A5046227302- Advanced Fluorescence Microscopy Techniques
- Genomics and Chromatin Dynamics
- Cancer, Hypoxia, and Metabolism
- DNA Repair Mechanisms
- Cytomegalovirus and herpesvirus research
- Single-cell and spatial transcriptomics
- High Altitude and Hypoxia
- Epigenetics and DNA Methylation
- Adipose Tissue and Metabolism
- Cell Image Analysis Techniques
- Herpesvirus Infections and Treatments
- Pluripotent Stem Cells Research
- Microtubule and mitosis dynamics
- CRISPR and Genetic Engineering
- Parvovirus B19 Infection Studies
- Gene expression and cancer classification
- Advanced Electron Microscopy Techniques and Applications
- Monoclonal and Polyclonal Antibodies Research
- DNA and Nucleic Acid Chemistry
- Advanced Biosensing Techniques and Applications
- Photosynthetic Processes and Mechanisms
- Microfluidic and Bio-sensing Technologies
- Click Chemistry and Applications
- Advanced biosensing and bioanalysis techniques
- Bacteriophages and microbial interactions
University of Cambridge
2014-2024
Lawn Tennis Association
2013
University of Sussex
2012
The Gurdon Institute
2007
The University of Adelaide
2000-2003
Centre for Cancer Biology
2003
Health Sciences and Nutrition
2002
Commonwealth Scientific and Industrial Research Organisation
2002
The University of Texas Southwestern Medical Center
2002
Institut Pasteur
1973
The hypoxia-inducible factors (HIFs) 1alpha and 2alpha are key mammalian transcription that exhibit dramatic increases in both protein stability intrinsic transcriptional potency during low-oxygen stress. This increased is due to the absence of proline hydroxylation, which normoxia promotes binding HIF von Hippel-Lindau (VHL tumor suppressor) ubiquitin ligase. We now show hypoxic induction COOH-terminal transactivation domain (CAD) occurs through abrogation hydroxylation a conserved...
Mammalian cells adapt to hypoxic conditions through a transcriptional response pathway mediated by the hypoxia-inducible factor, HIF. HIF activity is suppressed under normoxic hydroxylation of an asparagine residue within its C-terminal transactivation domain, blocking association with coactivators. Here we show that protein FIH-1, previously shown interact HIF, asparaginyl hydroxylase. Like known hydroxylase enzymes, FIH-1 Fe(II)-dependent enzyme uses molecular O 2 modify substrate....
The inheritance of the histone H3 variant CENP-A in nucleosomes at centromeres following DNA replication is mediated by an epigenetic mechanism. To understand process inheritance, or propagation histones and variants, as are disassembled reassembled living eukaryotic cells, we have explored feasibility exploiting photo-activated localization microscopy (PALM). PALM single molecules cells has potential to reveal new concepts cell biology, providing insights into stochastic variation cellular...
AlkB homolog 1 (ALKBH1) is one of nine members the family mammalian homologs. Most Alkbh1(-/-) mice die during embryonic development, and survivors are characterized by defects in tissues originating from ectodermal lineage. In this study, we show that deletion Alkbh1 prolonged expression pluripotency markers stem cells delayed induction genes involved early differentiation. vitro differentiation to neural progenitor (NPCs) displayed an increased rate apoptosis NPCs when compared with...
Hypoxia-inducible factor 1α (HIF-1α) and the HIF-like (HLF) are two highly related basic Helix-Loop-Helix/Per-Arnt-Sim (bHLH/PAS) homology transcription factors that undergo dramatically increased function at low oxygen levels. Despite strong similarities in their activation mechanisms (<i>e.g.</i> they both rapid hypoxia-induced protein stabilization, bind identical target DNA sequences, induce synthetic reporter genes to similar degrees), essential for embryo survival via distinct...
The nucleosome remodeling deacetylase (NuRD) complex is a highly conserved regulator of chromatin structure and transcription. Structural studies have shed light on this other modifying machines, but much less known about how they assemble whether stable functional sub-modules exist that retain enzymatic activity. Purification the endogenous Drosophila NuRD shows it consists core subunits, while others, in particular remodeler CHD4, associate transiently. To dissect assembly activity NuRD,...
The infectious units in native and alkalidenatured preparations of DNA herpes simplex virus were characterized with respect to their sensitivity Neurospora crassa endonuclease, sedimentation properties high-salt, neutral sucrose gradients, hydrodynamic shearing forces. Infectious molecules resistant N. sedimented at 56 S, highly sensitive After alkaline denaturation, became the enzyme, 200 relatively shear. We conclude that both intact duplex ([unk]100 x 10(6) daltons) single strands...
Enhancers bind transcription factors, chromatin regulators, and non-coding transcripts to modulate the expression of target genes. Here, we report 3D genome structures single mouse ES cells as they are induced exit pluripotency transition through a formative stage prior undergoing neuroectodermal differentiation. We find that there is remarkable reorganization structure where inter-chromosomal intermingling increases dramatically in state. This associated with formation large number multiway...
Abstract To understand how the nucleosome remodeling and deacetylase (NuRD) complex regulates enhancers enhancer–promoter interactions, we have developed an approach to segment extract key biophysical parameters from live-cell three-dimensional single-molecule trajectories. Unexpectedly, this has revealed that NuRD binds chromatin for minutes, decompacts structure increases enhancer dynamics. We also uncovered a rare fast-diffusing state of found restricts time spent in state. Hi-C...
We describe a microfluidic device designed specifically for the reversible immobilisation of Schizosaccharomyces pombe (Fission Yeast) cells to facilitate live cell super-resolution microscopy. Photo-Activation Localisation Microscopy (PALM) is used create detailed images within living with modal accuracy >25 nm in lateral dimensions. The novel flow design captures and holds well-defined array minimal effect on normal growth kinetics. Cells are held over several hours can continue grow...
Development of single-molecule localization microscopy techniques has allowed nanometre scale accuracy inside cells, permitting the resolution ultra-fine cell structure and elucidation crucial molecular mechanisms. Application these methodologies to understanding processes underlying DNA replication repair been limited defined in vitro biochemical analysis prokaryotic cells. In order expand eukaryotic systems, we have further developed a photo-activated microscopy-based method directly...
A major challenge in single-molecule imaging is tracking the dynamics of proteins or complexes for long periods time dense environments found living cells. Here, we introduce concept using FRET to enhance photophysical properties photo-modulatable (PM) fluorophores commonly used such studies. By developing novel pairs, consisting a PM donor fluorophore (either mEos3.2 PA-JF549) next photostable acceptor dye JF646, demonstrate that competes with normal photobleaching kinetic pathways increase...
In order to gain a more global view of the activity histone demethylases, we report here genome-wide studies fission yeast SWIRM and polyamine oxidase (PAO) domain homologues mammalian LSD1. Consistent with previous work find that two S. pombe proteins, which name Swm1 Swm2 (after SWIRM1 SWIRM2), associate together in complex. However, this complex specifically demethylates lysine 9 H3 (H3K9) both up- down-regulates expression different groups genes. Using chromatin-immunoprecipitation,...
The key to ensuring proper chromosome segregation during mitosis is the kinetochore (KT), a tightly regulated multiprotein complex that links centromeric chromatin spindle microtubules and as such leads process. Understanding its architecture, function, regulation therefore essential. However, due complexity dynamics, only individual subcomplexes could be studied in structural detail so far. In this study, we construct nanometer-precise situ map of human-like regional KT Schizosaccharomyces...
Single-cell chromosome conformation capture approaches are revealing the extent of cell-to-cell variability in organization and packaging genomes. These single-cell methods, unlike their multi-cell counterparts, allow straightforward computation realistic conformations that may be compared combined with other, independent, techniques to study 3D structure. Here we discuss how Hi-C subsequent genome structure determination allows comparison data from microscopy. We then carry out a systematic...
Abstract Enhancer-promoter dynamics are crucial for the spatiotemporal control of gene expression, but it remains unclear whether these controlled by chromatin regulators, such as nucleosome remodelling and deacetylase (NuRD) complex. The NuRD complex binds to all active enhancers modulate transcription here we use Hi-C experiments show that blurs TAD boundaries increases proximity intermediate-range (~1 Mb) genomic sequences enhancer-promoter interactions. To understand alters 3D genome...
Single-molecule super-resolution microscopy allows imaging of fluorescently-tagged proteins in live cells with a precision well below that the diffraction limit. Here, we demonstrate 3D sectioning single-molecule by making use fitting information is usually discarded to reject fluorophores emit from above or virtual-'light-sheet', thin volume centred on focal plane microscope. We describe an easy-to-use routine (implemented as open-source ImageJ plug-in) quickly analyse calibration sample...
Post-translational modification of histone proteins are known to play an important role in regulating chromatin structure. In effort find additional modifications we set out screen enzymes the 2-oxoglutarate and Fe(II)-dependent (2-OG-Fe(II)) dioxygenase family for activity towards histones. Here show that Schizosaccharomyces pombe 2-OG-Fe(II) domain containing protein-2 (Ofd2) is a H2A enzyme. Using combination peptide screening alanine scanning substitution analysis, identify HxxLR motif...
Summary Enhancers are genomic DNA sequences that bind transcription factors, chromatin regulators and non-coding transcripts to modulate the expression of target genes. They have been found act from different locations relative a gene activity promoters up ∼1 Mb away. Here we report first 3D genome structures single mouse ES cells as they induced exit pluripotency, transition through formative stage undergo neuroectodermal differentiation. In order directly study how interactions between...