Biology subjects

Brown, T.

Publications and source records attributed to Brown, T..

At least 55 records · Page 3Linked to original sources

bioRxiv2023

Chiropterans are a hotspot for horizontal transfer of DNA transposons in Mammalia

Horizontal transfer of transposable elements is an important mechanism contributing to genetic diversity and innovation. Bats (order Chiroptera) have repeatedly been shown to experience horizontal transfer of transposable elements at what appears to be a high rate compared to other mammals. We investigated the occurrence of horizontally transferred DNA transposons involving bats. We found over 200 putative horizontally transferred elements within bats; sixteen transposons were shared across distantly related mammalian clades and two other elements were shared with a fish and two lizard species. Our results indicate that bats are a hotspot for horizontal transfer of DNA transposons. These events broadly coincide with the diversification of several bat clades, supporting the hypothesis that DNA transposon invasions have contributed to genetic diversification of bats.

molecular biology↗

bioRxiv2022

Expanded olfactory system in fishes capable of terrestrial exploration

Smell abilities differ greatly among vertebrate species, as reflected by the exceptional size variability of gene families and brain regions involved in odour detection. However, key environmental factors shaping the molecular and phenotypic evolution of the olfactory sensory system remain mostly unknown. Here, we investigate the association between diverse ecological traits and the number of olfactory chemoreceptors in the genomes of more than two hundred ray-finned fishes. We found independent expansions producing large gene repertoires in nocturnal amphibious fishes, generally able to perform active terrestrial exploration. Furthermore, we reinforced this finding with an additional analysis of a clariid species (Channallabes apus), a fish lineage with chemosensory-based aerial orientation. Importantly, we also detected an augmented information-processing capacity in the olfactory bulb of examined nocturnal amphibious species. Overall, we report a convergent magnification of the olfactory system in amphibious fishes potentially enhancing terrestrial orientation, revealing the likelihood of an analogous event in tetrapod ancestors during the water-to-land transition.

evolutionary biology↗

bioRxiv2022

Sequencing and chromosome-scale assembly of the giant Pleurodeles waltl genome

The Iberian ribbed newt (Pleurodeles waltl) constitutes a central model for probing the basis of vertebrate regeneration. Here, we present the sequencing and chromosome-scale assembly of the 20.3Gb P. waltl genome, which exhibits the highest level of contiguity and completeness among giant genome assemblies. We uncover that DNA transposable elements are the major contributors to its expansion, with hAT transposons comprising a large portion of repeats. Several hATs are actively transcribed and differentially expressed during adult P. waltl limb regeneration, along with domesticated hAT transposons of the ZBED transcription factor family. Despite its size, syntenic relationships are conserved. As an example, we show the high degree of conservation of the regeneration-associated Tig1 locus with several neighbouring genes. Together, the P. waltl genome provides a fundamental resource for the study of regenerative, developmental and evolutionary principles.

developmental biology↗

bioRxiv2022

Pou3f1 orchestrates a gene regulatory network controlling contralateral retinogeniculate projections

The balance of contralateral and ipsilateral retinogeniculate projections is critical for the establishment of binocular vision, but the transcriptional programs regulating this process remain ill-defined. In this study, we show that the Pou class homeobox protein POU3F1 is selectively expressed in developing mouse contralateral retinal ganglion cells (cRGCs). Inactivation of Pou3f1 increases the number of ipsilateral RGCs produced, leading to abnormal ipsilateral to contralateral projection ratio at the optic chiasm, whereas expression of Pou3f1 in retinal progenitors increases the production of cRGCs. Using Cut&Run and RNA sequencing in wildtype and Pou3f1 mouse knockout retinas, we demonstrate that Pou3f1 is necessary for the establishment of a cRGC gene regulatory network through activation and repression of several known contralateral and ipsilateral determinants, respectively. Finally, we report that POU3F1 is sufficient to induce production of RGC-like cells sending projections to the optic nerve, even in late-stage retinal progenitors not normally competent to generate RGCs. This work uncovers POU3F1 as a master regulator of the cRGC transcriptional program and opens new possibilities for optic nerve regenerative therapies.

developmental biology↗

bioRxiv2022

Clinical mutations in WDR34 lead to diverse impacts on the assembly and function of dynein-2

The primary cilium is a sensory organelle, receiving signals from the external environment and relaying them into the cell. Mutations in proteins required for transport in the primary cilium result in ciliopathies, a group of genetic disorders that commonly lead to the malformation of organs such as the kidney, liver and eyes and skeletal dysplasias. Motor proteins dynein-2 and kinesin-2 mediate retrograde and anterograde transport respectively in the cilium. WDR34, a dynein-2 intermediate chain, is required for the maintenance of cilia function. Here, we investigated WDR34 mutations identified in Jeune syndrome, short-rib polydactyly syndrome or asphyxiating thoracic dysplasia patients. There is a poor correlation between genotype and phenotype in these cases making diagnosis and treatment highly complex. We set out to define the biological impacts on cilia formation and function of WDR34 mutations by stably expressing the mutant proteins in WDR34 knockout cells. WDR34 mutations led to different spectrums of phenotypes. Quantitative proteomics demonstrated changes in dynein-2 assembly, whereas initiation and extension of the axoneme, IFT-B protein localization, transition zone (TZ) integrity, and Hedgehog signalling were also affected. Summary statementDisease-associated mutations in WDR34 are found to have diverse impacts on ciliogenesis and cilia function following stable expression in a WDR34 knockout cell model.

cell biology↗

bioRxiv2022

Transient DNA binding to gapped DNA substrates links DNA sequence to the single-molecule kinetics of protein-DNA interactions

Protein interactions with nucleic acids are central to all genetic processes and many biotechnological applications. While many sequence-dependent protein-DNA interactions have been studied in detail using single-molecule methods, there is no standard high-throughput way to link the complex single-molecule kinetics of protein-DNA interactions with the DNA sequence of a single molecule. Here we provide the missing link by introducing a single-molecule imaging method (Gap-Seq) that interrogates DNA sequences via transient binding of short fluorescent DNA to a single DNA molecule previously used to characterise a protein-DNA interaction. In Gap-Seq, we identify a base by the degree of binding of 6-9 nt-long DNAs to surface-immobilised DNA substrates featuring a short single-stranded gap. To facilitate detection, we also developed a fluorescence quenching strategy that allows single-molecule detection at up to 500 nM of unbound fluorescent DNA. We link single-base differences on single DNA molecules to the kinetics of protein-DNA interactions by studying the interaction of a transcription activator with its cognate site. Finally, we show that our assay can address mixed sequences by distinguishing between two different sequences immobilised on the same field of view, paving the way for interrogation of sequence libraries for both mechanistic work and biotechnological applications.

biophysics↗

bioRxiv2022

Functional and effective connectivity between dorsolateral prefrontal and subgenual anterior cingulate cortex depends on the timing of transcranial magnetic stimulation relative to the phase of prefrontal alpha EEG

BACKGROUNDThe communication through coherence model posits that brain rhythms are synchronized across different frequency bands and that effective connectivity strength between interacting regions depends on their phase relation. Evidence to support the model comes mostly from electrophysiological recordings in animals while evidence from human data is limited. METHODSHere, an fMRI-EEG-TMS (fET) instrument capable of acquiring simultaneous fMRI and EEG during noninvasive single pulse TMS applied to dorsolateral prefrontal cortex (DLPFC) was used to test whether prefrontal EEG alpha phase moderates TMS-evoked top-down influences on subgenual, rostral and dorsal anterior cingulate cortex (ACC). Results in healthy volunteers (n=11) were compared to those from patients with major depressive disorder (MDD) (n=17) collected as part of a ongoing clinical trial investigation. RESULTSIn both groups, TMS-evoked functional connectivity between DLPFC and subgenual ACC (sgACC) depended on the EEG alpha phase. TMS-evoked DLPFC to sgACC effective connectivity (EC) was moderated by EEG alpha phase in healthy volunteers, but not in the MDD patients. Top-down EC was inhibitory for TMS onsets during the upward slope of the alpha wave relative to TMS timed to the downward slope of the alpha wave. Prefrontal EEG alpha phase dependent effects on TMS-evoked fMRI BOLD activation of the rostral anterior cingulate cortex were detected in the MDD patient group, but not in the healthy volunteer group. DISCUSSIONResults demonstrate that TMS-evoked top-down influences vary as a function of the prefrontal alpha rhythm, and suggest clinical applications whereby TMS is synchronized to the brains internal rhythms in order to more efficiently engage deep therapeutic targets.

neuroscience↗

bioRxiv2021

Striatal compartmentalization and clustering of different subtypes of astrocytes is altered in the zQ175 Huntington's disease mouse model

Huntingtons disease (HD) is a devastating neurodegenerative disease that primarily affects the striatum, a brain region that controls movement and some forms of cognition. Dysfunction and loss of medium spiny neurons of the striatum is accompanied by astrogliosis (increased astrocyte density and pathology). For decades, astrocytes were considered a homogeneous cell type, but recent transcriptomic analyses revealed astrocytes are a heterogeneous population classified into multiple subtypes depending on the expression of different gene markers. Here, we studied whether three different striatal astrocyte subtypes expressing glutamine synthetase (GS), glial fibrillary acidic protein (GFAP), or S100 calcium-binding protein B (S100B) are differentially altered in HD. We conducted a comparative immunofluorescence analysis in the striatum of WT and the heterozygous zQ175 HD mouse model and found that the expression and abundance of GFAP+ and S100B+ astrocytes increased in zQ175 mice, while GS+ astrocytes showed no alteration. We then explored whether there was a differential spatial distribution of any of these subtypes within the striatum. We developed a systematic brain compartmentalization approach and found that while GS+ and S100B+ astrocytes were more homogeneously distributed throughout the striatum in zQ175 mice, GFAP+ astrocytes preferentially accumulated in the dorsomedial and dorsolateral striatum, which are regions associated with goal-directed and habitual behaviors. Additionally, GFAP+ astrocytes in zQ175 mice showed increased clustering, a parameter that indicates increased proximity and that is associated with localized inflammation and/or neurodegeneration. Our data suggest a differential susceptibility in both increased density and striatal compartmentalization of different subtypes of astrocytes in zQ175. These results highlight new potential implications for our understanding of astrocyte pathology in HD.

neuroscience↗

bioRxiv2021

Gene losses in the common vampire bat illuminate molecular adaptations to blood feeding

Feeding exclusively on blood, vampire bats represent the only obligate sanguivorous lineage among mammals. To uncover genomic changes associated with adaptations to this unique dietary specialization, we generated a new haplotype-resolved reference-quality genome of the common vampire bat (Desmodus rotundus) and screened 26 bat species for genes that were specifically lost in the vampire bat lineage. We discovered previously-unknown gene losses that relate to metabolic and physiological changes, such as reduced insulin secretion (FFAR1, SLC30A8), limited glycogen stores (PPP1R3E), and a distinct gastric physiology (CTSE). Other gene losses likely reflect the biased nutrient composition (ERN2, CTRL) and distinct pathogen diversity of blood (RNASE7). Interestingly, the loss of REP15 likely helped vampire bats to adapt to high dietary iron levels by enhancing iron excretion and the loss of the 24S-hydroxycholesterol metabolizing enzyme CYP39A1 could contribute to their exceptional cognitive abilities. Finally, losses of key cone phototransduction genes (PDE6H, PDE6C) suggest that these strictly-nocturnal bats completely lack cone-based vision. These findings enhance our understanding of vampire bat biology and the genomic underpinnings of adaptations to sanguivory.

evolutionary biology↗

bioRxiv2021

Global and Gene-specific Transcriptional Responses to Acute Stress

Nucleosomes may regulate transcription by controlling access to promoters by transcription factors and RNA polymerase II (Pol2). Potentially active genes display nucleosome depleted regions flanked by positioned -1 and +1 nucleosomes. On yeast genes, the transcription start site (TSS) is on the upstream face of the +1 nucleosome, but whether precise +1 nucleosome positioning controls Pol2 access to the TSS remains unclear. Here, using acute nutrient starvation to rapidly reprogramme the genome, we show highly dynamic upstream or downstream shifts in the position of +1 nucleosomes, coincident with levels of transcriptionally engaged Pol2 at 58% of genes. Transcript level changes broadly reflect Pol2 occupancy changes with a delay but can be further influenced by Pub1 or Puf3 dependent changes in transcript degradation rates. The response to acute stress has a second component as we also observed genome-wide changes in Pol2 distribution on genes, independent of changes in Pol2 occupancy, with Pol2 accumulating upstream of a +170 nt stalling site. Mathematical modelling supports a global increase in promoter-proximal early transcription termination as a major component of the global stress response. Thus, we uncover a two-component transcriptional response to stress, one focused on the +1 nucleosome, the second on Pol2 itself. O_LIA two-component responses to acute stress involving a gene-specific response and a global response C_LIO_LIDynamic shifting of +1 nucleosome position with transcriptional activation or repression. C_LIO_LIGlobal targeting of Pol2 leading to early transcription termination on acute stress C_LI

molecular biology↗

bioRxiv2021

Mapping Human Transient Transcriptomes Using Single Nucleotide Resolution 4sU Sequencing (SNU-Seq)

Genomes are pervasively transcribed, leading to stable and unstable transcripts that influence 3-dimensional genome organisation and gene regulation. High sensitivity and nucleotide resolution are required to resolve mammalian nascent transcriptomes. Here, we exploit the sensitivity of 4-thio-uridine (4sU) metabolic pulse-labelling to develop two nucleotide-resolution methods: Single-Nucleotide resolution 4sU sequencing (SNU-Seq) and size-fractionated 4sU-Seq (sf4sU-Seq). sf4sU-Seq involves gel isolation of abundant 4sU-labelled promoter proximal nascent transcripts, enabling nucleotide resolution mapping of transcription start sites and promoter proximal pauses (PPPs) on the same transcript. SNU-Seq exploits 3 end RNA-Seq, using bacterial poly(A) polymerase (bPAP) to polyadenylate the 3 ends of nascent transcripts and create oligo(dT)-primed libraries. The artificial poly(A) tail marks the precise position of polymerase on a transcription unit. SNU-Seq read levels are similar at pre-mRNAs and enhancers genome-wide and read spikes in pre-mRNA outputs map pauses, PPPs and polyadenylation sites. SNU-Seq enables discovery of thousands of unannotated regions of divergent transcription and helps define hundreds of the more than 10,000 regions of primed non-transcribed acetylated open chromatin that induce divergent nascent transcripts within 0.5h of IFN-{gamma} treatment in Hep3B cells. Thus, combining chromatin analysis with SNU-Seq reveals the transcriptional responsiveness of an epigenetically primed human genome. HIGHLIGHTSO_LISNU-Seq maps nascent transcripts with bp resolution, high sensitivity and low cost C_LIO_LIsf4sU-Seq resolves TSS and PPP at the same gene, complementing SNU-Seq C_LIO_LI1000s of divergently transcribed enhancers resolved by SNU-Seq C_LIO_LIRapid IFN{gamma} dependent transcriptional induction from primed Hep3B epigenome C_LI GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=165 SRC="FIGDIR/small/452379v2_ufig1.gif" ALT="Figure 1"> View larger version (47K): org.highwire.dtl.DTLVardef@da6239org.highwire.dtl.DTLVardef@16692c6org.highwire.dtl.DTLVardef@12e30f7org.highwire.dtl.DTLVardef@190c98f_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2021

Spt4 Facilitates the Movement of RNA Polymerase II through the +2 Nucleosomal Barrier

Spt4 is a transcription elongation factor, with homologues in organisms with nucleosomes. Structural and in vitro studies implicate Spt4 in transcription through nucleosomes, yet the in vivo function of Spt4 is unclear. Here we assessed the precise position of Spt4 during transcription and the consequences of loss of Spt4 on RNA polymerase II (RNAPII) dynamics and nucleosome positioning in Saccharomyces cerevisiae. In the absence of Spt4, the spacing between gene-body nucleosomes increases and RNAPII accumulates upstream of the nucleosomal dyad, most dramatically at nucleosome +2. Spt4 associates with elongating RNAPII early in transcription and its association dynamically changes depending on nucleosome positions. Together, our data show that Spt4 regulates early elongation dynamics, participates in co-transcriptional nucleosome positioning, and promotes RNAPII movement through the gene-body nucleosomes, especially the +2 nucleosome.

genomics↗

bioRxiv2021

The Organelle in the Room: Under-annotated Mitochondrial Reads Bias Coral Microbiome Analysis

The genomes of mitochondria and chloroplasts contain ribosomal RNA (rRNA) genes, reflecting their evolutionary ancestry as free-living bacteria prior to endosymbiosis. In microbiome studies of animals, plants, or other eukaryotic hosts, these organellar rRNAs are often amplified. If identified, they can be discarded, merely reducing sequencing depth. However, incorrectly annotated mitochondrial reads may compromise statistical analysis by distorting relative abundances of free-living microbes. We quantified this by reanalyzing 7,459 samples from seven 16S rRNA sequencing studies, including the microbiomes of 927 unique animal genera. We find that under-annotation of cryptic mitochondrial reads affects multiple of these large-scale cross-species microbiome comparisons, and can be severe in some samples. It also varies between host species, potentially biasing cross-species microbiome comparisons. We propose a straightforward solution: by supplementing existing taxonomies with diverse mitochondrial rRNA sequences, we resolve up to 97% of unique unclassified sequences in some entire studies as mitochondrial (14% averaged across all studies), without increasing false positive annotations in mitochondria-free mock communities. Overall, improved annotation decreases the proportion of unknown sequences by [≥]10-fold in 2,262 of 7,459 samples (30%), including representatives from 5 of 7 studies examined. While standard DADA2 analyses are severely affected, the default positive filter in Deblur run through QIIME2 discards many divergent mitochondrial sequences, preventing bias in analysis, but also making analysis of these sequences more difficult. We recommend leveraging mitochondrial sequence diversity to better identify, remove and analyze mitochondrial rRNA gene sequences in microbiome studies.

bioinformatics↗

bioRxiv2021

Highly accurate barcode and UMI error correction using dual nucleotide dimer blocks allows direct single-cell nanopore transcriptome sequencing

Droplet-based single-cell sequencing techniques have provided unprecedented insight into cellular heterogeneities within tissues. However, these approaches only allow for the measurement of the distal parts of a transcript following short-read sequencing. Therefore, splicing and sequence diversity information is lost for the majority of the transcript. The application of long-read Nanopore sequencing to droplet-based methods is challenging because of the low base-calling accuracy currently associated with Nanopore sequencing. Although several approaches that use additional short-read sequencing to error-correct the barcode and UMI sequences have been developed, these techniques are limited by the requirement to sequence a library using both short- and long-read sequencing. Here we introduce a novel approach termed single-cell Barcode UMI Correction sequencing (scBUC-seq) to efficiently error-correct barcode and UMI oligonucleotide sequences synthesized by using blocks of dimeric nucleotides. The method can be applied to correct either short-read or long-read sequencing, thereby allowing users to recover more reads per cell and permits direct single-cell Nanopore sequencing for the first time. We illustrate our method by using species-mixing experiments to evaluate barcode assignment accuracy and evaluate differential isoform usage and fusion transcripts using myeloma and sarcoma cell line models.

genomics↗

bioRxiv2020

Single cell RNA sequence analysis of human bone marrow samples reveals new targets for isolation of skeletal stem cells using DNA-coated gold nanoparticles

There is a wealth of data indicating human bone marrow derived stromal cells (HBMSCs) contain the skeletal stem cell (SSC) with the potential to differentiate along the stromal osteogenic, adipogenic and chondrogenic lineages. However, despite these advances, current methods to isolate skeletal stem cells (SSCs) from human tissues have proved challenging as no single specific marker has been identified limiting understanding of SSC fate, immunophenotype and the widespread clinical application of these cells. While a number of cell surface markers can enrich for SSCs, none of the proposed markers, alone, provide a platform to isolate single cells with the ability to form bone, cartilage, and adipose tissue in humans. The current study details the application of oligonucleotide-coated nanoparticles, spherical nucleic acids (SNAs), to rapidly isolate human cells using mRNAs signatures detected in SSCs in real time, to identify stem and progenitor skeletal populations using single cell RNA sequencing. Based on scRNA-seq of samples from 11 patients, this method was able to identify novel targets for SSC enrichment, which were assessed in a total of 80 patients. This methodology was able to isolate potential SSCs found at a frequency of <1 in 1,000,000 in human bone marrow, with a capacity for tri-lineage differentiation in vitro. The current approach provides new targets and a platform to advance SSC isolation, enrichment with significant therapeutic impact therein.

cell biology↗

bioRxiv2020

Using a bistable animal opsin for switchable and scalable optogenetic inhibition of neurons

There is no consensus on the best optogenetic tool for neuronal inhibition. Lamprey parapinopsin ( Lamplight) is a Gi/o-coupled bistable animal opsin that can be activated and deactivated by short and long wavelength light, respectively. Since native mechanisms of neuronal inhibition frequently employ Gi/o signalling, we asked here whether Lamplight could be used for optogenetic silencing. We show that short (405nm) and long (525nm) wavelength pulses repeatedly switch Lamplight between stable signalling active and inactive states, and that combining these wavelengths can be used to achieve intermediate levels of activity. We demonstrate that these properties can be applied to produce switchable and scalable neuronal hyperpolarisation, and suppression of spontaneous spike firing in the mouse hypothalamic suprachiasmatic nucleus. We show that expressing Lamplight in (predominantly) ON bipolar cells can photosensitise retinas following advanced photoreceptor degeneration, and that 405 and 525nm stimuli can produce responses of opposite sign in output neurons of the retina. Lamplight-driven responses to both activating (405nm) and deactivating (525nm) light can occur within 500ms and be elicited by intensities at least 10x below threshold for available inhibitory optogenetic tools. We conclude that Lamplight can co-opt endogenous signalling mechanisms to allow optogenetic inhibition that is scalable, sustained and rapidly reversible.

neuroscience↗

bioRxiv2020

INSIGHT: a scalable isothermal NASBA-based platform for COVID-19 diagnosis

We present INSIGHT (Isothermal NASBA-Sequencing-based hIGH-througput Test): a two-stage COVID-19 testing strategy, using a barcoded isothermal NASBA reaction that combines point-of-care diagnosis with next generation sequencing, aiming to achieve population-scale COVID-19 testing. INSIGHT combines the advantages of near-patient with centralised testing. Stage 1 allows a quick decentralised readout for early isolation of pre-symptomatic or asymptomatic patients. The same reaction products can then be used in a highly multiplexed sequencing-based assay in Stage 2, confirming the near-patient testing results and facilitating centralised data collection. Based on experiments using commercially acquired human saliva with spiked-in viral RNA as input, the INSIGHT platform gives Stage 1 results within one to two hours, using either fluorescence detection or a lateral flow (dipstick) readout, whilst simultaneously incorporating sample-specific barcodes into the amplification product. INSIGHT Stage 2 can be performed by directly pooling and sequencing all post-amplification barcoded Stage 1 products from hundreds of thousands of samples with minimal sample preparation steps. The 95% limit of detection (LoD-95) for INSIGHT is estimated to be below 50 copies of viral RNA per 20 l of reaction. Our two-stage testing strategy is suitable for further development into a rapid home-based and point-of-care assay, and is potentially scalable to the population level.

molecular biology↗

bioRxiv2020

Targeted high-resolution chromosome conformation capture at genome-wide scale.

Chromosome conformation capture (3C) provides an adaptable tool for studying diverse biological questions. Current 3C methods provide either low-resolution interaction profiles across the entire genome, or high-resolution interaction profiles at up to several hundred loci. All 3C methods are affected to varying degrees by inefficiency, bias and noise. As such, generation of reproducible high-resolution interaction profiles has not been achieved at scale. To overcome this barrier, we systematically tested and improved upon current methods. We show that isolation of 3C libraries from intact nuclei, as well as shortening and titration of enrichment oligonucleotides used in high-resolution methods reduces noise and increases on-target sequencing. We combined these technical modifications into a new method Nuclear-Titrated (NuTi) Capture-C, which provides a >3-fold increase in informative sequencing content over current Capture-C protocols. Using NuTi Capture-C we target 8,061 promoters in triplicate, demonstrating that this method generates reproducible high-resolution genome-wide 3C interaction profiles at scale.

genomics↗