Biology subjects

Luo, X.

Publications and source records attributed to Luo, X..

At least 181 records · Page 10Linked to original sources

bioRxiv2021

Applying automated patch-clamp to disease modeling: recapitulate phenotypes of Brugada syndrome using iPSC-CMs

Recently, there have been great advances in cardiovascular channelopathy modeling and drug safety pharmacology using human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs). The automated patch-clamp (APC) technique overcomes the disadvantages of the manual patch-clamp (MPC) technique, which is labor intensive and gives low output. However, the application of the APC platform is still limited in iPSC-CM based research, due to the difficulty in maintaining the high quality of single iPSC-CMs during dissociation and recording. In this study, we improved the method for single iPSC-CM preparation by applying 2.5 M blebbistatin (BB, an excitation-contraction coupling uncoupler) throughout APC procedures (dissociation, filtration, storage, and recording). Under non-BB buffered condition, iPSC-CMs in suspension showed a severe bleb-like morphology. However, BB-supplement led to significant improvements in morphology and INa recording, and we even obtained several CMs that showed spontaneous action potentials with typical morphology. Furthermore, APC faithfully recapitulated the single-cell electrophysiological phenotypes of iPSC-CMs derived from Brugada syndrome patients, as detected with MPC. Our study indicates that APC is capable of replacing MPC in the modeling of cardiac channelopathies using human iPSC-CMs by providing high-quality data with higher throughput.

physiology↗

bioRxiv2021

A fluorescence-based genetic screen reveals diverse mechanisms silencing small RNA signaling in E. coli

As key players of gene regulation in many bacteria, small regulatory RNAs (sRNAs) associated with the RNA chaperone Hfq shape numerous phenotypic traits, including metabolism, stress response and adaptation as well as virulence. sRNAs can alter target mRNA translation and stability via base pairing. sRNA synthesis is generally under tight transcriptional regulation, but other levels of regulation of sRNA signaling are less well understood. Here we used a fluorescence-based functional screen to identify new regulators that can quench sRNA signaling of the iron-responsive sRNA RyhB in E. coli. The identified regulators fell into two classes, general regulators (affecting signaling by many sRNAs) and RyhB-specific regulators; we focused on the specific ones here. General regulators include three Hfq-interacting sRNAs, CyaR, ChiX and McaS, previously found to act through Hfq competition, RNase T, a 3'-5' exonuclease not previously implicated in sRNA degradation, and YhbS, a putative GCN5-related N-acetyltransferase (GNAT). Two new specific regulators were identified. AspX, a novel 3'end-derived small RNA, specifically represses RyhB signaling via an RNA sponging mechanism. YicC, a previously uncharacterized but widely conserved protein, triggers rapid RyhB degradation via collaboration with the exoribonuclease PNPase. These findings greatly expand our knowledge of regulation of bacterial sRNA signaling and suggest complex regulatory networks for controlling iron homeostasis in bacteria. The fluorescence-based genetic screen system described here is a powerful tool expected to accelerate the discovery of novel regulators of sRNA signaling in many bacteria. SignificanceThe ability to promptly switch genes on and off allows bacteria to adapt rapidly to changing environments for better survival. Many sRNAs, including RyhB, a sRNA made in response to iron starvation, are important switches in bacteria. We discovered new factors that can keep the sRNA switch off by using a facile genetic screen platform. These new factors include a new RNA sponge and an adaptor protein for a ribonuclease, providing new perspectives on controlling sRNA signaling in bacteria.

microbiology↗

bioRxiv2021

mRNA vaccine-induced SARS-CoV-2-specific T cells recognize B.1.1.7 and B.1.351 variants but differ in longevity and homing properties depending on prior infection status

While mRNA vaccines are proving highly efficacious against SARS-CoV-2, it is important to determine how booster doses and prior infection influence the immune defense they elicit, and whether they protect against variants. Focusing on the T cell response, we conducted a longitudinal study of infection-naive and COVID-19 convalescent donors before vaccination and after their first and second vaccine doses, using a high-parameter CyTOF analysis to phenotype their SARS-CoV-2-specific T cells. Vaccine-elicited spike-specific T cells responded similarly to stimulation by spike epitopes from the ancestral, B.1.1.7 and B.1.351 variant strains, both in terms of cell numbers and phenotypes. In infection-naive individuals, the second dose boosted the quantity and altered the phenotypic properties of SARS-CoV-2-specific T cells, while in convalescents the second dose changed neither. Spike-specific T cells from convalescent vaccinees differed strikingly from those of infection-naive vaccinees, with phenotypic features suggesting superior long-term persistence and ability to home to the respiratory tract including the nasopharynx. These results provide reassurance that vaccine-elicited T cells respond robustly to emerging viral variants, confirm that convalescents may not need a second vaccine dose, and suggest that vaccinated convalescents may have more persistent nasopharynx-homing SARS-CoV-2-specific T cells compared to their infection-naive counterparts.

immunology↗

bioRxiv2021

mRNA-decapping associated DcpS enzyme controls critical steps of neuronal development

Homozygous mutations in the gene encoding the scavenger mRNA-decapping enzyme, DcpS, have been shown to underlie developmental delay and intellectual disability. Intellectual disability is associated with both abnormal neocortical development and mRNA metabolism. However, the role of DcpS and its scavenger decapping activity in neuronal development is unknown. Here, we show that human neurons derived from patients with a DcpS mutation have compromised differentiation and neurite outgrowth. Moreover, in the developing mouse neocortex, DcpS is required for the radial migration, polarity, neurite outgrowth and identity of developing glutamatergic neurons. Collectively, these findings demonstrate that the scavenger mRNA decapping activity contributes to multiple pivotal roles in neural development, and further corroborate that mRNA metabolism and neocortical pathologies are associated with intellectual disability.

neuroscience↗

bioRxiv2021

Three bacterium-plasmid golden combinations facilitate the spread of ST11/CG258 carbapenemase-producing Klebsiella pneumoniae in China

Carbapenemase-producing Klebsiella pneumoniae (cpKP) poses serious threats to public health. Previous studies showed that only ST11/CG258-cpKP successfully disseminated in China, however, the underlying genetic bases are still unknown. We conducted a comprehensive genomic-epidemiology analysis on 420 cpKP isolates from 70 hospitals in 24 Chinese provinces during 2009-2017 based on short-/long-reads sequencing. Three golden combinations of host--blaKPC-carrying plasmids (Clade 3.1+3.2--IncFIIpHN7A8, Clade 3.1+3.2--IncFIIpHN7A8:IncR, Clade 3.3--IncFIIpHN7A8:IncpA1763-KPC) endowed cpKP with advantages both in genotypes (strong-correlation/co-evolution) and phenotypes (resistance/growth/competition), thereby facilitating nationwide spread of ST11/CG258-cpKP. Intriguingly, Bayesian skyline illustrated that the three golden combinations might directly lead to the strong population expansion during 2007-2008 and subsequent maintenance of the dissemination of ST11/CG258-cpKP after 2008. We tested drug-resistance profiles and proposed combination treatment regimens for CG258/non-CG258 cpKP. Our findings systematically revealed the molecular-epidemiology and genetic-basis for dissemination of Chinese ST11/CG258 cpKP and reminded us to monitor the golden combinations of cpKP- plasmid closely.

microbiology↗

bioRxiv2021

Exploration of clinical breakpoint of Danofloxacin for Glaesserella parasuis in plasma and in PELF

BackgroundTo establish the clinical breakpoint (CBP) of danofloxacin to G. parasuis, three cutoff values, including epidemiological cutoff value (ECV), pharmacodynamic cutoff value (COPD) and clinical cutoff value (COCL), was obtained in the present study. MethodsThe ECV was calculated using ECOFFinder base on MIC distribution of 347 G. parasuis collected from disease pigs. The COPD was established base on in vivo and ex vivo pharmacokinetic (PK) - pharmacodynamic (PD) modeling of danofloxacin both in plasma and pulmonary epithelial lining fluid (PELF) using Hill formula and Monte Carlo analysis. The COCL was established based on the relationship between possibility of cure (POC) and MIC in the clinical trials using "WindoW" approach, nonlinear regression and CART analysis. ResultsThe MIC50 and MIC90 of danofloxacin against 347 G. parasuis were 2 g/mL and 8 g/mL, respectively. The ECV value was set up as 8 g/mL using ECOFFinder. Concentration-time curve of danofloxacin indicated a two-compartment model for PK analysis. The PK parameters of the maximum concentration (Cmax) and area under concentration-time curve (AUC) in PELF were 3.67 {+/-} 0.25 g/mL and 24.28 {+/-} 2.70 h{middle dot}g/mL, higher than those in plasma (0.67 {+/-} 0.01g/mL and 4.47 {+/-} 0.51 h{middle dot}g/mL). The peak time (Tmax) in plasma was 0.23 {+/-} 0.07 h, shorter than that in PELF (1.61 {+/-} 0.15 h). The COPD in plasma and PELF were 0.125 g/mL and 0.5 g/mL, respectively. The COCL calculated by WindoW approach, nonlinear regression and CART analysis were 0.125[~]4 g/mL, 0.428 g/mL and 0.56 g/mL, respectively. The 0.5 g/mL was selected as eligible COCL. The ECV is much higher than the COPD and COCL, and the clinical breakpoint based on data in plasma was large different with that of in PELF. ConclusionsOur study firstly established three cutoff values of danofloxacin against G. parasuis. It suggested that epidemiological danofloxacin-resistant G. parasuis may lead to the ineffective treatment by danofloxacin. ImportanceG. parasuis, a gram-negative respiratory pathogen, can colonize in the upper respiratory tract in swine and cause Glassers disease. As the abuse of antibiotics, antimicrobial resistant G. parasuis emerged in different degrees, which brought serious threat to global economy and public health. Danofloxacin in quinolones are one of the best choices for treatment of G. parasuis infection, because of their strong bactericidal activity and good absorption into blood and great distribution in the lung. However, the clinical breakpoint (CBP) for danofloxacin against G. parasuis had not yet been established by clinical laboratory of standard Institute (CLSI) and European Commission of antimicrobial susceptibility testing (EUCAST). Our study firstly established three cutoff values of danofloxacin against G. parasuis. It suggested that epidemiological danofloxacin-resistant G. parasuis may lead to the ineffective treatment by danofloxacin.

microbiology↗

bioRxiv2021

High-quality reference genome of Fasciola gigantica: Insights into the genomic signatures of transposon-mediated evolution and specific parasitic adaption in tropical regions

Fasciola gigantica and Fasciola hepatica are causative pathogens of fascioliasis, with the widest latitudinal, longitudinal, and altitudinal distribution; however, among parasites, they have the largest sequenced genomes, hindering genomic research. In the present study, we used various sequencing and assembly technologies to generate a new high-quality Fasciola gigantica reference genome. We improved the integration of gene structure prediction, and identified two independent transposable element expansion events contributing to (1) the speciation between Fasciola and Fasciolopsis during the Cretaceous-Paleogene boundary mass extinction, and (2) the habitat switch to the liver during the Paleocene-Eocene Thermal Maximum, accompanied by gene length increment. Long interspersed element (LINE) duplication contributed to the second transposon-mediated alteration, showing an obvious trend of insertion into gene regions, regardless of strong purifying selection. Gene ontology analysis of genes with long LINE insertions identified membrane-associated and vesicle secretion process proteins, further implicating the functional alteration of the gene network. We identified 852 excretory/secretory proteins and 3300 protein-protein interactions between Fasciola gigantica and its host. Among them, copper/zinc superoxide dismutase genes, with specific gene copy number variations, might play a central role in the phase I detoxification process. Analysis of 559 single-copy orthologs suggested that Fasciola gigantica and Fasciola hepatica diverged at 11.8 Ma near the Middle and Late Miocene Epoch boundary. We identified 98 rapidly evolving gene families, including actin and aquaporin, which might explain the large body size and the parasitic adaptive character resulting in these liver flukes becoming epidemic in tropical and subtropical regions.

genomics↗

bioRxiv2021

High-resolution single-molecule long-fragment rRNA gene amplicon sequencing for uncultured bacterial and fungal communities

Although several large-scale environmental microbial projects have been initiated in the past two decades, understanding of the role of complex microbiotas is still constrained by problems of detecting and identifying unknown microorganisms1-6.Currently, hypervariable regions of rRNA genes as well as internal transcribed spacer regions are broadly used to identify bacteria and fungi within complex communities7,8, but taxonomic and phylogenetic resolution is hampered by insufficient sequencing length9-11. Direct sequencing of full length rRNA genes is currently limited by read length using second generation sequencing or sacrificed quality and throughput by using single molecule sequencing. We developed a novel method to sequence and assemble nearly full length rRNA genes using second generation sequencing.Benchmarking was performed on mock bacterial and fungal communities as well as two forest soil samples. The majority of rRNA gene sequences of all species in the mock community samples were successfully recovered with identities above 99.5% compared to the reference sequences. For soil samples we obtained exquisite coverage with identification of a large number of putative new species, as well as high abundance correlation between replicates. This approach provides a cost-effective method for obtaining extensive and accurate information on complex environmental microbial communities.

microbiology↗

bioRxiv2021

The CCL2 Chemokine Promotes Early Seeding of the Latent HIV Reservoir

HIV infects long-lived CD4 memory T cells establishing a latent viral reservoir that necessitates lifelong anti-retroviral therapy (ART). How this reservoir is formed so swiftly remains unknown. We now show the innate inflammatory response to HIV infection results in CCL2 chemokine release, which can drive recruitment of cells expressing the CCR2 receptor including a subset of central memory CD4 T cells. Supporting a role for the CCL2/CCR2 axis in rapid reservoir formation, we find 1) treatment of humanized mice with anti-CCL2 antibodies during HIV infection decreases reservoir seeding and 2) CCR2/5+ cells from the blood of HIV-infected individuals on long term ART contain significantly more provirus than CCR2/5-negative memory or naive cells. Together, these studies support a model where the hosts innate inflammatory CCL2 response to HIV infection recruits CCR2/5+ central memory CD4 T cells to zones of virus-associated inflammation likely contributing to rapid formation of the latent HIV reservoir. GRAPHICAL ABSTRACTWhy is the latent HIV reservoir established so early following infection? An innate immune response occurs during acute infection that establishes a "zone of inflammation" (step 1). The CCL2 chemokine is produced in part through IFI16 sensing of HIV DNA in abortively infected cells. CCL2 promotes rapid recruitment of CCR2/5+ memory CD4 T cells (step 2). Many of these cells become productively infected (step 3) and a fraction become latently infected (step 4). Thus, HIV hijacks the host inflammatory response to rapidly establish the latent reservoir. In support of this model, we find HIV reservoir reduction in humanized mice treated with anti-CCL2 antibodies during early infection. Further, we find that CCR2/5+ CD4 T cells harbor a substantial fraction of detectable proviruses in the blood of HIV-infected individuals on long-term suppressive ART. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=145 SRC="FIGDIR/small/432826v1_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@8d6da8org.highwire.dtl.DTLVardef@11a3a1corg.highwire.dtl.DTLVardef@dd05dborg.highwire.dtl.DTLVardef@ab84e6_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2021

Bone Marrow-Derived AXL Tyrosine Kinase Promotes Mitogenic Crosstalk and Cardiac Allograft Vasculopathy

Cardiac Allograft Vasculopathy (CAV) is a leading contributor to late transplant rejection. Although implicated, the mechanisms by which bone marrow-derived cells promote CAV remain unclear. Emerging evidence implicates the cell surface receptor tyrosine kinase AXL to be elevated in rejecting human allografts. AXL protein is found on multiple cell types, including bone marrow-derived myeloid cells. The causal role of AXL from this compartment and during transplant is largely unknown. This is important because AXL is a key regulator of myeloid inflammation. Utilizing experimental chimeras deficient in the bone marrow-derived Axl gene, we report that Axl antagonizes cardiac allograft survival and promotes CAV. Flow cytometric and histologic analyses of Axl-deficient transplant recipients revealed reductions in both allograft immune cell accumulation and vascular intimal thickness. Co-culture experiments designed to identify cell-intrinsic functions of Axl uncovered complementary cell-proliferative pathways by which Axl promotes CAV-associated inflammation. Specifically, Axl-deficient myeloid cells were less efficient at increasing the replication of both antigen-specific T cells and vascular smooth muscle cells (VSMCs), the latter a key hallmark of CAV. For the latter, we discovered that Axl-was required to amass the VSMC mitogen Platelet-Derived Growth Factor. Taken together, our studies reveal a new role for myeloid Axl in the progression of CAV and mitogenic crosstalk. Inhibition of AXL-protein, in combination with current standards of care, is a candidate strategy to prolong cardiac allograft survival.

immunology↗

bioRxiv2021

Drosophila functional screening of de novo variants in autism uncovers deleterious variants and facilitates discovery of rare neurodevelopmental diseases

Individuals with autism spectrum disorders (ASD) exhibit an increased burden of de novo variants in a broadening range of genes. We functionally tested the effects of ASD missense variants using Drosophila through humanization rescue and overexpression-based strategies. We studied 79 ASD variants in 74 genes identified in the Simons Simplex Collection and found 38% of them caused functional alterations. Moreover, we identified GLRA2 as the cause of a spectrum of neurodevelopmental phenotypes beyond ASD in eight previously undiagnosed subjects. Functional characterization of variants in ASD candidate genes point to conserved neurobiological mechanisms and facilitates gene discovery for rare neurodevelopmental diseases.

genetics↗

bioRxiv2020

Adducin Regulates Sarcomere Disassembly During Cardiomyocyte Mitosis

Recent interest in understanding cardiomyocyte cell-cycle has been driven by potential therapeutic applications in cardiomyopathy. However, despite recent advances, cardiomyocyte mitosis remains a poorly understood process. For example, it is unclear how sarcomeres are disassembled during mitosis to allow abscission of daughter cardiomyocytes. Here we identify adducin as a regulator of sarcomere disassembly during mammalian cardiomyocyte mitosis. /{gamma}-adducins are selectively expressed in neonatal mitotic cardiomyocytes, and their levels decline precipitously thereafter. Cardiomyocyte-specific overexpression of various splice isoforms and phosphoforms of -adducin in-vitro and in-vivo identified Thr445/Thr480 phosphorylation of a short isoform of adducin as a potent inducer of neonatal cardiomyocyte sarcomere disassembly. Concomitant overexpression of this -adducin variant along with {gamma}-adducin resulted in stabilization of the adducin complex and persistent sarcomere disassembly in adult mice, which is mediated by interaction with -actinin. These results highlight an important mechanism for coordination of cytoskeletal morphological changes during cardiomyocyte mitosis.

cell biology↗

bioRxiv2020

Integrative Analysis of Phenomic, Genomic, and Transcriptomic to Identify Potential Functional Genes of Yaks in Plain and Plateau

BackgroundThe yak is an important source of livelihood for the people living in the Qinghai-Tibet Plateau. Most genetics detection studies have focused on the comparison between different tissues of different breeds, both living in the Plateau and in the plains. The genetic background and complex regulatory relationship have frequently puzzled researchers. In this study, we divided a population of 10 yaks into two subgroups, namely Plateau (living in the Plateau) and Plain (living in the plains). Phenomic, genomic, and transcriptomic analyses were used to reveal the molecular genetic type in the body weight, slaughter, and beef quality of yaks. ResultsWe found a significant difference (P <0.01) between the third (60 days), fourth (90 days), fifth (120 days), and sixth (150 days) weights of Plateau and plain subpopulations. The difference in body weight was due to differences in kidney weight, meat weight, fur weight, and head weight. However, the beef quality was not significantly different. We identified 540 Differentially Expressed Genes (DEGs). Using weighted gene co-expression network analysis (WGCNA), we have constructed a co-express network, and the modules were strongly related to traits. In the genome-wide association studies (GWAS), we detected significant 156, 52, 33, 15, and 3 signals in the meat weight, head weight, fur weight, liver weight, and the last body weight traits. Based on the epigenome-wide association studies (eWAS) results, we created a link relationship between the DEGs expression level and genotype. ConclusionIn summary, our study demonstrated the effectiveness and representative of multidimensional data from a finite number of yak populations. The study highlights the underlying way, as well as a related network, to yield new information on genome genetics and pathogen-host interactions of both the living Plateau and plain yak populations.

genomics↗

bioRxiv2020

A core of functionally complementary bacteria colonizes oysters in Pacific Oyster Mortality Syndrome.

BackgroundThe Pacific oyster Crassostrea gigas is one of the main cultivated invertebrate species worldwide. Since 2008, oyster juveniles have been confronted with a lethal syndrome known as the Pacific Oyster Mortality Syndrome (POMS). POMS is a polymicrobial disease initiated by a primary infection with the herpesvirus OsHV-1 Var that creates an oyster immunocompromised state and evolves towards a secondary fatal bacteremia. In the present article, we describe the implementation of an unprecedented combination of metabarcoding and metatranscriptomic approaches to show that the sequence of events in POMS pathogenesis is conserved across infectious environments. We also identified a core bacterial consortium which, together with OsHV-1 Var, forms the POMS pathobiota. This bacterial consortium is characterized by high transcriptional activities and complementary metabolic functions to exploit hosts resources. A significant metabolic specificity was highlighted at the bacterial genus level, suggesting low competition for nutrients between members of the core bacteria. Lack of metabolic competition might favor complementary colonization of host tissues and contribute to the conservation of the POMS pathobiota across distinct infectious environments.

microbiology↗

bioRxiv2020

Brain-Restricted mTOR Inhibition with Binary Pharmacology

On-target-off-tissue drug engagement is an important source of adverse effects that constrains the therapeutic window of drug candidates. In diseases of the central nervous system, drugs with brain-restricted pharmacology are highly desirable. Here we report a strategy to achieve inhibition of mTOR while sparing mTOR activity elsewhere through the use of a brain-permeable mTOR inhibitor RapaLink-1 and brain-impermeable FKBP12 ligand RapaBlock. We show that this drug combination mitigates the systemic effects of mTOR inhibitors but retains the efficacy of RapaLink-1 in glioblastoma xenografts. We further present a general method to design cell-permeable, FKBP12-dependent kinase inhibitors from known drug scaffolds. These inhibitors are sensitive to deactivation by RapaBlock enabling the brain-restricted inhibition of their respective kinase targets.

cancer biology↗

bioRxiv2020

Implantable photonic neural probes for light-sheet fluorescence brain imaging

SignificanceLight-sheet fluorescence microscopy is a powerful technique for high-speed volumetric functional imaging. However, in typical light-sheet microscopes, the illumination and collection optics impose significant constraints upon the imaging of non-transparent brain tissues. Here, we demonstrate that these constraints can be surmounted using a new class of implantable photonic neural probes. AimMass manufacturable, silicon-based light-sheet photonic neural probes can generate planar patterned illumination at arbitrary depths in brain tissues without any additional micro-optic components. ApproachWe develop implantable photonic neural probes that generate light sheets in tissue. The probes were fabricated in a photonics foundry on 200 mm diameter silicon wafers. The light sheets were characterized in fluorescein and in free space. The probe-enabled imaging approach was tested in fixed and in vitro mouse brain tissues. Imaging tests were also performed using fluorescent beads suspended in agarose. ResultsThe probes had 5 to 10 addressable sheets and average sheet thicknesses < 16 m for propagation distances up to 300 m in free space. Imaging areas were as large as {approx} 240 m x 490 m in brain tissue. Image contrast was enhanced relative to epifluorescence microscopy. ConclusionsThe neural probes can lead to new variants of light-sheet fluorescence microscopy for deep brain imaging and experiments in freely-moving animals.

bioengineering↗

bioRxiv2020

Autophagy Compensates for Lkb1 Loss to Maintain Adult Mice Homeostasis and Survival

Liver Kinase B1 (LKB1) is the major energy sensor for cells to respond to metabolic stress. Autophagy degrades and recycles proteins, macromolecules, and organelles for cells to survive starvation. To access the role and cross-talk between autophagy and Lkb1 in normal tissue homeostasis, we generated genetically engineered mouse models where we can conditionally delete Lkb1 and autophagy essential gene, Atg7, respectively or simultaneously, throughout the adult mice. We found that Lkb1 was essential for the survival of adult mice, and autophagy activation could temporarily compensate for the acute loss of Lkb1 and extend mouse life span. We further found that acute deletion of Lkb1 in adult mice led to impaired intestinal barrier function, hypoglycemia, and abnormal serum metabolism, which was partly rescued by the Lkb1 loss-induced autophagy upregulation via inhibiting p53 induction. Taken together, we demonstrated that autophagy and Lkb1 work synergistically to maintain adult mouse homeostasis and survival.

cell biology↗

bioRxiv2020

Unraveling transcription factor functions through integrative inference of transcriptional networks in Arabidopsis thaliana yields novel regulators involved in reactive oxygen species stress signaling

Gene regulation is a dynamic process in which transcription factors (TFs) play an important role to control spatiotemporal gene expression. While gene regulatory networks describe the interactions between TFs and their target genes, our global knowledge about the complexity of TF control for different genes and biological processes is incomplete. To enhance our global understanding of regulatory interactions in Arabidopsis thaliana, different regulatory input networks capturing complementary information about DNA motifs, open chromatin, TF binding and expression-based regulatory interactions, were combined using a supervised learning approach, resulting in an integrated gene regulatory network (iGRN) covering 1,491 TFs and 31,393 target genes (1.7 million interactions). This iGRN outperforms the different input networks to predict known regulatory interactions and has a similar performance to recover functional interactions compared to state-of-the-art experimental methods like yeast one-hybrid and ChIP-seq. The iGRN correctly inferred known functions for 681 TFs and predicted new gene functions for hundreds of unknown TFs. For regulators predicted to be involved in reactive oxygen species stress regulation, we confirmed in total 75% of TFs with a function in ROS and/or physiological stress responses. This includes 13 novel ROS regulators, previously not connected to any ROS or stress function, that were experimentally validated in our ROS-specific phenotypic assays of loss- or gain-of-function lines. In conclusion, the presented iGRN offers a high-quality starting point to enhance our understanding of gene regulation in plants by integrating different experimental data types at the network level.

plant biology↗