Operating Cooperatively (OC) Sensor for Highly Specific Recognition of Nucleic Acids

Molecular Beacon (MB) probes have been extensively used for nucleic acid analysis because of their ability to produce fluorescent signal in solution instantly after hybridization. The indirect binding of MB probe to a target analyte offers several advantages, including: improved genotyping accuracy and the possibility to analyse folded nucleic acids. Here we report on a new design for MB-based sensor, called \u27Operating Cooperatively\u27 (OC), which takes advantage of indirect binding of MB probe to a target analyte. The sensor consists of two unmodified DNA strands, which hybridize to a universal MB probe and a nucleic acid analyte to form a fluorescent complex. OC sensors were designed to analyze two human SNPs and E.coli 16S rRNA. High specificity of the approach was demonstrated by the detection of true analyte in over 100 times excess amount of single base substituted analytes. Taking into account the flexibility in the design and the simplicity in optimization, we conclude that OC sensors may become versatile and efficient tools for instant DNA and RNA analysis in homogeneous solution

Comparative biochemical analysis of UHRF proteins reveals molecular mechanisms that uncouple UHRF2 from DNA methylation maintenance

UHRF1 is a histone- and DNA-binding E3 ubiquitin ligase that functions with DNMT1 to maintain mammalian DNA methylation. UHRF1 facilitates DNMT1 recruitment to replicating chromatin through a coordinated mechanism involving histone and DNA recognition and histone ubiquitination. UHRF2 shares structural homology with UHRF1, but surprisingly lacks functional redundancy to facilitate DNA methylation maintenance. Molecular mechanisms uncoupling UHRF2 from DNA methylation maintenance are poorly defined. Through comprehensive and comparative biochemical analysis of recombinant human UHRF1 and UHRF2 reader and writer activities, we reveal conserved modes of histone PTM recognition but divergent DNA binding properties. While UHRF1 and UHRF2 diverge in their affinities toward hemi-methylated DNA, we surprisingly show that both hemi-methylated and hemi-hydroxymethylated DNA oligonucleotides stimulate UHRF2 ubiquitin ligase activity toward histone H3 peptide substrates. This is the first example of an E3 ligase allosterically regulated by DNA hydroxymethylation. However, UHRF2 is not a productive histone E3 ligase toward purified mononucleosomes, suggesting UHRF2 has an intra-domain architecture distinct from UHRF1 that is conformationally constrained when bound to chromatin. Collectively, our studies reveal that uncoupling of UHRF2 from the DNA methylation maintenance program is linked to differences in the molecular readout of chromatin signatures that connect UHRF1 to ubiquitination of histone H3

Systematic comparison of monoclonal versus polyclonal antibodies for mapping histone modifications by ChIP-seq.

BackgroundThe robustness of ChIP-seq datasets is highly dependent upon the antibodies used. Currently, polyclonal antibodies are the standard despite several limitations: They are non-renewable, vary in performance between lots and need to be validated with each new lot. In contrast, monoclonal antibody lots are renewable and provide consistent performance. To increase ChIP-seq standardization, we investigated whether monoclonal antibodies could replace polyclonal antibodies. We compared monoclonal antibodies that target five key histone modifications (H3K4me1, H3K4me3, H3K9me3, H3K27ac and H3K27me3) to their polyclonal counterparts in both human and mouse cells.ResultsOverall performance was highly similar for four monoclonal/polyclonal pairs, including when we used two distinct lots of the same monoclonal antibody. In contrast, the binding patterns for H3K27ac differed substantially between polyclonal and monoclonal antibodies. However, this was most likely due to the distinct immunogen used rather than the clonality of the antibody.ConclusionsAltogether, we found that monoclonal antibodies as a class perform equivalently to polyclonal antibodies for the detection of histone post-translational modifications in both human and mouse. Accordingly, we recommend the use of monoclonal antibodies in ChIP-seq experiments

Lysine Methylation Regulators Moonlighting outside the Epigenome

Landmark discoveries made nearly two decades ago identified known transcriptional regulators as histone lysine methyltransferases; since then the field of lysine methylation signaling has been dominated by studies of how this small chemical posttranslational modification regulates gene expression and other chromatin-based processes. However, recent advances in mass spectrometry-based proteomics have revealed that histones are just a subset of the thousands of eukaryotic proteins marked by lysine methylation. As the writers, erasers, and readers of histone lysine methylation are emerging as a promising therapeutic target class for cancer and other diseases, a key challenge for the field is to define the full spectrum of activities for these proteins. Here we summarize recent discoveries implicating non-histone lysine methylation as a major regulator of diverse cellular processes. We further discuss recent technological innovations that are enabling the expanded study of lysine methylation signaling. Collectively, these findings are shaping our understanding of the fundamental mechanisms of non-histone protein regulation through this dynamic and multi-functional posttranslational modification

A physical basis for quantitative ChIP-sequencing

ChIP followed by next-generation sequencing (ChIP-Seq) is a key technique for mapping the distribution of histone posttranslational modifications (PTMs) and chromatin-associated factors across genomes. There is a perceived challenge to define a quantitative scale for ChIP-Seq data, and as such, several approaches making use of exogenous additives, or "spike-ins," have recently been developed. Herein, we report on the development of a quantitative, physical model defining ChIP-Seq. The quantitative scale on which ChIP-Seq results should be compared emerges from the model. To test the model and demonstrate the quantitative scale, we examine the impacts of an EZH2 inhibitor through the lens of ChIP-Seq. We report a significant increase in immunoprecipitation of presumed off-target histone PTMs after inhibitor treatment, a trend predicted by the model but contrary to spike-in-based indications. Our work also identifies a sensitivity issue in spike-in normalization that has not been considered in the literature, placing limitations on its utility and trustworthiness. We call our new approach the sans-spike-in method for quantitative ChIP-sequencing (siQ-ChIP). A number of changes in community practice of ChIP-Seq, data reporting, and analysis are motivated by this work

Brivaracetam to Treat Partial Onset Seizures in Adults.

PURPOSE OF REVIEW: Seizures are a hyperexcitable, and hypersynchronous imbalance between excitatory and inhibitory factors (E/I imbalance) in neurotransmission, and epilepsy is the recurrent manifestation of seizures within a reasonable time frame and without being attributable to a reversible cause. Brivaracetam is a derivative of the antiepileptic agent, levetiracetam, that is used as adjuvant therapy for focal onset seizures. It was approved by the FDA in 2016 and has shown promising results with minimal adverse effect reactions in clinical trials. RECENT FINDINGS: Brivaracetam has been used in multiple clinical trials at various dosages in adults that have partial-onset seizures refractory to conventional treatment. A meta-analysis in 2016 showed that brivaracetam as adjunctive therapy was statically significant in its reduction of adults with drug-refractory seizure frequency. CONCLUSION: The treatment of epilepsy with pharmacologic agents is a difficult task due to balancing the efficacy of the drug with the side effect profile that will allow for the best quality of life for the patient. There are approximately 30 antiepileptic agents for clinicians to choose from. Brivaracetam is a novel antiepileptic agent that was approved for use by the FDA in 2016 and is showing promising results as monotherapy and adjunctive therapy in individuals with drug-refractory focal seizures while minimizing adverse drug reactions

A Read/Write Mechanism Connects p300 Bromodomain Function to H2A.Z Acetylation

Acetylation of the histone variant H2A.Z (H2A.Zac) occurs at active regulatory regions associated with gene expression. Although the Tip60 complex is proposed to acetylate H2A.Z, functional studies suggest additional enzymes are involved. Here, we show that p300 acetylates H2A.Z at multiple lysines. In contrast, we found that although Tip60 does not efficiently acetylate H2A.Z in vitro, genetic inhibition of Tip60 reduces H2A.Zac in cells. Importantly, we found that interaction between the p300-bromodomain and H4 acetylation (H4ac) enhances p300-driven H2A.Zac. Indeed, H2A.Zac and H4ac show high genomic overlap, especially at active promoters. We also reveal unique chromatin features and transcriptional states at enhancers correlating with co-occurrence or exclusivity of H4ac and H2A.Zac. We propose that differential H4 and H2A.Z acetylation signatures can also define the enhancer state. In conclusion, we show both Tip60 and p300 contribute to H2A.Zac and reveal molecular mechanisms of writer/reader crosstalk between H2A.Z and H4 acetylation through p300

A DNA methylation reader complex that enhances gene transcription

[EN] DNA methylation generally functions as a repressive transcriptional signal, but it is also known to activate gene expression. In either case, the downstream factors remain largely unknown. By using comparative interactomics, we isolated proteins in Arabidopsis thaliana that associate with methylated DNA. Two SU(VAR)3-9 homologs, the transcriptional antisilencing factor SUVH1, and SUVH3, were among the methyl reader candidates. SUVH1 and SUVH3 bound methylated DNA in vitro, were associated with euchromatic methylation in vivo, and formed a complex with two DNAJ domain-containing homologs, DNAJ1 and DNAJ2. Ectopic recruitment of DNAJ1 enhanced gene transcription in plants, yeast, and mammals. Thus, the SUVH proteins bind to methylated DNA and recruit the DNAJ proteins to enhance proximal gene expression, thereby counteracting the repressive effects of transposon insertion near genes.This work was supported by grants NIH R01 GM60398 (to S.E.J.), NIH R01G M089778 (to J.A.W.) and NIH R35 GM124736 (to S.B.R), by an EMBO Long-Term Fellowship (ALTF 1138-2014) (to C.J.H), and by a Ruth L. Kirschstein National Research Service Award (GM007185) (to L.Y.). S.E.J. is an investigator of the Howard Hughes Medical Institute.Harris, CJ.; Scheibe, M.; Wongpalee, SP.; Liu, W.; Cornett, EM.; Vaughan, RM.; Li, X.... (2018). A DNA methylation reader complex that enhances gene transcription. Science. 362(6419):1182-1186. https://doi.org/10.1126/science.aar785411821186362641

Light-activated Binary Nucleotide Reagent For Inactivation Of Dna Polymerase

This work explores a binary reagent approach to increase the specificity of covalent inhibitors. In this approach, two ligand analogs equipped with inert pre-reactive groups specifically bind a target biopolymer. The binding event brings the pre-reactive groups in proximity with each other. The two groups react, generating active chemical intermediates that covalently modify and inactivate the target. In the present study we compare the new approach with the traditional single-component reagent strategy using DNA polymerase from bacteriophage T4 as a model target biopolymer. We report the design and synthesis of two analogs of deoxythymidine triphosphate, a natural DNA polymerase substrate. Together, the analogs function as a binary nucleotide reagent which is activated by light with wavelengths 365 nm and longer. However, the active analog functions as a traditional single component reagent when activated by light with wavelengths at 300 nm and longer. The traditional single-component reagent efficiently inactivated DNA polymerase. However, in the presence of non-target protein the inactivation efficiency is greatly diminished. Under the same conditions, the binary nucleotide reagent also inactivated DNA polymerase, and the inactivation efficiency is not affected by the presence of the non-target protein. Our results validate that a binary approach can be employed to design highly specific covalent inhibitors. The binary reagent strategy might be useful as a research tool for investigation of ligand-protein interactions in complex biological systems and for drug desig

Snp Analysis Using A Molecular Beacon-Based Operating Cooperatively (Oc) Sensor

Analysis of single-nucleotide polymorphisms (SNPs) is important for diagnosis of infectious and genetic diseases, for environment and population studies, as well as in forensic applications. Herein is a detailed description to design an operating cooperatively (OC) sensor for highly specific SNP analysis. OC sensors use two unmodified DNA adaptor strands and a molecular beacon probe to detect a nucleic acid targets with exceptional specificity towards SNPs. Genotyping can be accomplished at room temperature in a homogenous assay. The approach is easily adaptable for any nucleic acid target, and has been successfully used for analysis of targets with complex secondary structures. Additionally, OC sensors are an easy-to-design and cost-effective method for SNP analysis and nucleic acid detection. © 2013 Springer Science+Business Media, New York