add to growing evidence that there may be a role for 5mC in promoting transcription as well as its classical role in gene silencing

5-hydroxymethyl-cytosine enrichment of non-committed cells is not a universal feature of vertebrate development

5-hydroxymethyl-cytosine (5-hmc) is a cytosine modification that is relatively abundant in mammalian pre-implantation embryos and embryonic stem cells (Esc) derived from mammalian blastocysts. Recent observations imply that both 5-hmc and Tet1/2/3 proteins, catalyzing the conversion of 5-methyl-cytosine to 5-hmc, may play an important role in self renewal and differentiation of Escs. here we assessed the distribution of 5-hmc in zebrafish and chick embryos and found that, unlike in mammals, 5-hmc is immunochemically undetectable in these systems before the onset of organogenesis. In addition, Tet1/2/3 transcripts are either low or undetectable at corresponding stages of zebrafish development. however, 5-hmc is enriched in later zebrafish and chick embryos and exhibits tissue-specific distribution in adult zebrafish. Our findings show that 5-hmc enrichment of non-committed cells is not a universal feature of vertebrate development and give insights both into evolution of embryonic pluripotency and the potential role of 5-hmc in its regulation

Genomic mapping of 5-hydroxymethylcytosine in the human brain

Methylation at the 5-position of cytosine is a well-studied epigenetic pathway. In addition to 5-methylcytosine (5mC), substantial amounts of 5-hydroxymethylcytosine (5hmC) also referred to as the sixth DNA base have been detected in certain tissues, most notably the brain. However, the genomic distribution of this cytosine modification is unknown. Here, we have used an immunoprecipitation technique (5hmC-IP) to examine the occurrence of 5hmC in DNA from human brain frontal lobe tissue. The distribution of 5hmC was compared to that of 5mC. We show that 5hmC is more selectively targeted to genes than is 5mC. 5hmC is particularly enriched at promoters and in intragenic regions (gene bodies) but is largely absent from non-gene regions. 5hmC peaks at transcription start sites did not correlate with gene expression levels for promoters with intermediate or high CpG content. However, the presence of 5hmC in gene bodies was more positively correlated with gene expression levels than was the presence of 5mC. Promoters of testis-specific genes showed strong 5mC peaks in brain DNA but were almost completely devoid of 5hmC. Our data provide an overview of the genomic distribution of 5hmC in human brain and will set the stage for further functional characterization of this novel DNA modification

Targeted transcriptional activation of silent oct4 pluripotency gene by combining designer TALEs and inhibition of epigenetic modifiers

Specific control of gene activity is a valuable tool to study and engineer cellular functions. Recent studies uncovered the potential of transcription activator-like effector (TALE) proteins that can be tailored to activate user-defined target genes. It remains however unclear whether and how epigenetic modifications interfere with TALE-mediated transcriptional activation. We studied the activity of five designer TALEs (dTALEs) targeting the oct4 pluripotency gene. In vitro assays showed that the five dTALEs that target distinct sites in the oct4 promoter had the expected DNA specificity and comparable affinities to their corresponding DNA targets. In contrast to their similar in vitro properties, transcriptional activation of oct4 by these distinct dTALEs varied up to 25-fold. While dTALEs efficiently upregulated transcription of the active oct4 promoter in embryonic stem cells (ESCs) they failed to activate the silenced oct4 promoter in ESC-derived neural stem cells (NSCs), indicating that as for endogenous transcription factors also dTALE activity is limited by repressive epigenetic mechanisms. We therefore targeted the activity of epigenetic modulators and found that chemical inhibition of histone deacetylases by valproic acid or DNA methyltransferases by 5-aza-2′-deoxycytidine facilitated dTALE-mediated activation of the epigenetically silenced oct4 promoter in NSCs. Notably, demethylation of the oct4 promoter occurred only if chemical inhibitors and dTALEs were applied together but not upon treatment with inhibitors or dTALEs only. These results show that dTALEs in combination with chemical manipulation of epigenetic modifiers facilitate targeted transcriptional activation of epigenetically silenced target genes