Biology subjects

Moran, J.

Publications and source records attributed to Moran, J..

At least 19 recordsLinked to original sources

bioRxiv2026

ASAREE: An Analytical Sandbox for Agentic AI Research, Engineering, and Experimentation

Summary: Agentic AI platforms enable the engineering of autonomous workflows but are not designed for experimentation and hypothesis testing. ASAREE (Analytical Sandbox for Agentic AI Research, Engineering, and Experimentation), is an open-source platform to address this gap. ASAREE creates agents, connects to MCP servers and tools, and designs factorial experiments through a visual interface or Python SDK. It records a full provenance trace for every run and routes all model calls through a provider-agnostic bridge that supports local deployments, ensuring data privacy. As a use-case, we use ASAREE to evaluate key design choices in a mutli-agent machine learning pipeline. Across a 2 x 2 x 2 factorial design, more advanced models, greater reasoning effort, and critic agent use significantly increased compute time, token use, cost, and feature count without improving predictive performance. The lowest-cost baseline, Claude Sonnet 5 with medium effort and no critic, achieved the highest mean PR AUC while Claude Opus 5 with extra high effort and a critic agent cost 15.5x more (USD) and ran 13.1x longer while performing worse on average. These findings highlight ASAREE as a robust framework for evaluating agentic system performance and resource efficiency.

bioinformatics↗

bioRxiv2026

Dystrophic changes of nigrostriatal axons harboring a Synj1 Parkinson mutation suggest catastrophic failure of endocytic mechanisms

Synaptojanin 1 is a brain enriched phosphoinositide phosphatase implicated in endocytosis at the synapse. A mutation (R258Q) that selectively impairs its Sac1 phosphatase domain causes early onset familial Parkinsonism. Neurons of mice with this mutation display synaptic vesicle traffic defects across the brain, but selective dystrophic changes in a subset of dopaminergic axons in the dorsolateral striatum. Using correlative light microscopy-FIB-SEM of mutant mouse striata to visualize in 3D these abnormal structures we show that they represent clusters of focal axonal dilations harboring massive, onion-like DAT enriched plasma membrane infoldings, generally localized next to cell bodies of neighboring cells, often engulfing evaginations of such cells. This dysmorphia was associated with a deficit in dopamine release in the same striatal region. Given the involvement of Synj1 in endocytic mechanisms, these structures may reflect an imbalance between exocytosis and endocytosis. Their occurrence only in a subset of axons suggest a vulnerability threshold of these axons beyond which the expansion of the plasma membrane is not counteracted by compensatory mechanisms.

neuroscience↗

bioRxiv2026

Heritable single-cell gene expression states shape functional variability in innate immune responses

Activation of innate immunity at the single-cell level is inherently heterogeneous, yet the mechanisms underlying this variability remain incompletely understood. Here, we integrate transcriptomics, high-content imaging and mathematical modelling to quantify transcriptional heritability within the evolutionarily conserved Toll-like receptor (TLR) system. RNA-seq-based fluctuation tests identified a subset of TLR4-dependent genes, including cytokines and immune effectors, that retain transcriptional heritability for more than 25 cell divisions in clonal macrophage populations. High-content microscopy confirmed gene-specific propagation of heritable states and revealed that environmental context shapes their persistence and expression. CD36, a scavenger receptor involved in bacterial recognition and lipid uptake, exhibited a stable, cell density-reinforced heritable state, whereas the inflammatory programme exemplified by IL1{beta} was transient, with heritability decaying upon clonal expansion. The interplay between heritable transcriptional states and population context generated emergent spatial organisation in high-density populations, with CD36-high cells forming discrete pockets and IL1{beta}-high cells enriched in surrounding regions. Functionally, CD36 expression determined clonal susceptibility to Listeria monocytogenes infection, linking transcriptional heritability to heterogeneous infection outcomes. Together, these findings identify transcriptional heritability as a key determinant of innate immune heterogeneity and demonstrate how heritable cellular states interact with population context to generate complex immune behaviours. Key point summaryO_LIMemorySeq and scRNA-seq identify long-term heritable gene expression states within TLR4-induced macrophage populations. C_LIO_LIHigh-content imaging across thousands of clonal populations reveals gene-specific dynamics of heritable states at the protein level. C_LIO_LIIl1{beta} and Cd36 define mutually exclusive heritable states that are differently regulated by population context and drive spatial organisation in cellular monolayers. C_LIO_LIHeritable CD36 protein expression shapes heterogeneous outcomes during Listeria monocytogenes infection. C_LI

immunology↗

bioRxiv2026

Phosphoglycerate mutase 5 regulates lipid metabolism and mitochondrial homeostasis in hepatocellular cancer cells

The mitochondrial membrane protein phosphoglycerate mutase 5 (PGAM5) is a protein of interest in the complex transition from hepatic steatosis to hepatocellular carcinoma. PGAM5 is a serine/threonine/histidine phosphatase that plays a role in mitochondrial biogenesis, mitophagy, and multiple cell death pathways. Increased expression of PGAM5 in hepatocellular carcinoma is correlated with reduced patient survival. In this study, we demonstrate that loss of PGAM5 alters the bioenergetic landscape of liver cancer by promoting mitochondrial oxidant injury and suppressing the glycerophospholipid and lysophospholipid pathways, leading to accumulation of the bioactive phospholipid lysophosphatidylcholine. Additionally, PGAM5 deletion downregulates fatty acid biosynthesis, resulting in reduced cellular diacylglycerol concentrations through two probable mechanisms: attenuated long chain fatty acid uptake and suppressed de novo synthesis. These findings underscore the broad impact of a single phosphatase on mitochondrial function and provide a rationale for therapeutically targeting PGAM5 to disrupt lipid metabolism in hepatocellular carcinoma.

cancer biology↗

bioRxiv2026

Minding the gap: collective determinants of multiscale structure across interacting bacterial colonies

A bacterial colony rarely exists in isolation - in natural habitats, colonies interact to form spatially structured communities across length and time scales. Eco-evolutionary feedbacks link these scales, such that structure at one level can influence another, yet the interplay between single- and multi-colony organization remains poorly understood. As a step toward addressing this, we develop a high-throughput platform to track population dynamics across spatially extended networks of colonies. A common structural feature observed at the multi-colony scale is the formation of a stable gap region between colonies, even when they are isogenic. Numerous studies observe similar patterns of behavior across species, with few resolving the underlying mechanism. Here, we ask: what are the minimal ingredients shaping this multi-colony structure? We focus on colonies of the opportunistic pathogen Enterococcus faecalis, a model organism for which this behavior has yet to be reported. By combining modeling and experiments, we show that both nutrient competition and direct growth inhibition control colony morphology and expansion of interacting colonies. We identify distinct regimes of gap formation, relating intra- and inter-colony spatial patterns to ecological interactions mediated at the cellular scale. Together, our results suggest that antagonism, even between isogenic populations through self-inhibition, is likely a common behavior of bacterial species in general.

ecology↗

bioRxiv2025

A phylogenetic estimate of canine retrotransposition rates based on genome assembly comparisons

BackgroundDue to their history of domestication and breed formation, dogs are a powerful system for studying the phenotypic impact of genetic variation. Comparison of canine genome assemblies show that retrotransposons, mobile elements that mobilize via an RNA intermediate, are a major contributor to canine genetic diversity with an eightfold and 17-fold increase of LINE-1 and SINE differences, respectively, in dogs relative to that found among humans. The frequency of dimorphic retrotransposon insertions among dogs suggests these elements have mobilized at a high rate over recent canine evolution. However, the rate at which new insertions arise has yet to be determined. ResultsWe aligned a collection of genome assemblies derived from four breed dogs, a Dingo, and an American grey wolf to a Greenland grey wolf to identify dimorphic LINE-1 and SINEC insertions. Across our panel of seven canine assemblies, we identified and characterized 7,428 dimorphic LINE-1s and 51,572 dimorphic SINECs. Each assembly differs from the Greenland wolf genome by an average of 3,497 LINE-1s and 25,558 SINECs. Analysis of allele sharing among samples recapitulates known relationships and reveals substantial within-breed variation. Calibrating estimates using a previously estimated single nucleotide mutation rate of 4.5x10-9 per base pair per generation, we estimate that new LINE-1 and SINEC and insertions have occurred at a rate of 1/184 and 1/22 births over recent canine evolution. These estimates are largely consistent across assemblies and breeds. ConclusionsOur phylogenetic estimate of SINEC retrotransposition in canines is approximately twice as large as that estimated for Alu elements in humans, while the canine LINE-1 insertion rate is within the range of human estimates. These data suggest that although SINEC has been an outsized driver of canine genome evolution, the striking levels of canine LINE-1 and SINEC dimorphism mainly reflect high levels of long-standing genetic variation.

genomics↗

bioRxiv2024

ENaC contributes to macrophage dysfunction in cystic fibrosis

BackgroundCystic fibrosis (CF) is a chronic systemic disease caused by dysfunctional or absent cystic fibrosis transmembrane conductance regulator (CFTR). CFTR is expressed in human immune cells and plays a role in regulating innate immunity both directly and indirectly. Besides CFTR, research indicates that the epithelial sodium channel (ENaC) also contributes to dysfunction in CF airway epithelial cells. However, the impact of non-CFTR ion channel dysfunction on CF immune responses is not yet fully understood. A precise understanding of how CF immune function is regulated by ion channels may allow antibiotic-and mutation-agnostic treatment approaches to chronic bacterial infection and inflammation. Therefore, we hypothesized that ENaC is aberrantly expressed in CF macrophages and directly contributes to impaired phagocytic and inflammatory functions. MethodsENaC expression was characterized in human immune cells isolated from CF and non-CF blood donors. Monocyte-derived macrophage (MDM) function and bacterial killing was tested in the setting of ENaC modulation. ResultsBaseline expression of ENaC in human CF MDMs, lymphocytes, and granulocytes was increased at both the transcript and protein level relative to non-CF controls and persisted after exposure to bacteria. Inhibition of CFTR in non-CF MDMs resulted in ENaC overexpression. CFTR modulator treatment reduced but did not eliminate ENaC overexpression in CF MDMs. Interestingly, ENaC inhibition with Amiloride increased CFTR expression. Amiloride-treated CF MDMs also showed normalized ROS production, improved autophagy, and decreased pro-inflammatory cytokine production. Finally, results from an ion channel microarray indicated that sodium channel expression in CF MDMs normalized after Amiloride treatment with minimal effect on other ion channels. DiscussionENaC is overexpressed in CF immune cells and is associated with abnormal macrophage function. ENaC modulation in immune cells is a novel potential therapeutic target for infection control in CF, either in combination with CFTR modulators, or as a sole agent for patients not currently eligible for CFTR modulators.

immunology↗

bioRxiv2024

TDP43 autoregulation gives rise to shortened isoforms that are tightly controlled by both transcriptional and post-translational mechanisms

The nuclear RNA-binding protein TDP43 is integrally involved in the pathogenesis of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). Previous studies uncovered N-terminal TDP43 isoforms that are predominantly cytosolic in localization, highly prone to aggregation, and enriched in susceptible spinal motor neurons. In healthy cells, however, these shortened (s)TDP43 isoforms are difficult to detect in comparison to full-length (fl)TDP43, raising questions regarding their origin and selective regulation. Here, we show that sTDP43 is created as a byproduct of TDP43 autoregulation and cleared by nonsense mediated RNA decay (NMD). The sTDP43-encoding transcripts that escape NMD can lead to toxicity but are rapidly degraded post-translationally. Circumventing these regulatory mechanisms by overexpressing sTDP43 results in neurodegeneration in vitro and in vivo via N-terminal oligomerization and impairment of flTDP43 splicing activity, in addition to RNA binding-dependent gain-of-function toxicity. Collectively, these studies highlight endogenous mechanisms that tightly regulate sTDP43 expression and provide insight into the consequences of aberrant sTDP43 accumulation in disease.

neuroscience↗

bioRxiv2024

Variable patterns of retrotransposition in different HeLa strains provide mechanistic insights into SINE RNA mobilization processes

Alu elements are non-autonomous Short INterspersed Elements (SINEs) derived from the 7SL RNA gene that are present at over one million copies in human genomic DNA. Alu mobilizes by a mechanism known as retrotransposition, which requires the Long INterspersed Element-1 (LINE-1 or L1) ORF2-encoded protein (ORF2p). Here, we demonstrate that HeLa strains differ in their capacity to support Alu retrotransposition. Human Alu elements retrotranspose efficiently in HeLa-HA and HeLa-CCL2 (Alu-permissive) strains, but not in HeLa-JVM or HeLa-H1 (Alu-nonpermissive) strains. A similar pattern of retrotransposition was observed for other 7SL RNA-derived SINEs and tRNA-derived SINEs. In contrast, mammalian LINE-1s, a zebrafish LINE, a human SINE-VNTR-Alu (SVA) element, and an L1 ORF1-containing messenger RNA can retrotranspose in all four HeLa strains. Using an in vitro reverse transcriptase-based assay, we show that Alu RNAs associate with ORF2p and are converted into cDNAs in both Alu-permissive and Alu-nonpermissive HeLa strains, suggesting that 7SL- and tRNA-derived SINE RNAs use strategies to hijack' L1 ORF2p that are distinct from those used by SVA elements and ORF1-containing mRNAs. These data further suggest ORF2p associates with the Alu RNA poly(A) tract in both Alu-permissive and Alu-nonpermissive HeLa strains, but that Alu retrotransposition is blocked after this critical step in Alu-nonpermissive HeLa strains.

molecular biology↗

bioRxiv2024

Emergent predictability in microbial ecosystems

Microbial ecosystems exhibit a surprising amount of functionally relevant diversity at all levels of taxonomic resolution, presenting a significant challenge for most modeling frameworks. A long-standing hope of theoretical ecology is that some patterns might persist despite community complexity - or perhaps even emerge because of it. A deeper understanding of such "emergent simplicity" could enable new approaches for predicting the behaviors of the complex ecosystems in nature. However, the concept remains partly intuitive with no consistent definition, and most empirical examples described so far afford limited predictive power. Here, we propose an information-theoretic framework for defining and quantifying emergent simplicity in empirical data based on the ability of coarsened descriptions to predict community-level functional properties. Applying this framework to two published datasets, we demonstrate that all five properties measured across both experiments exhibit robust evidence of what we define as "emergent predictability": surprisingly, as community richness increases, simple compositional descriptions become more predictive. We show that standard theoretical models of high-diversity ecosystems fail to recapitulate this behavior. This is in contrast to simple self-averaging, which is well-understood and generic across models. We propose that, counterintuitively, emergent predictability arises when physiological or environmental feedbacks oppose statistical self-averaging along some axes of community variation. As a result, these axes of variation become increasingly predictive of community function at high richness. We demonstrate this mechanism in a minimal model, and argue that explaining and leveraging emergent predictability will require integrating large-N theoretical models with a minimal notion of physiology, which the dominant modeling frameworks currently omit.

ecology↗

bioRxiv2024

Application of chimeric antigens to paper-based diagnostics for detection of West Nile virus infections of Crocodylus porosus: a novel animal test case

Laboratory-based diagnostics like plaque reduction neutralization tests (PRNT) and ELISA are commonly used to detect seroconversion to flavivirus infections. However, faster, qualitative screening methods are needed for quicker diagnosis and better patient outcomes. Lateral flow assays (LFAs) can provide rapid results (5-15 mins) at the point-of-care, yet few commercial flavivirus antibody detection LFAs are available. We developed an LFA using novel chimeric viral antigens produced by genetically modifying the mosquito restricted Binjari virus (BinJV) to display the outer virion proteins of pathogenic viruses such as West Nile virus (WNV). The BinJV chimeric platform offers various advantages for diagnostic assay development, including rapid construction of new chimeras in response to emerging viral variants, safe, scalable antigen manufacturing, and structural indistinguishability to the wild-type pathogenic virion. As a demonstration of feasibility, we applied chimeric WNV (BinJV/WNV) antigen to LFA as the capture/test line reagent for detection of seroconversion of crocodilians to WNV - a virus affecting crocodilians on multiple continents. We verified the antigenic conservation of the chimera when applied to the LFA detection surface using monoclonal antibodies. Using well-characterised sera (n=60) from WNV seropositive or flavivirus naive Australian saltwater crocodiles (Crocodylus porosus), we illustrated 100% sensitivity and specificity, with results achieved in less than 15 minutes. The LFA further accurately detected seroconversion in animals experimentally infected with WNV. This qualitative screening method can be performed both inside and outside of a laboratory, and the assay design will guide the optimization of similar tests for vector borne virus infection detection in both humans and other animals.

biochemistry↗

bioRxiv2023

Modulation of Polar Auxin Transport Identifies the Molecular Determinants of Source-Sink Carbon Relationships and Sink Strength in Poplar

Source-to-sink carbon (C) allocation driven by the sink strength, i.e., the ability of a sink organ to import C, plays a central role in tissue growth and biomass productivity. However, molecular drivers of sink strength have not been thoroughly characterized in trees. Auxin, as a major plant phytohormone, regulates the mobilization of photoassimilates in source tissues and elevates the translocation of carbohydrates toward sink organs, including roots. In this study, we used an auxin-stimulated carbon sink approach to understand the molecular processes involved in the long-distance source-sink C allocation in poplar. Poplar cuttings were foliar sprayed with polar auxin transport modulators, including auxin enhancers (AE) (i.e., IBA and IAA) and auxin inhibitor (AI) (i.e., NPA), followed by a comprehensive analysis of leaf, stem, and root tissues using biomass evaluation, phenotyping, C isotope labeling, metabolomics, and transcriptomics approaches. Auxin modulators altered root dry weight and branching pattern, and AE increased photosynthetically fixed C allocation from leaf to root tissues. The transcriptome analysis identified highly expressed genes in root tissue under AE condition including transcripts encoding polygalacturonase and {beta}-amylase that could increase the sink size and activity. Metabolic analyses showed a shift in overall metabolism including an altered relative abundance levels of galactinol, and an opposite trend in citrate levels in root tissue under AE and AI conditions. In conclusion, we postulate a model suggesting that the source-sink C relationships in poplar could be fueled by mobile sugar alcohols, starch metabolism-derived sugars, and TCA-cycle intermediates as key molecular drivers of sink strength.

plant biology↗

bioRxiv2022

Single-cell imaging reveals non-cooperative and cooperative infection strategies of Listeria monocytogenes in macrophages

Pathogens have developed intricate strategies to overcome the hosts innate immune responses. In this paper we use live-cell microscopy with a single bacterium resolution to follow in real time interactions between the food-borne pathogen L. monocytogenes and host macrophages, a key event controlling the infection in vivo. We demonstrate that infection results in heterogeneous outcomes, with only a subset of bacteria able to establish a replicative invasion of macrophages. The fate of individual bacteria in the same host cell was independent from each other and non-cooperative, but a higher multiplicity of infection resulted in a reduced probability of replication. Using internalisation assays and conditional probabilities to mathematically describe the multi-stage invasion process, we demonstrate that the secreted Listeriolysin toxin (LLO) of the PrfA regulon regulates replication probability by compromising the ability to phagocytose bacteria. Using strains expressing fluorescent reporters to follow transcription of either the LLO-encoding hly or actA genes, we show that replicative bacteria exhibited higher PrfA regulon expression in comparison to those bacteria that did not replicate, however elevated PrfA expression per se was not sufficient to increase the probability of replication. Overall, this demonstrates a new role for the population-level, but not single cell PrfA-mediated cooperativity to regulate outcomes of host pathogen interactions. Key pointsO_LIL. monocytogenes invasion of innate immune macrophages results in heterogeneous infection outcomes at the single cell level C_LIO_LIFate of individual bacteria in the same host cell is independent from each other and non-cooperative C_LIO_LIBacterial populations coordinate host cell uptake via the rate of phagocytosis to reduce internalization at high MOI C_LIO_LIThe PrfA regulon system is necessary but not sufficient for L. monocytogenes replication, but population-level PrfA virulence regulates single cell outcome probability C_LI

microbiology↗

bioRxiv2022

Elaboration of a MALDI-TOF Mass Spectrometry-based Assay of Parkin Activity and High-Throughput screening platform for Parkin Activators

Parkinsons disease (PD) is a progressive neurological disorder that manifests clinically as alterations in movement (bradykinesia, postural instability, loss of balance, and resting tremors) as well as multiple non-motor symptoms including but not limited to cognitive and autonomic abnormalities. Mitochondrial dysfunction has been linked to sporadic PD and loss-of-function mutations in genes encoding the ubiquitin E3 ligase Parkin and protein kinase, PTEN-induced kinase 1 (PINK1), that regulate mitophagy, are causal for familial and juvenile PD1-3. Among several therapeutic approaches being explored to treat or improve PD patients prognosis, the use of small molecules able to reinstate or boost Parkin activity represents a potential pharmacological treatment strategy4. A major barrier is the lack of high throughput platforms based on robust and accurate quantification of Parkin activity in vitro. Here we present two different and complementary Matrix Assisted Laser Desorption/Ionization-Time of Flight mass spectrometry (MALDI-TOF MS) based approaches for the quantification of Parkin E3 ligase activity in vitro. These methods recapitulate distinct aspects of ubiquitin conjugation: Parkin auto-ubiquitylation and Parkin-catalysed discharge on lysine residues. Both approaches are scalable for high-throughput primary screening to facilitate the identification of Parkin modulators.

biochemistry↗

bioRxiv2021

Coarse-graining ecological dynamics in the face of unknown microscopic details

Despite their complexity, microbial ecosystems appear to be at least partially "coarse-grainable" in that some properties of interest can be adequately described by effective models of dimension much smaller than the number of interacting lineages (frequently in the dozens or hundreds). This is especially puzzling since recent studies demonstrate that a surprising amount of functionally relevant diversity is present at all levels of resolution, down to strains differing by 100 nucleotides or fewer. Rigorously defining coarse-grainability and understanding the conditions for its emergence is of critical importance for understanding microbial ecosystems. To begin addressing these questions, we propose a minimal model for investigating hierarchically structured ecosystems within the framework of resource competition. We use our model to operationally define coarse-graining quality based on reproducibility of the outcomes of a specified experiment and show that a coarse-graining can be operationally valid despite grouping together functionally diverse strains. We further show that, at least within our model, a high diversity of strains (while nominally more complex) may in fact facilitate coarse-grainability. However, this only applies if the pool of interacting strains is sampled from the appropriate "native" environment, as we discuss.

ecology↗

bioRxiv2019

Identification of a Novel Base J Binding Protein Complex Involved in RNA Polymerase II Transcription Termination in Trypanosomes

Base J, {beta}-D-glucosyl-hydroxymethyluracil, is a modification of thymine DNA base involved in RNA Polymerase (Pol) II transcription termination in kinetoplastid protozoa. Little is understood regarding how specific thymine residues are targeted for J-modification or the mechanism of J regulated transcription termination. To identify proteins involved in J-synthesis, we expressed a tagged version of the J-glucosyltransferase (JGT) in Leishmania tarentolae, and identified four co-purified proteins by mass spectrometry: protein phosphatase (PP1), a homolog of Wdr82, a potential PP1 regulatory protein (PNUTS) and a protein containing a J-DNA binding domain (named JBP3). Gel shift studies indicate JBP3 is a J-DNA binding protein. Reciprocal tagging, co-IP and sucrose gradient analyses indicate PP1, JGT, JBP3, Wdr82 and PNUTS form a multimeric complex in kinetoplastids, similar to the mammalian PTW/PP1 complex involved in transcription termination via PP1 mediated dephosphorylation of Pol II. Using RNAi and analysis of Pol II termination by RNA-seq and RT-PCR, we demonstrate that ablation of PNUTS, JBP3 and Wdr82 lead to defects in Pol II termination at the 3-end of polycistronic gene arrays in Trypanosoma brucei. Mutants also contain increased antisense RNA levels upstream of promoters, suggesting an additional role of the complex in regulating termination of bi-directional transcription. In addition, PNUTS loss causes derepression of silent Variant Surface Glycoprotein genes important for host immune evasion. Our results provide the first direct mechanistic link between base J and regulation of Pol II termination and suggest a novel molecular model for the role of the CTD of Pol II in terminating polycistronic transcription in trypanosomatids.\n\nAuthor SummaryTrypanosoma brucei is an early-diverged parasitic protozoan that causes African sleeping sickness in humans. The genome of T. brucei is organized into polycistronic gene clusters that contain multiple genes that are co-transcribed from a single promoter. We have recently described the presence of a modified DNA base J and variant of histone H3 (H3.V) at transcription termination sites within gene clusters where the loss of base J and H3.V leads to read-through transcription and the expression of downstream genes. We now identify a novel stable multimeric complex containing a J binding protein (JBP3), base J glucosyltransferase (JGT), PP1 phosphatase, PP1 interactive-regulatory protein (PNUTS) and Wdr82, which we refer to as PJW/PP1. A similar complex (PTW/PP1) has been shown to be involved in Pol II termination in humans and yeast. We demonstrate that PNUTS, JBP3 and Wdr82 mutants lead to read-through transcription in T. brucei. Our data suggest the PJW/PP1 complex regulates termination by recruitment to termination sites via JBP3-base J interactions and dephosphorylation of specific proteins (including Pol II and termination factors) by PP1. These findings significantly expand our understanding of mechanisms underlying transcription termination in eukaryotes, including divergent organisms that utilize polycistronic transcription and novel epigenetic marks such as base J and H3.V. The studies also provide the first direct mechanistic link between J modification of DNA at termination sites and regulated Pol II termination and gene expression in kinetoplastids.

biochemistry↗

bioRxiv2019

An integrative ENCODE resource for cancer genomics

ENCODE comprises thousands of functional genomics datasets, and the encyclopedia covers hundreds of cell types, providing a universal annotation for genome interpretation. However, for particular applications, it may be advantageous to use a customized annotation. Here, we develop such a custom annotation by leveraging advanced assays, such as eCLIP, Hi-C, and whole-genome STARR-seq on a number of data-rich ENCODE cell types. A key aspect of this annotation is comprehensive and experimentally derived networks of both transcription factors and RNA-binding proteins (TFs and RBPs). Cancer, a disease of system-wide dysregulation, is an ideal application for such a network-based annotation. Specifically, for cancer-associated cell types, we put regulators into hierarchies and measure their network change (rewiring) during oncogenesis. We also extensively survey TF-RBP crosstalk, highlighting how SUB1, a previously uncharacterized RBP, drives aberrant tumor expression and amplifies the effect of MYC, a well-known oncogenic TF. Furthermore, we show how our annotation allows us to place oncogenic transformations in the context of a broad cell space; here, many normal-to-tumor transitions move towards a stem-like state, while oncogene knockdowns show an opposing trend. Finally, we organize the resource into a coherent workflow to prioritize key elements and variants, in addition to regulators. We showcase the application of this prioritization to somatic burdening, cancer differential expression and GWAS. Targeted validations of the prioritized regulators, elements and variants using siRNA knockdowns, CRISPR-based editing, and luciferase assays demonstrate the value of the ENCODE resource.

genomics↗

bioRxiv2019

STARRPeaker: Uniform processing and accurate identification of whole human STARR-seq active regions

BackgroundHigh-throughput reporter assays, such as self-transcribing active regulatory region sequencing (STARR-seq), allow for unbiased and quantitative assessment of enhancers at a genome-wide scale. Recent advances in STARR-seq technology have employed progressively more complex genomic libraries and increased sequencing depths, to assay larger sized regions, up to the entire human genome. These advances necessitate a reliable processing pipeline and peak-calling algorithm. ResultsMost STARR-seq studies have relied on chromatin immunoprecipitation sequencing (ChIP-seq) processing pipelines. However, there are key differences in STARR-seq versus ChIP-seq. First, STARR-seq uses transcribed RNA to measure the activity of an enhancer, making an accurate determination of the basal transcription rate important. Second, STARR-seq coverage is highly non-uniform, overdispersed, and often confounded by sequencing biases, such as GC content and mappability. Lastly, here, we observed a clear correlation between RNA thermodynamic stability and STARR-seq readout, suggesting that STARR-seq may be sensitive to RNA secondary structure and stability. Considering these findings, we developed a negative-binomial regression framework for uniformly processing STARR-seq data, called STARRPeaker. In support of this, we generated whole-genome STARR-seq data from the HepG2 and K562 human cell lines and applied STARRPeaker to call enhancers. ConclusionsWe show STARRPeaker can unbiasedly detect active enhancers from both captured and whole-genome STARR-seq data. Specifically, we report [~]33,000 and [~]20,000 candidate enhancers from HepG2 and K562, respectively. Moreover, we show that STARRPeaker outperforms other peak callers in terms of identifying known enhancers with fewer false positives. Overall, we demonstrate an optimized processing framework for STARR-seq experiments can identify putative enhancers while addressing potential confounders.

bioinformatics↗