Acetylation of PCNA Sliding Surface by Eco1 Promotes Genome Stability through Homologous Recombination.

During DNA replication, proliferating cell nuclear antigen (PCNA) adopts a ring-shaped structure to promote processive DNA synthesis, acting as a sliding clamp for polymerases. Known posttranslational modifications function at the outer surface of the PCNA ring to favor DNA damage bypass. Here, we demonstrate that acetylation of lysine residues at the inner surface of PCNA is induced by DNA lesions. We show that cohesin acetyltransferase Eco1 targets lysine 20 at the sliding surface of the PCNA ring in vitro and in vivo in response to DNA damage. Mimicking constitutive acetylation stimulates homologous recombination and robustly suppresses the DNA damage sensitivity of mutations in damage tolerance pathways. In comparison to the unmodified trimer, structural differences are observed at the interface between protomers in the crystal structure of the PCNA-K20ac ring. Thus, acetylation regulates PCNA sliding on DNA in the presence of DNA damage, favoring homologous recombination linked to sister-chromatid cohesion.

UTX inhibition as selective epigenetic therapy against TAL1-driven T-cell acute lymphoblastic leukemia.

T-cell acute lymphoblastic leukemia (T-ALL) is a heterogeneous group of hematological tumors composed of distinct subtypes that vary in their genetic abnormalities, gene expression signatures, and prognoses. However, it remains unclear whether T-ALL subtypes differ at the functional level, and, as such, T-ALL treatments are uniformly applied across subtypes, leading to variable responses between patients. Here we reveal the existence of a subtype-specific epigenetic vulnerability in T-ALL by which a particular subgroup of T-ALL characterized by expression of the oncogenic transcription factor TAL1 is uniquely sensitive to variations in the dosage and activity of the histone 3 Lys27 (H3K27) demethylase UTX/KDM6A. Specifically, we identify UTX as a coactivator of TAL1 and show that it acts as a major regulator of the TAL1 leukemic gene expression program. Furthermore, we demonstrate that UTX, previously described as a tumor suppressor in T-ALL, is in fact a pro-oncogenic cofactor essential for leukemia maintenance in TAL1-positive (but not TAL1-negative) T-ALL. Exploiting this subtype-specific epigenetic vulnerability, we propose a novel therapeutic approach based on UTX inhibition through in vivo administration of an H3K27 demethylase inhibitor that efficiently kills TAL1-positive primary human leukemia. These findings provide the first opportunity to develop personalized epigenetic therapy for T-ALL patients.

A phosphorylation switch on RbBP5 regulates histone H3 Lys4 methylation.

The methyltransferase activity of the trithorax group (TrxG) protein MLL1 found within its COMPASS (complex associated with SET1)-like complex is allosterically regulated by a four-subunit complex composed of WDR5, RbBP5, Ash2L, and DPY30 (also referred to as WRAD). We report structural evidence showing that in WRAD, a concave surface of the Ash2L SPIa and ryanodine receptor (SPRY) domain binds to a cluster of acidic residues, referred to as the D/E box, in RbBP5. Mutational analysis shows that residues forming the Ash2L/RbBP5 interface are important for heterodimer formation, stimulation of MLL1 catalytic activity, and erythroid cell terminal differentiation. We also demonstrate that a phosphorylation switch on RbBP5 stimulates WRAD complex formation and significantly increases KMT2 (lysine [K] methyltransferase 2) enzyme methylation rates. Overall, our findings provide structural insights into the assembly of the WRAD complex and point to a novel regulatory mechanism controlling the activity of the KMT2/COMPASS family of lysine methyltransferases.

Constitutive Overexpression of RAM1 Leads to an Increase in Arbuscule Density in Brachypodium distachyon.

Arbuscular mycorrhizal (AM) symbiosis is a mutually beneficial association of plants and fungi of the subphylum Glomeromycotina. Endosymbiotic AM fungi colonize the inner cortical cells of the roots, where they form branched hyphae called arbuscules that function in nutrient exchange with the plant. To support arbuscule development and subsequent bidirectional nutrient exchange, the root cortical cells undergo substantial transcriptional reprogramming. REDUCED ARBUSCULAR MYCORRHIZA1 (RAM1), previously studied in several dicot plant species, is a major regulator of this cortical cell transcriptional program. Here, we generated ram1 mutants and RAM1 overexpressors in a monocot, Brachypodium distachyon. The AM phenotypes of two ram1 lines revealed that RAM1 is only partly required to enable arbuscule development in B. distachyon Transgenic lines constitutively overexpressing BdRAM1 showed constitutive expression of AM-inducible genes even in the shoots. Following inoculation with AM fungi, BdRAM1-overexpressing plants showed higher arbuscule densities relative to controls, indicating the potential to manipulate the relative proportion of symbiotic interfaces via modulation of RAM1 However, the overexpressors also show altered expression of hormone biosynthesis genes and aberrant growth patterns, including stunted bushy shoots and poor seed set. While these phenotypes possibly provide additional clues about the scope of influence of BdRAM1, they also indicate that directed approaches to increase the density of symbiotic interfaces will require a more focused, potentially cell type specific manipulation of transcription factor gene expression.

Diverse Sorghum bicolor accessions show marked variation in growth and transcriptional responses to arbuscular mycorrhizal fungi.

Sorghum is an important crop grown worldwide for feed and fibre. Like most plants, it has the capacity to benefit from symbioses with arbuscular mycorrhizal (AM) fungi, and its diverse genotypes likely vary in their responses. Currently, the genetic basis of mycorrhiza-responsiveness is largely unknown. Here, we investigated transcriptional and physiological responses of sorghum accessions, founders of a bioenergy nested association mapping panel, for their responses to four species of AM fungi. Transcriptome comparisons across four accessions identified mycorrhiza-inducible genes; stringent filtering criteria revealed 278 genes that show mycorrhiza-inducible expression independent of genotype and 55 genes whose expression varies with genotype. The latter suggests variation in phosphate transport and defence across these accessions. The mycorrhiza growth and nutrient responses of 18 sorghum accessions varied tremendously, ranging from mycorrhiza-dependent to negatively mycorrhiza-responsive. Additionally, accessions varied in the number of AM fungi to which they showed positive responses, from one to several fungal species. Mycorrhiza growth and phosphorus responses were positively correlated, whereas expression of two mycorrhiza-inducible phosphate transporters, SbPT8 and SbPT9, correlated negatively with mycorrhizal growth responses. AM fungi improve growth and mineral nutrition of sorghum, and the substantial variation between lines provides the potential to map loci influencing mycorrhiza responses.

Suppression of Arbuscule Degeneration in Medicago truncatula phosphate transporter4 Mutants is Dependent on the Ammonium Transporter 2 Family Protein AMT2;3.

During arbuscular mycorrhizal (AM) symbiosis, the plant gains access to phosphate (Pi) and nitrogen delivered by its fungal symbiont. Transfer of mineral nutrients occurs at the interface between branched hyphae called arbuscules and root cortical cells. In Medicago truncatula, a Pi transporter, PT4, is required for symbiotic Pi transport, and in pt4, symbiotic Pi transport fails, arbuscules degenerate prematurely, and the symbiosis is not maintained. Premature arbuscule degeneration (PAD) is suppressed when pt4 mutants are nitrogen-deprived, possibly the result of compensation by PT8, a second AM-induced Pi transporter. However, PAD is also suppressed in nitrogen-starved pt4 pt8 double mutants, negating this hypothesis and furthermore indicating that in this condition, neither of these symbiotic Pi transporters is required for symbiosis. In M. truncatula, three AMT2 family ammonium transporters are induced during AM symbiosis. To test the hypothesis that suppression of PAD involves AMT2 transporters, we analyzed double and triple Pi and ammonium transporter mutants. ATM2;3 but not AMT2;4 was required for suppression of PAD in pt4, while AMT2;4, but not AMT2;3, complemented growth of a yeast ammonium transporter mutant. In summary, arbuscule life span is influenced by PT4 and ATM2;3, and their relative importance varies with the nitrogen status of the plant.

Hyphal Branching during Arbuscule Development Requires Reduced Arbuscular Mycorrhiza1.

During arbuscular mycorrhizal symbiosis, arbuscule development in the root cortical cell and simultaneous deposition of the plant periarbuscular membrane generate the interface for symbiotic nutrient exchange. The transcriptional changes that accompany arbuscule development are extensive and well documented. By contrast, the transcriptional regulators that control these programs are largely unknown. Here, we provide a detailed characterization of an insertion allele of Medicago truncatula Reduced Arbuscular Mycorrhiza1 (RAM1), ram1-3, which reveals that RAM1 is not necessary to enable hyphopodium formation or hyphal entry into the root but is essential to support arbuscule branching. In ram1-3, arbuscules consist only of the arbuscule trunk and in some cases, a few initial thick hyphal branches. ram1-3 is also insensitive to phosphate-mediated regulation of the symbiosis. Transcript analysis of ram1-3 and ectopic expression of RAM1 indicate that RAM1 regulates expression of EXO70I and Stunted Arbuscule, two genes whose loss of function impacts arbuscule branching. Furthermore, RAM1 regulates expression of a transcription factor Required for Arbuscule Development (RAD1). RAD1 is also required for arbuscular mycorrhizal symbiosis, and rad1 mutants show reduced colonization. RAM1 itself is induced in colonized root cortical cells, and expression of RAM1 and RAD1 is modulated by DELLAs. Thus, the data suggest that DELLAs regulate arbuscule development through modulation of RAM1 and RAD1 and that the precise transcriptional control essential to place proteins in the periarbuscular membrane is controlled, at least in part, by RAM1.

DELLA proteins regulate arbuscule formation in arbuscular mycorrhizal symbiosis.

Most flowering plants are able to form endosymbioses with arbuscular mycorrhizal fungi. In this mutualistic association, the fungus colonizes the root cortex and establishes elaborately branched hyphae, called arbuscules, within the cortical cells. Arbuscule development requires the cellular reorganization of both symbionts, and the resulting symbiotic interface functions in nutrient exchange. A plant symbiosis signaling pathway controls the development of the symbiosis. Several components of the pathway have been identified, but transcriptional regulators that control downstream pathways for arbuscule formation are still unknown. Here we show that DELLA proteins, which are repressors of gibberellic acid (GA) signaling and function at the nexus of several signaling pathways, are required for arbuscule formation. Arbuscule formation is severely impaired in a Medicago truncatula Mtdella1/Mtdella2 double mutant; GA treatment of wild-type roots phenocopies the della double mutant, and a dominant DELLA protein (della1-Δ18) enables arbuscule formation in the presence of GA. Ectopic expression of della1-Δ18 suggests that DELLA activity in the vascular tissue and endodermis is sufficient to enable arbuscule formation in the inner cortical cells. In addition, expression of della1-Δ18 restores arbuscule formation in the symbiosis signaling pathway mutant cyclops/ipd3, indicating an intersection between DELLA and symbiosis signaling for arbuscule formation. GA signaling also influences arbuscule formation in monocots, and a Green Revolution wheat variety carrying dominant DELLA alleles shows enhanced colonization but a limited growth response to arbuscular mycorrhizal symbiosis.

Plumbagin reverses proliferation and resistance to apoptosis in experimental PAH.

Like cancer, pulmonary arterial hypertension (PAH) is characterised by a pro-proliferative and anti-apoptotic phenotype. In PAH, pulmonary artery smooth muscle cell (PASMC) proliferation is enhanced and apoptosis suppressed. The sustainability of this phenotype requires the activation of pro-survival transcription factors, such as signal transducer and activator of transcription (STAT)3 and nuclear factor of activated T-cells (NFAT). There are no drugs currently available that are able to efficiently and safely inhibit this axis. We hypothesised that plumbagin (PLB), a natural organic compound known to block STAT3 in cancer cells, would reverse experimental pulmonary hypertension. Using human PAH-PASMC, we demonstrated in vitro that PLB inhibits the activation of the STAT3/NFAT axis, increasing the voltage-gated K(+) current bone morphogenetic protein receptor type II (BMPR2), and decreasing intracellular Ca(2+) concentration ([Ca(2+)](i)), rho-associated coiled-coil containing protein kinase (ROCK)1 and interleukin (IL)-6, contributing to the inhibition of PAH-PASMC proliferation and resistance to apoptosis (proliferating cell nuclear antigen (PCNA), TUNEL, Ki67 and anexine V). In vivo, PLB oral administration decreases distal pulmonary artery remodelling, mean pulmonary artery pressure and right ventricular hypertrophy without affecting systemic circulation in both monocrotaline- and suden/chronic hypoxia-induced PAH in rats. This study demonstrates that the STAT3/NFAT axis can be therapeutically targeted by PLB in human PAH-PASMC and experimental PAH rat models. Thus, PLB could be considered a specific and attractive future therapeutic strategy for PAH.

Structural analysis of Atopobium parvulum SufS cysteine desulfurase linked to Crohn's disease.

Crohn's disease (CD) is characterized by the chronic inflammation of the gastrointestinal tract. A dysbiotic microbiome and a defective immune system are linked to CD, where hydrogen sulfide (H2 S) microbial producers positively correlate with the severity of the disease. Atopobium parvulum is a key H2 S producer from the microbiome of CD patients. In this study, the biochemical characterization of two Atopobium parvulum cysteine desulfurases, ApSufS and ApCsdB, shows that the enzymes are allosterically regulated. Structural analyses reveal that ApSufS forms a dimer with conserved characteristics observed in type II cysteine desulfurases. Four residues surrounding the active site are essential to catalyse cysteine desulfurylation, and a segment of short-chain residues grant access for substrate binding. A better understanding of ApSufS will help future avenues for CD treatment.

The genome of the forest insect pest Pissodes strobi reveals genome expansion and evidence of a Wolbachia endosymbiont.

The highly diverse insect family of true weevils, Curculionidae, includes many agricultural and forest pests. Pissodes strobi, commonly known as the spruce weevil or white pine weevil, is a major pest of spruce and pine forests in North America. Pissodes strobi larvae feed on the apical shoots of young trees, causing stunted growth and can destroy regenerating spruce or pine forests. Here, we describe the nuclear and mitochondrial Pissodes strobi genomes and their annotations, as well as the genome of an apparent Wolbachia endosymbiont. We report a substantial expansion of the weevil nuclear genome, relative to other Curculionidae species, possibly driven by an abundance of class II DNA transposons. The endosymbiont observed belongs to a group (supergroup A) of Wolbachia species that generally form parasitic relationships with their arthropod host.

Krüppel-like factor 5 contributes to pulmonary artery smooth muscle proliferation and resistance to apoptosis in human pulmonary arterial hypertension.

BACKGROUND: Pulmonary arterial hypertension (PAH) is a vascular remodeling disease characterized by enhanced proliferation of pulmonary artery smooth muscle cell (PASMC) and suppressed apoptosis. This phenotype has been associated with the upregulation of the oncoprotein survivin promoting mitochondrial membrane potential hyperpolarization (decreasing apoptosis) and the upregulation of growth factor and cytokines like PDGF, IL-6 and vasoactive agent like endothelin-1 (ET-1) promoting PASMC proliferation. Krüppel-like factor 5 (KLF5), is a zinc-finger-type transcription factor implicated in the regulation of cell differentiation, proliferation, migration and apoptosis. Recent studies have demonstrated the implication of KLF5 in tissue remodeling in cardiovascular diseases, such as atherosclerosis, restenosis, and cardiac hypertrophy. Nonetheless, the implication of KLF5 in pulmonary arterial hypertension (PAH) remains unknown. We hypothesized that KLF5 up-regulation in PAH triggers PASMC proliferation and resistance to apoptosis. METHODS AND RESULTS: We showed that KFL5 is upregulated in both human lung biopsies and cultured human PASMC isolated from distal pulmonary arteries from PAH patients compared to controls. Using stimulation experiments, we demonstrated that PDGF, ET-1 and IL-6 trigger KLF-5 activation in control PASMC to a level similar to the one seen in PAH-PASMC. Inhibition of the STAT3 pathway abrogates KLF5 activation in PAH-PASMC. Once activated, KLF5 promotes cyclin B1 upregulation and promotes PASMC proliferation and triggers survivin expression hyperpolarizing mitochondria membrane potential decreasing PASMC ability to undergo apoptosis. CONCLUSION: We demonstrated for the first time that KLF5 is activated in human PAH and implicated in the pro-proliferative and anti-apoptotic phenotype that characterize PAH-PASMC. We believe that our findings will open new avenues of investigation on the role of KLF5 in PAH and might lead to the identification of new therapeutic targets.

Conserved and reproducible bacterial communities associate with extraradical hyphae of arbuscular mycorrhizal fungi.

Extraradical hyphae (ERH) of arbuscular mycorrhizal fungi (AMF) extend from plant roots into the soil environment and interact with soil microbial communities. Evidence of positive and negative interactions between AMF and soil bacteria point to functionally important ERH-associated communities. To characterize communities associated with ERH and test controls on their establishment and composition, we utilized an in-growth core system containing a live soil-sand mixture that allowed manual extraction of ERH for 16S rRNA gene amplicon profiling. Across experiments and soils, consistent enrichment of members of the Betaproteobacteriales, Myxococcales, Fibrobacterales, Cytophagales, Chloroflexales, and Cellvibrionales was observed on ERH samples, while variation among samples from different soils was observed primarily at lower taxonomic ranks. The ERH-associated community was conserved between two fungal species assayed, Glomus versiforme and Rhizophagus irregularis, though R. irregularis exerted a stronger selection and showed greater enrichment for taxa in the Alphaproteobacteria and Gammaproteobacteria. A distinct community established within 14 days of hyphal access to the soil, while temporal patterns of establishment and turnover varied between taxonomic groups. Identification of a conserved ERH-associated community is consistent with the concept of an AMF microbiome and can aid the characterization of facilitative and antagonistic interactions influencing the plant-fungal symbiosis.

Cost-effectiveness of FreeO2 in patients with chronic obstructive pulmonary disease hospitalised for acute exacerbations: analysis of a pilot study in Quebec.

OBJECTIVE: Conduct a cost-effectiveness analysis of FreeO2 technology versus manual oxygen-titration technology for patients with chronic obstructive pulmonary disease (COPD) hospitalised for acute exacerbations. SETTING: Tertiary acute care hospital in Quebec, Canada. PARTICIPANTS: 47 patients with COPD hospitalised for acute exacerbations. INTERVENTION: An automated oxygen-titration and oxygen-weaning technology. METHODS AND OUTCOMES: The costs for hospitalisation and follow-up for 180 days were calculated using a microcosting approach and included the cost of FreeO2 technology. Incremental cost-effectiveness ratios (ICERs) were calculated using bootstrap resampling with 5000 replications. The main effect variable was the percentage of time spent at the target oxygen saturation (SpO2). The other two effect variables were the time spent in hyperoxia (target SpO2+5%) and in severe hypoxaemia (SpO2 <85%). The resamplings were based on data from a randomised controlled trial with 47 patients with COPD hospitalised for acute exacerbations. RESULTS: FreeO2 generated savings of 20.7% of the per-patient costs at 180 days (ie, -$C2959.71). This decrease is nevertheless not significant at the 95% threshold (P=0.13), but the effect variables all improved (P<0.001). The improvement in the time spent at the target SpO2 was 56.3%. The ICERs indicate that FreeO2 technology is more cost-effective than manual oxygen titration with a savings of -$C96.91 per percentage point of time spent at the target SpO2 (95% CI -301.26 to 116.96). CONCLUSION: FreeO2 technology could significantly enhance the efficiency of the health system by reducing per-patient costs at 180 days. A study with a larger patient sample needs to be carried out to confirm these preliminary results. TRIAL REGISTRATION NUMBER: NCT01393015; Post-results.

A complex of C9ORF72 and p62 uses arginine methylation to eliminate stress granules by autophagy.

Mutations in proteins like FUS which cause Amyotrophic Lateral Sclerosis (ALS) result in the aberrant formation of stress granules while ALS-linked mutations in other proteins impede elimination of stress granules. Repeat expansions in C9ORF72, the major cause of ALS, reduce C9ORF72 levels but how this impacts stress granules is uncertain. Here, we demonstrate that C9ORF72 associates with the autophagy receptor p62 and controls elimination of stress granules by autophagy. This requires p62 to associate via the Tudor protein SMN with proteins, including FUS, that are symmetrically methylated on arginines. Mice lacking p62 accumulate arginine-methylated proteins and alterations in FUS-dependent splicing. Patients with C9ORF72 repeat expansions accumulate symmetric arginine dimethylated proteins which co-localize with p62. This suggests that C9ORF72 initiates a cascade of ALS-linked proteins (C9ORF72, p62, SMN, FUS) to recognize stress granules for degradation by autophagy and hallmarks of a defect in this process are observable in ALS patients.

Cardiac Implantable Electronic Device Infection: Detailed Analysis of Cost Implications.

BACKGROUND: Infections of cardiac implantable electronic devices (CIED) are associated with significant morbidity and mortality. Despite many preventive measures, this condition is associated with significant costs for the health care system. METHODS: We retrospectively analyzed all infection cases referred for lead extraction at a single university hospital over 1 year (2015-2016). We then calculated all costs related to the infection episode per patient using hospital databases and charts review. RESULTS: Thirty-eight patients with CIED infections (29% women-mean age 71 ± 14 years) were referred for lead extraction (27 pocket infections, 11 endocarditis). Devices were mainly pacemakers (60%). When the pathogen was identified, Staphylococcus aureus methicillin sensitive was the main cause. Extraction was performed in all but 3 cases (92%). One death occurred in the nonextracted group. Respective durations of hospitalization and intravenous and antibiotic administration for patients undergoing extraction were 21 and 36 days. The calculated mean total cost for CIED infection management was CAD$29,907 (median: 26,879; range: CAD$4,827-$62,585). Mean hospital charges were CAD$12,291, accounting for 41% of the total costs. CONCLUSIONS: This study represents the first analysis of the direct costs associated with lead extraction in Canada. Device infections are associated with significant costs and increased morbidity. Any preventive measure will have a significant impact on the economic burden of the health care system and patient outcome after lead extraction.

A Transcriptional Program for Arbuscule Degeneration during AM Symbiosis Is Regulated by MYB1.

During the endosymbiosis formed between plants and arbuscular mycorrhizal (AM) fungi, the root cortical cells are colonized by branched hyphae called arbuscules, which function in nutrient exchange with the plant [1]. Despite their positive function, arbuscules are ephemeral structures, and their development is followed by a degeneration phase, in which the arbuscule and surrounding periarbuscular membrane and matrix gradually disappear from the root cell [2, 3]. Currently, the root cell's role in this process and the underlying regulatory mechanisms are unknown. Here, by using a Medicago truncatula pt4 mutant in which arbuscules degenerate prematurely [4], we identified arbuscule degeneration-associated genes, of which 38% are predicted to encode secreted hydrolases, suggesting a role in disassembly of the arbuscule and interface. Through RNAi and analysis of an insertion mutant, we identified a symbiosis-specific MYB-like transcription factor (MYB1) that suppresses arbuscule degeneration in mtpt4. In myb1, expression of several degeneration-associated genes is reduced. Conversely, in roots constitutively overexpressing MYB1, expression of degeneration-associated genes is increased and subsequent development of symbiosis is impaired. MYB1-regulated gene expression is enhanced by DELLA proteins and is dependent on NSP1 [5], but not NSP2 [6]. Furthermore, MYB1 interacts with DELLA and NSP1. Our data identify a transcriptional program for arbuscule degeneration and reveal that its regulators include MYB1 in association with two transcriptional regulators, NSP1 and DELLA, both of which function in preceding phases of the symbiosis. We propose that the combinatorial use of transcription factors enables the sequential expression of transcriptional programs for arbuscule development and degeneration.

DELLA proteins regulate expression of a subset of AM symbiosis-induced genes in Medicago truncatula.

The majority of the vascular flowering plants form symbiotic associations with fungi from the phylum Glomeromycota through which both partners gain access to nutrients, either mineral nutrients in the case of the plant, or carbon, in the case of the fungus. (1) The association develops in the roots and requires substantial remodeling of the root cortical cells where branched fungal hyphae, called arbuscules, are housed in a new membrane-bound apoplastic compartment. (2) Nutrient exchange between the symbionts occurs over this interface and its development and maintenance is critical for symbiosis. Previously, we showed that DELLA proteins, which are well known as repressors of gibberellic acid signaling, also regulate development of AM symbiosis and are necessary to enable arbuscule development. (3) Furthermore, constitutive overexpression of a dominant DELLA protein (della1-Δ18) is sufficient to induce transcripts of several AM symbiosis-induced genes, even in the absence of the fungal symbiont. (4) Here we further extend this approach and identify AM symbiosis genes that respond transcriptionally to constitutive expression of a dominant DELLA protein and also genes that do respond to this treatment. Additionally, we demonstrate that DELLAs interact with REQUIRED FOR ARBUSCULE DEVELOPMENT 1 (RAD1) which further extends our knowledge of GRAS factor complexes that have the potential to regulate gene expression during AM symbiosis.

Brighter Red Fluorescent Proteins by Rational Design of Triple-Decker Motif.

Red fluorescent proteins (RFPs) are used extensively in chemical biology research as fluorophores for live cell imaging, as partners in FRET pairs, and as signal transducers in biosensors. For all of these applications, brighter RFP variants are desired. Here, we used rational design to increase the quantum yield of monomeric RFPs in order to improve their brightness. We postulated that we could increase quantum yield by restricting the conformational degrees of freedom of the RFP chromophore. To test our hypothesis, we introduced aromatic residues above the chromophore of mRojoA, a dim RFP containing a π-stacked Tyr residue directly beneath the chromophore, in order to reduce chromophore conformational flexibility via improved packing and steric complementarity. The best mutant identified displayed an absolute quantum yield increase of 0.07, representing an over 3-fold improvement relative to mRojoA. Remarkably, this variant was isolated following the screening of only 48 mutants, a library size that is several orders of magnitude smaller than those previously used to achieve equivalent gains in quantum yield in other RFPs. The crystal structure of the highest quantum yield mutant showed that the chromophore is sandwiched between two Tyr residues in a triple-decker motif of aromatic rings. Presence of this motif increases chromophore rigidity, as evidenced by the significantly reduced temperature factors compared to dim RFPs. Overall, the approach presented here paves the way for the rapid development of fluorescent proteins with higher quantum yield and overall brightness.

Molecular basis for DPY-30 association to COMPASS-like and NURF complexes.

DPY-30 is a subunit of mammalian COMPASS-like complexes (complex of proteins associated with Set1) and regulates global histone H3 Lys-4 trimethylation. Here we report structural evidence showing that the incorporation of DPY-30 into COMPASS-like complexes is mediated by several hydrophobic interactions between an amphipathic α helix located on the C terminus of COMPASS subunit ASH2L and the inner surface of the DPY-30 dimerization/docking (D/D) module. Mutations impairing the interaction between ASH2L and DPY-30 result in a loss of histone H3K4me3 at the ß locus control region and cause a delay in erythroid cell terminal differentiation. Using overlay assays, we defined a consensus sequence for DPY-30 binding proteins and found that DPY-30 interacts with BAP18, a subunit of the nucleosome remodeling factor complex. Overall, our results indicate that the ASH2L/DPY-30 complex is important for cell differentiation and provide insights into the ability of DPY-30 to associate with functionally divergent multisubunit complexes.