Biology subjects

Davidovich, C.

Publications and source records attributed to Davidovich, C..

14 recordsLinked to original sources

bioRxiv2026

Extended-Spectrum and Last-Resort β-Lactams Exert Increasing Impacts on Chicken Manure-Derived Copiotrophic Microbiomes and Resistomes

Understanding the environmental dissemination of antimicrobial resistance (AMR) is crucial for mitigating its spread and for reducing the clinical burden of resistant infections. Within a One Health framework, animal husbandry systems, and particularly animal gut microbiomes, are widely used as models for agricultural environments, because their high densities and recurrent antibiotic exposure create strong selective pressures that can impact both environmental and human resistomes. In a previous study, we demonstrated that copiotrophic enrichment in medium that stimulates growth of gut-associated bacteria, can reveal clinically associated bacterial populations and associated antibiotic resistance genes (ARGs) that are not detected by direct environmental sampling. In the present study, we applied this enrichment approach to poultry manure to examine how {beta}-lactamase-resistant antibiotics (cefotaxime and meropenem) shape copiotrophic gut-like microbial community composition and resistance profiles relative to an earlier {beta}-lactam (ampicillin) and to antibiotic-free controls. Our results show that {beta}-lactamase-resistant antibiotics induced more pronounced shifts in both microbial diversity and ARG profiles than the early {beta}-lactam. When focusing on specific taxa, differential responses were observed correlating to antimicrobial range of action. Together, these findings underline how the diversity of pathogen and AMR indicators in gut microbiomes may be shaped by different selective pressures imposed by {beta}-lactamase-resistant antibiotics.

microbiology↗

bioRxiv2025

Structures of tRNA-bound CRISPR-Cas13 reveal universal HEPN RNase mechanisms

Ribonucleases (RNases) are ubiquitous drivers of RNA metabolism across life. Higher eukaryotes and prokaryotes nucleotide-binding (HEPN) domain-containing RNases form a nuclease superfamily whose mechanisms of substrate selection and catalysis remain poorly understood. Here, we report substrate preferences for diverse Cas13 HEPN RNases, revealing precise cleavage of tRNA anticodons and acceptor stems. Using cryo-EM, we present a series of activated Leptotrichia buccalis Cas13a (LbuCas13a) structures engaged with substrate tRNA. We show LbuCas13a captures tRNA through shape- and sequence-specific recognition, cleaving U-rich anticodons independently of tRNA modifications. Leveraging these insights, we reprogram Cas13 specificity and engineer variants with accelerated RNase activity. These findings establish the molecular basis for Cas13 tRNase activity and indicate that the HEPN RNase superfamily obeys universally conserved mechanisms of substrate recognition and catalysis.

biochemistry↗

bioRxiv2025

Accessory subunits of PRC2 mimic H3K27me3 to restrict the spread of Polycomb domains

The Polycomb repressive complex 2 (PRC2) is essential for normal development by maintaining developmental gene repression. PRC2 deposits the repressive chromatin mark H3 lysine 27 tri-methyl (H3K27me3) through a read-write loop that involves direct interactions between PRC2 and H3K27me3. According to current models, the PRC2-H3K27me3 read-write loop is initiated by the PRC2 subunits JARID2 and PALI1 that mimic H3K27me3. However, it is unknown what restricts the PRC2-H3K27me3 read-write loop from spreading H3K27me3 indefinitely. To answer this question, we generated mutant mice where PRC2 subunits cannot mimic H3K27me3. Unexpectedly, the mutations led to delayed Hox genes activation and a homeotic transformation characteristic of a Polycomb gain-of-function in vivo and the spread of H3K27me3 beyond Polycomb domains in stem cells. Collectively, we show that H3K27me3 mimicry evolved to compete against the PRC2-H3K27me3 read-write loop in a process that restrains PRC2 and restricts the spread of Polycomb domains. HighlightsO_LIH3K27me3 mimicry antagonises Polycomb function in vivo. C_LIO_LIJARID2 and PALI1 synergise to allosterically regulate PRC2 during development. C_LIO_LIH3K27me3 mimicry by JARID2 and PALI1 antagonises PRC2 in stem cells. C_LIO_LIJARID2 and PALI1 mimic H3K27me3 to restrict the spread of Polycomb domains. C_LI

molecular biology↗

bioRxiv2024

Modular cloning of multigene vectors for the baculovirus system and yeast

Recombinant macromolecular complexes are often produced by the baculovirus system, using multigene expression vectors. Yet, the construction of baculovirus-compatible multigene expression vectors is complicated and time-consuming. Furthermore, while the baculovirus and yeast are popular protein expression systems, no single method for multigene vector construction is compatible with both. Here we present the modular cloning (MoClo) Baculo toolkit for constructing multigene expression vectors for the baculovirus system and, through compatibility with the MoClo Yeast toolkit, also for yeast. Vector construction by MoClo Baculo utilises Golden Gate assembly, which does not require PCR, primers or the sequencing of intermediate products. As a proof of principle, MoClo Baculo was used to construct baculovirus and yeast multigene vectors expressing the four- and five-subunit human Polycomb Repressive Complex 2. We show that MoClo Baculo simplifies and expedites the construction of multigene expression vectors for the baculovirus system and provides compatibility with yeast as an alternative expression system.

biochemistry↗

bioRxiv2024

Occurrence of "under-the-radar" antibiotic resistance in anthropogenically affected produce

With global climate change, treated-wastewater (TWW) irrigation and manure amendment are becoming increasingly important in sustainable agriculture in water- and nutrient-stressed regions. Yet, these practices can potentially disseminate pathogens and antimicrobial resistance (AMR) determinants to crops, resulting in serious health risks to humans through the food chain. Previous studies demonstrated that pathogen and AMR indicators from wastewater and manure survive poorly in the environment, suggesting that ecological barriers prevent their dissemination. However, we recently found that these elements can persist below detection levels in low quality TWW-irrigated soil, and potentially proliferate under favorable conditions. This "under the radar" phenomenon was further investigated here, in TWW irrigated- and poultry litter-amended lettuce plants, using an enrichment platform that resembles gut conditions, and an analytical approach that combined molecular and cultivation-based techniques. Enrichment uncovered clinically-relevant multidrug resistant pathogen indicators and a myriad of antibiotic resistance genes in the litter amended and TWW-irrigated lettuce that were not detected by direct analyses, or in the enriched freshwater irrigated samples. Selected resistant E. coli isolates were capable of horizontally transferring plasmids carrying multiple resistance genes to a susceptible strain. Overall, our study underlines the hidden risks of under-the-radar pathogen and AMR determinants in anthropogenically affected agroenvironments, providing a novel platform to improve quantitative microbial risk assessment models in the future.

microbiology↗

bioRxiv2024

MORC2 phosphorylation fine tunes its DNA compaction activity

Variants in the poorly characterised oncoprotein, MORC2, a chromatin remodelling ATPase, lead to defects in epigenetic regulation and DNA damage response. The C-terminal domain (CTD) of MORC2, frequently phosphorylated in DNA damage, promotes cancer progression, but its role in chromatin remodelling remains unclear. Here, we report a molecular characterisation of full-length, phosphorylated MORC2, demonstrating its preference for binding open chromatin and functioning as a DNA sliding clamp. We identified a phosphate interacting motif within the CTD that dictates ATP hydrolysis rate and cooperative DNA binding. The DNA binding impacts several structural domains within the ATPase region. We provide the first visual proof that MORC2 induces chromatin remodelling through ATP hydrolysis-dependent DNA compaction, regulated by its phosphorylation state. These findings highlight phosphorylation of MORC2 CTD as a key modulator of chromatin remodelling, presenting it as a potential therapeutic target.

molecular biology↗

bioRxiv2024

A deep learning-based model integrating base-pairing patterns improves microRNA target site prediction

Micro RNAs (miRNAs) are short non-coding RNAs involved in various cellular processes, playing a crucial role in gene regulation. Identifying miRNA targets remains a central challenge and is pivotal for elucidating the complex gene regulatory networks. Traditional computational approaches have predominantly focused on identifying miRNA targets through perfect Watson-Crick base pairings within the seed region, referred to as canonical sites. However, emerging evidence suggests that perfect seed matches are not a prerequisite for miRNA-mediated regulation, underscoring the importance of also recognizing imperfect, or non-canonical, sites. To address this challenge, we propose Mimosa, a new computational approach that employs the Transformer framework to enhance the prediction of miRNA targets. Mimosa distinguishes itself by integrating contextual, positional, and base-pairing information to capture in-depth attributes, thereby improving its predictive capabilities. Its unique ability to identify non-canonical base-pairing patterns makes Mimosa a standout model, reducing the reliance on pre-selecting candidate targets. Mimosa achieves superior performance in gene-level predictions and also shows impressive performance in site-level predictions across various non-human species through extensive benchmarking tests. To facilitate research efforts in miRNA targeting, we have developed an easy-to-use web server for comprehensive end-to-end predictions, which is publicly available at http://monash.bioweb.cloud.edu.au/Mimosa/.

bioinformatics↗

bioRxiv2023

The apparent loss of PRC2 chromatin occupancy as an artefact of RNA depletion

RNA has been implicated in the recruitment of chromatin modifiers, and previous studies have provided evidence in favour and against this idea. RNase treatment of chromatin is a prevalent tool for the study of RNA-mediated regulation of chromatin modifiers, but the limitations of this approach remain unclear. RNase A treatment during chromatin immunoprecipitation (RNase-ChIP or rChIP) reduces chromatin occupancy of the H3K27me3 methyltransferase PRC2. This led to suggestions of an "RNA bridge" between PRC2 and chromatin. Here we show that RNase A treatment during chromatin immunoprecipitation leads to the apparent loss of all facultative heterochromatin, including both PRC2 and H3K27me3 genome wide. This phenomenon persists in mouse embryonic stem cells, human cancer cells and human-induced pluripotent stem cells. We track this observation to a gain of DNA from non-targeted chromatin, sequenced at the expense of DNA from facultative heterochromatin, which reduces ChIP signals. Our results point to substantial limitations in using RNase A treatment for mapping RNA-dependent chromatin occupancy and invalidate conclusions that were previously established for PRC2 based on this assay. HighlightsO_LIRNA degradation during ChIP-seq is insufficient to displace PRC2 from chromatin. C_LIO_LIRNA degradation led to the artificial depletion of ChIP-seq signals in multiple cell lines. C_LIO_LIArtificially reduced ChIP-seq signals are explained by a gain of non-targeted DNA. C_LIO_LIRNA is critical in maintaining the solubility of chromatin during experimentation. C_LI

molecular biology↗

bioRxiv2023

Dominant negative and directional dysregulation of Polycomb function in EZH2-mutant human growth disorders

Heterozygous missense mutations in EZH2 cause Weaver syndrome (WS), a developmental disorder characterized by intellectual disability and overgrowth. EZH2 encodes the enzymatic subunit of Polycomb Repressive Complex 2 (PRC2), which mediates mono-, di-, and tri-methylation of histone H3 lysine residue 27 (H3K27me1/2/3). Although the functional characterization of most WS-associated EZH2 variants is lacking, they are presumed loss of function. However, the dearth of reported early truncating mutations in EZH2 led us to hypothesise that a dominant negative mutational mechanism may underlie the development of WS. To test this, we performed a detailed structural analysis of all known WS-associated EZH2 variants, which provided initial support that they are dominant negative. Next, we isogenically modelled 10 representative WS-associated EZH2 variants in embryonic stem cells and showed they induce global reductions in H3K27me2 and H3K27me3 with concomitant global increases in H3K27me1, H3K27ac, and chromatin decompaction. Importantly, the reductions in H3K27me2/3 methylation revealed a pattern of dominant-negative interference to PRC2 activity. Comparative analysis of a gain-of-function EZH2 variant causing growth restriction highlighted the reciprocal nature of the chromatin changes in these opposing growth syndromes. Our findings detail the molecular effects of developmental-syndrome-associated EZH2 variants in cells and implicate imbalanced landscapes of H3K27 modification in their pathology.

genetics↗

bioRxiv2023

Histone H3.3 lysine 9 and 27 control repressive chromatin states at cryptic cis-regulatory elements and bivalent promoters in mouse embryonic stem cells

Histone modifications are associated with distinct transcriptional states, but it is unclear whether they instruct gene expression. To investigate this, we mutated histone H3.3 K9 and K27 residues in mouse embryonic stem cells (mESCs). Here, we find that H3.3K9 is essential for controlling specific distal intergenic regions and for proper H3K27me3 deposition at promoters. The H3.3K9A mutation resulted in decreased H3K9me3 at regions encompassing endogenous retroviruses and induced a gain of H3K27ac and nascent transcription. These changes in the chromatin environment unleashed cryptic enhancers, resulting in the activation of distinctive transcriptional programs and culminating in protein expression normally restricted to specialized immune cell types. The H3.3K27A mutant disrupted deposition and spreading of the repressive H3K27me3 mark, particularly impacting bivalent genes with higher basal level of H3.3 at promoters. Therefore, H3.3K9 and K27 crucially orchestrate repressive chromatin states at cis-regulatory elements and bivalent promoters, respectively, and instruct proper transcription in mESCs.

genomics↗

bioRxiv2023

Dynamic PRC1-CBX8 stabilizes a porous structure of chromatin condensates

The compaction of chromatin is a prevalent paradigm in gene repression. Chromatin compaction is commonly thought to repress transcription by restricting chromatin accessibility. However, the spatial organisation and dynamics of chromatin compacted by gene-repressing factors are unknown. Using cryo-electron tomography, we solved the three-dimensional structure of chromatin condensed by the Polycomb Repressive Complex 1 (PRC1) in a complex with CBX8. PRC1-condensed chromatin is porous and stabilised through multivalent dynamic interactions of PRC1 with chromatin. Mechanistically, positively charged residues on the internally disordered regions (IDRs) of CBX8 mask negative charges on the DNA to stabilize the condensed state of chromatin. Within condensates, PRC1 remains dynamic while maintaining a static chromatin structure. In differentiated mouse embryonic stem cells, CBX8-bound chromatin remains accessible. These findings challenge the idea of rigidly compacted polycomb domains and instead provides a mechanistic framework for dynamic and accessible PRC1-chromatin condensates.

biophysics↗

bioRxiv2023

CRISPR-ChIP delineates a Menin-dependent oncogenic DOT1L complex in MLL-leukaemia

The regulation of all chromatin-templated processes involves the selective recruitment of chromatin factors to facilitate DNA repair, replication, and transcription. Chromatin immunoprecipitation (ChIP) is a critical experimental method used to provide spatiotemporal evidence for the coordination of these chromatin-based events including the dynamic regulation of chromatin modifications at cis-regulatory elements. However, obtaining a global appreciation of all the factors that influence a specific chromatin event has remained challenging. Here, as a proof of concept we demonstrate the utility of coupling unbiased functional genomics with ChIP to identify the factors associated with active transcription. Specifically, we use this method to identify the major chromatin factors associated with the catalysis of two evolutionarily conserved histone modifications; H3K4me3 present at the transcriptional start site and H3K79me2 present through the gene body of actively transcribed genes. With CRISPR-ChIP, we identify all the non-redundant COMPASS complex members required for H3K4me3 and demonstrate that RNA polymerase II is dispensable for the maintenance of H3K4me3. As H3K79me2 has a putative oncogenic function in leukaemia cells driven by MLL-translocations, using CRISPR-ChIP we reveal a functional partitioning of H3K79 methylation into two distinct regulatory units. An oncogenic DOT1L complex, where the malignant driver directs the catalytic activity of DOT1L at MLL-Fusion target genes and a separate endogenous DOT1L complex, where catalytic activity is directed by MLLT10. This functional demarcation provides an explanation for the observed synergy with Menin and DOT1L inhibitors and why Menin inhibition surprisingly controls methylation of H3K79 at a critical subset of genes that sustain MLL-fusion leukaemia. Overall, CRISPR-ChIP provides a powerful tool for the unbiased interrogation of the mechanisms underpinning chromatin regulation.

molecular biology↗

bioRxiv2020

Structural basis of rotavirus RNA chaperone displacement and RNA annealing

Rotavirus genomes are distributed between eleven distinct RNA molecules, all of which must be selectively co-packaged during virus assembly. This likely occurs through sequence-specific RNA interactions facilitated by the RNA chaperone NSP2. Here, we report that NSP2 auto-regulates its chaperone activity through its C-terminal region (CTR) that promotes RNA-RNA interactions by limiting its helix-unwinding activity. Unexpectedly, structural proteomics data revealed that the CTR does not directly interact with RNA, whilst accelerating RNA release from NSP2. Cryo-electron microscopy reconstructions of an NSP2-RNA complex reveal a highly conserved acidic patch poised towards RNA. Virus replication was abrogated by charge-disrupting mutations within the acidic patch but completely restored by charge-preserving mutations. Mechanistic similarities between NSP2 and the unrelated bacterial RNA chaperone Hfq suggest that accelerating RNA dissociation whilst promoting inter-molecular RNA interactions may be a widespread strategy of RNA chaperone recycling.

biophysics↗

bioRxiv2020

Convergent evolution between PALI1 and JARID2 for the allosteric activation of PRC2

The polycomb repressive complex 2 (PRC2) is a histone methyltransferase that maintains cell identities. JARID2 is the only accessory subunit of PRC2 that known to trigger an allosteric activation of methyltransferase. Yet, this mechanism cannot be generalised to all PRC2 variants as, in vertebrates, JARID2 is mutually exclusive with most of the accessory subunits of PRC2. Here we provide functional and structural evidence that the vertebrate-specific PRC2 accessory subunit PALI1 emerged through a convergent evolution to mimic JARID2 at the molecular level. Mechanistically, PRC2 methylates PALI1 K1241, which then binds to the PRC2-regulatory subunit EED to allosterically activate PRC2. PALI1 K1241 is methylated in mouse and human cell lines and is essential for PALI1-induced allosteric activation of PRC2. High-resolution crystal structures revealed that PALI1 mimics the regulatory interactions formed between JARID2 and EED. Independently, PALI1 also facilitates DNA and nucleosome binding by PRC2. In acute myelogenous leukemia cells, overexpression of PALI1 leads to cell differentiation, with the phenotype abrogated by a separation-of-function PALI1 mutation, defective in allosteric activation and active in DNA binding. Collectively, we show that PALI1 facilitates catalysis and substrate binding by PRC2 and provide evidence that subunit-induced allosteric activation is a general property of holo-PRC2 complexes.

biochemistry↗