H3.1K27me1 maintains transcriptional silencing and genome stability by preventing GCN5-mediated histone acetylation.

Epigenetic mechanisms play diverse roles in the regulation of genome stability in eukaryotes. In Arabidopsis thaliana, genome stability is maintained during DNA replication by the H3.1K27 methyltransferases ARABIDOPSIS TRITHORAX-RELATED PROTEIN 5 (ATXR5) and ATXR6, which catalyze the deposition of K27me1 on replication-dependent H3.1 variants. The loss of H3.1K27me1 in atxr5 atxr6 double mutants leads to heterochromatin defects, including transcriptional de-repression and genomic instability, but the molecular mechanisms involved remain largely unknown. In this study, we identified the transcriptional co-activator and conserved histone acetyltransferase GCN5 as a mediator of transcriptional de-repression and genomic instability in the absence of H3.1K27me1. GCN5 is part of a SAGA-like complex in plants that requires the GCN5-interacting protein ADA2b and the chromatin remodeler CHR6 to mediate the heterochromatic defects in atxr5 atxr6 mutants. Our results also indicate that Arabidopsis GCN5 acetylates multiple lysine residues on H3.1 variants, but H3.1K27 and H3.1K36 play essential functions in inducing genomic instability in the absence of H3.1K27me1. Finally, we show that H3.1K36 acetylation by GCN5 is negatively regulated by H3.1K27me1 in vitro. Overall, this work reveals a key molecular role for H3.1K27me1 in maintaining transcriptional silencing and genome stability in heterochromatin by restricting GCN5-mediated histone acetylation in plants.

H3K36 methylation and DNA-binding both promote Ioc4 recruitment and Isw1b remodeler function.

The Isw1b chromatin-remodeling complex is specifically recruited to gene bodies to help retain pre-existing histones during transcription by RNA polymerase II. Recruitment is dependent on H3K36 methylation and the Isw1b subunit Ioc4, which contains an N-terminal PWWP domain. Here, we present the crystal structure of the Ioc4-PWWP domain, including a detailed functional characterization of the domain on its own as well as in the context of full-length Ioc4 and the Isw1b remodeler. The Ioc4-PWWP domain preferentially binds H3K36me3-containing nucleosomes. Its ability to bind DNA is required for nucleosome binding. It is also furthered by the unique insertion motif present in Ioc4-PWWP. The ability to bind H3K36me3 and DNA promotes the interaction of full-length Ioc4 with nucleosomes in vitro and they are necessary for its recruitment to gene bodies in vivo. Furthermore, a fully functional Ioc4-PWWP domain promotes efficient remodeling by Isw1b and the maintenance of ordered chromatin in vivo, thereby preventing the production of non-coding RNAs.

The domesticated transposase ALP2 mediates formation of a novel Polycomb protein complex by direct interaction with MSI1, a core subunit of Polycomb Repressive Complex 2 (PRC2).

A large fraction of plant genomes is composed of transposable elements (TE), which provide a potential source of novel genes through "domestication"-the process whereby the proteins encoded by TE diverge in sequence, lose their ability to catalyse transposition and instead acquire novel functions for their hosts. In Arabidopsis, ANTAGONIST OF LIKE HETEROCHROMATIN PROTEIN 1 (ALP1) arose by domestication of the nuclease component of Harbinger class TE and acquired a new function as a component of POLYCOMB REPRESSIVE COMPLEX 2 (PRC2), a histone H3K27me3 methyltransferase involved in regulation of host genes and in some cases TE. It was not clear how ALP1 associated with PRC2, nor what the functional consequence was. Here, we identify ALP2 genetically as a suppressor of Polycomb-group (PcG) mutant phenotypes and show that it arose from the second, DNA binding component of Harbinger transposases. Molecular analysis of PcG compromised backgrounds reveals that ALP genes oppose silencing and H3K27me3 deposition at key PcG target genes. Proteomic analysis reveals that ALP1 and ALP2 are components of a variant PRC2 complex that contains the four core components but lacks plant-specific accessory components such as the H3K27me3 reader LIKE HETEROCHROMATION PROTEIN 1 (LHP1). We show that the N-terminus of ALP2 interacts directly with ALP1, whereas the C-terminus of ALP2 interacts with MULTICOPY SUPPRESSOR OF IRA1 (MSI1), a core component of PRC2. Proteomic analysis reveals that in alp2 mutant backgrounds ALP1 protein no longer associates with PRC2, consistent with a role for ALP2 in recruitment of ALP1. We suggest that the propensity of Harbinger TE to insert in gene-rich regions of the genome, together with the modular two component nature of their transposases, has predisposed them for domestication and incorporation into chromatin modifying complexes.

INTEGRATED INSULIN PUMP AND CONTINUOUS GLUCOSE MONITORING TECHNOLOGY IN DIABETES CARE TODAY: A PERSPECTIVE OF REAL-LIFE EXPERIENCE WITH THE MINIMED™ 670G HYBRID CLOSED-LOOP SYSTEM.

OBJECTIVE: Intensive glucose management with insulin pump and continuous glucose monitoring therapy in insulin-treated patients with diabetes poses many challenges in all aspects of daily life. Automated insulin delivery (AID) is the ultimate goal of insulin replacement therapy to reduce the burden of managing this condition. Many systems are being tested in the clinical research setting, and one hybrid closed-loop (HCL) system has received Food and Drug Administration (FDA) approval for use in type 1 diabetes patients above the age of 14 years. METHODS: Literature review and clinical practice experience from the Diabetes and Technology Program at an academic medical center. RESULTS: This review outlines recent advances in AID systems, focusing on the FDA-approved MiniMed™ 670G HCL system and the real-life experience 1-year post-release in an academic medical center with over 60 patients on this system. The unique challenges of adapting to this new system outside the clinical trial setting are highlighted, and a training protocol designed specifically for the onboarding of first-time users is described. CONCLUSION: HCL insulin therapy offers several advantages, at the same time posing unique challenges to the user. Systematic training of patients with diabetes transitioning to this system is essential for retention and success of use. ABBREVIATIONS: AID = automated insulin delivery; CGM = continuous glucose monitoring; FDA = Food and Drug Administration; HbA1c = glycated hemoglobin; HCL = hybrid closed-loop; ICR = insulin to carbohydrate ratio; SAP = sensor augmented pump; T1DM = type 1 diabetes.

The U.S. Culture Collection Network Lays the Foundation for Progress in Preservation of Valuable Microbial Resources.

The U.S. Culture Collection Network was formed in 2012 by a group of culture collection scientists and stakeholders in order to continue the progress established previously through efforts of an ad hoc group. The network is supported by a Research Coordination Network grant from the U.S. National Science Foundation (NSF) and has the goals of promoting interaction among collections, encouraging the adoption of best practices, and protecting endangered or orphaned collections. After prior meetings to discuss best practices, shared data, and synergy with genome programs, the network held a meeting at the U.S. Department of Agriculture (USDA)-Agricultural Research Service (ARS) National Center for Genetic Resources Preservation (NCGRP) in Fort Collins, Colorado in October 2015 specifically to discuss collections that are vulnerable because of changes in funding programs, or are at risk of loss because of retirement or lack of funding. The meeting allowed collection curators who had already backed up their resources at the USDA NCGRP to visit the site, and brought collection owners, managers, and stakeholders together. Eight formal collections have established off-site backups with the USDA-ARS, ensuring that key material will be preserved for future research. All of the collections with backup at the NCGRP are public distributing collections including U.S. NSF-supported genetic stock centers, USDA-ARS collections, and university-supported collections. Facing the retirement of several pioneering researchers, the community discussed the value of preserving personal research collections and agreed that a mechanism to preserve these valuable collections was essential to any future national culture collection system. Additional input from curators of plant and animal collections emphasized that collections of every kind face similar challenges in developing long-range plans for sustainability.

Multilocus analysis using putative fungal effectors to describe a population of Fusarium oxysporum from sugar beet.

Sugar beet (Beta vulgaris) Fusarium yellows is caused by Fusarium oxysporum f. sp. betae and can lead to significant reductions in root yield, sucrose percentage, juice purity, and storability. F. oxysporum f. sp. betae can be highly variable and many F. oxysporum strains isolated from symptomatic sugar beet are nonpathogenic. Identifying pathogenicity factors and their diversity in the F. oxysporum f. sp. betae population could further understanding of how this pathogen causes disease and potentially provide molecular markers to rapidly identify pathogenic isolates. This study used several previously described fungal effector genes (Fmk1, Fow1, Pda1, PelA, PelD, Pep1, Prt1, Rho1, Sge1, Six1, Six6, Snf1, and Ste12) as genetic markers, in a population of 26 pathogenic and nonpathogenic isolates of F. oxysporum originally isolated from symptomatic sugar beet. Of the genes investigated, six were present in all F. oxysporum isolates from sugar beet (Fmk1, Fow1, PelA, Rho1, Snf1, and Ste12), and seven were found to be dispersed within the population (Pda1, PelD, Pep1, Prt1, Sge1, Six1, and Six6). Of these, Fmk1, Fow1, PelA, Rho1, Sge1, Snf1, and Ste12 were significant in relating clade designations and PelD, and Prt1 were significant for correlating with pathogenicity in F. oxysporum f. sp. betae.

Effects of intracellular Mn on the radiation resistance of the halophilic archaeon Halobacterium salinarum.

Ionizing radiation (IR) is of particular interest in biology because its exposure results in severe oxidative stress to the cell's macromolecules. Our recent work with extremophiles supports the idea that IR resistance is most likely achieved by a metabolic route, effected by manganese (Mn) antioxidants. Biochemical analysis of "super-IR resistant" mutants of H. salinarum, evolved over multiple cycles of exposure to high doses of IR, confirmed the key role for Mn antioxidants in the IR resistance of this organism. Analysis of the proteome of H. salinarum "super-IR resistant" mutants revealed increased expression for proteins involved in energy metabolism, replenishing the cell with reducing equivalents depleted by the oxidative stress inflicted by IR. Maintenance of redox homeostasis was also activated by the over-expression of coenzyme biosynthesis pathways involved in redox reactions. We propose that in H. salinarum, increased tolerance to IR is a combination of metabolic regulatory adjustments and the accumulation of Mn-antioxidant complexes.

Cross Pathogenicity and Vegetative Compatibility of Fusarium oxysporum Isolated from Sugar Beet.

Fusarium oxysporum f. sp. betae causes Fusarium yellows in sugar beet (Beta vulgaris). The F. oxysporum population from sugar beet can be highly variable in virulence and morphology and many isolates are nonpathogenic. Rapid and reliable methods to identify pathogenic isolates from nonpathogenic F. oxysporum generally are unavailable. Little is known about nonpathogenic isolates, including the role they may play in population diversity or virulence to sugar beet. Sugar beet is often grown in rotation with other crops, including dry edible bean (Phaseolus vulgaris) and onion (Allium cepa), with F. oxysporum able to cause disease on all three crops. Thirty-eight F. oxysporum isolates were collected from symptomatic sugar beet throughout the United States to investigate diversity of the F. oxysporum population and the influence of crop rotation on pathogenic variation. These isolates were characterized for pathogenicity to sugar beet, dry edible bean, and onion, as well as vegetative compatibility. Pathogenicity testing indicated that some F. oxysporum isolates from sugar beet may cause disease on onion and dry edible bean. Furthermore, vegetative compatibility testing supported previous reports that F. oxysporum f. sp. betae is polyphyletic and that pathogenic isolates cannot be differentiated from nonpathogenic F. oxysporum using vegetative compatibility.

Role of Mn2+ and compatible solutes in the radiation resistance of thermophilic bacteria and archaea.

Radiation-resistant bacteria have garnered a great deal of attention from scientists seeking to expose the mechanisms underlying their incredible survival abilities. Recent analyses showed that the resistance to ionizing radiation (IR) in the archaeon Halobacterium salinarum is dependent upon Mn-antioxidant complexes responsible for the scavenging of reactive oxygen species (ROS) generated by radiation. Here we examined the role of the compatible solutes trehalose, mannosylglycerate, and di-myo-inositol phosphate in the radiation resistance of aerobic and anaerobic thermophiles. We found that the IR resistance of the thermophilic bacteria Rubrobacter xylanophilus and Rubrobacter radiotolerans was highly correlated to the accumulation of high intracellular concentration of trehalose in association with Mn, supporting the model of Mn(2+)-dependent ROS scavenging in the aerobes. In contrast, the hyperthermophilic archaea Thermococcus gammatolerans and Pyrococcus furiosus did not contain significant amounts of intracellular Mn, and we found no significant antioxidant activity from mannosylglycerate and di-myo-inositol phosphate in vitro. We therefore propose that the low levels of IR-generated ROS under anaerobic conditions combined with highly constitutively expressed detoxification systems in these anaerobes are key to their radiation resistance and circumvent the need for the accumulation of Mn-antioxidant complexes in the cell.

Histone marks regulate the epithelial-to-mesenchymal transition via alternative splicing.

Histone modifications impact final splicing decisions. However, there is little evidence of the driving role of these marks in inducing cell-specific splicing changes. Using CRISPR epigenome editing tools, we show in an epithelial-to-mesenchymal cell reprogramming system (epithelial-to-mesenchymal transition [EMT]) that a single change in H3K27ac or H3K27me3 levels right at the alternatively spliced exon is necessary and sufficient to induce a splicing change capable of recapitulating important aspects of EMT, such as cell motility and invasiveness. This histone-mark-dependent splicing effect is highly dynamic and mediated by direct recruitment of the splicing regulator PTB to its RNA binding sites. These results support a role for H3K27 marks in inducing a change in the cell's phenotype via regulation of alternative splicing. We propose the dynamic nature of chromatin as a rapid and reversible mechanism to coordinate the splicing response to cell-extrinsic cues, such as induction of EMT.

Chloromethyl-triazole: a new motif for site-selective pseudo-acylation of proteins.

Rapid, site-selective modification of cysteine residues with chloromethyl-triazole derivatives generates pseudo-acyl sLys motifs, mimicking important post-translational modifications. Near-native biotinylation of peptide and protein substrates is shown to be site-selective and modified histone H4 retains functional activity.

Mid-Infrared (MIR) and Near-Infrared (NIR) Detection of Rhizoctonia solani AG 2-2 IIIB on Barley-Based Artificial Inoculum.

The amount of Rhizoctonia solani in the soil and how much must be present to cause disease in sugar beet (Beta vulgaris L.) is relatively unknown. This is mostly because of the usually low inoculum densities found naturally in soil and the low sensitivity of traditional serial dilution assays. We investigated the usefulness of Fourier transform mid-infrared (MIR) and near-infrared (NIR) spectroscopic properties in identifying the artificial colonization of barley grains with R. solani AG 2-2 IIIB and in detecting R. solani populations in plant tissues and inoculants. The objectives of this study were to compare the ability of traditional plating assays to NIR and MIR spectroscopies to identify R. solani in different-size fractions of colonized ground barley (used as an artificial inoculum) and to differentiate colonized from non-inoculated barley. We found that NIR and MIR spectroscopies were sensitive in resolving different barley particle sizes, with particles that were <0.25 and 0.25-0.5 mm having different spectral properties than coarser particles. Moreover, we found that barley colonized with R. solani had different MIR spectral properties than the non-inoculated samples for the larger fractions (0.5-1.0, 1.0-2.0, and >2.0 mm) of the ground barley. This colonization was confirmed using traditional plating assays. Comparisons with the spectra from pure fungal cultures and non-inoculated barley suggest that the MIR spectrum of colonized barley is different because of the consumption of C substrates by the fungus rather than because of the presence of fungal bands in the spectra of the colonized samples. We found that MIR was better than NIR spectroscopy in differentiating the colonized from the control samples.

Proteomic Profiling of Sugar Beet (Beta vulgaris) Leaves during Rhizomania Compatible Interactions.

Rhizomania, caused by Beet necrotic yellow vein virus (BNYVV), severely impacts sugar beet (Beta vulgaris) production throughout the world, and is widely prevalent in most production regions. Initial efforts to characterize proteome changes focused primarily on identifying putative host factors that elicit resistant interactions with BNYVV, but as resistance breaking strains become more prevalent, effective disease control strategies will require the application of novel methods based on better understanding of disease susceptibility and symptom development. Herein, proteomic profiling was conducted on susceptible sugar beet, infected with two strains of BNYVV, to clarify the types of proteins prevalent during compatible virus-host plant interactions. Total protein was extracted from sugar beet leaf tissue infected with BNYVV, quantified, and analyzed by mass spectrometry. A total of 203 proteins were confidently identified, with a predominance of proteins associated with photosynthesis and energy, metabolism, and response to stimulus. Many proteins identified in this study are typically associated with systemic acquired resistance and general plant defense responses. These results expand on relatively limited proteomic data available for sugar beet and provide the ground work for additional studies focused on understanding the interaction of BNYVV with sugar beet.

Mass spectrometry contamination from Tinuvin 770, a common additive in laboratory plastics.

The superior sensitivity of current mass spectrometers makes them prone to contamination issues, which can have deleterious effects on sample analysis. Here, bis(2,2,6,6-tetramethyl-4-piperidyl) sebacate (marketed under the name Tinuvin 770) is identified as a major contaminant in applications using liquid chromatography coupled with mass spectrometry (LC-MS). Tinuvin 770 is often added to laboratory and medical plastics as a UV stabilizer. One particular lot of microcentrifuge tubes was found to have an excess of this compound that would leach into samples and drastically interfere with LC-MS data acquisition. Further analysis found that Tinuvin 770 readily leached into polar and nonpolar solvents from the contaminated tube lot. Efforts to remove Tinuvin 770 from contaminated samples were unsuccessful. A prescreening method using MALDI-TOF MS is presented to prevent system contamination and sample loss.