Biology subjects

Burdett, H.

Publications and source records attributed to Burdett, H..

11 recordsLinked to original sources

bioRxiv2026

The DNA demethylase TET1 recognises chromatin via nucleosome engagement of its N-terminal region

TET1 is a DNA demethylase critical for mammalian embryonic development, yet how it is recruited to its chromatin substrate remains poorly understood. Here we identify a previously uncharacterized N-terminal region of TET1 that directly binds nucleosomes and promotes chromatin binding in mouse embryonic stem cells. Robust chromatin association was evidenced by fluorescence recovery after photobleaching during interphase and chromosome retention throughout mitosis. A 2.47 angstroms cryo-EM structure of the TET1-nucleosome complex reveals recognition of the nucleosome acidic patch through a canonical arginine anchor centred on R75, with additional contacts extending along the H2B surface. Biochemical and cellular analyses validate this interaction and demonstrate its contribution to TET1 chromatin engagement. The region is absent from TET2 and TET3 and displays strong conservation within mammals, indicating that direct nucleosome recognition is a specialized feature of TET1. Together, these findings uncover a previously unknown chromatin-binding module in TET1 and establish a structural basis for its interaction with nucleosomes.

molecular biology↗

bioRxiv2026

Structural basis of canonical TIR-NLR activation in plant innate immunity

In plants, intracellular NLRs (nucleotide-binding leucine-rich repeat receptors) detect pathogen effector proteins, form oligomeric resistosomes, and activate ETI (effector-triggered immunity). NLRs contain N-terminal signaling, central NB-ARC (nucleotide-binding) and C-terminal LRR (leucine-rich repeat) domains. NLRs with N-terminal TIR (Toll/interleukin-1 receptor) domains (TNLs) hydrolyze NAD+ (nicotinamide adenine dinucleotide) to generate signaling molecules. We determined cryo-EM structures of flax M, a canonical non C-JID (C-terminal jellyroll/Ig-like domain) TNL, in both monomeric autoinhibited conformation, and tetrameric resistosome after activation by its rust fungal effector AvrM-A. AvrM-A homodimers dissociate into monomers to bind directly to the LRR and NB-ARC domains in the M resistosome. The resistosome structure includes a non-hydrolyzable NAD+ analogue, revealing the substrate NAD+ recognition mechanism by the TIR domains. M cleaves NAD+ and generates the same signaling compounds as the related flax TNL, L6. Our findings explain the mechanism of TNL signaling, and provide a basis for rational engineering of disease-resistant crops.

plant biology↗

bioRxiv2025

Tetramerisation governs SALL transcription factor function in development and disease

Spalt-like (SALL) proteins are C2H2 zinc-finger transcription factors important for embryogenesis, with mutations in SALL1 and SALL4 causing rare congenital disorders Townes-Brocks and Okihiro syndromes, respectively. While SALL proteins are known to associate with one another, the biological significance of the resulting complexes is unknown. Here we define a conserved glutamine-rich region that mediates SALL1/4 homo- and heterotetramerisation and find that complex formation is indispensable for DNA binding. Modelling a patient mutation that abolishes SALL4 multimerisation led to gene misregulation and, in mice, embryonic lethality, therefore phenocopying a complete Sall4 knockout. Furthermore, a common disease-causing SALL1 truncation, which retains multimerisation but lacks DNA-binding domains, sequesters SALL4 into heterotetramers that are defective in DNA binding, thereby providing a mechanistic explanation for the dominant-negative effects of many Townes-Brocks mutations. Together, our findings establish tetramerisation as a prerequisite for SALL function, linking complex formation to developmental gene regulation and human disease.

molecular biology↗

bioRxiv2025

Trypanosome histone variants H3.V and H4.V promote nucleosome plasticity in repressed chromatin.

Histone variants define distinct chromatin states by modulating the biophysical properties of nucleosomes. Variants play a particularly important role in the parasitic protist Trypanosoma brucei, which has unusual chromatin and lacks a canonical repressive heterochromatin system. Instead, T. brucei utilises specialised divergent histone variants H3.V and H4.V. However, the biochemical basis of their repressive functions is unknown. Here, we determined the structure of the H3.V-H4.V nucleosome core particle and biochemically characterised variant-containing nucleosomes and nucleosome arrays probing their unique properties. We discovered that surprisingly for repressive-state nucleosomes, H3.V promotes pronounced DNA splaying, largely via its N-terminal tail region, whilst retaining overall stability that is comparable to canonical nucleosomes. In contrast, H4.V exhibits near-identical binding to DNA but mediates a slight increase in histone octamer stability. The surface of the H3.V-H4.V nucleosome is altered and provides a differential platform for chromatin binding proteins, linking the variants to parasite pathogenicity.

biochemistry↗

bioRxiv2025

Recognition-dependent activation of the RRS1-R/RPS4 immune receptor complex

The Arabidopsis TIR-NLR immune receptors RPS4 and RRS1 function together to enable recognition of multiple effector proteins including AvrRps4 and PopP2. We show here that both in the presence and absence of effector, RPS4 and RRS1 form an oligomer that does not change in size upon effector provision. Oligomer formation involves interactions between the RPS4 and RRS1 TIR domains and requires nucleotide binding capacity in RPS4. RPS4 mutants that lose TIR domain NADase activity abrogate immune activation but retain oligomerization. A cysteine residue in the RPS4 LRR domain contributes to oligomer stabilization. We propose that upon effector recognition, conformational changes in the complex relieve inhibition of RPS4 TIR domains by RRS1 TIR domains, enabling proximity between RPS4 TIR domains to create NADase activity.

plant biology↗

bioRxiv2023

The N-terminal region of DNMT3A combines multiple chromatin reading motifs to guide recruitment.

DNA methyltransferase 3A (DNMT3A) plays a critical role in establishing and maintaining DNA methylation patterns. However, the mechanisms underlying DNMT3A recruitment to and function within different chromatin environments remain unclear. Using a combination of biochemical and structural approaches we find that DNMT3A interacts using multiple interfaces with chromatin; directly binding generic nucleosome features as well as site-specific post-translational histone modifications. The N-terminal region, unique to the DNMT3A1 isoform, is essential for these interactions and stabilises H3K36me2-nucleosome recruitment. Intriguingly, in the same region critical for nucleosome binding we also map a ubiquitylation-dependent recruitment motif (UDR). The UDR binds specifically to ubiquitylated H2AK119, explaining the previously observed recruitment to Polycomb-occupied heterochromatin. A cryo-EM structure of DNMT3A1-DNMT3L with a modified nucleosome reveals that the UDR interacts with the nucleosome surface including the acidic patch. Previously unexplained disease-associated mutations are present in the UDR and ablate nucleosome interactions. This leads to an increased understanding of how DNMT3A1 recruitment occurs in the genome and highlights the importance of multivalent binding of DNMT3A to histone modifications and the nucleosome.

biochemistry↗

bioRxiv2023

Histone divergence in Trypanosoma brucei results in unique alterations to nucleosome structure

Eukaryotes have a multitude of diverse mechanisms for organising and using their genomes, but the histones that make up chromatin are highly conserved. Unusually, histones from kinetoplastids are highly divergent. The structural and functional consequences of this variation are unknown. Here, we have biochemically and structurally characterised nucleosome core particles (NCPs) from the kinetoplastid parasite Trypanosoma brucei. A structure of the T. brucei NCP reveals that global histone architecture is conserved, but specific sequence alterations lead to distinct DNA and protein interaction interfaces. The T. brucei NCP is unstable and has weakened overall DNA binding. However, dramatic changes at the H2A-H2B interface introduce local reinforcement of DNA contacts. The T. brucei acidic patch has altered topology and is refractory to known binders, indicating that the nature of chromatin interactions in T. brucei may be unique. Overall, our results provide a detailed molecular basis for understanding evolutionary divergence in chromatin structure.

biochemistry↗

bioRxiv2023

BRCA1-BARD1 combines multiple chromatin recognition modules to bridge nascent nucleosomes

Chromatin association of the BRCA1-BARD1 heterodimer is critical to promote homologous recombination repair of DNA double-strand breaks (DSBs) in S/G2. How the BRCA1-BARD1 complex interacts with chromatin that contains both damage induced histone H2A ubiquitin and inhibitory H4AK20 methylation is not fully understood. We characterised BRCA1-BARD1 binding and enzymatic activity to an array of mono- and di-nucleosome substrates using biochemical, structural, and single molecule imaging approaches. We find that the BRCA1-BARD1 complex preferentially interacts and modifies di-nucleosomes over mono-nucleosomes, allowing integration of H2A Lys-15 ubiquitylation signals with other chromatin modifications and features. Using high speed-AFM to provide real-time visualization of BRCA1-BARD1 complex recognising chromatin, we show a highly dynamic complex that bridges two nucleosomes and associates with the DNA linker region. Bridging is aided by multivalent cross-nucleosome interactions that enhance BRCA1-BARD1 E3 ubiqiutin ligase catalytic activity. Multivalent interactions across nucleosomes explains how BRCA1-BARD1 can recognize chromatin that retains partial di-methylation at H4 Lys-20 (H4K20me2), a parental histone mark that blocks BRCA1-BARD1 interaction with nucleosomes, to promote its enzymatic and DNA repair activities.

molecular biology↗

bioRxiv2022

Chemical structures of cyclic ADP ribose (cADPR) isomers and the molecular basis of their production and signaling

Cyclic ADP ribose (cADPR) isomers are important signaling molecules produced by bacterial and plant Toll/interleukin-1 receptor (TIR) domains via NAD+ hydrolysis, yet their chemical structures are unknown. We show that v-cADPR (2cADPR) and v2-cADPR (3cADPR) isomers are cyclized by O-glycosidic bond formation between the ribose moieties in ADPR. Structures of v-cADPR (2cADPR)-producing TIR domains reveal that conformational changes are required for the formation of the active assembly that resembles those of Toll-like receptor adaptor TIR domains, and mutagenesis data demonstrate that a conserved tryptophan is essential for cyclization. We show that v2-cADPR (3cADPR) is a potent activator of ThsA effector proteins from Thoeris anti-phage defence systems and is responsible for suppression of plant immunity by the effector HopAM1. Collectively, our results define new enzymatic activities of TIR domains, reveal the molecular basis of cADPR isomer production, and establish v2-cADPR (3cADPR) as an antiviral signaling molecule and an effector-mediated signaling molecule for plant immunity suppression. One-Sentence SummaryThe chemical structures of two O-glycosidic bond-containing cyclic ADP ribose isomers, the molecular basis of their production, and their function in antiviral and plant immunity suppression by bacteria are reported.

biochemistry↗

bioRxiv2021

Self-association configures the NAD+-binding site of plant NLR TIR domains

TIR domains are signalling domains present in plant nucleotide-binding leucine-rich repeat receptors (NLRs), with key roles in plant innate immunity. They are required for the induction of a hypersensitive response (HR) in effector-triggered immunity, but the mechanism by which this occurs is not yet fully understood. It has been recently shown that the TIR domains from several plant NLRs possess NADase activity. The oligomeric structure of TIR-containing NLRs ROQ1 and RPP1 reveals how the TIR domains arrange into an active conformation, but low resolution around the NAD+ binding sites leaves questions unanswered about the molecular mechanisms linking self-association and NADase activity. In this study, a number of crystal structures of the TIR domain from the grapevine NLR RUN1 reveal how self-association and enzymatic activity may be linked. Structural features previously proposed to play roles involve the "AE interface" (mediated by helices A and E), the "BB-loop" (connecting {beta}-strand B and helix B in the structure), and the "BE interface" (mediated by the BB-loop from one TIR and the "DE surface" of another). We demonstrate that self-association through the AE interface induces conformational changes in the NAD+-binding site, shifting the BB-loop away from the catalytic site and allowing NAD+ to access the active site. We propose that an intact "DE surface" is necessary for production of the signalling product (variant cyclic ADPR), as it constitutes part of the active site. Addition of NAD+ or NADP+ is not sufficient to induce self-association, suggesting that NAD+ binding occurs after TIR self-association. Our study identifies a mechanistic link between TIR self-association and NADase activity.

biochemistry↗

bioRxiv2019

Low-cost, open-access refractive index mapping of algal cells using the transport of intensity equation

Phase contrast microscopy allows stain free imaging of transparent biological samples. One technique, using the transport of intensity equation (TIE), can be performed without dedicated hardware by simply processing pairs of images taken at known spacings within the sample. The resulting TIE images are quantitative phase maps of unstained biological samples. Therefore, spatially resolved refractive index information can also be determined.\n\nUsing low-cost, open-source hardware, we applied the TIE to living algal cells to measure their refractive index. We obtained refractive index values that were repeatable within species and differed by distinct amounts depending on the species being measured. We suggest TIE imaging as a method of discrimination between different algal species and, potentially, non-biological materials, based on refractive index. Potential applications in biogeochemical modelling and climate sciences are suggested.

biophysics↗