Biology subjects

Marshall, O. J.

Publications and source records attributed to Marshall, O. J..

11 recordsLinked to original sources

bioRxiv2026

damidBind: an R/Bioconductor package for differential DamID analysis and data exploration

SummaryDamID, and its cell-type specific adaptations, including Targeted DamID (TaDa) and Chromatin Accessibility TaDa (CATaDa), are now widely-adopted as techniques for the genome-wide profiling of DNA binding proteins. Despite this popularity, no dedicated software solution exists for identifying differentially bound or accessible loci, or differentially transcribed genes, between cell types using DamID. The R/Bioconductor package damidBind provides these functions, allowing an end-user to move from processed binding profiles to identifying differentially-bound loci in a reproducible, statistically appropriate and straightforward workflow. Availability and ImplementationdamidBind is an open-source R/Bioconductor package and freely available from Bioconductor at https://bioconductor.org/packages/-damidBind/, and from GitHub at https://github.com/marshall-lab/damidBind. It is released under the GPLv3 licence. ContactOwen Marshall (owen.marshall@utas.edu.au)

bioinformatics↗

bioRxiv2024

The transcription factor Chronophage/BCL11A/B promotes intestinal stem cell proliferation and endocrine differentiation in the Drosophila intestine

Tissue-resident Adult Stem Cells (ASCs) need to continuously adapt their rate of division and differentiation based on their tissue environment. However, the gene regulatory networks that govern these decisions in ASCs and how they respond to challenges such as infection are often not fully understood. We identify a novel role for the transcription factor (TF) Chronophage (Cph) in ISC proliferation and entero-endocrine (EE) cell differentiation. Cph is a Z2H2 zinc TF orthologous to mammalian BCL11A/B that are involved in regulating adult stem cell fate in various contexts. We show here that Cph is expressed in ISCs and EEs in the Drosophila intestine. Increased levels of Cph correlates with increased ISC proliferation and EE differentiation. cph loss-of-function leads to impaired ISC proliferation. Cph levels are elevated during tumourigenesis as well as in ageing and infection conditions. Knockdown of Cph in a Notch-mutant tumour model reduces tumour size and incidence and extends lifespan. Mechanistically, Cph overexpression leads to an increase in enteroendocrine (EE) cells and DamID DNA-binding and qRT-PCR analysis reveals that Cph directly regulates the levels of key EE regulatory genes such as Prospero (pros) and Phyllopod (phyl). In addition, Cph directly regulates core cell cycle regulators such as E2F1 as well as the TF Nerfin-1 that controls ISC proliferation and maintenance. Together, these data support a role for Cph in finetuning the balance between differentiation and proliferation during entero-endocrine differentiation.

developmental biology↗

bioRxiv2024

Dynamic changes in neuronal and glial GAL4 driver expression during Drosophila ageing

Understanding how diverse cell types come together to form a functioning brain relies on the ability to specifically target these cells. This is often done using genetic tools such as the GAL4/UAS system in Drosophila melanogaster. Surprisingly, despite its extensive usage during studies of the ageing brain, detailed spatio-temporal characterisation of GAL4 driver lines in adult flies has been lacking. Here we show that three commonly used neuronal drivers (elav[C155]-GAL4, nSyb[R57C10]-GAL4 and ChAT-GAL4) and the commonly used glial driver repo-GAL4 all show rapid and pronounced decreases in activity over the first 1.5 weeks of adult life, with activity becoming undetectable in some regions after 30 days. In addition to an overall decrease in GAL4 activity over time, we found notable differences in spatial patterns, mostly occurring soon after eclosion. Although all lines showed these changes, the nSyb-GAL4 line exhibited the most consistent and stable expression patterns over ageing. Our findings suggest that gene transcription of key loci decreases in the aged brain, a finding broadly similar to previous work in mammalian brains. Our results also raise questions over past work on long-term expression of disease models in the brain, and stress the need to find better genetic tools for ageing studies.

neuroscience↗

bioRxiv2023

KDM5-mediated activation of genes required for mitochondrial biology is necessary for viability in Drosophila

The precise coordination of gene expression is critical for developmental programs, and histone modifying proteins play important, conserved roles in fine-tuning transcription for these processes. One such family of proteins are KDM5 enzymes that interact with chromatin through demethylating H3K4me3 as well as demethylase-independent mechanisms that remain less understood. The single kdm5 ortholog in Drosophila is an essential gene that has crucial developmental roles in a neuroendocrine tissue, the prothoracic gland. To characterize the regulatory functions of KDM5, we examined its role in coordinating gene expression programs critical to cellular homeostasis and organismal viability in larval prothoracic gland cells. Utilizing targeted genetic experiments, we analyzed the relationship between critical cell signaling pathways, particularly MAPK, and the lethality caused by loss of kdm5. Integrating KDM5 genome binding and transcriptomic data revealed conserved and tissue-specific transcriptional programs regulated by KDM5. These experiments highlighted a role for KDM5 in regulating the expression of a set of genes critical for the function and maintenance of mitochondria. This gene expression program is key to the essential functions of KDM5, as expression of the mitochondrial biogenesis transcription factor Ets97D/Delg, the Drosophila homolog of GABP, in prothoracic gland cells suppressed the lethality of kdm5 null animals. Consistent with this, we observed morphological changes to mitochondria in the prothoracic gland of kdm5 null mutant animals. Together, these data establish KDM5-mediated cellular functions that are both important for normal development and could also contribute to KDM5-linked disorders when dysregulated.

genetics↗

bioRxiv2023

Optogenetic inhibition of Gα signalling alters and regulates circuit functionality and early circuit formation

Optogenetic techniques provide genetically targeted, spatially and temporally precise approaches to correlate cellular activities and physiological outcomes. In the nervous system, G-protein-coupled receptors (GPCRs) have essential neuromodulatory functions through binding extracellular ligands to induce intracellular signaling cascades. In this work, we develop and validate a new optogenetic tool that disrupt Gq signaling through membrane recruitment of a minimal Regulator of G-protein signaling (RGS) domain. This approach, Photo-induced Modulation of G protein - Inhibition of Gq (PiGM-Iq), exhibited potent and selective inhibition of Gq signaling. We alter the behavior of C. elegans and Drosophila with outcomes consistent with GPCR-Gq disruption. PiGM-Iq also changes axon guidance in culture dorsal root ganglia neurons in response to serotonin. PiGM-Iq activation leads to developmental deficits in zebrafish embryos and larvae resulting in altered neuronal wiring and behavior. By altering the choice of minimal RGS domain, we also show that this approach is amenable to Gi signaling.

neuroscience↗

bioRxiv2022

Eight principal chromatin states functionally segregate the fly genome into developmental and housekeeping roles

Different chromatin forms, or states, represent a fundamental means of controlling gene regulation. Chromatin states have been studied through either the distribution of histone modifications (e.g. 1-5) or more rarely via the occupancy of chromatin proteins 6-8. However, these two approaches disagree on the nature and composition of active chromatin states 2,9 and modelling chromatin via both histone marks and chromatin proteins has been lacking. Here, combining protein and histone mark profiles, we show that chromatin in Drosophila melanogaster is organised into eight principle chromatin states that have consistent forms and constituents across cell types. These states form through the association of the Swi/Snf chromatin remodelling complex, Polycomb Group (PcG)/H3K27me3, HP1a/H3K9me3 or H3K36me3 complexes with either active complexes (RNA Pol/COM-PASS/H3K4me3/NuRF) or repressive marks (histone H1 and nuclear lamin occupancy). Enhancers, core promoters, transcription factor motifs, and gene bodies show distinct chromatin state preferences that separate by developmental and housekeeping/metabolic gene ontology. Within the 3D genome, chromatin states add an additional level of compartmentalisation through self-association of topologically associated domains (TADs) of the same state. Our results suggest that the epigenetic landscape is organised by the binding of chromatin remodellers and repressive complexes, and that through chromatin states the genome is fundamentally segregated into developmental and housekeeping/metabolic roles.

genomics↗

bioRxiv2022

Cell cycle and temporal transcription factors regulate proliferation and neuronal diversity of dedifferentiation-derived neural stem cells

Dedifferentiation is the reversion of differentiated cells to a stem cell like fate, whereby, the gene expression program of mature cells is altered and genes associated with multipotency are expressed. Appropriate terminal differentiation of NSCs is essential for restricting the overall number of neurons produced; in addition, faithful production of neuronal subtypes that populate the brain is important for NSC function. Both characteristics of NSCs are specified through temporal patterning of the NSCs driven by the successive expression of temporal transcription factors (tTFs). In this study, we found that ectopic NSCs induced via bHLH transcription factor Deadpan (Dpn) expression fail to undergo timely expression of temporal transcription factors (tTFs), where they express mid-tTF, Sloppy-paired 1 (Slp-1) and fail to express late-tTF Tailless (Tll); consequently generating an excess of Twin of eyeless (Toy) positive neurons at the expense of Reversed polarity (Repo) positive glial cells. In addition to disrupted production of neuronal/glial progeny, Dpn overexpression also resulted in stalled progression through the cell cycle, and a failure to undergo timely terminal differentiation. Mechanistically, DamID studies demonstrated that Dpn directly binds to both Dichaete (D), a Sox-box transcription factor known to repress Slp-1, as well as a number of cell cycle genes. Promoting cell cycle progression or overexpression of D were able to re-trigger the progression of the temporal series in dedifferentiated NBs, restoring both neuronal diversity and timely NB terminal differentiation.

developmental biology↗

bioRxiv2020

The Drosophila histone demethylase KDM5 is required during early neurodevelopment for proper mushroom body formation and cognitive function

Mutations in the lysine demethylase 5 (KDM5) family of transcriptional regulators are associated with intellectual disability, yet little is known regarding the spatiotemporal requirements or neurodevelopmental contributions of KDM5 proteins. Utilizing the mushroom body (MB), a major learning and memory center within the Drosophila brain, we demonstrate that KDM5 is specifically required within ganglion mother cells and immature neurons for proper neurodevelopment and cognitive function. Within this cellular subpopulation, we identify a core network of KDM5-regulated genes that are critical modulators of neurodevelopment. Significantly, we find that a majority of these genes are direct targets of Prospero (Pros), a transcription factor with well-established roles in neurodevelopment in other neuronal contexts. We demonstrate that Pros is essential for MB development and functions downstream of KDM5 to regulate MB morphology. We therefore provide evidence for a KDM5-Pros axis that orchestrates a transcriptional program critical for proper axonal development and cognitive function.

neuroscience↗

bioRxiv2020

FlyORF-TaDa allows rapid generation of new lines for in vivo cell-type specific profiling of protein-DNA interactions in Drosophila melanogaster

Targeted DamID (TaDa) is an increasingly popular method of generating cell-type specific DNA binding profiles in vivo. Although sensitive and versatile, TaDa requires the generation of new transgenic fly lines for every protein that is profiled, which is both time-consuming and costly. Here, we describe the FlyORF-TaDa system for converting an existing FlyORF library of inducible open reading frames (ORFs) to TaDa lines via a genetic cross, with recombinant progeny easily identifiable by eye colour. Profiling the binding of the H3K36me3-associated chromatin protein MRG15 in larval neural stem cells using both FlyORF-TaDa and conventional TaDa demonstrates that new lines generated using this system provide accurate and highly-reproducible DamID binding profiles. Our data further show that MRG15 binds to a subset of active chromatin domains in vivo. Courtesy of the large coverage of the FlyORF library, the FlyORF-TaDa system enables the easy creation of TaDa lines for 74% of all transcription factors and chromatin modifying proteins within the Drosophila genome.

molecular biology↗

bioRxiv2020

Psi promotes Drosophila wing growth through transcriptional repression of key developmental networks

Psi, the sole FUSE Binding Protein (FUBP) family single stranded DNA/RNA binding protein in Drosophila, is essential for proper cell and tissue growth, however its mechanism of function remains unclear. Here we use Targeted DamID combined with RNA-sequencing to generate the first genome-wide binding and expression profiles for Psi. Surprisingly, we demonstrate Psi drives growth in the Drosophila wing through transcriptional repression of key developmental pathways (e.g. Wnt, Notch and TGF{beta}). Thus, Psi patterns tissue growth by directly repressing transcription of developmental growth suppressors. Analysis of direct Psi targets identified novel growth inhibitors, including Tolkin (Zinc metallopeptidase implicated in TGF{beta} signalling), Ephexin (Rho-GEF) and emp (CD36 scavenger receptor-related protein). Their depletion not only suppressed impaired growth associated with Psi knockdown, but alone was sufficient to drive wing overgrowth. Thus, Psi drives wing growth twofold, through direct activation of Myc and through transcriptional repression of growth inhibitors comprising core developmental pathways.

developmental biology↗

bioRxiv2020

Membrane-bound GFP-labelled vectors for Targeted DamID allow simultaneous profiling of expression domains and DNA binding

Targeted DamID (TaDa) allows highly efficient cell-type-specific profiling of protein-DNA interactions. Cell-type-specificity, however, is governed by the GAL4/UAS system, which can exhibit differences in expression patterns depending upon the genomic insertion site and the UAS promoter strength. The TaDa system uses a bicistronic transcript to reduce the translation rates of Dam-fusion proteins, presenting the possibility of using the primary ORF within in the transcript to label expression domains and precisely identified the profiled cell populations in experimental samples. Here, we describe new TaDa vectors, pTaDaG, pTaDaG2 and pTaDaM, that use myristoylated-GFP or myristoylated-mCherry as the primary ORF. Differing lengths of the myristoylation sequence between the two GFP plasmids allows additional translational control. The mCherry plasmid allows profiled cells to be visualised when using the NanoDam system, in which an anti-GFP nanobody is fused to Dam to profile the localisation of GFP-fusion proteins. Fly lines created with this system allow easy visualisation of expression domains under both fluorescent dissecting and confocal microscopes without the use of antibody staining, whilst faithfully profiling protein-DNA interactions via Targeted DamID.

genomics↗