ORCID Profile
0000-0002-1838-4976
Current Organisation
University of New South Wales
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Animal cell and molecular biology | Gene expression (incl. microarray and other genome-wide approaches) | Systems biology | Genetics |
Publisher: Elsevier BV
Date: 06-2022
DOI: 10.1016/J.CELS.2022.04.005
Abstract: RNA concentration homeostasis involves coordinating RNA abundance and synthesis rates with cell size. Here, we study this in human cells by combining genome-wide perturbations with quantitative single-cell measurements. Despite relative ease in perturbing RNA synthesis, we find that RNA concentrations generally remain highly constant. Perturbations that would be expected to increase nuclear mRNA levels, including those targeting nuclear mRNA degradation or export, result in downregulation of RNA synthesis. This is associated with reduced abundance of transcription-associated proteins and protein states that are normally coordinated with RNA production in single cells, including RNA polymerase II (RNA Pol II) itself. Acute perturbations, elevation of nuclear mRNA levels, and mathematical modeling indicate that mammalian cells achieve robust mRNA concentration homeostasis by the mRNA-based negative feedback on transcriptional activity in the nucleus. This ultimately acts to coordinate RNA Pol II abundance with nuclear mRNA degradation and export rates and may underpin the scaling of mRNA abundance with cell size.
Publisher: Cold Spring Harbor Laboratory
Date: 17-05-2021
DOI: 10.1101/2021.05.17.444432
Abstract: Unlike its DNA template, RNA abundance and synthesis rates increase with cell size, as part of a mechanism of cellular RNA concentration homeostasis. Here, we study this scaling phenomenon in human cells by combining genome-wide perturbations with quantitative single-cell measurements. Despite relative ease in perturbing RNA synthesis, we find that RNA concentrations remain highly constant. Systems-level analysis indicates that perturbations that would lead to increased nuclear mRNA abundance result in downregulation of mRNA synthesis. This is associated with reduced levels of several transcription-associated proteins and protein states that are normally coordinated with RNA production in single cells, including RNA polymerase II (Pol II) itself. Acute shut-down of nuclear RNA degradation, elevation of nuclear mRNA levels, and mathematical modelling indicate that mammalian cells achieve RNA concentration homeostasis by an mRNA-based negative feedback on transcriptional activity in the nucleus. Ultimately, this acts to robustly scale Pol II abundance with cell volume and coordinate mRNA synthesis and degradation.
Publisher: Wiley
Date: 29-05-2015
DOI: 10.1111/TPJ.12869
Publisher: Elsevier BV
Date: 10-2011
Publisher: eLife Sciences Publications, Ltd
Date: 28-04-2015
Publisher: Cold Spring Harbor Laboratory
Date: 25-05-2022
DOI: 10.1101/2022.05.25.493370
Abstract: Arrayed CRISPR libraries extend the scope of gene-perturbation screens but require large numbers of efficacious sgRNA-expressing vectors. Using a newly invented liquid-phase plasmid cloning methodology, we constructed genome-wide arrayed libraries for human gene ablation (19,936 plasmids), activation, and epigenetic silencing (22,442 plasmids). At least 76% of each plasmid preparation encoded an intact array of 4 non-overlapping sgRNAs designed to tolerate most human DNA polymorphisms. We achieved perturbation efficacies of 75-99%, 76-92% and up to 10,000x in deletion, silencing and activation experiments, respectively. Upon conversion into massively parallel lentiviral vectors, an arrayed activation screen of 1,634 human transcription factors yielded 11 novel regulators of the cellular prion protein PrP C . Furthermore, a screen using a pooled version of the ablation library identified 5 novel modifiers of autophagy that went undetected with either of two 1sgRNA libraries. The CRISPR libraries described here represent a powerful resource for the targeted perturbation of human protein-coding genes.
Publisher: Oxford University Press (OUP)
Date: 2017
DOI: 10.1039/C7IB00112F
Abstract: Exclusion-based S le Preparation enables simple multiplexed RNA extractions to enhance detection of rare, multiply spliced HIV RNA events.
Publisher: Cold Spring Harbor Laboratory
Date: 08-05-2022
DOI: 10.1101/2022.05.07.490900
Abstract: Highly multiplexed quantitative subcellular imaging holds enormous promise for understanding how spatial context shapes the activity of our genome and its products at multiple scales. Yet unbiased analysis of subcellular organisation across experimental conditions remains challenging, because differences in molecular profiles between conditions confound differences in molecular profiles across space. Here, we introduce a deep-learning framework called CAMPA (Conditional Autoencoder for Multiplexed Pixel Analysis), which uses a variational autoencoder conditioned on cellular states and perturbations to learn consistent molecular signatures. Clustering the learned representations into subcellular landmarks allows quantitative comparisons of landmark sizes, shapes, molecular compositions and relative spatial organisation between conditions. By performing high-resolution multiplexed immunofluorescence on human cells, we use CAMPA to reveal how subnuclear organisation changes upon different perturbations of RNA production or processing, and how different membraneless organelles scale with cell size. Furthermore, by integrating information across the cellular and subcellular scales, we uncover new links between the molecular composition of membraneless organelles and bulk RNA synthesis rates of single cells. We anticipate that CAMPA will greatly accelerate the systematic mapping of multiscale atlases of biological organisation to identify the rules by which context shapes physiology and disease.
Publisher: Proceedings of the National Academy of Sciences
Date: 14-04-2014
Abstract: Lipopolysaccharides (LPSs) are unique glycolipids that are characteristic components of outer membranes in Gram negative bacteria and play important roles in pathogenesis. Many LPSs contain a long-chain polysaccharide (known as the O antigen) whose length can be an important factor in bacterial resistance to complement-mediated killing. While components involved in chain-length determination are known in many systems, the underlying regulatory mechanism is not. Here we apply a mathematical modeling approach that integrates the existing structural and biochemical data to develop a new model, variable geometry, for chain-length regulation using the prototype for O antigens whose synthesis involves the widespread ATP-binding cassette transporter-dependent pathway.
Publisher: Elsevier BV
Date: 04-2017
Publisher: American Physical Society (APS)
Date: 14-04-2011
Publisher: Springer Science and Business Media LLC
Date: 28-06-2021
DOI: 10.1038/S41597-021-00944-5
Abstract: Coordination of RNA abundance and production rate with cell size has been observed in erse organisms and cell populations. However, how cells achieve such ‘scaling’ of transcription with size is unknown. Here we describe a genome-wide siRNA screen to identify regulators of global RNA production rates in HeLa cells. We quantify the single-cell RNA production rate using metabolic pulse-labelling of RNA and subsequent high-content imaging. Our quantitative, single-cell measurements of DNA, nascent RNA, proliferating cell nuclear antigen (PCNA), and total protein, as well as cell morphology and population-context, capture a detailed cellular phenotype. This allows us to account for changes in cell size and cell-cycle distribution (G1/S/G2) in perturbation conditions, which indirectly affect global RNA production. We also take advantage of the subcellular information to distinguish between nascent RNA localised in the nucleolus and nucleoplasm, to approximate ribosomal and non-ribosomal RNA contributions to perturbation phenotypes. Perturbations uncovered through this screen provide a resource for exploring the mechanisms of regulation of global RNA metabolism and its coordination with cellular states.
Publisher: American Physical Society (APS)
Date: 26-10-2010
Publisher: Elsevier BV
Date: 08-2022
DOI: 10.1016/J.TCB.2022.05.003
Abstract: For most genes, mRNA transcript abundance scales with cell size to ensure a constant concentration. Scaling of mRNA synthesis rates with cell size plays an important role, with regulation of the activity and abundance of RNA polymerase II (Pol II) now emerging as a key point of control. However, there is also considerable evidence for feedback mechanisms that kinetically couple the rates of mRNA synthesis, nuclear export, and degradation to allow cells to compensate for changes in one by adjusting the others. Researchers are beginning to integrate results from these different fields to reveal the mechanisms underlying transcript homeostasis. This will be crucial for moving beyond our current understanding of relative gene expression towards an appreciation of how absolute transcript levels are linked to other aspects of the cellular phenotype.
Publisher: Cold Spring Harbor Laboratory
Date: 11-2017
Abstract: Epigenetic maintenance of gene repression is essential for development. Polycomb complexes are central to this memory, but many aspects of the underlying mechanism remain unclear. LIKE HETEROCHROMATIN PROTEIN 1 (LHP1) binds Polycomb-deposited H3K27me3 and is required for repression of many Polycomb target genes in Arabidopsis . Here we show that LHP1 binds RNA in vitro through the intrinsically disordered hinge region. By independently perturbing the RNA-binding hinge region and H3K27me3 (trimethylation of histone H3 at Lys27) recognition, we found that both facilitate LHP1 localization and H3K27me3 maintenance. Disruption of the RNA-binding hinge region also prevented formation of subnuclear foci, structures potentially important for epigenetic repression.
Publisher: eLife Sciences Publications, Ltd
Date: 08-05-2015
DOI: 10.7554/ELIFE.07205
Abstract: Inheritance of gene expression states is fundamental for cells to ‘remember’ past events, such as environmental or developmental cues. The conserved Polycomb Repressive Complex 2 (PRC2) maintains epigenetic repression of many genes in animals and plants and modifies chromatin at its targets. Histones modified by PRC2 can be inherited through cell ision. However, it remains unclear whether this inheritance can direct long-term memory of in idual gene expression states (cis memory) or instead if local chromatin states are dictated by the concentrations of diffusible factors (trans memory). By monitoring the expression of two copies of the Arabidopsis Polycomb target gene FLOWERING LOCUS C (FLC) in the same plants, we show that one copy can be repressed while the other is active. Furthermore, this ‘mixed’ expression state is inherited through many cell isions as plants develop. These data demonstrate that epigenetic memory of FLC expression is stored not in trans but in cis.
Publisher: eLife Sciences Publications, Ltd
Date: 19-07-2023
DOI: 10.7554/ELIFE.79743
Abstract: Quantitative gene regulation at the cell population level can be achieved by two fundamentally different modes of regulation at in idual gene copies. A ‘digital’ mode involves binary ON/OFF expression states, with population-level variation arising from the proportion of gene copies in each state, while an ‘analog’ mode involves graded expression levels at each gene copy. At the Arabidopsis floral repressor FLOWERING LOCUS C (FLC), ‘digital’ Polycomb silencing is known to facilitate quantitative epigenetic memory in response to cold. However, whether FLC regulation before cold involves analog or digital modes is unknown. Using quantitative fluorescent imaging of FLC mRNA and protein, together with mathematical modeling, we find that FLC expression before cold is regulated by both analog and digital modes. We observe a temporal separation between the two modes, with analog preceding digital. The analog mode can maintain intermediate expression levels at in idual FLC gene copies, before subsequent digital silencing, consistent with the copies switching OFF stochastically and heritably without cold. This switch leads to a slow reduction in FLC expression at the cell population level. These data present a new paradigm for gradual repression, elucidating how analog transcriptional and digital epigenetic memory pathways can be integrated.
Publisher: Springer Science and Business Media LLC
Date: 29-05-2023
DOI: 10.1038/S41592-023-01894-Z
Abstract: Highly multiplexed imaging holds enormous promise for understanding how spatial context shapes the activity of the genome and its products at multiple length scales. Here, we introduce a deep learning framework called CAMPA (Conditional Autoencoder for Multiplexed Pixel Analysis), which uses a conditional variational autoencoder to learn representations of molecular pixel profiles that are consistent across heterogeneous cell populations and experimental perturbations. Clustering these pixel-level representations identifies consistent subcellular landmarks, which can be quantitatively compared in terms of their size, shape, molecular composition and relative spatial organization. Using high-resolution multiplexed immunofluorescence, this reveals how subcellular organization changes upon perturbation of RNA synthesis, RNA processing or cell size, and uncovers links between the molecular composition of membraneless organelles and cell-to-cell variability in bulk RNA synthesis rates. By capturing interpretable cellular phenotypes, we anticipate that CAMPA will greatly accelerate the systematic mapping of multiscale atlases of biological organization to identify the rules by which context shapes physiology and disease.
Publisher: Springer Science and Business Media LLC
Date: 02-12-2021
Publisher: American Association for the Advancement of Science (AAAS)
Date: 15-09-2017
Abstract: A multifactorial strategy establishes and maintains repressive chromatin marks in plants in response to environmental signals.
Publisher: Cold Spring Harbor Laboratory
Date: 2012
DOI: 10.1101/SQB.2013.77.015941
Abstract: In the past few years, mathematical modeling approaches in biology have begun to fulfill their promise by assisting in the dissection of complex biological systems. Here, we review two recent ex les of predictive mathematical modeling in plant biology. The first involves the quantitative epigenetic silencing of the floral repressor gene FLC in Arabidopsis, mediated by a Polycomb-based system. The second involves the spatiotemporal dynamics of telomere bouquet formation in wheat-rye meiosis. Although both the biology and the modeling framework of the two systems are different, both exemplify how mathematical modeling can help to accelerate discovery of the underlying mechanisms in complex biological systems. In both cases, the models that developed were relatively minimal, including only essential features, but both nevertheless yielded fundamental insights. We also briefly review the current state of mathematical modeling in biology, difficulties inherent in its application, and its potential future development.
Publisher: eLife Sciences Publications, Ltd
Date: 10-03-2023
Location: United Kingdom of Great Britain and Northern Ireland
Start Date: 2017
End Date: 2021
Funder: Human Frontier Science Program
View Funded ActivityStart Date: 2017
End Date: 2018
Funder: European Molecular Biology Organization
View Funded ActivityStart Date: 2023
End Date: 2025
Funder: Australian Research Council
View Funded ActivityStart Date: 2023
End Date: 12-2025
Amount: $463,618.00
Funder: Australian Research Council
View Funded Activity