Minerva Elements Records
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ItemCombinatorial Smad2/3 Activities Downstream of Nodal Signaling Maintain Embryonic/Extra-Embryonic Cell Identities during Lineage Priming(CELL PRESS, 2018-08-21)Epiblast cells in the early post-implantation stage mammalian embryo undergo a transition described as lineage priming before cell fate allocation, but signaling pathways acting upstream remain ill defined. Genetic studies demonstrate that Smad2/3 double-mutant mouse embryos die shortly after implantation. To learn more about the molecular disturbances underlying this abrupt failure, here we characterized Smad2/3-deficient embryonic stem cells (ESCs). We found that Smad2/3 double-knockout ESCs induced to form epiblast-like cells (EpiLCs) display changes in naive and primed pluripotency marker gene expression, associated with the disruption of Oct4-bound distal regulatory elements. In the absence of Smad2/3, we observed enhanced Bmp target gene expression and de-repression of extra-embryonic gene expression. Cell fate allocation into all three embryonic germ layers is disrupted. Collectively, these experiments demonstrate that combinatorial Smad2/3 functional activities are required to maintain distinct embryonic and/or extra-embryonic cell identity during lineage priming in the epiblast before gastrulation.
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ItemRYBP stimulates PRC1 to shape chromatin-based communication between Polycomb repressive complexes(ELIFE SCIENCES PUBLICATIONS LTD, 2016-10-05)Polycomb group (PcG) proteins function as chromatin-based transcriptional repressors that are essential for normal gene regulation during development. However, how these systems function to achieve transcriptional regulation remains very poorly understood. Here, we discover that the histone H2AK119 E3 ubiquitin ligase activity of Polycomb repressive complex 1 (PRC1) is defined by the composition of its catalytic subunits and is highly regulated by RYBP/YAF2-dependent stimulation. In mouse embryonic stem cells, RYBP plays a central role in shaping H2AK119 mono-ubiquitylation at PcG targets and underpins an activity-based communication between PRC1 and Polycomb repressive complex 2 (PRC2) which is required for normal histone H3 lysine 27 trimethylation (H3K27me3). Without normal histone modification-dependent communication between PRC1 and PRC2, repressive Polycomb chromatin domains can erode, rendering target genes susceptible to inappropriate gene expression signals. This suggests that activity-based communication and histone modification-dependent thresholds create a localized form of epigenetic memory required for normal PcG chromatin domain function in gene regulation.
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ItemOphthalmic Antimicrobial Prescribing in Australian Healthcare Facilities(MDPI, 2022-05-01)The National Antimicrobial Prescribing Survey (NAPS) is a web-based, standardized tool, widely adopted in Australian healthcare facilities to assess the reasons for, the quantity of, and the quality of antimicrobial prescribing. It consists of multiple modules tailored towards the needs of a variety of healthcare facilities. Data regarding ophthalmological antimicrobial use from Hospital NAPS, Surgical NAPS, and Aged Care NAPS were analysed. In Hospital NAPS, the most common reasons for inappropriate prescribing were incorrect dose or frequency and incorrect duration. Prolonged duration was also common in Aged Care prescribing: about one quarter of all antimicrobials had been prescribed for greater than 6 months. All three modules found chloramphenicol to be the most prescribed antimicrobial with a high rate of inappropriate prescribing, usually for conjunctivitis.
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ItemGerms and germlines: how "public" B-cell clones evolve in the gut(WILEY, 2020-05-16)Chen et al. describe how B-cell clones observed in the gut of many different individuals (recurrent or "public" clonotypes) are shaped by the combined influences of common microbial antigens and underlying genomic recombination biases.
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ItemThe SET1 Complex Selects Actively Transcribed Target Genes via Multivalent Interaction with CpG Island Chromatin(CELL PRESS, 2017-09-05)Chromatin modifications and the promoter-associated epigenome are important for the regulation of gene expression. However, the mechanisms by which chromatin-modifying complexes are targeted to the appropriate gene promoters in vertebrates and how they influence gene expression have remained poorly defined. Here, using a combination of live-cell imaging and functional genomics, we discover that the vertebrate SET1 complex is targeted to actively transcribed gene promoters through CFP1, which engages in a form of multivalent chromatin reading that involves recognition of non-methylated DNA and histone H3 lysine 4 trimethylation (H3K4me3). CFP1 defines SET1 complex occupancy on chromatin, and its multivalent interactions are required for the SET1 complex to place H3K4me3. In the absence of CFP1, gene expression is perturbed, suggesting that normal targeting and function of the SET1 complex are central to creating an appropriately functioning vertebrate promoter-associated epigenome.
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ItemKDM2 proteins constrain transcription from CpG island gene promoters independently of their histone demethylase activity(OXFORD UNIV PRESS, 2019-09-26)CpG islands (CGIs) are associated with the majority of mammalian gene promoters and function to recruit chromatin modifying enzymes. It has therefore been proposed that CGIs regulate gene expression through chromatin-based mechanisms, however in most cases this has not been directly tested. Here, we reveal that the histone H3 lysine 36 (H3K36) demethylase activity of the CGI-binding KDM2 proteins contributes only modestly to the H3K36me2-depleted state at CGI-associated gene promoters and is dispensable for normal gene expression. Instead, we discover that KDM2 proteins play a widespread and demethylase-independent role in constraining gene expression from CGI-associated gene promoters. We further show that KDM2 proteins shape RNA Polymerase II occupancy but not chromatin accessibility at CGI-associated promoters. Together this reveals a demethylase-independent role for KDM2 proteins in transcriptional repression and uncovers a new function for CGIs in constraining gene expression.
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ItemVariant PRC1 Complex-Dependent H2A Ubiquitylation Drives PRC2 Recruitment and Polycomb Domain Formation(CELL PRESS, 2014-06-05)Chromatin modifying activities inherent to polycomb repressive complexes PRC1 and PRC2 play an essential role in gene regulation, cellular differentiation, and development. However, the mechanisms by which these complexes recognize their target sites and function together to form repressive chromatin domains remain poorly understood. Recruitment of PRC1 to target sites has been proposed to occur through a hierarchical process, dependent on prior nucleation of PRC2 and placement of H3K27me3. Here, using a de novo targeting assay in mouse embryonic stem cells we unexpectedly discover that PRC1-dependent H2AK119ub1 leads to recruitment of PRC2 and H3K27me3 to effectively initiate a polycomb domain. This activity is restricted to variant PRC1 complexes, and genetic ablation experiments reveal that targeting of the variant PCGF1/PRC1 complex by KDM2B to CpG islands is required for normal polycomb domain formation and mouse development. These observations provide a surprising PRC1-dependent logic for PRC2 occupancy at target sites in vivo.
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ItemThe ENJOY MAP for HEALTH: Exercise interveNtion outdoor proJect in the cOmmunitY for older people-More Active People for HEALTHier communities: a study protocol(BMC, 2022-05-21)BACKGROUND: Physical activity is important to maintain health in older age, with physical activity in the outdoors providing mental and physical health benefits for all age groups. One way by which older people can engage in physical activity in the outdoors is through using suitable age-friendly outdoor exercise equipment, the Seniors Exercise Park. The ENJOY MAP for HEALTH aims to evaluate the effect of the Seniors Exercise Park installation and associated capacity building activities on park visitation, park-based physical activity by older people and delivery of community physical activity programs. METHOD: This study is a quasi-experimental (natural experiment) with pre and post study design evaluating the effect of age-friendly outdoor spaces with specialised outdoor exercise equipment on older people's physical activity and wellbeing in six Victorian municipalities (local governments/councils). Each council will undergo four stages (site construction and development, promotion and marketing, capacity building and training, evaluation and sustainability). Several activities and methods will be employed from stage one through stage four to evaluate the potential impact of the age-friendly outdoor spaces on physical activity and wellbeing and will comprise the following elements: site observation and equipment utilisation, face to face intercept surveys, development of an online access monitor and community building activities. DISCUSSION: The project is expected to result in a significant change in the physical outdoor environment for the participating councils and communities whereby older people and other community members will be able to engage in safe physical and social activity programs, socialise more and hence improve the overall wellbeing of older people. TRIAL REGISTRATION: This trial is retrospectively registered with the Australian New Zealand Clinical Trials Registry. Trial registration number ACTRN12621000965808 . Date registered 23/07/2021.
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ItemProtection of CpG islands from DNA methylation is DNA-encoded and evolutionarily conserved(OXFORD UNIV PRESS, 2016-08-19)DNA methylation is a repressive epigenetic modification that covers vertebrate genomes. Regions known as CpG islands (CGIs), which are refractory to DNA methylation, are often associated with gene promoters and play central roles in gene regulation. Yet how CGIs in their normal genomic context evade the DNA methylation machinery and whether these mechanisms are evolutionarily conserved remains enigmatic. To address these fundamental questions we exploited a transchromosomic animal model and genomic approaches to understand how the hypomethylated state is formed in vivo and to discover whether mechanisms governing CGI formation are evolutionarily conserved. Strikingly, insertion of a human chromosome into mouse revealed that promoter-associated CGIs are refractory to DNA methylation regardless of host species, demonstrating that DNA sequence plays a central role in specifying the hypomethylated state through evolutionarily conserved mechanisms. In contrast, elements distal to gene promoters exhibited more variable methylation between host species, uncovering a widespread dependence on nucleotide frequency and occupancy of DNA-binding transcription factors in shaping the DNA methylation landscape away from gene promoters. This was exemplified by young CpG rich lineage-restricted repeat sequences that evaded DNA methylation in the absence of co-evolved mechanisms targeting methylation to these sequences, and species specific DNA binding events that protected against DNA methylation in CpG poor regions. Finally, transplantation of mouse chromosomal fragments into the evolutionarily distant zebrafish uncovered the existence of a mechanistically conserved and DNA-encoded logic which shapes CGI formation across vertebrate species.
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ItemThe first reptilian allergen and major allergen for fish-allergic patients: Crocodile beta-parvalbumin(WILEY, 2022-05-01)BACKGROUND: Clinical cross-reactivity between bony fish, cartilaginous fish, frog, and chicken muscle has previously been demonstrated in fish-allergic patients. In indicative studies, two reports of anaphylaxis following the consumption of crocodile meat and IgE-cross-binding were linked to the major fish allergen parvalbumin (PV). This study investigates IgE-binding proteins in crocodile meat with a focus on PV and their clinical relevance. METHODS: Proteins were extracted from muscle tissue of crocodile, three bony fish, and two cartilaginous fish. A cohort of fish-allergic pediatric patients (n = 77) underwent allergen skin prick testing (SPT) to three fish preparations (n = 77) and crocodile (n = 12). IgE-binding proteins were identified and quantified by SDS-PAGE, mass spectrometric analyses, and immunoblotting using commercial and in-house antibodies, as well as individual and pooled patients' serum. PV isoforms were purified or recombinantly expressed before immunological analyses, including human mast cell degranulation assay. RESULTS: Of the tissues analyzed, PV was most abundant in heated crocodile preparation, triggering an SPT of ≥3 mm in 8 of 12 (67%) fish-allergic patients. Seventy percent (31 of 44) of fish PV-sensitized patients demonstrated IgE-binding to crocodile PV. Crocodile β-PV was the major IgE-binding protein but 20-fold less abundant than α-PV. Cellular reactivity was demonstrated for β-PV and epitopes predicted, explaining frequent IgE-cross-binding of β-PVs. Both PV isoforms are now registered as the first reptile allergens with the WHO/IUIS (β-PV as Cro p 1 and α-PV as Cro p 2). CONCLUSION: Fish-allergic individuals may be at risk of an allergy to crocodile and should seek specialist advice before consuming crocodilian meat.